ILT7-targeted antibodies and their use

Abstract

Disclosed herein are anti-ILT7 antibodies and antigen-binding fragments, polynucleotides encoding said antibodies and antigen-binding fragments, and pharmaceutical compositions comprising said 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

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to International Patent Application PCT / CN2023 / 082006, filed on 16 March 2023, the entire disclosure thereof incorporated in whole by reference for all purposes.

[0002] Reference to electronically submitted sequence listings This application incorporates, by reference, an array listing as an XML file titled "110961-1435499-018-001-02PCT", created on March 15, 2024, and having a size of 65,204 bytes.

[0003] field This invention relates to molecular biology, cell biology, and immunology. Provided herein are anti-ILT7 antibodies and their use in the treatment of plasmacytoid dendritic cells (pDCs) or type I interferon (type I IFN)-associated immunological disorders. [Background technology]

[0004] background Plasmacytoid dendritic cells (pDCs), which are involved in the production of type I interferons (IFNs) and pro-inflammatory cytokines, are drivers of both innate and adaptive immune responses. Both pDCs and type I IFNs are involved in multiple immunological disorders. ILT7, a member of the immunoglobulin-like transcript (ILT) or leukocyte immunoglobulin-like receptor (LIR) gene family, is selectively expressed in pDCs. Therefore, for example, to treat and prevent autoimmune diseases, there is a need for ILT7 targeting agents that can suppress the release of pDC-associated type I IFNs. However, success in the development of such ILT7 targeting agents has been limited. The compositions and methods provided herein meet these needs and offer relative advantages. [Overview of the project]

[0005] overview As used herein, the terms “invention,” “the invention,” “this invention,” and “the present invention” are intended to broadly refer to the subject matter of this patent application and all of the following claims. Any statements containing these terms should be understood not to limit the subject matter described herein, nor to restrict the meaning or scope of the following claims. The embodiments covered by the invention are defined by the claims, not by the summary of this invention. The summary of this invention is an overview of various aspects of the invention and introduces some of the concepts described and illustrated herein and in the accompanying drawings. The summary of this invention is not intended to identify any important 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. The subject matter should be understood by referring to the entirety of this specification, any or all of the drawings, and the appropriate portion of each claim. Some of the exemplary embodiments of the invention are described below.

[0006] Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human ILT7, wherein the antibody or antigen-binding fragment is (1) 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 NOs. 11, 12, and 13, respectively, or a variant thereof having up to 5 amino acid substitutions, additions, and / or deletions in the VL CDR, 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 NOs. 14, 15, and 16, respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VH CDR, or (2) defined by Chothia, (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, or VL A variant thereof having up to five amino acid substitutions, additions, and / or deletions in the CDR, and / or (b) a VH comprising 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 approximately five amino acid substitutions, additions, and / or deletions in the VH CDR.

[0007] In some embodiments, the antibody or antigen-binding fragments provided herein include 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. In some embodiments, the antibody or antigen-binding fragments provided herein include 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.

[0008] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human ILT7, and the antibody or antigen-binding fragment comprises (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. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a VL and a VH having the amino acid sequences of SEQ ID NO: 9 and 10, respectively.

[0009] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human ILT7, and these antibodies or antigen-binding fragments include (a) VL comprising VL CDR1, VL CDR2, and VL CDR3 from VL having the amino acid sequence of SEQ ID NO: 9, and / or (b) VH comprising VH CDR1, VH CDR2, and VH CDR3 from VH having the amino acid sequence of SEQ ID NO: 10.

[0010] 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.

[0011] 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) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to an amino acid sequence selected from the group consisting 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28.

[0012] In some embodiments, the humanized anti-ILT7 antibody or antigen-binding fragment is (1) SEQ ID NOs. 19 and 23, respectively, (2) SEQ ID NOs. 19 and 24, respectively, (3) SEQ ID NOs. 19 and 25, respectively, (4) SEQ ID NOs. 19 and 26, respectively, (5) SEQ ID NOs. 19 and 27, respectively, (6) SEQ ID NOs. 19 and 28, respectively, (7) SEQ ID NOs. 20 and 23, respectively, (8) SEQ ID NOs. 20 and 24, respectively, (9) SEQ ID NOs. 20 and 25, respectively, (10) SEQ ID NOs. 20 and 26, respectively, (11) SEQ ID NOs. 20 and 27, respectively, (12) SEQ ID NOs. 20 and 28, respectively. (13) VL and VH having the amino acid sequences of SEQ ID NOs. 21 and 23 respectively, (14) SEQ ID NOs. 21 and 24 respectively, (15) SEQ ID NOs. 21 and 25 respectively, (16) SEQ ID NOs. 21 and 26 respectively, (17) SEQ ID NOs. 21 and 27 respectively, (18) SEQ ID NOs. 21 and 28 respectively, (19) SEQ ID NOs. 22 and 23 respectively, (20) SEQ ID NOs. 22 and 24 respectively, (21) SEQ ID NOs. 22 and 25 respectively, (22) SEQ ID NOs. 22 and 26 respectively, (23) SEQ ID NOs. 22 and 27 respectively, or (24) SEQ ID NOs. 22 and 28 respectively.

[0013] 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.

[0014] In some embodiments, the anti-ILT7 antibody provided herein is an IgG1 antibody. In some embodiments, the anti-ILT7 IgG1 antibody provided herein includes a light chain constant region (CL) having at least 85% sequence identity with kappa CL (Cκ, SEQ ID NO: 29). In some embodiments, the anti-ILT7 IgG1 antibody provided herein includes a light chain constant region (CL) having at least 85% sequence identity with lambda CL (Cλ, SEQ ID NO: 30).

[0015] In some embodiments, the anti-ILT7 IgG1 antibodies provided herein include a heavy chain constant region (CH) having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 and 40-44.

[0016] In some embodiments of the anti-ILT7 IgG1 antibodies provided herein, the heavy chain constant region (CH) contains either a wild-type IgG1 CH or 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 in 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. In some embodiments, the CH region of the IgG1 antibody provided herein has amino acid substitutions that are 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. Anti-ILT7 antibody provided herein In some embodiments of the IgG1 antibody, the CH region is numbered according to the EU index: (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; and (xi)D270E, K326D, A330M, and K334E are used to modify the amino acid substitutions selected from the group. In some embodiments, the CH region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-64.

[0017] In some embodiments of the anti-ILT7 IgG1 antibodies provided herein, Fc is defucosylated.

[0018] Provided herein are antibodies or antigen-binding fragments that compete with the antibodies or antigen-binding fragments described herein for binding to human ILT7.

[0019] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a bispecific or multispecific antibody.

[0020] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a monoclonal antibody or an antigen-binding fragment thereof.

[0021] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is (1) measured by SPR, with a K content of 500 nM or less. 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, and LILB5, (3) inhibits interferon alpha (IFNα) release by 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) to (7).

[0022] Also provided herein are anti-ILT7 antibodies or their antigen-binding fragments, (1) measured by SPR, with a K content of 500 nM or less. 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, and LILB5, (3) inhibits IFNα release by 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) to (7).

[0023] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein may (1) inhibit IFNα release by CpG-stimulated PBMCs in vitro with an EC50 of 1 nM or less, (2) exhibit NK-dependent ADCC activity against ILT7-expressing cells with an EC50 of 0.01 nM or less, (3) exhibit neutrophil-dependent ADCC activity against ILT7-expressing cells with an EC50 of 100 nM or less, (4) exhibit macrophage-dependent ADCP activity against ILT7-expressing cells with an EC50 of 10 nM or less, or (5) exhibit macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index of 20% or more, or any combination of (1) to (5).

[0024] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein may (1) inhibit IFNα release by PBMCs at an EC50 in the range of 0.01 nM to 0.1 nM, (2) exhibit NK-dependent ADCC activity against ILT7-expressing cells at an EC50 in the range of 0.001 nM to 0.01 nM, (3) exhibit neutrophil-dependent ADCC activity against ILT7-expressing cells at an EC50 in the range of 1 nM to 50 nM, (4) exhibit macrophage-dependent ADCP activity against ILT7-expressing cells at an EC50 in the range of 0.5 nM to 5 nM, or (5) exhibit macrophage-dependent ADCP activity against ILT7-expressing cells at a maximum phagocytic index in the range of 20% to 80%, or any combination of (1) to (5).

[0025] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein exhibits neutrophil-dependent ADCC activity.

[0026] Also provided herein are polynucleotides encoding polypeptides of anti-ILT7 antibodies or antigen-binding fragments provided herein. Also provided herein are vectors containing the polynucleotides described herein.

[0027] Also provided herein are host cells containing the polynucleotides or vectors described herein. In some embodiments, the host cells described herein are (1) overexpressing N-acetylglucosaminyltransferase III (GnTIII), (2) lacking α-1,6-fucosyltransferase (FUT8), or (3) having a low fucose content, or any combination of (1) to (3).

[0028] Also provided herein are methods for producing the anti-ILT7 antibody or antigen-binding fragment described herein, the methods comprising culturing the host cells described herein under conditions that enable the expression of the antibody or antibody fragment. In some embodiments, the methods provided herein include isolating the antibody from the culture.

[0029] Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein and a pharmaceutically acceptable carrier.

[0030] Also provided herein are methods for reducing type I interferon (IFN) levels in subjects where such reduction is necessary, the methods comprising administering to the subject a therapeutically effective dose of an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the type I interferon is IFNα.

[0031] Also provided herein is a method for suppressing or depleting pDCs in subjects where such suppression or depletion is required, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein.

[0032] Also provided herein is a method for reducing autoimmunity in subjects that require such reduction, the method comprising administering to the subject an effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein.

[0033] In some embodiments of the methods provided herein, the subjects have an autoimmune disease.

[0034] Also provided herein is a method for treating an autoimmune disease associated with type I IFN or pDC in a subject who needs treatment, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein.

[0035] In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE).

[0036] In some embodiments, the methods provided herein further include administering an additional therapy to the subject.

[0037] In some embodiments of the methods provided herein, the subject is a human.

[0038] In some embodiments, provided herein is the use of the anti-ILT7 antibody or antigen-binding fragment described herein in reducing type I IFN. Also provided herein is the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the preparation of pharmaceuticals for reducing type I IFN. In some embodiments of the use provided herein, type I IFN is IFNα.

[0039] In some embodiments, the use of the anti-ILT7 antibody or antigen-binding fragment described herein in the suppression or depletion of pDCs is provided herein. Also provided herein is the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the preparation of a pharmacopoeia for suppressing or depleting pDCs.

[0040] In some embodiments, the use of the anti-ILT7 antibody or antigen-binding fragment described herein in reducing autoimmunity is provided herein. In some embodiments, the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the preparation of a pharmaceutical for reducing autoimmunity is provided herein.

[0041] In some embodiments, the use of the anti-ILT7 antibody or antigen-binding fragment described herein in treating an autoimmune disease associated with type I IFN or pDC. In some embodiments, the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the preparation of a medicament for treating an autoimmune disease associated with type I IFN or pDC. In some embodiments, the autoimmune disease is SLE. [Brief explanation of the drawing]

[0042] [Figure 1]This paper provides ELISA results showing the binding of an ILT7 chimeric antibody, a reference antibody (Tab1), and a negative control antibody (hIgG1) to the human ILT7 protein. [Figure 2A] Figures 2A and 2B provide flow cytometry results showing the binding of ILT7 chimeric antibody, reference antibody (Tab1), and negative control antibody (hIgG1) to 293F-human ILT7 cells (Figure 2A) and CHOK1-cynomolgus monkey ILT7 cells (Figure 2B). [Figure 2B] Please refer to the explanation in Figure 2A. [Figure 3] This report presents representative results from an IFNα release assay in CpG-stimulated PBMCs. The inhibitory activity of an ILT7 chimeric antibody against IFNα release was measured, and results from both a reference antibody (Tab1) and a negative control antibody (hIgG1) are also shown. [Figure 4A] Figures 4A-4B provide flow cytometry results showing the binding of the humanized antibody cmAb12 (Hu12) and the reference antibody (Tab1) to 293F-human ILT7 cells (Figure 4A) and CHOK1-cynomolgus monkey ILT7 cells (Figure 4B). [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 5A] Figures 5A–5B show representative results of the IFN-α release assay in CpG-stimulated PBMCs. The inhibitory activity of four humanized antibodies against IFN-α release was measured for cmAb12, as well as for cmAb12, the reference antibody (Tab1), and the negative control antibody (hIgG1). [Figure 5B] Please refer to the explanation in Figure 5A. [Figure 6] This report provides representative results from ELISA measurements of the binding affinity of the hu-cmAb12 antibody, reference antibody (Tab1), and negative control antibody (hIgG1) to the human ILT7 protein. [Figure 7A] Figures 7A-7B provide representative flow cytometry results showing the binding of hu-cmAb12, a reference antibody (Tab1), and a negative control antibody (isotype) to 293F-human ILT7 cells (Figure 7A) and CHOK1-cynomolgus monkey ILT7 cells (Figure 7B). [Figure 7B] Please refer to the explanation in Figure 7A. [Figure 8] This provides representative flow cytometry results showing the binding of hu-cmAb12, a reference antibody (Tab1), and a negative control antibody (isotype) to cell surface ILT7 on pDCs and other immune cells in human PBMCs. [Figure 9A] Figures 9A-9B show representative results from IFNα release assays on CpG-stimulated PBMCs (from four donors) (graph and summary table, respectively). The inhibitory activity of hu-cmAb12 and the reference antibody (Tab1) on IFNα release was measured. [Figure 9B] Please refer to the explanation in Figure 9A. [Figure 10] This report provides representative results from a cytotoxic activity assay demonstrating NK cell-dependent ADCC activity of hu-cmAb12, a reference antibody (Tab1), and a negative control antibody (isotype). [Figure 11] This report provides representative results from a cytotoxic activity assay demonstrating neutrophil-dependent ADCC activity of hu-cmAb12, a reference antibody (Tab1), and a negative control antibody (isotype). [Figure 12A] Figures 12A-12B provide representative results of phagocytic assays showing macrophage-dependent ADCP activity for hu-cmAb12, a reference antibody (Tab1), and a negative control antibody (isotype) in three donors (graph and summary table, respectively). [Figure 12B] Please refer to the explanation in Figure 12A. [Figure 13A] Figures 13A-13B provide representative results of blood cell changes in humanized mice (Figure 13A) and cynomolgus monkeys (Figure 13B) after administration of hu-cmAb12, negative control antibody (isotype), or PBS infusion. [Figure 13B] Please refer to the explanation in Figure 13A. [Modes for carrying out the invention]

[0043] Detailed explanation This disclosure provides a novel antibody comprising an antigen-binding fragment that specifically binds to ILT7 (e.g., human ILT7). Pharmaceutical compositions comprising a therapeutically effective amount of such antibody or antigen-binding fragment are also disclosed herein. Also disclosed herein is the use of such pharmaceutical compositions for the treatment of autoimmune diseases associated with plasmacytoid dendritic cells (pDCs) and / or type I interferon (IFN).

[0044] pDCs are a subpopulation of dendritic cells (DCs) in peripheral blood and secondary lymphoid organs. Although they constitute only about 0.1–0.8% of peripheral blood mononuclear cells (PBMCs), these cells are drivers of both innate and adaptive immune responses. pDCs enhance the innate immune response 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 natural killer (NK) cell recruitment, activation, and cytotoxicity. pDCs also promote the adaptive immune response by facilitating antigen presentation, supporting the activation and expansion of antigen-specific CD4+ Th cells, driving CD4+ T cells to differentiate into Th2 and Treg cells, promoting CD8+ T cell survival and activity, and improving B cell survival, maturation, differentiation, and autoantibody production.

[0045] Importantly, pDCs are a major source of type I IFN(α / β), which promotes the function of NK cells, B cells, T cells, and bone marrow DCs. Both pDCs and type I IFN are known to be involved in the development of immunological disorders such as autoimmune diseases. See, 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.

[0046] Immunoglobulin-like transcript-7 (ILT7), also known as leukocyte immunoglobulin-like receptor A4 (LIRA4 or LILRA4) or CD85g, 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 important for interacting with the ILT7 ligand, bone marrow stromal cell antigen 2 (BST2), and the transmembrane domain of ILT7 contains positively charged residues that allow it to bind to FcεRIγ and inhibit pDC function via the ITAM-mediated signaling pathway.

[0047] 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 the matured protein), an extracellular domain (amino acids 24-446), a transmembrane domain (amino acids 447-467), and a cytoplasmic domain (amino acids 468-499). The extracellular domain contains four immunoglobulin-like C2 domains (amino acids 24-118, 123-213, 224-313, and 324-413). MTLILTSLLFFGLSLGPRTRVQAENLPKPILWAEPGPVITWHNPVTIWCQGTLEAQGYRLDKEGNSMSRHILKTLESENKVKLSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPT LSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLNSHQHNHGKFQALFPMGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVTPGENLTLQCGSDVG YIRYTLYKEGADGLPQRPGRQPQAGLSQANFTLSPVSRSYGGQYRCYGAHNVSSEWSAPSDPLDILIAGQISDRPSLSVQPGPTVTSGEKVTLLCQSWDPMFTFLLTKEGAAHPPLRLRSMYGAH KYQAEFPMSPVTSAHAGTYRCYGSRSSNPYLLSHPSEPLELVVSGATETLNPAQKKSDSKTAPHLQDYTVENLIRMGVAGLVLLFLGILLFEAQHSQRSPPRCSQEANSRKDNAPFRVVEPWEQI (Sequence ID 1)

[0048] Further information on human ILT7 can be found in public databases under the following IDs: HGNC:15503, NCBI Entrez Gene:23547, Ensembl:ENSG00000239961, OMIM(registered trademark):607517, UniProtKB / Swiss-Prot:Q8IZF0. Two alternatively spliced ​​transcriptional variants encoding different isoforms of the human ILT7 gene have been described (Uniprot NO:P59901-1, P59901-2).

[0049] The sequence of ILT7 in cynomolgus monkeys is provided below. MTPILTTLLCFGLSLGPRTCLQAENLLKPILWAEPGPIIWKKPVTIWCQGTLEAQEYRLDKEGNSMLRHMLKTLESENKAKFSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPS LSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLDSHQHNHGKFQALFPVGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVAPGDNLTLQCGSDVG YIRYALYKEGGDGLPQRPGQQSQAGLSQASFTLNPVRGSHGQYRCYGAHNVSSKWSAPSDPLDILIAGQIPDRPSLSVQLGPTVASGEKVTLLCQSWGPMFTFLLAKEGAAHPPLRLRSTYRAQ QYQAEFPMSPVTSAHAGTYRCYGSRSSDPYLLSHSSEPLELVVSEATETLNPAQNKSDSKTAPHLQDYTVENLIRMGIAGLVLVFLGILLFEAQQSQRSPTRCSQEVNSREDNAPFRVVEPWEQI (Sequence 2)

[0050] ILT7 is selectively expressed on the surface of human pDCs but not on myeloid DCs or other peripheral blood leukocytes. ILT7 transcripts are minimally detectable in most human tissues but are moderately enriched in lymphoid organs where pDCs are present. While not theoretically bound, the anti-ILT7 antibodies or antigen-binding fragments provided herein are useful for reducing pDC activity via NK / neutrophil-mediated ADCC (antibody-dependent cytotoxicity) and / or macrophage-mediated ADCP (antibody-dependent phagocytosis), and therefore are useful, for example, in the treatment and prevention of autoimmune diseases.

[0051] Before further description of this disclosure, it should be understood that this disclosure is not limited to the specific embodiments shown herein, and the terminology used herein is intended to describe, and not limit, specific embodiments.

[0052] A.Definition Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms. Generally, the terminology and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art.

[0053] The term "a" or "an" entity refers to one or more of those entities; for example, "antibody" is understood to represent one or more antibodies.

[0054] Where used herein, the term "and / or" should be interpreted as a specific disclosure of each of two designated features or components, with or without the other. Accordingly, where "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, where "and / or" is used in phrases such as "A, B, and / or C," it is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0055] As used herein, the term “approximately” is used to indicate that a value includes the inherent variation of the device’s error, the method used to determine the value, or the variation present among the subjects of study. The term “approximately” encompasses the exact numbers listed. In some embodiments, “approximately” means within plus or minus 10% of a given value or range. In certain embodiments, “approximately” means that the variation is ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that “approximately” refers to. In some embodiments, “approximately” means that the variation is ±1%, ±0.5%, ±0.2%, or ±0.1% of the value that “approximately” refers to.

[0056] As used herein, the term “antibody” and its grammatical equivalent refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, via at least one antigen-binding site, the antigen-binding site of which is typically located within the variable region of the immunoglobulin molecule. As used herein, this term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAb; e.g., camel antibodies, alpaca antibodies), single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAb), light-chain antibodies (LCAb), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecules, including those with an antigen-binding site (e.g., a bivariable-domain immunoglobulin molecule), as long as 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. An antibody 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), are based on the identity of their heavy chain constant domains, which are called alpha, delta, epsilon, gamma, and mu, respectively. Unless otherwise expressly indicated, the term “antibody” as used herein includes the “antigen-binding fragment” of an intact antibody. The term “antigen-binding fragment” as used herein refers to a portion or fragment of an intact antibody that is the antigen-determining variable region of the intact 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-bonded scFv (dsscFv), diabodies, tribodies, tetrabodies, minibodies, bivariable-domain antibodies (DVD), single-variable-domain antibodies (sdAb; e.g., camel antibody, alpaca antibody), and single-variable-domains of heavy-chain antibodies (VHH), as well as bispecific or multispecific antibodies formed from antibody fragments.A "bispecific" antibody is an artificial hybrid antibody that has two different antigen-binding sites that recognize and specifically bind to two different targets. Bispecific antibodies can be produced by various methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990) and Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0057] As used herein, the term “humanized antibody” refers to a form of non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof containing minimal non-human sequences. Typically, a humanized antibody is a human immunoglobulin. In some cases, Fv framework region residues of a human immunoglobulin are replaced with corresponding residues in an antibody derived from a non-human species. In some cases, CDR residues are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, hamster, camel) that have the desired specificity, affinity, and / or binding ability. Humanized antibodies can be further modified by substitution of any further residues within the Fv framework region and / or within the replaced non-human residues to purify and optimize antibody specificity, affinity, and / or binding ability. As used herein, the term “human antibody” refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to a human-produced antibody made using any of the techniques known in the art.

[0058] When used in relation to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50–70 kDa, with an amino-terminal portion containing a variable region of approximately 120–130 or more amino acids and a carboxy-terminal portion containing a constant region. The constant region can be one of five distinct types, designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. The different heavy chains differ in size, with α, δ, and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. When combined with a light chain, these different types of heavy chains produce five well-known classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each containing four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0059] When used in relation to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, with an amino-terminal region containing a variable region of approximately 100 to 110 or more amino acids and a carboxy-terminal region containing 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 lambda (λ) and kappa (κ). Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0060] The term “variable domain” or “variable region” generally refers to a portion of the light or heavy chain of an antibody, located at the amino terminus of the light or heavy chain, having a length of approximately 120–130 amino acids in the heavy chain and approximately 100–110 amino acids in the light chain, and used in the binding and specificity of each particular antibody to its particular antigen. Variable domains differ significantly in sequence between different antibodies. Sequence variability is concentrated in the CDR, while less variable portions within the variable domain are referred to as framework regions (FRs). The CDRs of the light and heavy chains are primarily involved in antibody-antigen interactions. The amino acid position numbering used herein follows the EU index, as in Kabat et al. (1991) Sequences of proteins of immunological interest. (USD Department of Health and Human Services, Washington, DC) 5th ed. Variable regions may be human variable regions.

[0061] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. Therefore, CDRs are variable region sequences scattered within the framework region sequence. CDR regions are well known to those skilled in the art and are defined by various methods / systems. These systems and / or definitions have been developed and improved over many years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defines most hypervariable regions within the antibody variable (V) domain (Kabat et al, J. Biol. Chem. 252:6609-6616 (1977), Kabat, Adv. Prot. Chem. 32:1-75 (1978)). Chothia's definition is based on the position of the structural loop region, defining the CDR region sequence as residues that are not part of the conserved β-sheet framework, and therefore different conformations can be adapted (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). Both terms are well recognized in the art. Additionally, the IMGT system is based on the sequence variability and position within the structure of 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 for antibody sequence analysis and CDR determination (e.g., abYsis) are available and known to those skilled in the art. The position of the CDR within the canonical antibody variable domain has been determined by comparing numerous structures (Al-Lazikani et al., J.Mol.Biol.273:927-948 (1997), Morea et al., Methods 20:267-279 (2000)). Since the number of residues in the hypervariable region varies with different antibodies, additional residues relative to the canonical position are traditionally numbered a, b, c, etc., next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., above (1997)).Such nomenclature is equally well known to those skilled in the art.

[0062] For example, the following table shows CDRs defined according to either the Kabat (highly variable) or Chothia (structure) designation. JPEG2026510891000002.jpg55170

[0063] One or more CDRs can also be incorporated into a molecule either covalently or noncovalently to form an immunoadhesin. The immunoadhesin can incorporate the CDR as part of a larger polypeptide chain, covalently to another polypeptide chain, or noncovalently. The CDR enables the immunoadhesin to bind to a specific antigen of interest. The CDR region can be analyzed, for example, by the abysis website (abysis.org).

[0064] The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to a site on the surface of a target molecule to which an antibody or antigen-binding fragment binds, for example, a localized region on the surface of an antigen. Target molecules may include proteins, peptides, nucleic acids, carbohydrates, or lipids. An immunogenic epitope is a portion of a target molecule that elicits an immune response in an animal. An antigenic epitope of a target molecule is a portion of the target molecule to which an antibody binds, as determined by any method well known in the art, including immunoassays. Antigenic epitopes do not necessarily have to be immunogenic. Epitopes often consist of a chemically active surface classification of a molecule, such as an amino acid or sugar side chain, and have specific three-dimensional structural properties as well as specific charge properties. The term “epitope” includes linear epitopes and conformational epitopes. The region of the target molecule (e.g., polypeptide) contributing to an epitope may be a sequence of amino acids in the polypeptide, or the epitope may be formed from two or more discontinuous regions of the target molecule. Epitopes may or may not be three-dimensional surface features of a target molecule. Epitopes formed from continuous amino acids (also called linear epitopes) are typically retained during protein denaturation, while epitopes formed by tertiary folding (also called structural epitopes) are typically lost during protein denaturation. Epitopes typically contain at least three, more commonly, at least five, six, seven, or eight to ten amino acids within their unique spatial conformation.

[0065] As used herein, the term “specifically binds” means that a polypeptide or molecule interacts with an epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or in some combination of the above, than alternative substances, including related and unrelated proteins. A binding site (e.g., an antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified, for example, by immunoassays, ELISA, biolayer interference ("BLI"), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, a particular reaction is at least twice the background signal or noise and can be more than 10 times the background. For example, see Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York, at pages 332–336 for a discussion regarding antibody specificity. A binding site that specifically binds to a target molecule can bind to the target molecule with a higher affinity than its affinity to other molecules. In some embodiments, the binding moiety that specifically binds to the target molecule can bind to the target molecule with an affinity at least 20, 30, 40, 50, 60, 70, 80, 90, or at least 100 times greater than its affinity to a different molecule. In some embodiments, the binding moiety that specifically binds to a particular target molecule binds to a different molecule with such a low affinity that the binding cannot be detected using assays described herein or otherwise known in the art. In some embodiments, "specifically binds" means, for example, that the binding moiety has a K content of about 0.1 mM or less. D This means binding to a molecular target. In some embodiments, "specifically binding" means that the polypeptide or molecule has a K content of about 10 μM or less or about 1 μM or less. Dmeans binding to a target. In some embodiments, "specifically binds" means that a polypeptide or molecule has a K of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less D means binding to a target. Due to sequence identity between homologous proteins in different species, specific binding can include polypeptides or molecules that recognize proteins or targets in two or more species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include polypeptides or molecules that recognize two or more proteins or targets. In some embodiments, it is understood that a binding moiety (e.g., an antibody) that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding, i.e., binding to a single target (although it can include it). Thus, a binding moiety (e.g., an antibody) can specifically bind to two or more targets in some embodiments. For example, an antibody can, in certain instances, include two identical antigen-binding sites that each specifically bind to the same epitope on two or more proteins. In certain alternative embodiments, an antibody can be bispecific and include at least two antigen-binding sites with different specificities.

[0066] As used herein, the term "binding affinity" generally refers to the overall strength of the non-covalent binding interactions between a binding moiety and a target molecule (e.g., an antigen). The binding between a binding moiety and a target molecule is a reversible process, and the binding affinity is typically reported as the equilibrium dissociation constant (K D ). K D is the ratio of the dissociation rate (k off or k d ) to the association rate (k on or k a ). The lower the K D of a binding pair, the higher the affinity. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present disclosure. Specific exemplary embodiments include the following. In some embodiments, "KD " or "K D The value can be measured by assays known in the art, such as binding assays. D This may also be measured in a radiolabeled antigen binding assay (RIA) (Chen, et al., (1999) J. Mol Biol 293:865-881). K D or K D The values ​​can also be measured using biolayer interferometry (BLI) with a system such as the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). D or K D The value can also be measured using a surface plasmon resonance assay (SPR) with Biacore, for example, using BIAcore(trademark)-2000 or BIAcore(trademark)-3000 (BIAcore, Inc., Piscataway, NJ). Binding affinity is the concentration of the ligand in which half of the target is bound in the binding assay, EC 50 It can also be quantified using this method.

[0067] Where used interchangeably herein, the terms “polypeptide,” “peptide,” and “protein,” and their grammatical equivalents, refer to polymers of amino acids of any length, which may be linear or branched. They may contain unnatural or modified amino acids, or may be interrupted by non-amino acids. Polypeptides, peptides, or proteins may also be modified, for example, by the formation of disulfide bonds, glycosylation, lipid addition, acetylation, phosphorylation, or any other operation or modification.

[0068] As used herein with respect to a protein or polypeptide having specific sequence features ("reference protein" or "reference polypeptide"), the term "variant" refers to a different protein or polypeptide having one or more amino acid substitutions, deletions, and / or additions (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, etc.) compared to the reference protein or reference polypeptide. Changes to the amino acid sequence may be amino acid substitutions. Changes to the amino acid sequence may be conserved amino acid substitutions. Changes to the amino acid sequence may be amino acid deletions. A variant may be a fragment of the reference protein or polypeptide. Functional variants of a protein or polypeptide maintain the basic structural and functional properties of the reference protein or polypeptide.

[0069] The terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents, when used interchangeably herein, refer to polymers or oligomers of nucleotides of any length. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (such as methylated, hydroxymethylated, or glycosylated), non-natural nucleotides, non-nucleotide building blocks exhibiting similar structure and / or function as natural nucleotides (i.e., “nucleotide analogs”), and / or any substrate that can be incorporated into polymers by DNA or RNA polymerase. 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. In addition, nucleic acids may be DNA or RNA, or mixtures thereof, and can exist permanently or transitionally in single-stranded or double-stranded forms, including homo-double-stranded, hetero-double-stranded, and hybrid states. Nucleic acid structures also include, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholino nucleic acids (see, e.g., Braasch and Corey, Biochemistry, 4(14):4503-4510 (2002) and U.S. Patent Nos. 5,034,506), loc nucleic acids (LNAs, see Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, Am. Chem. Soc., 122:8595-8602 (2000)), and / or ribozymes.

[0070] The terms “identical,” “percent “identity,” and their grammatical equivalents as used herein with respect to two or more polynucleotides or polypeptides refer to two or more sequences or subsequences having a specific percentage of nucleotide or amino acid residues that are identical or identical when compared and aligned (with gaps introduced as necessary) for maximum match, without considering any conserved amino acid substitutions as part of sequence identity. The identity percentage can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and their variants. In some embodiments, two polynucleotides or polypeptides provided herein are substantially identical, meaning that when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection, 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. In some embodiments, the identity exists over a region of amino acid sequence that is 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 between them. In some embodiments, the identity exists over a region longer than 60–80 residues, such as at least about 80–100 residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequence being compared, such as the coding region of a target protein or antibody.In some embodiments, identity exists over a region of nucleotide sequence that is 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 in between. In some embodiments, identity exists over a region longer than 60–80 bases, such as at least about 80–1000 bases or more, and in some embodiments, the sequence is substantially identical over the entire length of the sequence being compared, such as a nucleotide sequence encoding the protein in question.

[0071] The term “vector” and its grammatical equivalent as used herein refer to a vehicle used to carry genetic material (e.g., polynucleotide sequences) that can be introduced into a host cell, which can then be replicated and / or expressed. Applicable vectors for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain selectable sequences or markers that can be activated for stable integration into the chromosomes of the host cell. In addition, a vector may contain one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that may be included may, for example, provide resistance to antibiotics or toxins, complement malnutrition deficiencies, or supply essential nutrients not present in the culture medium. Expression control sequences may include constitutive and inductive promoters, transcription enhancers, transcription terminators, etc., which are well known in the art. When two or more polynucleotides are co-expressed, both polynucleotides may be inserted, for example, into a single expression vector or into separate expression vectors. In the case of single-vector expression, the coding polynucleotide can be operatively ligated to one common expression control sequence, or to different expression control sequences, such as one inductive promoter and one constitutive promoter. The introduction of the polynucleotide into host cells can be confirmed using methods well known in the art. It will be understood by those skilled in the art that the polynucleotide is expressed in an amount sufficient to produce the desired product (e.g., the anti-ILT7 antibody or antigen-binding fragment described herein), and it will be further understood that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

[0072] As used herein, the term “code” and its grammatical equivalents refer to the inherent properties of a particular sequence of nucleotides in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, and the biological properties arising therefrom, which serve as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein. Unless otherwise specified, “nucleotide sequences that code for an amino acid sequence” include all nucleotide sequences that code for the same amino acid sequence, including degenerate versions of each other. Nucleotide sequences that code for proteins and RNA may contain introns.

[0073] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions include polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions that have been purified to the extent that they are no longer found in nature. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

[0074] As used herein in relation to a disease or condition, or an object having a disease or condition, the term “treatment” and its grammatical equivalents refer to an action that suppresses, eliminates, reduces, and / or improves symptoms, the severity of symptoms, and / or the frequency of symptoms associated with the disease or disorder being treated.

[0075] As used herein, the term “administer” and its grammatical equivalents refer to the act of delivering or causing to be delivered a therapeutic or pharmaceutical composition to a subject by means of a method described herein or known in the art. A therapeutic may be a compound, polypeptide, antibody, cell, or population of cells. Administering a therapeutic or pharmaceutical composition involves formulating a therapeutic or pharmaceutical composition to be delivered into a subject's body. 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 such as creams, jellies, powders, or patches; oral dosage forms; inhaled powders, sprays, suspensions, and rectal suppositories.

[0076] As used herein, the terms “effective dose,” “therapeutic dose,” and their grammatical equivalents refer to administering a drug to a subject in an amount that, when administered to the subject, will have any detectable positive effect on any symptom, aspect, or characteristic of any disease, disorder, or condition, either alone or as part of a pharmaceutical composition, and in a single dose or as part of a series of doses. The therapeutic dose can be determined by measuring the relevant physiological effect. The exact amount required will vary from subject to subject, depending on the subject’s age, weight, and general condition, the severity of the condition being treated, the clinician’s judgment, etc. The appropriate “effective dose” in any individual case can be determined by those skilled in the art using routine experiments.

[0077] The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refer to a material suitable for administering a drug to an individual together with an activator without causing undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition.

[0078] As used herein, the term “subject” refers to any animal (e.g., mammal) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, dogs, cats, rodents, etc. A subject may be human. A subject may have a particular disease or condition.

[0079] Scope: Throughout this disclosure, various aspects of the present invention can be presented in range form. It should be understood that the range form description is merely for convenience and conciseness and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, the range description should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0080] Exemplary genes and polypeptides are described herein with reference to their GenBank numbers, GI numbers, and / or sequence numbers. Those skilled in the art will understand that homologous sequences can be readily identified by referencing sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0081] B. Anti-ILT7 antibody and antigen-binding fragment Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 (e.g., human ILT7). In some embodiments, provided herein are 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.

[0082] In some embodiments, the antigen-binding fragments provided herein are anti-ILT7 antibodies. In some embodiments, the antigen-binding fragments provided herein may be single-domain antibodies (sdAb), heavy-chain antibodies (HCAb), Fab, Fab', F(ab')2, Fv, single-chain variable fragments (scFv), or (scFv)2. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is a single-domain antibody (sdAb). In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is a heavy-chain antibody (HCAb). In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is Fab. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is Fab'. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is F(ab')2. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is Fv. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is scFv. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is a disulfide-bonded scFv[(scFv)2]. In some embodiments, the antigen-binding fragment of the anti-ILT7 antibody is a diabody (dAb).

[0083] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a recombinant antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a monoclonal antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a polyclonal antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises an antibody or antigen-binding fragment from a camelid animal (e.g., camel, dromedary, and llama). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a chimeric antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a humanized antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a human antibody or antigen-binding fragment. In some embodiments, provided herein is an anti-ILT7 human scFv.

[0084] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is isolated. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is substantially pure.

[0085] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a multispecific antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a bispecific antibody or antigen-binding fragment. 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.

[0086] 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 single-specific binding site. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes a bivalent binding site.

[0087] 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 approach is to screen a protein expression library, e.g., a phage or ribosome display library. Phage displays are described, for example, in Ladner et al., U.S. Patent No. 5,223,409, Smith (1985) Science 228:1315-1317, and WO92 / 18619. In some embodiments, a recombinant monoclonal antibody is isolated from a phage display library expressing a variable region or CDR of a desired species. Screening of phage libraries can be achieved by various techniques known in the art.

[0088] In some embodiments, monoclonal antibodies are modified by generating surrogate antibodies using recombinant DNA technology. In some embodiments, the constant domains of the light and heavy chains of a mouse monoclonal antibody are replaced with the constant region of a human antibody to generate a chimeric antibody. In some embodiments, the constant region is cleaved or removed to generate a desired antibody fragment of the monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region is used to optimize the specificity and / or affinity of the monoclonal antibody.

[0089] In some embodiments, the anti-ILT7 antibody clone Ab12 is provided herein. Its sequence characteristics are described below. Specific CDR sequences as defined herein are generally based on either Kabat or Chothia's definitions. However, general references to the heavy-chain CDR(pl) and / or light-chain CDR(pl) of a particular antibody are understood to encompass all CDR definitions known to those skilled in the art.

[0090] [Table 1]

[0091] [Table 2]

[0092] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises one, two, three, four, five, and / or six CDRs of any one of the antibodies described herein. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a light chain variable region (VL) containing one, two, and / or three light chain CDRs (VL CDRs) from Table 1. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises a heavy chain variable region (VH) containing one, two, and / or three heavy chain CDRs (VH CDRs) from Table 2. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein comprises one, two, and / or three VL CDRs from Table 1 and one, two, and / or three VH CDRs from Table 2.

[0093] In some embodiments, provided herein are antibodies or antigen-binding fragments that specifically bind to ILT7, comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 11, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 12, and / or (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions of the VL CDR, and / or VH CDR1 having an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14 or 17, (2) VH CDR2 having the amino acid sequence of SEQ ID NO: 15 or 18, and / or (3) VH CDR3 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions of the VH CDR.

[0094] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, and include VLs comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 11, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 12, or (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 13, 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, and include VLs comprising (1) VL CDR1 having the amino acid sequence of SEQ ID NO: 11, (2) VL CDR2 having the amino acid sequence of SEQ ID NO: 12, and (3) VL CDR3 having the amino acid sequence of SEQ ID NO: 13, 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR.

[0095] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 having a VL, the VL including VL CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 having a VL, the VL including VL CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR.

[0096] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, and include VH having (1) an amino acid sequence selected from the group consisting of SEQ ID NO: 14 or 17 (VH CDR1), (2) an amino acid sequence of SEQ ID NO: 15 or 18 (VH CDR2), or (3) an amino acid sequence selected from the group consisting of SEQ ID NO: 16 (VH CDR3), 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, and include VH having an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 14 or 17 (VH CDR1), (2) SEQ ID NO: 15 or 18 (VH CDR2), and (3) SEQ ID NO: 16 (VH CDR3), 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0097] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 having VH, where VH includes VH CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 14, 15, and 16, 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 VH CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 having VH, where VH includes VH CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NOs. 17, 18, and 16, respectively, 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 CDR. In some embodiments, the variant has up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDR.

[0098] In some embodiments, as used herein, an antibody or antigen-binding fragment thereof that specifically binds to ILT7, and which, as defined by Kabat, includes (a) VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 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 CDR, and / or (b) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs. 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 CDR.

[0099] In some embodiments, provided herein are ILT7s including 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, 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 CDRs.

[0100] In some embodiments, provided herein are antibodies or antigen-binding fragments that specifically bind to ILT7, and include, as defined by Chothia, (a) VLs comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 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 CDR, and / or (b) VHs comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of numbers 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 CDR.

[0101] In some embodiments, provided herein are antibodies or antigen-binding fragments that specifically bind to ILT7, comprising 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, 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 CDR.

[0102] [Table 3]

[0103] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 containing 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 to the amino acid sequence of SEQ ID NO: 9. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7 containing 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 to the amino acid sequence of SEQ ID NO: 10.

[0104] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, 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 to the amino acid sequence of SEQ ID NO: 9, and (b) 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 to the amino acid sequence of SEQ ID NO: 10. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VL and VH, where VL and VH have the amino acid sequences of SEQ ID NOs: 9 and 10, respectively.

[0105] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, including 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, the 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, the 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, the 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, the 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, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising a VL having the amino acid sequence of SEQ ID NO: 9.

[0106] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein VH has sequence identity of 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% with respect to SEQ ID NO: 10. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof has VH having sequence identity of at least 85% with respect to SEQ ID NO: 10. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof has VH having sequence identity of at least 90% with respect to SEQ ID NO: 10. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof has VH having sequence identity of at least 95% with respect to SEQ ID NO: 10. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment has a VH having at least 98% sequence identity with SEQ ID NO: 10. In some embodiments, provided herein is an antibody or its antigen-binding fragment that specifically binds to ILT7, comprising a VH having the amino acid sequence of SEQ ID NO: 10.

[0107] 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. The variant of the anti-ILT7 antibody or antigen-binding fragment may contain 1 to 30 amino acid substitutions, additions, and / or deletions to the anti-ILT7 antibody or antigen-binding fragment. The variant of the anti-ILT7 antibody or antigen-binding fragment may contain 1 to 25 amino acid substitutions, additions, and / or deletions to the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, the variant of the anti-ILT7 antibody or antigen-binding fragment contains 1 to 20 substitutions, additions, and / or deletions to the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment variant contains 1 to 15 substitutions, additions, and / or deletions in the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment variant contains 1 to 10 substitutions, additions, and / or deletions in the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment variant contains 1 to 5 conserved amino acid substitutions, additions, and / or deletions in the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment variant contains 1 to 3 substitutions, additions, and / or deletions in 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 located within the CDR of the antibody or antigen-binding fragment. In some embodiments, the conserved amino acid substitutions are not located within the CDR of the antibody or antigen-binding fragment. In some embodiments, the conservative amino acid substitution is located within the framework region of the antibody or antigen-binding fragment.

[0108] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28, and / or (b) 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28. In some embodiments, the humanized antibody or its antigen-binding fragment includes 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 respect to the amino acid sequence of SEQ ID NO: 19, and 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 respect to the amino acid sequence of SEQ ID NO: 26.

[0109] In some embodiments, provided herein are (a) VLs 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-22, and VLs having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively, as defined by Kabat or Chothia, VL CDR1, VL CDR2, and VL A humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, comprising (1) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs. 14, 15, and 16 as defined by Kabat, or (2) VH having the amino acid sequences of SEQ ID NOs. 17, 18, and 16 as defined by Chothia, respectively.In some embodiments, the 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 respect to the amino acid sequence of SEQ ID NO: 19, and the VL also has the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively, as defined by Kabat or Chothia, and the VL also has the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively, as VL CDR1, VL CDR2, and VL VH has CDR3 and 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 respect to the amino acid sequence of SEQ ID NO: 26, and VH also has (1) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively, as defined by Kabat, or (2) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 17, 18, and 16, respectively.

[0110] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NOs. 19 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 19 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 19 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 19 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 19 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 19 and 28, respectively. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NOs. 20 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 20 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 20 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 20 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 20 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 20 and 28, respectively. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that specifically binds to ILT7, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NOs. 21 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 21 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 21 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 21 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 21 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 21 and 28, respectively.In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NOs. 22 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 22 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 22 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 22 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 22 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NOs. 22 and 28, respectively.

[0111] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, including 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 its antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 19. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 19. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 19. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 19. In some embodiments, provided herein are humanized antibodies or their antigen-binding fragments that specifically bind to ILT7, comprising a VL having the amino acid sequence of SEQ ID NO: 19.

[0112] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, including 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 its antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 20. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 20. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 20. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 20. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and includes a VL having the amino acid sequence of SEQ ID NO: 20.

[0113] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, including 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. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 21. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and includes a VL having the amino acid sequence of SEQ ID NO: 21.

[0114] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, including 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 its antigen-binding fragment may have a VL having at least 85% sequence identity with SEQ ID NO: 22. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 90% sequence identity with SEQ ID NO: 22. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 95% sequence identity with SEQ ID NO: 22. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VL having at least 98% sequence identity with SEQ ID NO: 22. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and includes a VL having the amino acid sequence of SEQ ID NO: 22.

[0115] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein 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 VH having at least 85% sequence identity with SEQ ID NO: 23. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 90% sequence identity with SEQ ID NO: 23. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 95% sequence identity with SEQ ID NO: 23. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 23. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and contains a VH having the amino acid sequence of SEQ ID NO: 23.

[0116] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein 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 VH having at least 85% sequence identity with SEQ ID NO: 24. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 90% sequence identity with SEQ ID NO: 24. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 95% sequence identity with SEQ ID NO: 24. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with respect to SEQ ID NO: 24. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and contains a VH having the amino acid sequence of SEQ ID NO: 24.

[0117] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein 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 its antigen-binding fragment may have VH having at least 85% sequence identity with SEQ ID NO: 25. A humanized anti-ILT7 antibody or its antigen-binding fragment may have VH having at least 90% sequence identity with SEQ ID NO: 25. A humanized anti-ILT7 antibody or its antigen-binding fragment may have VH having at least 95% sequence identity with SEQ ID NO: 25. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with respect to SEQ ID NO: 25. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and contains a VH having the amino acid sequence of SEQ ID NO: 25.

[0118] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein 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 its antigen-binding fragment may have VH having at least 85% sequence identity with SEQ ID NO: 26. A humanized anti-ILT7 antibody or its antigen-binding fragment may have VH having at least 90% sequence identity with SEQ ID NO: 26. A humanized anti-ILT7 antibody or its antigen-binding fragment may have VH having at least 95% sequence identity with SEQ ID NO: 26. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 26. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and contains a VH having the amino acid sequence of SEQ ID NO: 26.

[0119] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein 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 VH having at least 85% sequence identity with SEQ ID NO: 27. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 90% sequence identity with SEQ ID NO: 27. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 95% sequence identity with SEQ ID NO: 27. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 27. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and contains a VH having the amino acid sequence of SEQ ID NO: 27.

[0120] In some embodiments, provided herein are humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising VH, wherein 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 VH having at least 85% sequence identity with SEQ ID NO: 28. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 90% sequence identity with SEQ ID NO: 28. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have VH having at least 95% sequence identity with SEQ ID NO: 28. A humanized anti-ILT7 antibody or its antigen-binding fragment may have a VH having at least 98% sequence identity with SEQ ID NO: 28. In some embodiments, provided herein are a humanized antibody or its antigen-binding fragment that specifically binds to ILT7 and contains a VH having the amino acid sequence of SEQ ID NO: 28.

[0121] An anti-ILT7 antibody or its antigen-binding fragment may include a combination of any VL disclosed herein and any VH disclosed herein. In some embodiments, the VL and VH are linked by a linker. The linker may be a flexible linker or a rigid linker. In some embodiments, the linker has an amino acid sequence (GGGGS)n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 35). In some embodiments, the linker has an amino acid sequence (EAAAK)n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 36). In some embodiments, the linker has an amino acid sequence (PA)nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 37).

[0122] In some embodiments, provided herein are anti-ILT7 antibodies or antigen-binding fragments thereof, comprising VL CDRs from VL as described herein (SEQ ID NOs: 9, 19, 20, 21, or 22) and / or VH CDRs from VH as described herein (SEQ ID NOs: 10, 23, 24, 25, 26, 27, or 28). Methods for identifying CDRs are well known in the art. For example, a publicly available software program (abYsis) on a website is known to those skilled in the art for antibody sequence analysis and CDR determination.

[0123] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising (a) a VL containing VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 9, and / or (b) a VH containing VH CDR1, 2, and 3 from a VH having the amino acid sequence of SEQ ID NO: 10.

[0124] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to ILT7, comprising (a) a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-22, comprising VL CDR1, 2, and 3, and / or (b) a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28, comprising VH CDR1, 2, and 3.

[0125] In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, including a VL, and the VL includes VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, including a VL, and the VL includes VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 19. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, including a VL, and the VL includes VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 20. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, including a VL, and the VL includes VL CDR1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 21. In some embodiments, the Specified herein provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, comprising VL, wherein VL includes VL CDR1, 2, and 3 from VL having the amino acid sequence of SEQ ID NO: 22.

[0126] In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 23. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the Specified provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7 containing VH, wherein VH includes VH CDR1, 2, and 3 from VH having the amino acid sequence of SEQ ID NO: 28.

[0127] In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein is an antibody designated as cmAb12 (chimeric Ab12). In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein has a VL from cmAb12 (SEQ ID NO: 9). In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein has a VH from cmAb12 (SEQ ID NO: 10). The anti-ILT7 antibody or its antigen-binding fragment provided herein may have both a VL and a VH from cmAb12. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein has a VL containing VL CDR1, 2, and 3 from the VL of cmAb12 (SEQ ID NO: 9). In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein has a VH containing VH CDR1, 2, and 3 from the VH of cmAb12 (SEQ ID NO: 10). The anti-ILT7 antibodies or antigen-binding fragments provided herein may have VL containing VL CDR1, 2, and 3 and VH containing VH CDR1, 2, and 3, respectively, from VL and VH of cmAb12. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments provided herein are variants of cmAb12. The cmAb12 variant may have VL which is a variant of 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 in SEQ ID NO: 9. The cmAb12 variant may have VL which is a variant of VL of cmAb12 having up to 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 9. A cmAb12 variant may have a VH variant of cmAb12, which has up to approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 10. A cmAb12 variant may have a VH variant of cmAb12, which has up to 5 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 10. Amino acid substitutions, additions, and / or deletions may be present in the VH CDR or VL CDR.In some embodiments, amino acid substitutions, additions, and / or deletions are not present in the CDR. In some embodiments, variants of cmAb12 have up to about five conserved amino acid substitutions. In some embodiments, variants of cmAb12 have up to three conserved amino acid substitutions. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a humanized antibody or antigen-binding fragment derived from cmAb12. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a human antibody or antigen-binding fragment derived from cmAb12.

[0128] In some embodiments, provided herein are humanized antibodies of Ab12 (e.g., humanized Ab12, hu-cmAb12, hu-Ab12, or hu-12). In some embodiments, the humanized anti-ILT7 antibody or its antigen-binding fragment provided herein includes a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs. 19 to 22. In some embodiments, the humanized anti-ILT7 antibody or its antigen-binding fragment provided herein includes a VH having an amino acid sequence selected from SEQ ID NOs. 23 to 28. In some embodiments, the humanized anti-ILT7 antibody or its antigen-binding fragment provided herein includes a VL having an amino acid sequence selected from the group consisting of SEQ ID NOs. 19 to 22, and a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs. 23 to 28. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein is a variant of humanized Ab12 provided herein. The variant may have a VL variant of humanized Ab12 VL having up to approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-22. The variant may have a VL variant of humanized Ab12 VL having up to approximately 5 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-22. The variant may have a VH variant of humanized Ab12 VH having up to approximately 3, 5, 8, 10, 12, or 15 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28. The variant may have a VH variant of humanized Ab12 VH having up to approximately 5 amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-28. In some embodiments, variants of humanized Ab12 have up to approximately five conserved amino acid substitutions. In some embodiments, the humanized antibody is the antibody "hu-cmAb12," which comprises VL having the amino acid sequence of SEQ ID NO: 19 and VH having the amino acid sequence of SEQ ID NO: 26.The selected properties of the humanized antibody hu-cmAB12 are demonstrated in Examples 8-17.

[0129] 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.

[0130] In some embodiments, the anti-ILT7 antibodies provided herein include a light chain and a heavy chain. The light chain may include a light chain constant domain (CL) and a light chain variable domain (VL). The heavy chain may include a heavy chain variable domain (VH) and a heavy chain constant domain (CH). VL / VH can be any VL / VH disclosed herein. In some embodiments, the light chain constant domain (CL) is kappa CL (Cκ, SEQ ID NO: 29). In some embodiments, the light chain constant domain (CL) is lambda CL (Cλ, SEQ ID NO: 30). In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgA. In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgD. In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgE. In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgG. In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgM. In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgG1 (e.g., SEQ ID NO: 31). In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgG2 (e.g., SEQ ID NO: 32). In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgG3 (e.g., SEQ ID NO: 33). In some embodiments, the heavy chain includes a heavy chain constant domain (CH) from human IgG4 (e.g., SEQ ID NO: 34). What is expressly intended herein is any and all combinations of the VL / VH pairs disclosed herein that specifically bind to ILT7 (e.g., human ILT7) and the CL / CH pairs disclosed herein or otherwise known in the art. JPEG2026510891000006.jpg38170JPEG2026510891000007.jpg169170

[0131] In some embodiments, the antibodies provided herein have 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 antibodies provided herein have a CL having the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibodies provided herein have 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 antibodies provided herein have a CL having the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibodies provided herein have 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 antibodies provided herein have 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.

[0132] In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with the antibodies 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" means an antibody that (partially or completely) inhibits the binding of the other antibody to its target. Whether two antibodies compete with each other for binding to a target, i.e., whether one antibody inhibits the binding of the other antibody to its target, and to what extent, can be determined using known competition experiments, e.g., BIACORE® surface plasmon resonance (SPR) analysis. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment competes with another antibody or antigen-binding fragment and inhibits binding to ILT7 by at least 50%, 60%, 70%, 80%, 90%, or 100%. Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harbor Protoc; 2006; doi:l0.H0l / pdb.prot4277 or Chapter 11 of “Using Antibodies” Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999.

[0133] In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with anti-ILT7 antibodies or antigen-binding fragments disclosed herein for binding to ILT7 (e.g., human ILT7). In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with chimeric Ab12 for binding to ILT7 (e.g., human ILT7). In some embodiments, provided herein are antibodies or antigen-binding fragments that compete with humanized Ab12 disclosed herein for binding to ILT7 (e.g., human ILT7).

[0134] Epitope mapping is a method for identifying binding sites, regions, or epitopes on target proteins to which antibodies bind. Various methods for mapping epitopes on target proteins are known in the art. These methods include, but are not limited to, shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology); visualization methods (e.g., phage display, microbial display, and ribosome / mRNA display); methods including proteolysis and mass spectrometry; and mutagenesis, including structural determination (e.g., X-ray crystallography and NMR). In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion-exchange chromatography, and papain digestion.

[0135] As will be provided in more detail in the experimental section below, Ab12 does not compete with the benchmark antibody daxidrimab for binding to human ILT7. In some embodiments, the anti-ILT7 antibodies and antigen-binding fragments provided herein do not compete with daxidrimab for binding to human ILT7.

[0136] The anti-ILT7 antibodies or antigen-binding fragments of this disclosure can be analyzed for their physical, chemical, and / or biological properties by various methods known in the art. In some embodiments, the anti-ILT7 antibody is tested for its ability to bind to ILT7 (e.g., human ILT7). In some embodiments, the anti-ILT7 antibody is tested for its ability to bind to FcγR. In some embodiments, the anti-ILT7 antibody is tested for its ability to bind to FcγRIIA / CD32A. In some embodiments, the anti-ILT7 antibody is tested for its ability to bind to FcγRIIIA / CD16A. Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. In addition, the antibodies can be evaluated for solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency.

[0137] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has high affinity, for example, 10 -6 M or less, 5×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 10 -9 M or less, 5×10 -10 M or less, or 10 -10 K below M D It binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is 5 × 10 -7 K below M D It binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is, for example, about 10 -6 M, about 5 x 10 -7 M, about 10 -7 M, about 5 x 10 -8 M, about 10 -8 M, about 5 x 10 -9 M, about 10 -9 M, about 5 x 10 -10 M, or about 10 -10 M's KD Therefore, it binds to human ILT7 with high affinity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is about 10 -7 M's K D It binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is 10 -10 M~10 -6 M, 10 -9 M~10 -6 M, 10 -8 M, 10 -6 M, 10 -7 M~10 -6 M, 10 -10 M~5×10 -7 M, 10 -9 M~5×10 -7 M, 10 -8 M~5×10 -7 M, or 10 -7 M~5×10 -7 K in the range of M D It binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has high affinity, for example, 10 -7 M~10 -6 M's K D It binds to human ILT7. In some embodiments, K D This is determined by BLI. In some embodiments, K D This is determined by SPR. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has high affinity, for example, when measured by SPR, 10 -6 M or less, 5×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 10 -9 M or less, 5×10 -10 M or less, or 10 -10 K below M D or 10 -10 M~10 -6 M, 10 -9 M~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 It binds to human ILT7 within the range of M.

[0138] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein binds to both human ILT7 and cynomolgus monkey ILT7.

[0139] 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 to other LILR family member proteins is equivalent to that of an isotype antibody (e.g., hIgG1). 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.

[0140] In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein inhibit the release of IFNα by PBMC. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein inhibit the release of IFNα by CpG-stimulated PBMC in vitro. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein inhibit the release of IFNα by PBMC in vivo. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein reduce IFNα levels in vivo. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein have an EC 50 such that it inhibits the release of IFNα by CpG-stimulated PBMC in vitro. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein have an EC of 0.1 nM or less 50 such that it inhibits the release of IFNα by CpG-stimulated PBMC in vitro. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein have an EC 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 such that it inhibits the release of IFNα by CpG-stimulated PBMC in vitro. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein have an EC of about 0.05 nM 50 such that it inhibits the release of IFNα by CpG-stimulated PBMC in vitro. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein have an EC in the range of 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 such that it inhibits the release of IFNα by CpG-stimulated PBMC in vitro. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein have an EC of 0.01 nM to 0.1 nM 50This inhibits the release of IFNα by CpG-stimulated PBMCs in vitro. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is 0.01 nM to 1 nM EC 50 This inhibits the release of IFNα by CpG-stimulated PBMCs in vitro. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used to control the EC of the reference antibody daxzirimab. 50 EC is 60% or less, 50% or less, 40% or less, or 30% or less. 50 This inhibits the release of IFNα by CpG-stimulated PBMCs in vitro. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used to control the EC of the reference antibody daxzirimab. 50 less than 50% of e-commerce 50 This inhibits the release of IFNα by CpG-stimulated PBMCs in vitro. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used to control the EC of the reference antibody daxzirimab. 50 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% EC 50 Then, in vitro, CpG-stimulated PBMC-mediated release of IFNα is inhibited. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used to control the EC of the reference antibody daxzirimab. 50 10-50% of e-commerce 50 Therefore, it inhibits the release of IFNα by CpG-stimulated PBMCs in vitro.

[0141] 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, and monocytes in PBMCs.

[0142] In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein exhibit ADCC activity and ADCP activity against ILT7-expressing cells such as pDCs. The ADCC activity may be NK-dependent ADCC. The ADCC activity may be neutrophil-dependent ADCC. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein deplete pDCs in vivo.

[0143] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC 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 has an EC of 0.01 nM or less. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC of 0.008 nM or less. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC of 0.005 nM or less. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is present in concentrations of 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 of EC 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is approximately 0.008 nM EC 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is approximately 0.005 nM EC 50In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits NK-dependent ADCC activity. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC in the range of about 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 the EC of the reference antibody daxdilimab. 50 EC is 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% of 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is 10-50% of the reference antibody daxdilimab EC 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is the EC of the reference antibody daxdilimab. 50 E-commerce accounts for approximately 40% of the total. 50 It exhibits NK-dependent ADCC activity.

[0144] 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 has an EC 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 has an EC of 100 nM or less. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC of 50 nM or less. 50It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC in the range of 1nM-500nM, 1nM-200nM, 1nM-100nM, 1nM-80nM, 1nM-50nM, 1nM-20nM, 5nM-500nM, 5nM-200nM, 5nM-100nM, 5nM-80nM, 5nM-50nM, or 5nM-20nM. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC in the range of about 1 nM to 50 nM. 50 It exhibits neutrophil-dependent ADCC activity having the following characteristics. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC of about 500 nM, about 200 nM, about 100 nM, about 80 nM, about 50 nM, about 20 nM, about 10 nM, about 5 nM, or about 1 nM. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is approximately 10 nM EC 50 It exhibits neutrophil-dependent ADCC activity.

[0145] 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 has an EC 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. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC of 10 nM or less. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC of 8 nM or less. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is present in concentrations of about 10 nM, about 8 nM, about 5 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.2 nM, or about 0.1 nM of EC 50It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is approximately 1 nM EC 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC in the range of 0.01-100 nM, 0.01-50 nM, 0.01-10 nM, 0.1-100 nM, 0.1-50 nM, 0.1-10 nM, or 0.1-5 nM. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC in the range of about 0.1 to 10 nM. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has an EC in the range of about 0.5 to 5 nM. 50 It exhibits macrophage-dependent ADCP activity.

[0146] 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 in the range of 20-80%, 20-70%, 20-60%, 30-80%, 30-70%, or 30-60%. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index in the range of 20-80%. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index in the range of 30-60%.

[0147] C. Variants and Conjugates This disclosure further intends to describe the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies described herein, or additional variants and equivalents that are substantially homologous to their antibody fragments. 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, thermal stability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that changes in amino acids can alter the post-translational processes of an antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0148] A variation can be a substitution, deletion, or insertion of one or more nucleotides encoding an antibody or polypeptide that results in a change in the amino acid sequence compared to the native antibody or polypeptide sequence. In some embodiments, an amino acid substitution is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as a serine substitution of leucine, e.g., a conservative amino acid substitution. Insertions or deletions can range from about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the parent molecule. In some embodiments, a variation in a biologically useful and / or relevant amino acid sequence can be determined by systematically performing insertions, deletions, or substitutions within the sequence and testing the activity of the resulting variant protein against the parent protein.

[0149] In some embodiments, what is provided herein is a variant of the anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, what is provided herein is a variant of the anti-ILT7 antibody clone Ab12 (cmAb12 or hu-Ab12). In some embodiments, the variant comprises 1 to 30 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises 1 to 25 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises 1 to 20 substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises 1 to 15 substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises 1 to 10 substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises 1 to 5 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the variant includes 1 to 3 amino acid substitutions, additions, and / or deletions in the parent antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions are located within the CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions are not located within the CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions are located within the framework region of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and / or deletions are conservative amino acid substitutions.

[0150] It is known in the art that the constant region of an antibody mediates several effector functions, and these effector functions can vary depending on the antibody isotype. For example, the binding of the complement C1 component to the Fc region of an IgG or IgM antibody (binding to an antigen) activates the complement system. Complement activation is important in opsonization and lysis of cellular pathogens. Complement activation can also stimulate inflammatory responses and be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind to cells that express Fc receptors (FcRs). There are several Fc receptors that are specific to different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). The binding of antibodies to Fc receptors on the cell surface triggers several important and diverse biological responses, including the entrapment and disruption of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production.

[0151] As is known in the art, allotypes are polymorphic markers of IG subclasses that correspond to amino acid changes and are detected serologically by antibody reagents. In particular, the allotype of the human heavy gamma chain of IgG is designated as Gm ("gamma marker"). Allotypes G1m, G2m, and G3m are carried by the constant regions of the gamma 1, gamma 2, and gamma 3 chains, encoded by the IHG1, IHG2, and IHG3 genes, respectively. The gamma 1 chain can express the G1m allele (combination 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 IHG1 allele is known in the art, for example, in Lefranc, Chapter 26 - IMGT (registered trademark) Immunoglobulin Repertoire Analysis and Antibody Humanization, Molecular Biology of B Cells (Second Edition), Academic Press, 2015, Pages 481-514 (Table 7). In IHG1 CH1, the lysine at position 120 (K120) of chain G corresponds to the G1m17 allotype. Isoleucine I103 (chain F) is specific to the gamma 1 chain isotype. When arginine is expressed at position 120 (R120), the simultaneous presence of R120 and I103 corresponds to the expression of the G1m3 allotype. For gamma 3 and gamma 4 isotypes (which have R120 instead of T at position 103), R120 corresponds only to the expression of the nG1m17 isoallotype (the isoallotype or nGm is detected by antibody reagents that identify this marker as an allotype in one IgG subclass and as an isotype for other subclasses).In IHG1 CH3, aspartate D12 and leucine L14 (chain A) correspond to G1m1, while glutamate E12 and methionine M14 correspond to the nG1m1 isoallotype. Glycine at position 110 corresponds to G1m2, but alanine does not correspond to any allotype (G1m2-negative chain).

[0152] Please refer to the following exemplary allotypes of human IgG1 heavy chain constant region (IgG1 CH). In some embodiments, provided herein are IgG1 antibodies having a heavy chain constant region (CH) having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs. 31 and 40-44. In some embodiments, the IgG1 antibodies provided herein have a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 31. In some embodiments, the IgG1 antibodies provided herein have a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 40. In some embodiments, the IgG1 antibodies provided herein have a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NOs. 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 having 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 having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 44. JPEG2026510891000008.jpg242170

[0153] The gamma 2 chain can express the G2m allele. Position 45.1 (the first position on the transverse CD chain) corresponds to the presence (G2m23) or absence (G2m..) of the only identified G2m allotype. Valine V45.1 corresponds to G2m.., while methionine corresponds to G2m23.

[0154] The gamma 3 chain can express G3m alleles (combinations of G3m allotypes). G3m16(W83), G3m21(L82), and nG3m21(P82) are located on CH2. Other G3m allotypes form two mosaics 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 mosaic. G3m11(S44), nG3m11(N44), G3m10(S44, I101), G3m24(S44, V101), G3m27(I101), G3m6(S44, E98), and G3m13(S44, Q98) form a second mosaic.

[0155] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgA antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgD antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgE antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgG antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgM antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgG1 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgG2 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes a constant region of a human IgG3 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes 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, the IgG1 antibody may be any allotype known in the art. In some embodiments, the IgG1 antibody is 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 is allotype G1m3. In some embodiments, the IgG1 antibody is allotype G1m3,1. In some embodiments, the IgG1 antibody is allotype G1m17,1. In some embodiments, the IgG1 antibody is allotype G1m17,1,2. In some embodiments, the IgG1 antibody is allotype Gm17,1,28. In some embodiments, the IgG1 antibody is of 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, the IgG2 antibody may be any allotype known in the art.In some embodiments, the IgG2 antibody is allotype G2m23. In some embodiments, the IgG2 antibody is allotype G2m. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG3 antibody, and the IgG3 antibody can be any allotype known in the art. In some embodiments, the IgG3 antibody is allotype G3m16, G3m21, G3m26, G3m5, G3m28, G3m14, G3m15, G3m11, G3m10, G3m24, G3m27, G3m6, or G3m13. In some embodiments, the IgG3 antibody is allotype G3m16. In some embodiments, the IgG3 antibody is allotype G3m21. In some embodiments, the IgG3 antibody is allotype G3m26. In some embodiments, the IgG3 antibody is allotype G3m5. In some embodiments, the IgG3 antibody is allotype G3m28. In some embodiments, the IgG3 antibody is allotype G3m14. In some embodiments, the IgG3 antibody is allotype G3m15. In some embodiments, the IgG3 antibody is allotype G3m11. In some embodiments, the IgG3 antibody is allotype G3m10. In some embodiments, the IgG3 antibody is allotype G3m24. In some embodiments, the IgG3 antibody is allotype G3m27. In some embodiments, the IgG3 antibody is allotype G3m6. In some embodiments, the IgG3 antibody is allotype G3m13.

[0156] In some embodiments, at least one of the constant regions is modified or deleted in the anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the antibody includes modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or modifications to the light chain constant region (CL).

[0157] 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 two or more human constant regions. In some embodiments, modification to 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 entirely deleted from the constant region of the modified antibody. In some embodiments, an entire CH2 domain is 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 deleted constant region. In some embodiments, the modified antibody includes a CH3 domain directly fused 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 domain.

[0158] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes an Fc region. In some embodiments, the Fc region is fused via a hinge. The hinge can 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 mutations have been identified in native antibodies. In some embodiments, a modified antibody (e.g., a modified Fc region) then provides an altered effector function that affects the antibody's biological profile. For example, in some embodiments, deletion or inactivation of the constant region (via point mutation or other means) reduces its Fc receptor binding as the modified antibody circulates. 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 decreases the serum half-life of the antibody. In some embodiments, constant region modification enhances the ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, constant region modification enhances the antibody-dependent phagocytosis (ADCP) of the antibody. In some embodiments, constant region modification reduces or eliminates the ADCC and / or CDC of the antibody. In some embodiments, specific amino acid substitutions within the human IgG1 Fc region having corresponding IgG2 or IgG4 residues reduce effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody has no effector function (e.g., an "effectorless" antibody). In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody has no effector function. In some embodiments, constant region modification increases or enhances the ADCC and / or ADCP of the antibody. In some embodiments, the constant region is modified to remove a disulfide bond or oligosaccharide moiety. In some embodiments, the constant region is modified by adding / substituting one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites.In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes a variant Fc region that is manipulated by substitution at specific amino acid positions compared to the native Fc region.

[0159] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region containing one or more amino acid substitutions selected from the group consisting of L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, A330, I332, K326, E333, K334, and P396, numbered according to the EU index.

[0160] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes an IgG1 heavy chain constant region comprising at least one amino acid substitution. The IgG1 heavy chain constant region may include an L234 substitution, for example, L234Y. The IgG1 heavy chain constant region may include an L235 substitution, for example, L235Q or L235V. The IgG1 heavy chain constant region may include a G236 substitution, for example, G236A or G236W. The IgG1 heavy chain constant region may include an S239 substitution, for example, S239D or S239M. The IgG1 heavy chain constant region may include an F243 substitution, for example, F243L. The IgG1 heavy chain constant region may include an H268 substitution, for example, H268D. The IgG1 heavy chain constant region may contain a D270 substitution. For example, the D270 substitution may be D270E. The IgG1 heavy chain constant region may contain an R292 substitution. For example, the R292 substitution may be R292P. The IgG1 heavy chain constant region may contain an S298 substitution. For example, the S298 substitution may be S298A. The IgG1 heavy chain constant region may contain a Y300 substitution. For example, the Y300 substitution may be Y300L. The IgG1 heavy chain constant region may contain a V305 substitution. For example, the V305 substitution may be V305I. The IgG1 heavy chain constant region may contain a K326 substitution. For example, the K326 substitution may be K326D. The IgG1 heavy chain constant region may contain an A330 substitution. For example, the A330 substitution may be A330M or A330L. The IgG1 heavy chain constant region may contain an I332 substitution. For example, the I332 substitution may be I332E. The IgG1 heavy chain constant region may contain an E333 substitution. For example, the E333 substitution may be E333A. The IgG1 heavy chain constant region may contain a K334 substitution. For example, the K334 substitution may be K334A or K334E.The IgG1 heavy chain constant region may contain a P396 substitution. The P396 substitution may be, for example, P396L.

[0161] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein includes an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, and P396L, numbered according to the EU index. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M, numbered according to the EU index. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions S298A, E333A, and K334A. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions S239D and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions S239D, A330L, and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitution G236A. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein includes variants of the human IgG1 heavy chain constant region modified by amino acid substitutions G236A, S239D, and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions G236A, A330L, and I332E.In some embodiments, the anti-ILT7 antibodies and antigen-binding fragments described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions F243L, R292P, Y300L, V305I, and P396L. In some embodiments, the anti-ILT7 antibodies and antigen-binding fragments described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions L235V, F243L, R292P, Y300L, and P396L. In some embodiments, the anti-ILT7 antibodies and antigen-binding fragments described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, the anti-ILT7 antibodies and antigen-binding fragments described herein include variants of the human IgG1 heavy chain constant region modified by amino acid substitutions D270E, K326D, A330M, and K334E. All are numbered according to the EU index. Provided below are exemplary IgG1 allotype heavy chain constant regions (CHs) having different mutations that increase or enhance the ADCC and / or ADCP of the antibodies. Expressly intended for inclusion in the antibodies disclosed herein are heavy chain constant regions (CHs) of any immunoglobulin (e.g., human IgG1) disclosed herein or otherwise known in the art, having any combination of mutations known in the art to increase or enhance the ADCC and / or ADCP of the antibodies disclosed herein or otherwise. JPEG2026510891000009.jpg209170JPEG2026510891000010.jpg237170JPEG2026510891000011.jpg236170JPEG2026510891000012.jpg119170

[0162] In some embodiments, the herein provides an IgG1 antibody having a heavy chain constant region (CH) having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 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 to SEQ ID NOs. 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 to SEQ ID NOs. 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 to SEQ ID NOs. 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 to SEQ ID NOs. 48. 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: 49. 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: 50. 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: 51. 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: 52.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: 53. 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: 54. 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: 55. 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: 56. 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: 57. 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: 58. 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: 59. 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: 60. 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: 61.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: 62. 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: 63. 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: 64.

[0163] In some embodiments, the provided antibody or antigen-binding fragment includes CH having the amino acid sequence of SEQ ID NO: 55. In some embodiments (for example, embodiments where the antibody or antigen-binding fragment is the humanized antibody hu-cmAb12), the antibody or antigen-binding fragment includes 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 includes 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 demonstrated in Examples 8-17.

[0164] In some embodiments, the variant may include the addition of amino acid residues at the amino acid terminus and / or carboxyl terminus of an antibody or polypeptide. The length of the additional amino acid residues may range from one to more than 100 residues. In some embodiments, the variant includes an N-terminal methionyl residue. In some embodiments, the variant includes an additional polypeptide / protein (e.g., an Fc region) for creating a fusion protein. In some embodiments, the variant may be manipulated to be detectable and may include a detectable label and / or protein (e.g., a fluorescent tag or enzyme).

[0165] 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 scanning mutagenesis, and PCR mutagenesis. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), Kunkel et al., Methods Enzymol. 54:367-382 (1987), Sambrook et al. (1989) Molecular Cloning: A LABORATORY MANUAL (Cold Spring Harbor, NY), U.S. Patent No. 4,873,192, and the references cited herein (incorporated herein by reference). Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target polypeptide can be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), pp. 345–352 (the entire model is incorporated herein by reference). The Dayhoff et al. model uses a point-receptor mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conserved amino acid substitutions. Conservative substitutions, such as replacing one amino acid with another amino acid having similar properties, can be beneficial. Examples of conserved amino acid substitutions taught by the PAM 250 matrix in the Dayhoff et al. model include, but are not limited to, Gly→Ala, Val→Ile→Leu, Asp→Glu, Lys→Arg, Asn→Gln, and Phe→Trp→Tyr.

[0166] In the construction of variants of anti-ILT7 binding molecules, such as antibodies or their antigen-binding fragments, variants, or derivatives, modifications are made such that the variant possesses desired properties, such as the ability to specifically bind to ILT7, inhibit IFN-alpha release in certain embodiments, and / or deplete pDCs in vivo. Clearly, any mutations made in the DNA encoding the variant polypeptide should not cause the sequence to deviate from the read frame. In some embodiments, mutations made in the DNA do not create complementary regions capable of producing secondary mRNA structures.

[0167] In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment disclosed herein may retain the ability to bind to ILT7 to the same degree, the same degree, or a higher degree than the parent antibody or antigen-binding fragment. In some embodiments, the variant may be identical to the parent antibody or antigen-binding fragment in its amino acid sequence by at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more. In certain embodiments, the variant of the anti-ILT7 antibody or antigen-binding fragment comprises 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 include those known in the art in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties.

[0168] In some embodiments, a variant of an anti-ILT7 antibody or antigen-binding fragment comprises the amino acid sequence of a parent antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions. In some embodiments, a variant of an anti-ILT7 antibody or antigen-binding fragment comprises the amino acid sequence of a parent-binding antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions, and 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 certain embodiments, one or more conservative amino acid substitutions and / or 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 that of the parent antibody or antigen-binding fragment.

[0169] In some embodiments, the variant has 1, 2, 3, 4, or 5 amino acid substitutions in the binding CDR (e.g., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3).

[0170] In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein are chemically modified either naturally or by intervention. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linkage to cellular ligands or other proteins. Any of the numerous chemical modifications can be carried out by known techniques. The anti-ILT7 antibodies or antigen-binding fragments may include one or more analogues of amino acids (e.g., including non-natural amino acids), as well as other modifications known in the art.

[0171] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment disclosed herein is conjugated to at least one drug to form an antibody conjugate. The conjugate may be an antibody conjugated to, for example, another protein, carbohydrate, lipid, steroid, immunosuppressant, or mixed partial molecule. Such antibody conjugates include, but are not limited to, modifications involving the conjugation of the antibody to one or more polymers. For example, an antibody or antigen-binding fragment may be conjugated to one or more water-soluble polymers. Conjugation with a water-soluble polymer reduces the likelihood of the antibody or antigen-binding fragment precipitating 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 profile of the antibody (e.g., half-life, dosage, activity, antigenicity, and / or other factors).

[0172] Conventional methods for enhancing the effectiveness of antibody molecules as diagnostic or therapeutic agents involve ligating, covalently bonding, or conjugating at least one desired molecule or moiety. Such molecules or moieties may, but are not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules conjugated to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antivirals, 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 conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, enzymes (e.g., catalyzing colorimetric, fluorescent, or bioluminescent reactions), substrates, and solid matrices (e.g., biotin). Antibodies may contain one, two, or more of these labels.

[0173] In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein are chemically modified either naturally or by intervention. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments are chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linkage to cellular ligands or other proteins. Any of the numerous chemical modifications can be carried out by known techniques. The anti-ILT7 antibodies or antigen-binding fragments may include one or more analogues of amino acids (e.g., including non-natural amino acids), as well as other modifications known in the art.

[0174] Antibody conjugates can be used to deliver cytotoxic agents to target cells. This type of cytotoxic agent can enhance antibody-mediated cytotoxicity and includes components such as cytokines, radioisotopes, chemotherapeutic agents (including prodrugs), bacterial toxins (e.g., Pseudomonas exotoxin, diphtheria toxin, etc.), plant toxins (e.g., lysine, geronin, etc.), chemical conjugates (e.g., meitansinoid toxins, calicheamicin, etc.), radioconjugates, and enzyme conjugates (e.g., RNase conjugates, granzyme antibody-directed enzyme / prodrug therapy).

[0175] Antibody conjugates are also used as diagnostic agents. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated into a detectable substance or molecule that enables the agent to be used for diagnosis and / or detection. Detectable substances may include, but are not limited to, enzymes, artificial groups (e.g., biotin and flavin), fluorescent materials, bioluminescent materials such as luciferase, radioactive materials, positron-emitting metals, and magnetic metal ions.

[0176] Generally, antibody diagnostics are classified into two classes: those for use in in vitro diagnostics such as various immunoassays, and those for use in in vivo diagnostic protocols commonly known as “antibody-directed imaging.” Many suitable imaging agents are known in the art, as are methods for their attachment 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, fluorochromes, NMR detectables, MR hyperpolarizing molecules, targeted ultrasonic bubbles, and X-ray imaging agents.

[0177] Paramagnetic ions intended for use as conjugates include chromium(III), manganese(II), iron(III), iron(II), 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 useful in other contexts such as X-ray imaging include, but are not limited to, lanthanum(III), gold(III), lead(II), and bismuth(III). Alternatively useful isotopes include carbon-13 and silica-29, which are used in hyperpolarized MRI.

[0178] Radioisotopes intended for use in imaging and radiotherapy as conjugates or covalents 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, iodine-131, and indium-11. This includes 1, 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 often preferred for PET imaging. Lu-177, At-211, and Yt-90 are often preferred for radiotherapy. The radiolabeled monoclonal antibodies and antibody fragments of this disclosure can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide and / or potassium iodide, and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. Monoclonal antibodies according to this disclosure can be labeled with technetium-99m by a ligand exchange process, for example, by reducing the pertechnetium salt with a tin solution, chelating the reduced technetium onto a Sephadex column, and applying the antibody to this column. Alternatively, a direct labeling technique can be used, for example, by incubating the antibody with a pertechnetium salt, a reducing agent such as SNCl2, a buffer such as a sodium phthalate-potassium phthalate solution, etc.Intermediate functional groups that incorporate chelating agents, often used to bind radioactive isotopes existing as metal ions to antibodies, include diethylenetriamine-pentaacetic acid (DTPA), ethylenediamine-tetraacetic acid (EDTA), monomeric or dendritic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazaicyclononane-1,4,7-triacetic acid (NOTA), deferoxamine (DFO), or 1-hydroxy-2(1H)-pyridinone derivatives (e.g., 3,4,3-LI(1,2-HOPO) or HOPO).

[0179] Fluorescent labels intended 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, Cyanine (Cy3), Cy5,6-FAM, Dansyl Chloride, Dichlorotriazinylamine Fluorescein, Fluorescein Isothiocyanate (FITC), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine Isothiocyanate (TRITC), Texas Red, and / or Umbelliferone.

[0180] The additional types of antibodies envisioned in this disclosure are primarily intended for in vitro use, and the antibodies are linked to secondary ligands and / or enzymes (enzyme tags) that produce a colored product upon contact with a chromogenic substrate. Examples of preferred enzymes include beta-galactosidase, acetylcholinesterase, urease, alkaline phosphatase, (wasabi) hydrogen peroxidase, or glucose oxidase. Preferred secondary ligands are biotin and avidin and streptavidin compounds.

[0181] Several methods for attaching or conjugating antibodies to their conjugate portions are known in the art. Some attachment methods involve the use of metal chelate complexes with organic chelating agents such as diethylene-triamine-pentaacetic anhydride (DTPA), ethylene-diamine-tetraacetic acid, monomers or dendrimers of 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-diphenylglycolyl-3 attached to the antibody (U.S. Patents 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or perate. Conjugates containing fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Patent No. 4,938,948, imaging of breast tumors is achieved using a monoclonal antibody, and the detectable imaging portion is conjugated to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0182] Another known method for site-specific attachment of molecules to antibodies involves the reaction of the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids within the antigen-binding site, thereby disrupting this site and blocking the specific antigen reaction.

[0183] Molecules containing azide groups can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light. In particular, 2- and 8-azide analogs of purine nucleotides have been used as site-specific photoprobes for identifying nucleotide-binding proteins in crude cell extracts. 2- and 8-azide nucleotides have also been used to map nucleotide-binding domains in purified proteins and can be used as antibody conjugates.

[0184] Derivatization of immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody binding site is also being considered. Antibody conjugates produced according to this method have been disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which a reporter or effector molecule is conjugated to a carbohydrate residue within the Fc region, has also been disclosed in the literature. This approach has been reported to produce diagnostic and therapeutically promising antibodies currently undergoing clinical evaluation.

[0185] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated to a steroid or immunosuppressant. In some embodiments, the antibody or antigen-binding fragment is conjugated to 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 to 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 to an immunosuppressant which may be an antimalarial agent (e.g., hydroxychloroquine, chloroquine), an antimetabolite (e.g., methotrexate, azathioprine, mercaptopurine), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), mycophenolic acid, mycophenolate mofetil, thalidomide, or acitretin.

[0186] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated to a cytotoxic agent or part thereof. 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 potent biopharmaceuticals designed as targeted therapies. An ADC consists of an antibody (an antibody fragment such as a whole mAb or scFv) conjugated to a biologically active cytotoxic / antiviral payload or drug via a stable chemical linker with an unstable binding. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates. By combining the unique targeting ability of monoclonal antibodies with cytotoxic agents, ADCs enable a delicate distinction between healthy and affected tissue. This means that, in contrast to conventional systemic approaches, ADCs target and attack affected cells so that healthy cells are less severely affected.

[0187] In the development of ADC-based antitumor therapies, the warhead (e.g., cytotoxin) is coupled to an antibody that specifically targets a particular cellular marker (e.g., a protein found only within or on affected cells). The antibody targets these proteins in the body and attaches itself to the surface of affected cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal within the target cell, which then absorbs or internalizes the antibody along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cells or impairing cell replication. In other cases, the linker can be cleaved on the surface of the target cell or early endosome, and therefore complete internalization is not required. For this targeting, ideally, the drug has fewer side effects and offers a broader therapeutic concentration range than other drugs.

[0188] In some embodiments, the cytotoxic moiety of an ADC having an 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, duocalmycin, daunorubicin, pyrrolobenzodiazepine (PBD), or other intercalating agents. In some embodiments, the cytotoxic moiety is a microtubule inhibitor including, but not limited to, auristatin, maytansinoids (e.g., DM1 and DM4), and tubulicin. In some embodiments, the cytotoxic moiety is an enzyme-active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof, including but not limited to diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain, lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, geronin, mitogenin, restrictoctocin, phenomycin, enomycin, and trichothecenes. In some embodiments, the antibody or antigen-binding fragment is conjugated to one or more small molecule toxins such as calicheamicin, meitansinoid, trichothecenes, and CC1065.

[0189] Stable binding between the antibody and the cytotoxic agent is a crucial aspect of ADCs. 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 types of linkers have been proven safe in preclinical and clinical trials. The availability of better, more stable linkers has altered the function of the chemical bond. The type of cleavable or non-cleavable linker imparts specific properties to cytotoxic (e.g., anticancer) drugs. For example, non-cleavable linkers retain the drug within the cell. As a result, the entire antibody, linker, and cytotoxic agent enter the target cell where the antibody is broken down to the amino acid level. The resulting complex (amino acids, linker, and cytotoxic agent) then becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes within or on the host cell, thereby releasing the cytotoxic agent. Commonly used mechanisms for linker cleavage include protease-sensitive, pH-sensitive, and glutathione-sensitive mechanisms. Another type of cleavable linker adds an extra molecule between the cytotoxic drug and the cleavage site. This linker technique allows researchers to create more flexible ADCs without altering cleavage kinetics. Novel peptide cleavage methods based on Edman degradation have also been developed. Future directions in ADC development include site-directed conjugations (TDCs) to further improve stability and therapeutic index, as well as the development of α-luminescent immunoconjugates and antibody-conjugated nanoparticles.

[0190] The anti-ILT7 antibodies or antigen-binding fragments described herein can be attached to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized anti-ILT7 antibodies or antigen-binding fragments are used in immunoassays. In some embodiments, the immobilized anti-ILT7 antibodies or antigen-binding fragments are used in the purification of a target antigen (e.g., human ILT7).

[0191] D. Polynucleotides and vectors Polynucleotides encoding polypeptides described herein (e.g., anti-ILT7 antibodies or antigen-binding fragments) are also provided herein. The term “polynucleotide encoding polypeptide” encompasses polynucleotides comprising only the coding sequence of the polypeptide, as well as polynucleotides comprising additional coding and / or non-coding sequences. The polynucleotides of this disclosure 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. The single-stranded DNA may be a coding strand or a non-coding (antisense) strand. The polynucleotides of this disclosure may be mRNA.

[0192] The express intent of this specification is a polynucleotide encoding any anti-ILT7 antibody or antigen-binding fragment disclosed herein. For illustrative purposes, in some embodiments, the polynucleotides provided herein include (1) 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 five amino acid substitutions, additions, and / or deletions in the VL CDR, as defined by Kabat, 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 NOs. 14, 15, and 16, respectively, or a variant thereof having up to about five amino acid substitutions, additions, and / or deletions in the VH CDR, as defined by Chothia, or VL The encoding of an anti-ILT7 antibody or antigen-binding fragment comprising a variant thereof having up to five amino acid substitutions, additions, and / or deletions in the CDR, and / or (b) VH comprising 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 approximately five amino acid substitutions, additions, and / or deletions in the VH CDR.

[0193] In some embodiments, the polynucleotides provided herein encode an anti-ILT7 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. The polynucleotide may be in the form of DNA. The polynucleotide may be in the form of mRNA.

[0194] In some embodiments, the polynucleotides provided herein encode an anti-ILT7 antibody or antigen-binding fragment disclosed herein, comprising VL and VH, where VL comprises VL CDR1, CDR2 and CDR3, and VH comprises VH CDR1, CDR2 and CDR3, and VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 each have (1) the amino acid sequences of SEQ ID NOs. 11, 12, 13, 14, 15 and 16, respectively, or (2) the amino acid sequences of SEQ ID NOs. 11, 12, 13, 17, 18 and 16, respectively, or variants thereof having up to about five substitutions, additions and / or deletions to the CDR. The polynucleotides may be in the form of DNA. The polynucleotides may be in the form of mRNA.

[0195] In some embodiments, the polynucleotides provided herein encode an anti-ILT7 antibody or antigen-binding fragment disclosed herein, comprising VL and VH, wherein VL and VH are (1) SEQ ID NOs. 9 and 10, respectively, (2) SEQ ID NOs. 19 and 23, respectively, (3) SEQ ID NOs. 19 and 24, respectively, (4) SEQ ID NOs. 19 and 25, respectively, (5) SEQ ID NOs. 19 and 26, respectively, (6) SEQ ID NOs. 19 and 27, respectively, (7) SEQ ID NOs. 19 and 28, respectively, (8) SEQ ID NOs. 20 and 23, respectively, (9) SEQ ID NOs. 20 and 24, respectively, (10) SEQ ID NOs. 20 and 25, respectively, (11) SEQ ID NOs. 20 and 26, respectively. (12) Having the amino acid sequences of SEQ ID NOs. 20 and 27 respectively, (13) SEQ ID NOs. 20 and 28 respectively, (14) SEQ ID NOs. 21 and 23 respectively, (15) SEQ ID NOs. 21 and 24 respectively, (16) SEQ ID NOs. 21 and 25 respectively, (17) SEQ ID NOs. 21 and 26 respectively, (18) SEQ ID NOs. 21 and 27 respectively, (19) SEQ ID NOs. 21 and 28 respectively, (20) SEQ ID NOs. 22 and 23 respectively, (21) SEQ ID NOs. 22 and 24 respectively, (22) SEQ ID NOs. 22 and 25 respectively, (23) SEQ ID NOs. 22 and 26 respectively, (24) SEQ ID NOs. 22 and 27 respectively, or (25) SEQ ID NOs. 22 and 28 respectively. Polynucleotides can be in the form of DNA. Polynucleotides can be in the form of mRNA.

[0196] In some embodiments, VL and VH are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker has an amino acid sequence (GGGGS)n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 35). In some embodiments, the linker has an amino acid sequence (EAAAK)n, n=1, 2, 3, 4, or 5 (SEQ ID NO: 36). In some embodiments, the linker has an amino acid sequence (PA)nP, n=1, 2, 3, 4, or 5 (SEQ ID NO: 37).

[0197] This disclosure also provides variants of the polynucleotides described herein, which encode, for example, fragments, analogs, and / or derivatives of the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, this disclosure provides polynucleotides having nucleotide sequences that are at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the polynucleotide sequences encoding the anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, this disclosure provides polynucleotides having nucleotide sequences that are at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or at least about 99% identical to the polynucleotide sequences encoding the anti-ILT7 antibody or antigen-binding fragment described herein.

[0198] As used herein, the phrase "polynucleotide having a nucleotide sequence identical to at least about 95% of the polynucleotide sequence" means that the nucleotide sequence of the polynucleotide is identical to that of the reference sequence, except that the polynucleotide sequence may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence identical to at least 95% of the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, 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' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, and may be individually scattered between nucleotides in the reference sequence or scattered within one or more consecutive groups in the reference sequence.

[0199] Polynucleotide variants may include modifications to coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants include modifications that produce silent substitutions, additions, or deletions but do not alter the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants include silent substitutions that do not result in a change to the amino acid sequence of the polypeptide (due to genetic coding degeneracy). Polynucleotide variants may be produced for a variety of reasons, for example, to optimize codon expression for a particular host (e.g., to change codons in human mRNA to those preferred by a bacterial host such as Escherichia coli (E. coli)). In some embodiments, polynucleotide variants include at least one silent mutation in the non-coding region or coding region of the sequence.

[0200] In some embodiments, polynucleotide variants are produced to modulate or alter the expression (or expression level) of the encoded polypeptide. In some embodiments, polynucleotide variants are produced to increase the expression of the encoded polypeptide. In some embodiments, polynucleotide variants are produced to decrease the expression of the encoded polypeptide. In some embodiments, the polynucleotide variant increased the expression of the encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, the polynucleotide variant decreased the expression of the encoded polypeptide compared to the parent polynucleotide sequence.

[0201] In some embodiments, the polynucleotide comprises a coding sequence of a polypeptide (e.g., an antibody) fused within the same read frame to the polynucleotide, which assists in the expression and secretion of the polypeptide (e.g., a leader sequence that functions as a secretion sequence to control polypeptide transport) from a host cell. The polypeptide may have a leader sequence cleaved by the host cell, forming a “mature” form of the polypeptide.

[0202] In some embodiments, the polynucleotide includes the coding sequence of a polypeptide (e.g., an antibody) fused to the 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) that enables efficient purification of the polypeptide fused to the marker. In some embodiments, the marker sequence is a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG® tag. In some embodiments, the marker is used in conjunction with other markers or tags.

[0203] In some embodiments, the polynucleotides are isolated. In some embodiments, the polynucleotides are substantially pure.

[0204] Vectors and cells containing the polynucleotides described herein are also provided. In some embodiments, what is provided herein is a vector containing the polynucleotides provided herein. The vector may be an expression vector. In some embodiments, the vector provided herein contains a polynucleotide encoding an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the vector provided herein contains a polynucleotide encoding a polypeptide that is part of an anti-ILT7 antibody or antigen-binding fragment described herein.

[0205] In some embodiments, provided herein are recombinant expression vectors that can be used to amplify and express polynucleotides encoding anti-ILT7 antibodies or antigen-binding fragments described herein. For example, a recombinant expression vector may be a replicable DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding an anti-ILT7 antibody polypeptide chain, operably linked to a suitable transcription and / or translation regulatory element derived from a mammalian, microorganism, viral, or insect gene. In some embodiments, a viral vector is used. DNA regions are “operably linked” if they are functionally related to one another. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence, or a ribosome-binding site is operably linked to a coding sequence if it is positioned to enable translation. In some embodiments, structural elements intended for use in a particular expression system include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In some embodiments, in situations where the recombinant protein is expressed without a leader or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0206] A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli plasmids containing pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA phages. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed from one or more vectors.

[0207] Provided herein are host cells containing the vectors described herein. In some embodiments, the host cells are used for recombinant expression of the anti-ILT7 antibody described herein. The host cells may include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein production, including antibody production, are well known in the art.

[0208] Examples of suitable mammalian host cells include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may include non-transcription elements, such as origins of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' adjacent non-transcription sequences, as well as 5' or 3' untranslated sequences, such as required ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded, biologically functional proteins. Baculovirus systems for the production of heterologous proteins in insect cells are well known to those skilled in the art.

[0209] This disclosure also provides host cells comprising polypeptides described herein, polynucleotides encoding polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, provided herein are host cells comprising a vector comprising a polynucleotide disclosed herein. In some embodiments, the host cells provided herein comprise a vector comprising a polynucleotide encoding an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the host cells provided herein comprise a vector comprising a polynucleotide encoding a polypeptide that is part of an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the host cells provided herein comprise a polynucleotide encoding an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the cells produce an anti-ILT7 antibody or antigen-binding fragment described herein.

[0210] E. Manufacturing Method Also provided herein are methods for producing anti-ILT7 antibodies and their antigen-binding fragments, including but not limited to monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, and their antigen-binding fragments.

[0211] 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) and Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981) (each of these is incorporated herein by reference in whole). In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to those skilled in the art. For example, mice, rats, rabbits, hamsters, or other suitable host animals are immunized as described above using hybridoma methods. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof.

[0212] After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, for example, using polyethylene glycol. Hybridoma cells are selected using specialized media known in the art, and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas producing monoclonal antibodies directed to the selected antigen can be identified by a variety of methods, including but not limited to immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells producing antibodies of desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting dilution or by other techniques. Hybridomas can be grown in vitro using standard methods or in vivo as ascites tumors in animals. Monoclonal antibodies can be purified from media or ascites according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0213] In some embodiments, monoclonal antibodies are prepared using recombinant DNA techniques known to those skilled in the art. For example, the polynucleotides encoding the antibody are isolated from mature B cells or hybridoma cells by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, 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 that produces monoclonal antibodies when transfected into host cells such as E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.

[0214] The polynucleotides of antibody or antigen-binding fragments provided herein can be prepared, manipulated, and / or expressed using any of the 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 include plasmids, autonomously replicating sequences, and transposable factors. Exemplary transposon systems such as Sleeping Beauty and PiggyBac can be used and stably incorporated into the genome (e.g., Ivics et al., Cell, 91(4):501-510 (1997), Cadinanos et al., (2007) Nucleic Acids Research. 35(12):e87). Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of animal virus categories useful 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 papovaviruses (e.g., SV40). Examples of expression vectors include the pCl-neo vector (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST(trademark), pLenti6 / V5-DEST(trademark), and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells.

[0215] In some embodiments, the vector is an episomal vector or vector maintained outside the chromosome. As used herein, the term “episome” means a vector that can replicate without integration into the host’s chromosomal DNA and without progressive loss from the dividing host cell, and also means that the vector replicates outside the chromosome or in an episome. The vector is engineered to possess a sequence encoding DNA replication or the origin of “ori” from a lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically, the origin of replication of a lymphotropic herpesvirus or gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotropic herpesvirus is Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), herpesvirus thymili (HS), or Malek’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.

[0216] The "expression regulatory sequences," "regulatory elements," or "regulatory sequences" present within an expression vector are the vector-derived untranslated regions of replication, selection cassettes, promoters, enhancers, translation initiation signal (Shine Dalgarno or Kozak sequence) introns, polyadenylated sequences, and 5' and 3' untranslated regions, which interact with host cell proteins to perform transcription and translation. Such elements can vary in their intensity and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, can be used.

[0217] Exemplary ubiquitous expression regulatory sequences that can be used in this disclosure include the cytomegalovirus (CMV) immediate early promoter, the Virmianvirus 40 (SV40) promoter (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Roussarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and the P11 promoter from vaccinia virus, elongation This includes, but is not limited to, the factor 1-alpha (EF1a) promoter, early growth 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 beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 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.

[0218] Examples of inductive promoters / systems include, but are not limited to, steroid-inductive promoters, e.g., promoters of genes encoding glucocorticoids or estrogen receptors (induced by treatment with the corresponding hormone), metallothione promoters (induced by treatment with various heavy metals), MX-1 promoters (induced by interferon), the "GeneSwitch" mifepristone regulatory system (Sirin et al., 2003, Gene, 323:67), the kmet-inductive gene switch (WO2002 / 088346), and tetracycline-dependent regulatory systems. The anti-ILT7 antibodies or antigen-binding fragments described herein can be produced by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise indicated, the invention will be carried out using molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and conventional techniques in the relevant fields within the art.

[0219] The polypeptides described herein can be prepared using a wide variety of techniques known in the art, including the use of hybridomas and recombinant technologies, or combinations thereof. In some embodiments, recombinant expression vectors are used to express polynucleotides encoding the polypeptides described herein. For example, a recombinant expression vector may be a replicable DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding a polypeptide, operably linked to a suitable transcription and / or translation regulatory element derived from a mammalian, microorganism, virus, or insect gene. In some embodiments, the coding sequences of the polypeptides disclosed herein may be ligated into such expression vectors for their expression in mammalian cells. In some embodiments, viral vectors are used. DNA regions are “operably linked” if they are functionally related to one another. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence, or a ribosome-binding site is operably linked to a coding sequence if it is positioned to enable translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In some embodiments, in situations where the recombinant protein is expressed without a leader or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0220] A wide variety of expression host / vector combinations can be used. Suitable host cells for expression can include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells, under the control of an appropriate promoter. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein production, including antibody production, are well known in the art. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli plasmids containing pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA phages.

[0221] Examples of expression vectors useful for eukaryotic hosts include vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may include non-transcription elements, such as origins of replication, suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' adjacent non-transcription sequences, as well as 5' or 3' untranslated sequences, such as required ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Recombinant protein expression in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded, biologically functional proteins. Baculovirus systems for heterologous protein production in insect cells are well known to those skilled in the art.

[0222] To produce defucosylated anti-ILT7 antibodies and antigen-binding fragments, (1) host cells overexpressing N-acetylglucosaminyltransferase III (GnTIII), (2) host cells lacking α-1,6-fucosyltransferase (FUT8), (3) host cells having a low fucose content, or any combination of (1) to (3) can be used. In some embodiments, host cells overexpressing N-acetylglucosaminyltransferase III (GnTIII) are used herein. In some embodiments, host cells lacking 1,6-fucosyltransferase (FUT8) are used herein. In some embodiments, host cells having a low fucose content are used herein. In some embodiments, CHO host cells are used.

[0223] Peptides can be synthesized whole or partially using chemical methods (see, e.g., Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215, Horn (1980), and Banga, AK, Therapeutic Peptides and Proteins, Formula, Processing and Delivery Systems (1995), Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be carried out using various solid-phase techniques (see, e.g., Roberge, Science 269:202 (1995), Merrifield, Methods. Enzymol. 289:3 (1997)), and automated synthesis may be achieved, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Peptides can also be synthesized using combination methodologies. Synthetic residues and polypeptides can be synthesized using a variety of procedures and methodologies known in the art (see, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, e.g., Belousov, Nucleic Acids Res. 25:3440 (1997), Frenkel, Free Radic. Biol. Med. 19:373 (1995), and Blommers, Biochemistry 33:7886 (1994)). Modification, derivative, substitution, and alteration of peptide sequences can also be performed using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis.Site-specific mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986), Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)), and other techniques can be performed on cloned DNA to generate the peptide sequences, variants, fusions, and chimeras of the invention, as well as their modifications, derivatives, substitutions, and alterations.

[0224] For in vivo use of antibodies in humans, it may be preferable to use human antibodies. Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be made by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements of these techniques. See also U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT Publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741 (each of which 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 sources of human DNA.

[0225] 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 immunized individuals. In either case, cells producing antibodies directed against a target antigen can be generated and isolated. In some embodiments, human antibodies are selected from a phage library that expresses human antibodies. Alternatively, human antibodies and antibody fragments can be produced in vitro from an immunoglobulin variable region gene repertoire from an unimmunized donor using phage display techniques. Techniques for generating and using antibody phage libraries are well known in the art. Once the antibody is identified, higher affinity human antibodies can be produced using affinity maturation strategies known in the art, including, but not limited to, chain shuffling and site-directed mutagenesis. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. Upon immunization, these mice can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[0226] Human antibodies can also be produced using transgenic mice that can express human immunoglobulin genes but cannot express functional endogenous immunoglobulins. For example, the human heavy and light chain immunoglobulin gene complex can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional separately or simultaneously by homologous recombination along with the introduction of human immunoglobulin loci. For example, homozygous deletion of the antibody heavy chain junction region (JH) gene in chimeric and germline mutant mice has been shown to result in complete inhibition of endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then fed to produce homozygotes that express human antibodies. Transgenic mice are immunized in a normal manner with a selected antigen, for example, all or part of the polypeptide of the present invention. For example, an anti-ILT7 antibody directed against the human ILT7 antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene possessed by the transgenic mouse is rearranged during B cell differentiation and subsequently undergoes class switching and somatic mutation. Thus, it is possible to produce therapeutically useful IgG, IgA, IgM, and IgE antibodies, including but not limited to IgG1 (gamma 1) and IgG3, using such techniques. For an overview of this technique for producing human antibodies, see Lonberg and Huszar (Int. Rev. Immunol., 13:65-93 (1995)).For a detailed description of this technology for producing human antibodies and human monoclonal antibodies and the protocols for producing such antibodies, see, for example, PCT Publications WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735, and U.S. Patents Nos. 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 which is incorporated herein by reference in its entirety). In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can use technologies similar to those described above to provide human antibodies directed against selected antigens. For a specific discussion of the transfer of the human germline immunoglobulin gene array in germline mutant mice that results in the production of human antibodies upon antigen challenge, see, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993), Jakobovits et al., Nature, 362:2-55-258 (1993), Bruggermann et al., Year in Immunol., 7:33 (1993), and Duchosal et al., Nature, 355:258 (1992).

[0227] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J.Mol.Biol., 227:381 (1991), Marks et al., J.Mol.Biol., 222:581-597 (1991), Vaughan et al., Nature Biotech., 14:309 (1996)). Using phage display technology (McCafferty et al., Nature, 348:552-553 (1990)), human antibodies and antibody fragments can be produced in vitro from an immunoglobulin variable (V) domain gene repertoire from unimmunized donors. According to this technique, the antibody V domain gene is cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage such as M13 or fd, and displayed as a functional antibody fragment on the surface of a phage particle. Since fibrous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of antibodies also leads to the selection of genes encoding antibodies that exhibit those properties. Thus, phages mimic some of the properties of B cells. Phage display can be performed in various formats; for an overview of these, see, for example, Johnson and Chiswell, Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated diverse anti-oxazolone antibodies from a small, randomly combined library of V genes derived from the spleen of unimmunized mice. A repertoire of V genes derived from unimmunized human donors can be constructed, and antibodies against a variety of antigens (including autoantigens) can be isolated essentially according to the techniques described by Marks et al., J.Mol.Biol., 222:581-597 (1991) or Griffith et al., EMBO J., 12:725-734 (1993).See also U.S. Patent Nos. 5,565,332 and 5,573,905 (each of which is incorporated herein by reference in whole).

[0228] Human antibodies can also be produced in vitro by activated B cells (see U.S. Patents No. 5,567,610 and No. 5,229,275, each of which is incorporated herein by reference in whole). Human antibodies can also be produced in vitro using hybridoma techniques, but are not limited to those described by Roder et al. (Methods Enzymol., 121:140-167 (1986)).

[0229] Alternatively, in some embodiments, non-human antibodies are humanized, and specific sequences or regions of the antibody are modified to increase their similarity to antibodies naturally produced in humans. In some embodiments, the antigen-binding domain portion is humanized. Various methods for generating humanized antibodies are known in the art. Methods for achieving high-affinity binding with humanized antibodies are also known in the art. Non-limiting examples of such methods include hypermutation of variable regions and selection of cells expressing such high-affinity antibodies (affinity maturation). In addition to the use of display libraries, non-human animals, such as rodents, can be immunized using specific antigens (e.g., recombinant ILT7 or its epitope). In certain 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 antigens (e.g., recombinant ILT7 or its epitope).

[0230] Humanized antibodies may be used, but are not limited to, CDR implantation (see, e.g., European Patent No. 239,400, International Publication No. 91 / 09967, and U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089), veneering, or resurfacing (see, e.g., European Patents No. 592,106 and 519,596, Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering, 7(6):805-814, and Roguska et al.) al., 1994, PNAS, 91:969-973 (each of these is incorporated herein by reference in its entirety), chain shuffling (e.g., U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety), as well as, for example, U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. 9317105, Tan et al., J.Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et It can be produced using techniques including those disclosed in al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994) (each of these is incorporated herein in whole by reference).In many cases, framework residues within the framework region can be substituted with corresponding residues from the CDR donor antibody to alter, and preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by modeling the interaction between the CDR and framework residues to identify abnormal framework residues at specific locations, to identify framework residues important for antigen binding and sequence comparison (see, for example, Queen et al., U.S. Patent No. 5,585,089, and Riechmann et al., 1988, Nature, 332:323 (both incorporated herein by reference)).

[0231] 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, and are typically extracted from an "import" variable domain. Thus, humanized antibodies contain one or more CDRs derived from non-human immunoglobulin molecules and a human-derived framework region. Antibody humanization is well known in the art and is essentially the work of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.). This can be done by replacing a rodent CDR or CDR sequence with the corresponding sequence of a human antibody, i.e., by CDR transplantation, according to the method in al. Science, 239:1534-1536 (1988) (EP239,400, PCT Publication No. WO91 / 09967, and U.S. Patents No. 4,816,567, No. 6,331,415, No. 5,225,539, No. 5,530,101, No. 5,585,089, No. 6,548,640, the contents of which are referred to in their entirety). (These are incorporated herein by reference). In such humanized chimeric antibodies, substantially less than the intact human variable domain is substituted with the corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which several CDR residues, and possibly several FR residues, are substituted with residues from similar sites in rodent antibodies. Antibody humanization can also be achieved by veneering or resurfacing (EP592,106, EP519,596, Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498, Protein Engineering, 7(6):805-814 (1994), and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332) (these are incorporated herein by reference in their entirety).

[0232] The selection of light and heavy human variable domains used in the production of humanized antibodies aims to reduce antigenicity. The sequences of variable domains of rodent antibodies are screened against an entire library of known human variable domain sequences according to a so-called "best fit" method. The human sequence most closely resembling the rodent sequence is then accepted as the human framework (FR) for the humanized antibody (Sims et al. J.Immunol., 151:2296 (1993), Chothia et al. J.Mol.Biol., 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Alternatively, a specific framework is used, derived from the consensus sequences of all human antibodies in a particular subgroup of the light or heavy chain. The same framework may be used for several different humanized antibodies (as described in Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety).

[0233] Antibodies can be humanized while retaining high affinity for target antigens and other desirable biological properties. For example, humanized antibodies can be prepared by analytical processes of the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional structures of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of 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 import sequences, resulting in the achievement of desired antibody properties, such as improved affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0234] Humanized antibodies retain similar antigen specificity to the original antibody, for example, the ability to bind to the human ILT7 antigen. However, using certain humanization methods, the affinity and / or specificity of the antibody binding to a particular antigen can be increased using the “directed evolution” method described by Wu et al. J. Mol. Biol., 294:151 (1999) (the entire content of which is incorporated herein by reference).

[0235] The anti-ILT7 antibodies or antigen-binding fragments described herein can be tested for binding to human ILT7, for example, by standard ELISA. Briefly, a microtiter plate is coated with purified ILT7 and then blocked with bovine serum albumin. A diluted antibody (e.g., a diluted plasma 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., in the case of human antibodies, a goat-anti-human IgG Fc-specific polyclonal reagent). After washing, the plate can be unfolded and analyzed by spectrophotometer. Subsequently, serum from immunized mice can be further screened by flow cytometry for binding to cell lines expressing human ILT7, but not for binding to control cell lines that do not express ILT7. Briefly, the binding of anti-ILT7 antibodies can be evaluated by incubation of ILT7-expressing CHO cells with the anti-ILT7 antibody. The cells can be washed, and binding can be detected with anti-human IgG Ab. Flow cytometry analysis can be performed using a FACScan flow cytometry instrument (Becton Dickinson, San Jose, CA). Mice that develop the highest titer can be used for fusion.

[0236] Using the ELISA assay described above, antibodies that produce antibodies positively reactive with the ILT7 immunogen, and therefore hybridomas, can be screened. Hybridomas that produce antibodies that bind to ILT7 with high affinity can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parent cell (by ELISA) can then be selected for cell banking and antibody purification.

[0237] To purify anti-ILT7 antibodies, selected hybridomas can be grown for monoclonal antibody purification. The supernatant can be filtered and concentrated before affinity chromatography. Eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be changed to determine the concentration. The monoclonal antibodies can be divided and stored.

[0238] To determine whether a selected anti-ILT7 monoclonal antibody binds to its own epitope, each antibody can be biotinylated using a commercially available reagent (Pierce, Rockford, IL). Biotinylated MAb binding can be detected with a streptavidin-labeled probe. Competitive studies using unlabeled and biotinylated monoclonal antibodies can be performed using ILT7-coated ELISA plates as described above.

[0239] To determine the isotype of a purified antibody, isotype ELISA can be performed using reagents specific to the antibody of 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 purified isotype control antibody at ambient temperature for 1-2 hours. The wells can then be reacted with either a human IgG1 or human IgM-specific alkaline phosphatase conjugate probe. The plate is then unfolded and analyzed as described above.

[0240] To test the binding of monoclonal antibodies to living 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. The samples can be analyzed by a FACScan instrument using light and lateral scattering properties to gate on single cells, and binding of the labeled antibody can be determined. An alternative assay using fluorescence microscopy can be used (in addition to, or instead of) the flow cytometry assay. Cells can be stained precisely as described above and examined by fluorescence microscopy. This method allows for the visualization of individual cells, but the sensitivity can be reduced depending on the antigen density.

[0241] Anti-ILT7 antibodies or antigen-binding fragments can be further tested for reactivity with the ILT7 antigen by Western blotting. Briefly, cell extracts can be prepared from cells expressing ILT7 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 probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase, which can be developed with BCIP / NBT substrate tablets (Sigma Chem Co., St. Louis, MO).

[0242] 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 the Gator system (Probe Life) or Octet-96 system (Sartorius AG), or BIACORE® surface plasmon resonance (SPR) analysis using the BIACORE® 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

[0243] F. Pharmaceutical Compositions Provided herein are 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 is useful in suppressing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the pharmaceutical composition is useful in treating diseases or disorders associated with type I IFN (e.g., IFNα) or pDC.

[0244] In some embodiments, the pharmaceutical compositions provided herein comprise an anti-ILT7 antibody or antigen-binding fragment provided herein. The anti-ILT7 antibody or antigen-binding fragment can be present at various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise the soluble anti-ILT7 antibody or antigen-binding fragment provided herein at a concentration of 1 to 1000 mg / mL. The dosage can be readily adjusted by those skilled in the art; for example, a decrease in purity may require an increase in dosage.

[0245] Also provided herein is a kit for the preparation of a pharmaceutical composition having an 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 can comprise the anti-ILT7 antibody or antigen-binding fragment disclosed herein for administration to a subject. In certain embodiments, the kit comprises instructions regarding the preparation and / or administration of the anti-ILT7 antibody or antigen-binding fragment.

[0246] In some embodiments, provided herein is a pharmaceutical composition comprising an anti-ILT7 antibody or antigen-binding fragment or cell provided herein, which composition is suitable for topical administration.

[0247] Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., injection or infusion). Depending on the route of administration, the active ingredient (i.e., the anti-ILT7 antibody or antigen-binding fragment) can be coated in the material to protect the active ingredient from the action of acids and other natural conditions that can inactivate the active ingredient.

[0248] Also provided herein are pharmaceutical compositions or formulations that improve the stability of anti-ILT7 antibodies or antigen-binding fragments, enabling their 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 volume extender, and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C.

[0249] A buffer useful in a pharmaceutical composition or formulation disclosed herein may be a weak acid or base used to maintain the acidity (pH) of the solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may 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 includes histidine (e.g., L-histidine) together with an isotonic agent and potentially pH adjustment with an acid or base known in the art. In certain embodiments, the buffer is L-histidine. In certain embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0250] Stabilizers are added to pharmaceuticals to stabilize the product. Such agents can stabilize proteins in different 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; glycerol, mannitol, sorbitol, cyclodextrin, or dextrans of any type and molecular weight, or polyols such as PEG. In some embodiments, the stabilizer is selected to maximize the stability of the polypeptide in the lyophilized preparation. In certain embodiments, the stabilizer is sucrose and / or arginine.

[0251] Volume extenders can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating its accurate measurement and handling. Common volume extenders include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0252] A surfactant is an amphiphilic substance having a hydrophilic group and a hydrophobic group. 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, cocamide MEA, cocamide DEA, polysorbate, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0253] The pharmaceutical compositions disclosed herein may further comprise one or more of buffer systems, preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, and 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. See Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0254] 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.

[0255] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized, and the physician or patient adds a solvent and / or diluent before use.

[0256] The pharmaceutical compositions disclosed herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0257] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (e.g., 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 fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0258] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial presence can be ensured by the sterilization procedures described above, as well as by the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, sustained absorption of the injectable pharmaceutical form can be achieved by the inclusion of absorption-delaying agents such as aluminum monostearate and gelatin.

[0259] Pharmaceutically acceptable carriers include sterile aqueous solutions, or dispersions and sterile powders for the immediate preparation of sterile injections or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, their use in the pharmaceutical compositions described herein is intended. The pharmaceutical compositions or formulations may or may not contain preservatives. Auxiliary active compounds may be incorporated into the compositions.

[0260] Pharmaceutical compositions or formulations must typically 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. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Often, compositions may contain isotonic agents, such as sugars, polyhydric alcohols (e.g., mannitol, sorbitol), or sodium chloride. Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents in the composition, such as monostearate and gelatin.

[0261] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing one or a combination thereof of the components listed above, as needed, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other components as needed from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze-drying (lyophilization), which yield powders of the active component and any additional desired components from a pre-sterile filtered solution.

[0262] The amount of active ingredient that can be combined with a carrier material in a pharmaceutical composition or formulation disclosed herein may 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. Generally, out of 100 percent, this amount is in the range of about 0.01 percent to about 99 percent, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent of the active ingredient combined with a pharmaceutically acceptable carrier.

[0263] The pharmaceutical compositions disclosed herein can be prepared with a carrier that protects the active ingredient from rapid release, such as an implant, a transdermal patch, and a controlled-release formulation including a microencapsulation delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyoxides, 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, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0264] In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein are formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the activating components described herein cross the BBB, they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811, 5,374,548, and 5,399,331. Liposomes may contain one or more portions that are selectively transported to specific cells or organs, thus enhancing targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.), mannoside (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PGBloeman et al. (1995) FEBS Lett. 357:140, M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), and pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090), as well as K. Keinanen; M. Laukkanen (1994) FEBS See also Lett.346:123 and JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0265] G. Method and Use The antibodies or antigen-binding fragments, compositions, and methods described herein have numerous in vitro and in vivo applications, including, for example, reducing the release of type I IFN by ILT7-expressing cells such as pDCs. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments described herein are humanized antibodies or antigen-binding fragments. For example, the anti-ILT7 antibodies or antigen-binding fragments described herein can be administered to cells in culture in vitro or ex vivo, or, for example, to a human subject in vivo, to selectively inhibit type I IFN release or suppress pDC activity in various diseases. Provided herein is a method for reducing autoimmunity in a subject, the method comprising administering to the subject the anti-ILT7 antibody or antigen-binding moiety described herein to reduce type I IFN or pDC-associated autoimmunity in the subject.

[0266] This disclosure also provides methods for using anti-ILT7 antibodies or antigen-binding fragments, polynucleotides encoding such anti-ILT7 antibodies or antigen-binding fragments, vectors comprising such polynucleotides, or pharmaceutical compositions having such antibodies or antigen-binding fragments disclosed herein, in order to reduce cytokine (e.g., type I IFN) release by ILT7-expressing cells such as pDCs, or to treat diseases or disorders associated with type I IFN-related diseases or pDC-related diseases, such as autoimmune diseases.

[0267] In some embodiments, the Specified provides a method for reducing type I IFN (e.g., IFNα) in a subject, 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, the Specified 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, the Specified provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for the preparation of a pharmaceutical for reducing type I IFN (e.g., IFNα). In some embodiments, the Specified provides a method for reducing type I IFN (e.g., IFNα) in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the Specified provides the use of the pharmaceutical composition disclosed herein for reducing type I IFN (e.g., IFNα). In some embodiments, what is provided herein is the use of pharmaceutical compositions provided herein for preparing a pharmacopoeia for reducing type I IFN (e.g., IFNα).

[0268] In some embodiments, the foregoing provides a method for suppressing or depleting pDCs in a subject, 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, the foregoing provides the use of the anti-ILT7 antibody or antigen-binding fragment described herein for suppressing or depleting pDCs. In some embodiments, the foregoing provides the use of the anti-ILT7 antibody or antigen-binding fragment provided herein for the preparation of a drug for suppressing or depleting pDCs. In some embodiments, the foregoing provides a method for suppressing or depleting pDCs in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the foregoing provides the use of the pharmaceutical composition disclosed herein for suppressing or depleting pDCs. In some embodiments, the foregoing provides the use of the pharmaceutical composition disclosed herein for the preparation of a drug for suppressing or depleting pDCs. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment disclosed herein depletes pDCs in the target where this is necessary.

[0269] In some embodiments, provided herein are methods for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC in subjects, 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, provided herein are 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, provided herein are the use of the anti-ILT7 antibody or antigen-binding fragment provided herein for the preparation of a pharmaceutically effective amount for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, provided herein are methods for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC in subjects, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the use of the pharmaceutical compositions disclosed herein for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the use of the pharmaceutical compositions disclosed herein for preparing a pharmacopoeia for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC.

[0270] In some embodiments, the foregoing provides a method for reducing autoimmune diseases associated with type I IFN (e.g., IFNα) or pDCs in subjects where such reduction is needed, and the method comprises administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, the foregoing provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for treating autoimmune diseases associated with type I IFN (e.g., IFNα) or pDCs. In some embodiments, the foregoing provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for the preparation of a pharmaceutically effective amount for treating autoimmune diseases associated with type I IFN (e.g., IFNα) or pDCs. In some embodiments, the foregoing provides a method for treating autoimmune diseases associated with type I IFN (e.g., IFNα) or pDCs in subjects where such treatment is needed, and the method comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, the use of the pharmaceutical compositions disclosed herein for treating autoimmune diseases associated with type I IFNs (e.g., IFNα) or pDCs. In some embodiments, the use of the pharmaceutical compositions disclosed herein for preparing pharmaceuticals for treating autoimmune diseases associated with type I IFNs (e.g., IFNα) or pDCs.

[0271] As is known in the art, dysregulation or excessive activation of pDCs, or release of type I IFNs (e.g., IFNα), results in undesirable activation of the immune system, which is involved in the pathogenesis of a wide variety of diseases, including autoimmune diseases. In some embodiments, diseases or disorders 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 disorder 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 disorder 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 disorder 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 disorder that can be treated with the anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition provided herein is DLE. In some embodiments, the disease or disorder that can be treated with the anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition provided herein is LN.

[0272] In some embodiments, the methods provided herein promote beneficial therapeutic responses with respect to autoimmune responses. 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 decrease in one or more other symptoms associated with the disease. Thus, for example, improvement of the disease can be characterized as a complete response. In some embodiments, the clinical response can be evaluated using screening techniques such as magnetic resonance imaging (MRI) scanning, X-ray imaging, computed tomography (CT) scanning, flow cytometry or fluorescence-activated cell sorting (FACS) analysis, histology, macroscopic pathology, and hematochemistry, including but not limited to techniques for measuring changes detectable by ELISA, RIA, chromatography, etc.

[0273] The actual dose levels 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 the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dose level depends on various pharmacokinetic factors, including the activity of the particular composition described herein, the route of administration, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, as well as similar factors well known in the medical field.

[0274] Anti-ILT7 antibodies or antigen-binding fragments can be administered as sustained-release formulations, in which case 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. For therapeutic purposes, relatively high doses at relatively short intervals may be required until disease progression is reduced or terminated, and until the patient shows partial or complete improvement in disease symptoms.

[0275] The anti-ILT7 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, including, but not limited to, pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intramuscular administration, intradermal administration, subarachnoid administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal administration, or other parenteral administration routes such as injection or infusion, or direct administration to the thymus. As used herein, the term “parenteral administration” usually means, but is not limited to, administration methods other than enteral and topical administration by injection, and includes, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrafascial injections and infusions. In some embodiments, subcutaneous administration is employed. In some embodiments, intravenous administration is employed. In some embodiments, oral administration is employed. In one embodiment, 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.

[0276] In the methods disclosed herein, a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition disclosed herein is administered to a subject who can benefit from a reduction in IFNα levels. The subject may have undesirable, unregulated, or excessive activation of pDCs. The subject may be a mammal. In some embodiments, the subject is human.

[0277] The anti-ILT7 antibodies, antigen-binding fragments, or pharmaceutical compositions provided herein can be administered in conjunction with medical devices known in the art. For example, in some embodiments, needle-free subcutaneous injection devices such as those disclosed in U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556 may be used. Examples of well-known implants and modules for use described herein include U.S. Patent No. 4,487,603 (disclosing an implantable microinfusion pump for distributing drugs at a controlled rate), U.S. Patent No. 4,486,194 (disclosing a therapeutic device for administering drugs through the skin), U.S. Patent No. 4,447,233 (disclosing a drug infusion pump for delivering drugs at a precise infusion rate), U.S. Patent No. 4,447,224 (disclosing a variable-flow implantable infusion device for continuous drug delivery), U.S. Patent No. 4,439,196 (disclosing an osmotic drug delivery system having multiple chamber compartments), and U.S. Patent No. 4,475,196 (disclosing an osmotic 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.

[0278] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition provided herein is administered in conjunction with additional therapy. The additional therapy may be administered before, simultaneously with, or after the administration of the anti-ILT7 antibody or antigen-binding fragment, cells, or pharmaceutical composition described herein. Concomitant administration may include concomitant administration using a single pharmaceutical formulation or separate formulations, or sequential administration in any order, but generally may include sequential administration within a period such that all active agents can exert their biological activity simultaneously. Those skilled in the art can readily determine, based on the needs of the subject being treated, an appropriate regimen for the combined administration of the pharmaceutical composition and additional therapy described herein, including the timing and dosage of the additional agent used in the concomitant therapy.

[0279] The antibodies or antigen-binding fragments provided herein can also be used for the detection of ILT7. Furthermore, methods for detecting the presence of human ILT7 antigen in a sample, or measuring the amount of human ILT7 antigen, are also included, which involve contacting a sample and a control sample with a monoclonal antibody that specifically binds to human ILT7, such as a humanized monoclonal antibody, or its antigen-binding moiety, under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment and human ILT7. The formation of the complex is then detected, and the difference in complex formation between the sample and the control sample indicates the presence of human ILT7 antigen in the sample. Moreover, human ILT7 can be purified via immunoaffinity purification using the anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used to detect ILT7-expressing cells such as pDCs. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used to quantify ILT7 antigen or ILT7-expressing cells.

[0280] H. Exemplary Embodiments Embodiment 1: An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, wherein (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 NOs. 11, 12, and 13, respectively, or a variant thereof having up to 5 amino acid substitutions, additions, and / or deletions in the VL CDR, 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 NOs. 14, 15, and 16, respectively, or a variant thereof having up to approximately 5 amino acid substitutions, additions, and / or deletions in the VH CDR, or (2) as defined by Chothia, (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, or VL An antibody or antigen-binding fragment thereof comprising a variant thereof having up to five amino acid substitutions, additions, and / or deletions in the CDR, and / or (b) VH comprising 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 approximately five amino acid substitutions, additions, and / or deletions in the VH CDR.

[0281] Embodiment 2: The antibody or antigen-binding fragment according to Embodiment 1, comprising 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.

[0282] Embodiment 3: The antibody or antigen-binding fragment according to Embodiment 1, comprising 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.

[0283] Embodiment 4: An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, comprising (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with respect 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 with respect to the amino acid sequence of SEQ ID NO: 10.

[0284] Embodiment 5: The antibody or antigen-binding fragment according to Embodiment 4, comprising VL and VH having the amino acid sequences of SEQ ID NOs: 9 and 10, respectively.

[0285] Embodiment 6: An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, comprising (a) VL including VL CDR1, VL CDR2, and VL CDR3 from VL having the amino acid sequence of SEQ ID NO: 9, and / or (b) VH including VH CDR1, VH CDR2, and VH CDR3 from VH having the amino acid sequence of SEQ ID NO: 10.

[0286] Embodiment 7: The antibody or antigen-binding fragment according to any one of Embodiments 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.

[0287] Embodiment 8: The antibody or antigen-binding fragment according to Embodiment 7, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment.

[0288] Embodiment 9: The antibody or antigen-binding fragment according to Embodiment 8, comprising (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one amino acid sequence among SEQ ID NOs: 19 to 22, and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one amino acid sequence among SEQ ID NOs: 23 to 28.

[0289] Embodiment 10: The antibody or antigen-binding fragment according to Embodiment 9, comprising (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 19, and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 26.

[0290] Embodiment 11: The antibody or antigen-binding fragment according to Embodiment 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 including VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 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, and including (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 CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 17, 18, and 16, respectively.

[0291] Embodiment 12: (1) Sequence IDs 19 and 23, respectively, (2) Sequence IDs 19 and 24, (3) Sequence IDs 19 and 25, respectively, (4) Sequence IDs 19 and 26, (5) Sequence IDs 19 and 27, respectively, (6) Sequence IDs 19 and 28, respectively, (7) Sequence IDs 20 and 23, respectively, (8) Sequence IDs 20 and 24, (9) Sequence IDs 20 and 25, respectively, (10) Sequence IDs 20 and 26, respectively, (11) Sequence IDs 20 and 27, respectively, (12) Sequence IDs 20 and 28, respectively, (13) Sequence IDs 21 and 23, (14 (15)Sequence IDs 21 and 25, (16)Sequence IDs 21 and 26, (17)Sequence IDs 21 and 27, (18)Sequence IDs 21 and 28, (19)Sequence IDs 22 and 23, (20)Sequence IDs 22 and 24, (21)Sequence IDs 22 and 25, (22)Sequence IDs 22 and 26, (23)Sequence IDs 22 and 27, or (24)Sequence IDs 22 and 28, respectively, comprising VL and VH according to Embodiment 9.

[0292] Embodiment 13: The antibody or antigen-binding fragment according to Embodiment 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.

[0293] Embodiment 14: The antibody or antigen-binding fragment according to any one of Embodiments 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).

[0294] Embodiment 15: The antibody or antigen-binding fragment according to any one of Embodiments 1 to 13, wherein the antibody or antigen-binding fragment is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0295] Embodiment 16: The antibody or antigen-binding fragment according to Embodiment 15, wherein the antibody is an IgG1 antibody.

[0296] Embodiment 17: The antibody or antigen-binding fragment according to Embodiment 16, comprising a light chain constant region (CL) having at least 85% sequence identity with kappa CL (Cκ, SEQ ID NO: 29).

[0297] Embodiment 18: The antibody or antigen-binding fragment according to Embodiment 16, comprising a light chain constant region (CL) having at least 85% sequence identity with lambda CL (Cλ, SEQ ID NO: 30).

[0298] Embodiment 19: The antibody or antigen-binding fragment according to Embodiment 16, comprising a heavy chain constant region (CH) having at least 85% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 31 and 40-44.

[0299] Embodiment 20: The antibody or antigen-binding fragment according to any one of Embodiments 16 to 19, wherein the heavy chain constant region (CH) of the IgG1 antibody contains wild-type IgG1 CH or contains at least one amino acid mutation that enhances the antibody's ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent phagocytosis).

[0300] Embodiment 21: The antibody or antigen-binding fragment according to Embodiment 20, wherein the CH region of the IgG1 antibody has amino acid substitutions in 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.

[0301] Embodiment 22: The antibody or antigen-binding fragment according to Embodiment 20, wherein the CH region of the IgG1 antibody has amino acid substitutions that are 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.

[0302] Embodiment 23: The CH region of the IgG1 antibody is numbered according to the EU index: (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 The antibody or antigen-binding fragment according to Embodiment 20, modified by amino acid substitutions such as (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.

[0303] Embodiment 24: The antibody or antigen-binding fragment according to 23, wherein the CH region has an amino acid sequence selected from the group consisting of SEQ ID NOs. 45 to 64.

[0304] Embodiment 25: The antibody or antigen-binding fragment according to Embodiment 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.

[0305] Embodiment 26: The antibody or antigen-binding fragment according to Embodiment 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.

[0306] Embodiment 27: The antibody or antigen-binding fragment according to any one of Embodiments 16 to 26, wherein the Fc region of the IgG1 antibody is defucosylated.

[0307] Embodiment 28: An antibody or antigen-binding fragment thereof that competes with any one of Embodiments 1 to 27 for binding to human ILT7.

[0308] Embodiment 29: The antibody or antigen-binding fragment according to any one of Embodiments 1 to 28, wherein the antibody or antigen-binding fragment is a bispecific antibody or a multispecific antibody.

[0309] Embodiment 30: The antibody or antigen-binding fragment according to any one of Embodiments 1 to 29, wherein the antibody or antigen-binding fragment is a monoclonal antibody or an antigen-binding fragment.

[0310] Embodiment 31: The antibody or antigen-binding fragment (1) has a K content of 500 nM or less, as measured 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, and LILB5, (3) inhibits interferon alpha (IFNα) release by 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) to (7), as described in any one of Embodiments 1 to 30.

[0311] Embodiment 32: An antibody or antigen-binding fragment thereof that specifically binds to the protease domain of human ILT7, wherein (1) the K content is 500 nM or less, as measured by SPR. DAn antibody or its antigen-binding fragment that either (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, and LILB5, (3) inhibits IFNα release by 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) to (7).

[0312] Embodiment 33: The antibody or antigen-binding fragment according to Embodiment 31 or 32, wherein the antibody or antigen-binding fragment (1) inhibits IFNα release by 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 more, or any combination of (1) to (5).

[0313] Embodiment 34: The antibody or antigen-binding fragment according to Embodiment 33, which (1) inhibits IFNα release by PBMCs with an EC50 in the range of 0.01 nM to 0.1 nM, (2) exhibits NK-dependent ADCC activity against ILT7-expressing cells with an EC50 in the range of 0.001 nM to 0.01 nM, (3) exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells with an EC50 in the range of 1 nM to 50 nM, (4) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with an EC50 in the range of 0.5 nM to 5 nM, or (5) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index in the range of 20% to 80%, or any combination of (1) to (5).

[0314] Embodiment 35: The antibody or antigen-binding fragment according to any one of Embodiments 31 to 34, wherein the antibody or antigen-binding fragment exhibits neutrophil-dependent ADCC activity.

[0315] Embodiment 36: A polynucleotide encoding a polypeptide of an antibody or antigen-binding fragment described in any one of Embodiments 1 to 35.

[0316] Embodiment 37: A vector comprising the polynucleotide described in Embodiment 36.

[0317] Embodiment 38: A host cell comprising the polynucleotide described in Embodiment 36, or the vector described in Embodiment 37.

[0318] Embodiment 39: The host cell according to Embodiment 38, wherein the host cell (1) overexpresses N-acetylglucosaminyltransferase III (GnTIII), (2) lacks α-1,6-fucosyltransferase (FUT8), or (3) has a low fucose content, or any combination of (1) to (3).

[0319] Embodiment 40: A method for producing an antibody or antigen-binding fragment thereof that specifically binds to human ILT7, comprising culturing the host cells described in Embodiment 38 or 39 in a culture medium under conditions that enable the expression of the antibody or antibody fragment.

[0320] Embodiment 41: The method according to Embodiment 40, further comprising isolating the antibody from the culture.

[0321] Embodiment 42: A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment described in any one of Embodiments 1 to 35, and a pharmaceutically acceptable carrier.

[0322] Embodiment 43: A method for reducing type I interferon (IFN) in a subject where such reduction is necessary, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment described in any one of Embodiments 1 to 35.

[0323] Embodiment 44: The method according to Embodiment 43, wherein the type I interferon is IFNα.

[0324] Embodiment 45: A method for suppressing or depleting pDCs in a subject where such suppression is necessary, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment described in any one of Embodiments 1 to 35.

[0325] Embodiment 46: A method for reducing autoimmunity in a subject in need, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment described in any one of Embodiments 1 to 35.

[0326] Embodiment 47: The method according to any one of Embodiments 43 to 46, wherein the subject has an autoimmune disease.

[0327] Embodiment 48: A method for treating an autoimmune disease associated with type I IFN or pDC in a subject in need, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment described in any one of Embodiments 1 to 35.

[0328] Embodiment 49: The method according to Embodiment 47 or 48, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

[0329] Embodiment 50: The method according to any one of Embodiments 43 to 49, further comprising administering an additional therapy to the subject.

[0330] Embodiment 51: The method according to any one of Embodiments 43 to 50, wherein the subject is a human.

[0331] Embodiment 52: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 in reducing type I IFN.

[0332] Embodiment 53: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 for the preparation of a pharmaceutical for reducing type I IFN.

[0333] Embodiment 54: The use described in Embodiment 52 or 53, wherein the type I IFN is IFNα.

[0334] Embodiment 55: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 in suppressing or depleting pDCs.

[0335] Embodiment 56: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 for the preparation of a pharmaceutical for inhibiting or depleting pDCs.

[0336] Embodiment 57: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 in reducing autoimmunity.

[0337] Embodiment 58: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 for the preparation of a pharmaceutical product for reducing autoimmunity.

[0338] Embodiment 59: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 in treating an autoimmune disease associated with type I IFN or pDC.

[0339] Embodiment 60: Use of an antibody or antigen-binding fragment according to any one of Embodiments 1 to 35 for the preparation of a pharmaceutical for treating an autoimmune disease associated with type I IFN or pDC.

[0340] Embodiment 61: The use according to Embodiment 59 or 60, wherein the autoimmune disease is SLE.

[0341] The implementation of the present invention will utilize, unless otherwise indicated, conventional techniques in molecular biology, cell biology, microbiology, gene analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the art. These techniques are described in and fully explained in the references cited herein.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, Second Edition, Cold Spring Harbor Laboratory Press, Sambrook et al. Press,Cold Spring Harbor,NY,Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley&Sons(1987 and annual updates),CURRENT PROTOCOLS IN IMMUNOLOGY,John Wiley&Sons(1987 and annual updates)Gait(ed.)(1984)OLIGONUCLEOTIDE SYNTHESIS:A PRACTICAL APPROACH,IRL See also Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practice Approach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering, Second Edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology); Vol. 248, Humana Press, Inc. (Each of these is incorporated herein by reference in whole). [Examples]

[0342] The embodiments provided below are for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Therefore, the invention should not be construed as being limited in any way to the embodiments below, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0343] In short, the results of the studies described below show that cmAb12 and its humanized version bind to human ILT7 with high affinity and are more potent than the benchmark antibody daxzirimab ("Tab1", heavy chain: SEQ ID NO: 38; and light chain: SEQ ID NO: 39) in blocking IFNα release and suppressing pDCs.

[0344] Example 1: Immunization and antibody screening Methods: Balb / c and SJL mice were immunized using three different strategies to produce anti-ILT7 antibodies. First, human ILT7-his protein was generated by fusing the 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, thereby obtaining 293F-human ILT7 cells. All of the above materials were used as immunogens. In particular, human ILT7-his protein was administered by Hoch injection. Serum titers were examined for the first round of screening, and antibodies from mice with a strong immune response to ILT7 were selected for hybridoma generation. Next, the selected monoclonal antibodies were re-screened for their binding affinity to ILT7 using FACS and ELISA, as well as their activity in reducing IFNα release using an IFNα release assay.

[0345] Example 2: Generation and Characterization of Chimeric Antibodies Methods: Twenty-five antibodies were expressed and purified from hybridoma screening. Vectors expressing the heavy and light chains of the chimeric antibodies were constructed. The heavy chain expression vector contained the coding sequence of the heavy chain variable domain of the chimeric antibody, linked to the coding sequence of the heavy chain constant region of human IgG1. Similarly, the light chain expression vector contained the coding sequence for the light chain variable domain of the chimeric antibody and the coding sequence for the constant region of the κ light chain.

[0346] Example 3: Binding of cmAb12 to ILT7 as measured by ELISA. Methods: The binding of chimeric anti-ILT7 antibodies, including cmAb12, identified from screening to the human ILT7-his protein was measured by ELISA along with the isotype antibody hIgG1 and the reference anti-ILT7 antibody Tab1 as negative controls. 96-well ELISA plates were coated overnight at 4°C with human ILT7-his in PBS (2 μg / ml). Chimeric antibodies were added to the wells and co-incubated with the coated protein at 37°C for 1 hour, followed by the addition of secondary HRP-labeled antibodies. After further 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. Absorbance at 450 nm was measured using a microplate reader.

[0347] Results and conclusions: As shown in Figure 1, cmAb12 has high affinity (EC2) (0.07513 nM). 50 It bound to the human ILT7 protein.

[0348] Example 4: Binding of cmAb12 to ILT7-expressing cells as measured by flow cytometry. Methods: 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. CHOK1-cynomolgus monkey ILT7 stable cell lines were constructed in the same manner as 293F-human ILT7 cells. Both cell lines were seeded, resuspended, and incubated with the chimeric antibody at 4°C for 1 hour. The 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.

[0349] Results and Conclusions: As shown by the concentration-MFI ("Central Fluorescence Intensity") curves in Figures 2A-2B, cmAb12 binds in a dose-dependent manner to 293F-human ILT7 cells (Figure 2A) and CHOK1-cynomolgus monkey ILT7 cells (Figure 2B). EC of cmAb12 50 The levels were 0.3121 nM in 293F-human ILT7 cells and 0.9385 nM in CHOK1-cynomolgus monkey ILT7 cells.

[0350] Example 5: Lack of cross-reactivity of cmAb12 to other LILR family members Methods: The binding specificity of a chimeric antibody containing cmAb12 to ILT7 and 10 related LILR superfamily member proteins was evaluated by ELISA. As shown, the chimeric antibody was incubated at 37°C for 1 hour on a plate coated with 1 μg / ml of LILR superfamily protein, followed by incubation of the secondary antibody. Detailed OD450 measurements are listed in Table 4.

[0351] Results and Conclusion: As shown in Table 4, cmAb12 did not specifically bind to other LILR family members (affinity comparable to IgG-negative controls), demonstrating its high specificity for ILT7.

[0352] [Table 4]

[0353] Example 6: Blocking CpG-induced IFNα production in human PBMCs by cmAb12 Methods: The activity of anti-ILT7 chimeric antibodies disclosed herein, including cmAb12, 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 of IL-2 and pre-incubated with the antibody at 4°C, 5% CO2 for 6 hours as shown. Then, ODN2216 solution (CpG-containing DNA) was added and the cells were incubated for a further 18 hours. Subsequently, the cell culture supernatant was collected and IFNα levels were measured by the Human IFNα pan ELISA Development Kit (HRP).

[0354] Results and conclusions: As shown in Figure 3, cmAb12 inhibited IFNα release by CpG-stimulated PBMCs in a dose-dependent manner.

[0355] Example 7: Epitope Binning Methods: Epitope binning of anti-ILT7 antibodies disclosed herein, including cmAb12, was performed by competitive FACS. Specifically, human ILT7-expressing CHOK1 cells were incubated with an equal volume of reference antibody (Tab1-Alexa488) and the antibody under test at 4°C for 1 hour. Isotyped antibody hIgG1 was used as a negative control. Cells were washed with PBS, and relative MFI of different fluorescence signals was analyzed by flow cytometry.

[0356] The relative inhibition rate of each test antibody against Tab1 was calculated as follows: [F(hIgG) - F(test antibody)] * 100% / F(hIgG). F(hIgG) refers to the signal intensity of Tab1-Alexa488 in the presence of hIgG, and F(test antibody) refers to the signal intensity of Tab1-Alexa488 in the presence of the test antibody. Therefore, the positive inhibition rate indicates that the test antibody and Tab1 competitively bind to the same or overlapping epitopes on ILT7 grouped in the bin. The results of the epitope binning analysis are shown in Table 5.

[0357] Results and Conclusions: As shown in Table 5, the binding of Tab1-Alexa488 did not affect the binding of cmAb12, indicating that cmAb12 and Tab1 bind to different epitopes on human ILT7.

[0358] [Table 5]

[0359] Example 8: Humanization of cmAb12 Methods: For the humanization of cmAb12, the most appropriate human framework for transplanting rodent CDRs was selected using NCBI-derived IgBLAST. Variable regions with high amino acid sequence identity to the rodent variable region (homologous matching or best fit) were used. cmAb12 was humanized by transplanting three VL CDRs into human VL cells that were as homologous as possible to mouse VL cells. Similarly, three VH CDRs were transplanted into human VH cells that were as homologous as possible to mouse VH cells. Numbering was followed according to Kabat and Chothia. Furthermore, exemplary humanized antibody sequences are listed in Table 3. The binding and function of humanized antibodies derived from cmAb12 were evaluated using the same experimental procedure as described above.

[0360] Results and conclusions: As shown in Figures 4A-4B, all humanized cmAb12 antibodies maintained high binding affinity to 293F-human ILT7 cells (Figure 4A) and CHOK1-cynomolgus monkey ILT7 cells (Figure 4B).

[0361] Example 9: Blocking of CpG-induced IFNα production in human PBMCs by a humanized antibody against cmAb12. Methods: The function of blocking CpG-induced IFN-α secretion was evaluated for four humanized cmAb12 antibodies, cmAb12, a positive control antibody (Tab1), and a negative control antibody (hIgG1) using the same experimental procedure as described in Example 6.

[0362] Results and Conclusions: As shown in Figures 5A and 5B, four humanized antibodies of cmAb12 dose-dependently inhibited IFNα secretion in CpG-stimulated PBMCs.

[0363] Example 10: Binding of hu-cmAb12 to ILT7 as measured by Biacore and ELISA. Methods: The binding affinity of the humanized cmAb12 antibody, hu-cmAb12, was further measured by surface plasmon resonance (SPR) technology using 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 contains VL with the amino acid sequence of SEQ ID NO: 19, VH with the amino acid sequence of SEQ ID NO: 26, and CH with the amino acid sequence of SEQ ID NO: 55. Hu-cmAb12 or Tab1 was immobilized on a CM-5 chip. The assay was performed at 25°C, and the electrophoresis buffer was 1×HEPES (10mM HEPES, 150mM NaCl, 3mM EDTA) containing 0.005% Tween-20, pH 7.4. Diluted antibodies were captured on the sensor chip via the Fc capture method. Human ILT7-his was used as the analyte, and electrophoresis buffer was used as the dissociation phase. Similarly, ELISA was performed as described above. Isotype control antibody and reference anti-ILT7 antibody Tab1 were used as controls.

[0364] Results and conclusions: The hu-cmAb12 antibody showed a KD of 111 nM when measured by Biacore (data not shown) and an EC of 0.07911 μg / ml when measured by ELISA. 50 (Figure 6) showed that the cells bound to human ILT7, and all were equivalent to Tab1.

[0365] Example 11: Binding of hu-cmAb12 to ILT7-expressing cells as measured by flow cytometry. Methods: Both 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, followed by the addition of a secondary antibody, and incubation at 4°C for 1 hour. Isotype control antibodies and reference anti-ILT7 antibody Tab1 were used as negative and positive controls. The concentration-MFI curves of FACS were then determined.

[0366] Results and Conclusion: Representative results are shown in Figures 7A-7B, which show that the hu-cmAb12 antibody produced EC2 levels of 0.3434 nM and 1.649 nM, respectively. 50 This shows that it bound to 293F-human ILT7 cells (Figure 7A) and CHOK1-cynomolgus monkey ILT7 cells (Figure 7B) (comparable to Tab1).

[0367] Example 12: Binding of hu-cmAb12 to human PBMCs Methods: The hu-cmAb12 antibody was conjugated with the fluorescent dye Alexa488, and PBMC cells (Allcells, frozen) were seeded in assay medium (1640 medium + 10% FBS). All cells were first stained with a live / dead dye, and then specific binding of hu-cmAb12 was detected in T cells, B cells, natural killer cells, natural killer T cells, monocytes, and plasmacytoid dendritic cells. In particular, plasmacytoid dendritic cells were identified as having low CD11c, being HLA-DR positive, and being CD123 positive.

[0368] Results and Conclusions: Representative results are provided in Figure 8. As shown, similar to Tab1, the hu-cmAb12 antibody specifically bound to pDCs in human PBMCs, but not to T cells, B cells, NK cells, NKT cells, or monocytes.

[0369] Example 13: Blocking CpG-induced IFNα production in human PBMCs by hu-cmAb12 Methods: The hu-cmAb12 antibody was also tested for its activity in reducing IFNα release by an IFNα release assay using the same experimental procedure as above. PBMCs obtained from four different donors were tested.

[0370] Results and Conclusions: Representative results are provided in Figures 9A-9B. As shown, hu-cmAb12 strongly inhibited IFNα release in CpG-stimulated PBMCs obtained from all four donors. Compared to Tab1, hu-cmAb12 resulted in lower EC. 50 It also showed a higher inhibition rate.

[0371] Example 14: NK cell-dependent ADCC activity of hu-cmAb12 Methods: 293F-human ILT7 cells were used as target cells. After labeling with DELFIA BATDA reagent (Perkin Elmer, AD0116) at 37°C for 20 minutes, the cells were washed and resuspended in assay medium (RPMI1640 medium, 2% FBS without phenol red). Meanwhile, NK effector cells (NK-92 CD16a 176V) were also resuspended in assay medium. The target cells and effector cells were then mixed and seeded in a 5:1 ratio. Subsequently, hu-cmAb12 antibody was added, and the plate was incubated at 37°C and 5% CO2 for 4 hours. The supernatant was transferred to a DELFIA® yellow 96-well plate (Perkin Elmer, catalog no. AAAND-0001) pre-filled with europium solution, and the fluorescence absorbance at 615 nm was measured. The results of ADCC were demonstrated by cytotoxicity %.

[0372] Results and Conclusions: Representative results are provided in Figure 10. As shown, hu-cmAb12 has a lower EC of 0.0038 nM than Tab1 (0.0103 nM). 50 Then, we depleted ILT7-expressing cells via NK-dependent ADCC activity.

[0373] Example 15: Neutrophil-dependent ADCC activity of hu-cmAb12 Methods: 293F-human ILT7 cells were used as target cells. The cells were washed and resuspended in assay medium (RPMI1640 medium, 2% FBS, without phenol red). Meanwhile, neutrophils were isolated from whole blood using density gradient centrifugation and resuspended in assay medium containing GM-CSF (50 U / mL). Target cells and effector cells were mixed and seeded in an 80:1 ratio. Subsequently, hu-cmAb12 antibody was added, and the plates were incubated at 37°C and 5% CO2 for 3 hours. LDH released in the supernatant was detected using an LDH assay kit. The neutrophil-mediated killing effect on target cells was demonstrated by cytotoxicity percentage.

[0374] Results and Conclusions: Representative results are provided in Figure 11. As shown, hu-cmAb12 has an EC of 16.15 nM. 50 This depleted ILT7-expressing cells via neutrophil-dependent ADCC activity. In contrast, Tab1 lacked neutrophil-dependent ADCC activity.

[0375] Example 16: Macrophage-dependent ADCP activity of hu-cmAb12 Methods: PBMCs were obtained from three different donors and seeded in FBS-free 1640 medium for 2 hours. Non-adherent cells were then cultured in induction medium (1640 + 10% FBS + 20 ng / mL rhuM-CSF). Half of the induction medium was replaced with fresh induction medium every two days. On day 8, monocyte-derived macrophages (MDMs) were digested and pre-seeded as effector cells. 293F-human ILT7 cells were used as target cells. Cells were labeled with 2 μM CFSE in PBS at 37°C for 8 minutes, washed, and resuspended. Cultured MDMs were added with hu-cmAb12, antibody, followed by an equal volume of target cells, and incubated at 37°C for 4 hours. All cells were then isolated and stained with APC mouse anti-hCD11b for flow cytometry analysis. Phagocytosis % reflected the percentage of MDMs that engulfed target cells. The phagocytosis % curve for ADCPs is shown.

[0376] Results and Conclusions: Representative results are provided in Figures 12A-12B. As shown, using PBMCs from three different donors, hu-cmAb12 yielded EC values ​​of 0.881 nM (Donor 1), 2.171 nM (Donor 2), and 1.577 nM (Donor 3), respectively. 50 Furthermore, it depleted ILT7-expressing cells via macrophage-dependent ADCP activity at maximum phagocytic indices of 57.844% (Donor 1), 45.687% (Donor 2), and 32.126% (Donor 3), demonstrating better phagocytic activity than Tab1.

[0377] Example 17: Specific pDC depletion by hu-cmAb12 in humanized mice and cynomolgus monkeys Methods: Eighteen NOG-EXL mice with at least eight weeks of immunoreconstitution were used. 5 μg of FLT3L protein was intravenously injected into the mice three times a week for two weeks. Two days after the last FLT3L injection was defined as the day before Day 0 (Day-1). Blood samples were then taken from all mice, and the proportions of T cells, B cells, NK cells, pDCs, and monocytes were detected by flow cytometry analysis. Each mouse had 5 × 10⁶ cells. 4After intravenous administration of human NK cells, mice were intraperitoneally injected with either a 5 mg / kg dose of the isotype control antibody hIgG1 or a 1 mg / kg or 5 mg / kg dose of hu-cmAb12 antibody on day 0. Peripheral blood was sampled from all mice, and 24 hours after antibody injection, the proportions of T cells, B cells, NK cells, pDCs, and monocytes were detected by flow cytometry analysis. In particular, human pDCs in mice were identified as hCD45-positive, mouse CD45-negative, CD3-negative, CD19-negative, CD14-negative, HLA-DR-positive, and CD123-positive.

[0378] Cynomolgus monkeys were also used. Specifically, a vehicle or hu-cmAb12 antibody at a dose of 30 mg / kg was intravenously injected on day 0. Peripheral blood was sampled from all monkeys, and the proportions of T cells, B cells, NK cells, pDCs, and monocytes were detected by flow cytometry analysis on day 0 (before antibody injection), day 1 (24 hours after antibody injection), day 7 (168 hours after antibody injection), and day 28 (672 hours after antibody injection). In particular, pDCs in cynomolgus monkeys were identified as CD45-positive, CD19-negative, CD3-negative, CD159a-negative, HLA-DR-positive, CD14-negative, CD1c-negative, and CD123-positive.

[0379] Results and Conclusions: Representative results are provided in Figures 13A-13B. As shown, the hu-cmAb12 antibody specifically and rapidly depleted pDCs 24 hours after injection in humanized mice and cynomolgus monkeys, while other cells remained unaffected. In humanized mice, hu-cmAb12 antibodies at doses of 1 mg / kg and 5 mg / kg reduced pDCs by 80% and 87%, respectively, 24 hours after injection, compared to an 18% reduction in the IgG1 isotype group (Figure 13A). In cynomolgus monkeys, the hu-cmAb12 antibody reduced pDCs by 95% 24 hours after injection, compared to a 38% reduction in the control (Figure 13B).

[0380] All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined below. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques and / or substitutions of equivalent techniques that would be obvious to those skilled in the art.

[0381] All patents, patent publications, patent applications, journal articles, books, technical references, etc., discussed herein are incorporated herein by reference in their entirety for all purposes.

[0382] The foregoing description includes numerous specific details to provide a more complete understanding of the Disclosure. However, it will be apparent to those skilled in the art that the embodiments described herein may be carried out without using one or more of these specific details. In other examples, features and procedures well known to those skilled in the art are not described in order to avoid obscuring the Disclosure. The embodiments described herein are described for illustrative purposes only, not limiting purposes. While the Disclosure is described primarily by reference to specific embodiments, it is anticipated that other embodiments will become...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, (1) When defined by Kabat, (a) Light chain variable regions (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 11, 12, and 13, respectively, or variants thereof having up to about five amino acid substitutions, additions, and / or deletions in the VL CDR, and / or (b) Heavy chain variable regions (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs. 14, 15, and 16, respectively, or variants thereof having up to about five amino acid substitutions, additions, and / or deletions in the VH CDR, (2) When defined by Chothia, (a) VLs comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs. 11, 12, and 13 respectively, or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VL CDR, and / or (b) VH comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs. 17, 18, and 16, respectively, or variants thereof having up to approximately five amino acid substitutions, additions, and / or deletions in the VH CDR. An antibody or its antigen-binding fragment, including the above.

2. The antibody or antigen-binding fragment according to claim 1, comprising 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 according to claim 1, comprising 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, (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with respect 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 with respect to the amino acid sequence of SEQ ID NO: 10 An antibody or its antigen-binding fragment, including the above.

5. The antibody or antigen-binding fragment according to claim 4, comprising VL and 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, (a) VLs comprising VL CDR1, VL CDR2, and VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 9, and / or (b) VH CDR1, VH CDR2, and VH CDR3 from VH having the amino acid sequence of SEQ ID NO: 10 An antibody or its antigen-binding fragment, including the above.

7. The antibody or antigen-binding fragment according to any one of claims 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.

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

9. (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one amino acid sequence among 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 with respect to any one amino acid among SEQ ID NOs. 23-28 The antibody or antigen-binding fragment according to claim 8, comprising:

10. (a) VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with respect 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 with the amino acid sequence of SEQ ID NO: 26 The antibody or antigen-binding fragment according to claim 9, comprising:

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

12. (1) Sequence IDs 19 and 23 respectively, (2) Sequence IDs 19 and 24 respectively, (3) Sequence IDs 19 and 25 respectively, (4) Sequence IDs 19 and 26 respectively, (5) Sequence IDs 19 and 27 respectively, (6) Sequence IDs 19 and 28 respectively, (7) Sequence IDs 20 and 23 respectively, (8) Sequence IDs 20 and 24 respectively, (9) Sequence IDs 20 and 25 respectively, (10) Sequence IDs 20 and 26 respectively, (11) Sequence IDs 20 and 27 respectively, (12) Sequence IDs 20 and 28 respectively, (13) Sequence IDs 21 and 23 respectively, (14) The antibody or antigen-binding fragment according to claim 9, comprising VL and VH having the amino acid sequences of 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 according to claim 12, comprising VL and VH having the amino acid sequences of SEQ ID NOs. 19 and 26, respectively.

14. The antibody or antigen-binding fragment according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment is 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 according to 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 according to claim 15, wherein the antibody is an IgG1 antibody.

17. The antibody or antigen-binding fragment according to claim 16, comprising a light chain constant region (CL) having at least 85% sequence identity with kappa CL (Cκ, SEQ ID NO: 29).

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

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

20. The antibody or antigen-binding fragment according to any one of claims 16 to 19, wherein the heavy chain constant region (CH) of the IgG1 antibody contains a wild-type IgG1 CH, or contains at least one amino acid mutation that enhances the ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent phagocytosis) of the antibody.

21. The antibody or antigen-binding fragment according to claim 20, wherein the CH region of the IgG1 antibody has an amino acid substitution in 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 according to claim 20, wherein the CH region of the IgG1 antibody has an amino acid substitution which 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, as numbered according to the EU index.

23. The CH region of the IgG1 antibody is numbered according to the EU index as follows: (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; (viiii) 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, modified by amino acid substitutions, the antibody or antigen-binding fragment according to claim 20.

24. The antibody or antigen-binding fragment according to claim 23, wherein the CH region has one amino acid sequence from sequence numbers 45 to 64.

25. VL having the amino acid sequence of SEQ ID NO: 19, VH having the amino acid sequence of SEQ ID NO: 26, CH having the amino acid sequence of SEQ ID NO: 55 The antibody or antigen-binding fragment according to claim 1, comprising:

26. 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, CH having the amino acid sequence of SEQ ID NO: 55 The antibody or antigen-binding fragment according to claim 1, comprising:

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 antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment described in 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 an antigen-binding fragment.

31. The antibody or antigen-binding fragment is (1) When measured by SPR, K is 500 nM or less Ｄ So does it bind to human ILT7, (2) Not specifically binding to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, and LILB5, (3) Inhibit the release of interferon alpha (IFNα) by peripheral blood mononuclear cells (PBMCs), (4) Selectively bind to plasmacytoid dendritic cells (pDCs) in human PBMCs, (5) Does it exhibit 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) to (7), The antibody or antigen-binding fragment according to any one of claims 1 to 30.

32. An antibody or its antigen-binding fragment that specifically binds to the protease domain of human ILT7, (1) When measured by SPR, K is 500 nM or less Ｄ So does it bind to human ILT7, (2) Not specifically binding to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, and LILB5, (3) Inhibit the release of IFNα by PBMC, (4) Selectively binds to pDCs in human PBMCs, (5) Does it show 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) to (7), An antibody or its antigen-binding fragment.

33. The antibody or antigen-binding fragment is (1) Inhibit IFNα release by CpG-stimulated PBMCs in vitro with an EC50 of 1 nM or less, (2) Does it show NK-dependent ADCC activity against ILT7-expressing cells at an EC50 of 0.01 nM or less? (3) Does it show neutrophil-dependent ADCC activity against ILT7-expressing cells at an EC50 of 100 nM or less? (4) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells at an EC50 of 10 nM or less, or (5) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells at a maximum phagocytic index of 20% or more, or any combination of (1) to (5). The antibody or antigen-binding fragment according to claim 31 or 32.

34. The antibody or antigen-binding fragment is (1) Inhibit IFNα release by PBMCs at an EC50 in the range of 0.01 nM to 0.1 nM, (2) Whether it exhibits NK-dependent ADCC activity against ILT7-expressing cells at an EC50 in the range of 0.001 nM to 0.01 nM, (3) Whether the EC50 ranges from 1 nM to 50 nM and exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells, (4) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells at an EC50 in the range of 0.5 nM to 5 nM, or (5) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells at a maximum phagocytic index in the range of 20% to 80%, or any combination of (1) to (5). The antibody or antigen-binding fragment according to claim 33.

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 described in claim 36.

38. A host cell comprising the polynucleotide described in claim 36, or the vector described in claim 37.

39. The aforementioned host cells (1) Overexpress N-acetylglucosaminyltransferase III (GnTIII), (2) Lacking a-1,6-fucosyltransferase (FUT8), or (3) Having a low fucose content, or any combination of (1) to (3), The host cell according to claim 38.

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

41. The method according to 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 for which such reduction is necessary, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment described in any one of claims 1 to 35.

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

45. A method for suppressing or depleting pDCs in a subject where such suppression is necessary, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment described in any one of claims 1 to 35.

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

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

48. A method for treating an autoimmune disease associated with type I IFN or pDC in a subject in need, 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. The use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 in 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 pharmaceutical 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 in suppressing or depleting pDCs.

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

57. The use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 in 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 pharmaceutical product for reducing autoimmunity.

59. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 in treating an autoimmune disease 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 pharmaceutical for treating an autoimmune disease associated with type I IFN or pDC.

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

Images