Antibody

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof capable of binding to CD1a. The antibodies or antigen-binding fragments thereof may be chimeric or humanized and may be used to treat one or more inflammatory skin or mucosal disorders or diseases, or one or more related systemic diseases or disorders, or one or more inflammatory drug reactions manifesting systemically, or CD1a-expressing malignancies.

Description

[Technical Field]

[0001] The present invention relates to antibodies and their use in the treatment, prevention, diagnosis, or monitoring of inflammatory skin and mucosal diseases or disorders, or related systemic diseases or disorders, or inflammatory drug reactions, or CD1a-expressing malignancies. [Background technology]

[0002] Antigen presentation is one of the fundamental pillars of host immunity, allowing the immune system to detect threats, including infection, tissue damage, and disease, and orchestrate a coordinated defense. Antigen presentation involves antigen internalization, processing, and presentation by presentation molecules on the surface of specialized antigen-presenting cells (APCs). Antigen presentation is designed to achieve optimal activation of the immune response, targeting the antigen source and eliminating the threat. Antigens encompass a wide range of molecules, including peptides, lipids, and metabolites. MHC1 and MHC2 are proteins expressed on the surface of APCs that bind peptide antigens and are primarily present on CD8+ and CD4+ T cells, respectively. These T cell subsets are induced to exert their effector functions upon recognition of MHC-bound peptide antigens by cell surface T cell receptors (TCRs), enabling immunity against pathogens and cancer. However, dysregulation of the presentation of harmless antigens, such as allergens in allergic diseases or self-proteins in autoimmunity, leads to host damage, inflammation, and disease. Therefore, targeting antigen presentation pathways is a powerful means of modulating the subsequent immune response.

[0003] CD1 molecules constitute a family of antigen-presenting molecules structurally similar to MHC II. In contrast, CD1 molecules are relatively non-polymorphic, and the CD1 antigen-binding groove is rich in hydrophobic amino acids, allowing for the presentation of lipid species. Lipids are important antigens, forming key components of host and pathogen cell membranes and less susceptible to mutation than protein-derived peptide antigens. The CD1 family consists of the cell surface group 1 molecules CD1a / b / c, group 2 molecule CD1d, and group 3 molecule CD1e. Most of our understanding of CD1 lipid presentation and T cell responses has come from studies of invariant natural killer T cell recognition of glycolipid-bound CD1d. This is in part because CD1d is the only CD1 molecule normally expressed by mice. While CD1d and MHC II molecules are widely expressed, expression of MHC II and group 1 CD1 is relatively restricted to APCs. However, unique among these molecules, CD1a is highly specific for the skin and mucous membranes. CD1a is constitutively expressed by Langerhans cells (LCs) in the epidermis of skin and mucosa (1) and is commonly used, in addition to Langerin, as an identifying marker for LCs. Furthermore, CD1a is expressed at lower levels on a subset of dermal dendritic cells (2-4), and can be expressed and upregulated on cutaneous innate lymphoid cells (ILCs), particularly ILC2s (5). Importantly, CD1a was first described on the surface of immature thymocytes, but its expression is typically lost upon T cell maturation (6). High levels of constitutive expression of CD1a in skin indicate an important physiological role for CD1a-dependent surveillance and T cell activation in healthy and diseased human skin. Furthermore, increased CD1a expression in atopic dermatitis skin may underlie the increased activation of CD1a-reactive T cell populations in inflammatory skin diseases.

[0004] T cell responses directed by CD1a, CD1b, or CD1c molecules presenting mycobacterial lipid-based antigens are involved in human immune responses to Mycobacterium tuberculosis and Mycobacterium leprae infections. Recognition of other, more common pathogenic or commensal bacterial lipids by CD1a-restricted T cells is the subject of ongoing research, but some data are presented here. While TCR recognition of peptide antigens by MHC-restricted T cells is generally highly specific for peptide antigens, CD1-mode TCR recognition is more diverse, involving highly lipid-specific responses (7) and cross-reactivity, as in the case of CD1a-autoreactive T cells, or even apparently lipid-independent signaling mediated by direct TCR-CD1 interactions (8-10). CD1a-autoreactive T cells are, in some cases, activated upon recognition of CD1a bearing small, hydrophobic, host-derived lipids that fit within the antigen-binding groove and do not protrude, allowing the TCR to interact with the CD1a protein itself rather than the lipid. In this case, attachment of lipids with large or charged head groups would prevent the interaction between autoreactive TCRs and CD1a, thereby preventing T cell activation (11, 12).

[0005] CD1a is relatively non-polymorphic and therefore has potential across the population in the prevention and / or treatment of inflammatory skin and mucous membrane diseases and disorders such as atopic dermatitis, psoriasis, lupus erythematosus, or related systemic diseases or disorders, or systemic inflammatory drug reactions. In this case, the frequency of subsets of CD1a-expressing dendritic cells changes, and the migration patterns of LCs or responsive T cells are altered (13-15). Furthermore, CD1a has been associated with inflammatory bowel disease, multiple sclerosis, Guillain-Barré syndrome, thyroiditis, and other systemic disorders, including neurodegeneration (Al-amodi, Inflammatory Bowel Diseases 2018 24:1225-1236; H. Caporale, J Neuroimmunol 2006 177:112-8; Jamshidian, Immunological Investigations 2010 3:874-889; Roura-Mir, J Immunol 2005 174:3773-80; Wang, Aging 2019 11:4521-4535). Furthermore, CD1a may be expressed by certain malignancies, including rare descriptions of other malignancies such as Langerhans cell histiocytosis, Langerhans cell sarcoma, subsets of T-cell lymphoma, subsets of thymoma, and subsets of mastocytosis.

[0006] The object of the present invention is to provide anti-CD1a antibodies. Such antibodies are particularly useful in treating or preventing inflammatory diseases or disorders of the skin or mucous membranes (e.g., psoriasis, dermatitis, lupus erythematosus, or drug reactions that manifest as inflammatory skin or mucous membrane diseases or disorders). Such antibodies may also be beneficial in treating or preventing related systemic diseases or disorders, or systemic inflammatory drug reactions, or in treating CD1a-expressing malignancies. Summary of the Invention

[0007] The present invention relates to an antibody or antigen-binding fragment thereof capable of binding to CD1a. The antibody or antigen-binding fragment thereof can specifically bind to CD1a. The antibody or antigen-binding fragment thereof can preferentially bind to CD1a. The antibody or antigen-binding fragment thereof can induce cell death of cells expressing CD1a. The antibody or antigen-binding fragment thereof can block binding of a ligand to CD1a.

[0008] In one embodiment, the antibody or its antigen-binding fragment is a) CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38, Or a light chain variable region containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, b) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of sequence number 3, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6, Or a light chain variable region containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, c) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or CDR1 of sequence number 12, CDR2 of sequence number 13, and CDR3 of sequence number 14, Or a light chain variable region containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, d) CDR1 of sequence number 17, CDR2 of SEQ ID NO: 18, and CDR3 of sequence number 19, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or CDR1 of sequence number 20, CDR2 of SEQ ID NO: 21, and CDR3 of sequence number 22, Or a light chain variable region containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, e) CDR1 of sequence number 25, CDR2 of sequence number 26, and CDR3 of sequence number 27, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or CDR1 of sequence number 28, CDR2 of sequence number 29, and CDR3 of sequence number 30, Or a light chain variable region containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, f) CDR1 with sequence number 91, CDR2 of sequence number 92, and CDR3 of SEQ ID NO: 93, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or CDR1 of SEQ ID NO: 94, CDR2 of SEQ ID NO: 95, and CDR3 of SEQ ID NO: 96, Alternatively, it may be a chimeric antibody comprising or consisting of a light chain variable region containing a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with those sequences.

[0009] In any of the above, an antibody that is a chimeric antibody of any combination of CDRs or an antigen-binding fragment thereof may be used. Alternatively, the antibody that is a chimeric antibody or an antigen-binding fragment thereof may consist only of the listed CDR3 heavy and light chain variable regions described above.

[0010] In another aspect, the antibody or antigen-binding fragment thereof (a) heavy chains containing or consisting of sequences such as SEQ ID NO: 211, SEQ ID NO: 212, or SEQ ID NO: 213, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and / or SEQ ID NO: 210 or a light chain comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (b) Heavy chains containing or consisting of sequences identical to, or having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with, SEQ ID NO: 215, SEQ ID NO: 216, or SEQ ID NO: 217, and / or SEQ ID NO: 214 or a light chain comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (c) Heavy chains containing or consisting of sequences identical to, or having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with, SEQ ID NO: 219, SEQ ID NO: 220, or SEQ ID NO: 221, and / or SEQ ID NO: 218 or a light chain comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (d) Heavy chains containing or consisting of sequences that are at least 80%, 90%, 95%, 98%, 99%, or 100% identical to sequence number 254, sequence number 255, or sequence number 256, and / or SEQ ID NO: 253 Alternatively, it may include a light chain having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with the sequence, or a chimeric antibody comprising such a sequence.

[0011] The antibody or antigen-binding fragment thereof is a) Heavy chains containing or consisting of Sequence ID No. 211, Sequence ID No. 212, or Sequence ID No. 213, and b) It may be a chimeric antibody containing or comprising a light chain comprising SEQ ID NO: 210.

[0012] The antibody or antigen-binding fragment thereof is a) Heavy chains containing or consisting of Sequence ID No. 215, Sequence ID No. 216, or Sequence ID No. 217, and b) It may be a chimeric antibody containing or comprising a light chain comprising SEQ ID NO: 214.

[0013] The antibody or antigen-binding fragment thereof is a) Heavy chains containing or consisting of Sequence ID No. 219, Sequence ID No. 220, or Sequence ID No. 221, and b) It may be a chimeric antibody containing or comprising a light chain comprising SEQ ID NO: 218.

[0014] The antibody or antigen-binding fragment thereof is a) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or b) CDR1 of sequence number 12, CDR2 of sequence number 13, and CDR3 of SEQ ID NO: 14, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109; Alternatively, the antibody may be a humanized antibody comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0015] The antibody or antigen-binding fragment thereof is a) CDR1 of sequence number 17, CDR2 of SEQ ID NO: 18, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134, and CDR3 of SEQ ID NO: 19 or SEQ ID NO: 110, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or b) CDR1 of sequence number 20, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO:22, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, or SEQ ID NO:119; Alternatively, the antibody may be a humanized antibody comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0016] The antibody or antigen-binding fragment thereof is a) CDR1 of sequence number 25, CDR2 of sequence number 26, and CDR3 of sequence number 27, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or b) CDR1 of sequence number 28, CDR2 of sequence number 29, and CDR3 of SEQ ID NO: 30, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122; Alternatively, the antibody may be a humanized antibody comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0017] The antibody or antigen-binding fragment thereof is a) CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or b) CDR1 of SEQ ID NO: 36, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 13, or SEQ ID NO: 139; CDR2 of SEQ ID NO: 37, and CDR3 of sequence number 38, sequence number 123, sequence number 124, sequence number 125, sequence number 126, sequence number 127, sequence number 128, sequence number 129, sequence number 130, or sequence number 131, Alternatively, the antibody may be a humanized antibody comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0018] The antibody or antigen-binding fragment thereof is a) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NOs. 2, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, or 251, and CDR3 of sequence number 3, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or b) CDR1 of SEQ ID NO: 4, CDR2 of Sequence ID 5, Sequence ID 140, or Sequence ID 141, and CDR3 of SEQ ID NO: 6, Alternatively, the antibody may be a humanized antibody comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0019] The antibody or antigen-binding fragment thereof is a) CDR1 with sequence number 91, CDR2 of sequence number 92, sequence number 257, sequence number 258, or sequence number 259, and CDR3 of SEQ ID NO: 93, Or heavy chain variable regions containing sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, and / or b) CDR1 of SEQ ID NO: 94, CDR2 of SEQ ID NO: 95, and CDR3 of SEQ ID NO: 96, Alternatively, the antibody may be a humanized antibody comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0020] Any antibody or antigen-binding fragment CDR disclosed herein may associate with any framework region. Preferably, the framework region is of human origin.

[0021] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO:7, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, or SEQ ID NO:209, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or b) SEQ ID NO: 8, SEQ ID NO: 195, SEQ ID NO: 196, or SEQ ID NO: 197; Alternatively, it may be a humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with the sequence, or comprising the same.

[0022] The antibody or antigen-binding fragment thereof is a) Heavy chain variable regions containing or consisting of sequences identical to or at least 80%, 90%, 95%, 98%, 99%, or 100% of sequence 97, 261, 262, 263, or 264, and / or b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 98 or sequence number 260.

[0023] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 157, and b) A humanized antibody that includes or comprises a light chain variable region containing or consisting of SEQ ID NO: 16, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, or SEQ ID NO: 156.

[0024] The antibody or antigen-binding fragment thereof is a) Heavy chain variable regions comprising or including sequences that are at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO:24, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, or SEQ ID NO:167, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0025] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 31 or sequence number 178, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence 32, sequence 174, sequence 175, sequence 176, or sequence 177.

[0026] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 39 or sequence number 194, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO:40, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, or SEQ ID NO:193, or a sequence with at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0027] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO:7, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, or SEQ ID NO:209, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 8, sequence number 195, sequence number 196, or sequence number 197.

[0028] The antibody or antigen-binding fragment thereof is a) Heavy chain variable regions comprising or consisting of sequences that are at least 80%, 90%, 95%, 98%, 99%, or 100% identical to sequence 97, sequence 261, sequence 262, sequence 263, or sequence 264, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 98 or sequence number 260.

[0029] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 179, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith.

[0030] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 180, or a light chain variable region comprising such sequence.

[0031] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 181, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith.

[0032] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 182, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0033] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 183, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0034] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 184, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0035] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 185, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0036] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 186, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith.

[0037] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 187, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith.

[0038] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 188, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith.

[0039] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 189, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith.

[0040] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 190, or a light chain variable region comprising such sequence.

[0041] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 191, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with it.

[0042] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) A humanized antibody comprising a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 192, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with it.

[0043] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 193, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0044] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 198, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0045] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 198, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0046] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 198, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0047] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 199, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0048] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 199, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0049] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 199, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0050] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 200, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0051] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 200, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0052] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 200, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0053] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 201, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0054] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 201, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0055] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 201, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0056] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 202, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0057] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 202, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0058] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 202, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0059] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 203, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0060] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 203, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0061] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 203, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0062] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 204, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0063] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 204, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0064] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 204, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0065] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 205, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0066] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 205, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0067] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 205, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0068] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 206, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0069] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 206, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0070] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 206, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0071] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 207, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0072] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 207, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0073] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 207, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0074] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 208, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0075] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 208, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0076] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 208, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0077] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 209, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0078] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 209, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0079] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 209, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0080] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 210, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0081] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 210, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0082] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 210, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0083] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 211, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0084] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 211, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0085] The antibody or antigen-binding fragment thereof is a) A heavy chain variable region comprising or consisting of sequence sequence number 211, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0086] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 212, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 195, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0087] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 212, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 196, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0088] The antibody or antigen-binding fragment thereof is a) a heavy chain variable region comprising or consisting of SEQ ID NO: 212, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain variable region comprising, or consisting of, SEQ ID NO: 197, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0089] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 266, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0090] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 267, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0091] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 268, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0092] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 269, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0093] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 270, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0094] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 271, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0095] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 272, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0096] The antibody or antigen-binding fragment thereof is a) A Fab region comprising, or consisting of, the heavy chain portion of SEQ ID NO: 265 and the light chain portion of SEQ ID NO: 273, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, b) a humanized antibody comprising or consisting of an Fc region.

[0097] The antibody or antigen-binding fragment thereof is a) A Fab region comprising, or consisting of, the heavy chain portion of SEQ ID NO: 265 and the light chain portion of SEQ ID NO: 274, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, b) a humanized antibody comprising or consisting of an Fc region.

[0098] The antibody or antigen-binding fragment thereof is a) A Fab region comprising, or consisting of, the heavy chain portion of SEQ ID NO: 265 and the light chain portion of SEQ ID NO: 275, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, b) a humanized antibody comprising or consisting of an Fc region.

[0099] The antibody or antigen-binding fragment thereof is a) A Fab region comprising, or consisting of, the heavy chain portion of SEQ ID NO: 265 and the light chain portion of SEQ ID NO: 276, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, b) a humanized antibody comprising or consisting of an Fc region.

[0100] The antibody or antigen-binding fragment thereof is a) A Fab region comprising, or consisting of, the heavy chain portion of SEQ ID NO: 265 and the light chain portion of SEQ ID NO: 277, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with them, b) a humanized antibody comprising or consisting of an Fc region.

[0101] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 279, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0102] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 265 and a light chain portion of SEQ ID NO: 280, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0103] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 281 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0104] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 281 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0105] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 281 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0106] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 285 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0107] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 285 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0108] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 285 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0109] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 286 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0110] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 286 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0111] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 286 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0112] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 287 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0113] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 287 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0114] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 287 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0115] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 288 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0116] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 288 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0117] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 288 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0118] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 289 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0119] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 289 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0120] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 289 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0121] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 290 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0122] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 290 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0123] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 290 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0124] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 291 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0125] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 291 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0126] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 291 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0127] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 292 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0128] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 292 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0129] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 292 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0130] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 293 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0131] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 292 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0132] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 293 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0133] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 294 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0134] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 294 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0135] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 294 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0136] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 295 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0137] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 295 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0138] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 295 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0139] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 296 and a light chain portion of SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0140] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 296 and a light chain portion of SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0141] The antibody or antigen-binding fragment thereof is a) a Fab region comprising or consisting of a heavy chain portion of SEQ ID NO: 296 and a light chain portion of SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; b) a humanized antibody comprising or consisting of an Fc region.

[0142] The antibody may comprise a human Fc region. More particularly, the antibody or antigen-binding fragment thereof may be a full-length antibody. More particularly, the antibody may be of the IgG isotype. More particularly, the antibody may be an IgG1 or IgG4. Most preferably, the Fc region comprises or consists of SEQ ID NO: 297.

[0143] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 266, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0144] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising or consisting of a light chain comprising or consisting of SEQ ID NO: 267, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0145] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 268, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0146] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 269, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0147] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and 270 b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: YY, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0148] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising or consisting of a light chain comprising or consisting of SEQ ID NO: 271, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0149] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 272, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0150] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 273, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0151] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising or consisting of a light chain comprising or consisting of SEQ ID NO: 274, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0152] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising or consisting of a light chain comprising or consisting of SEQ ID NO: 275, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0153] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 276, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0154] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) A humanized antibody comprising a light chain containing or having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 277.

[0155] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) A humanized antibody comprising a light chain containing or having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 278.

[0156] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) A humanized antibody comprising a light chain containing or having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 279.

[0157] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 280, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0158] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 301, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0159] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 301, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0160] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 301, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0161] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 302, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0162] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 302, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0163] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 302, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0164] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 303, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0165] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 303, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0166] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 303, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0167] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 304, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0168] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 304, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0169] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 304, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0170] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 305, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0171] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 305, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0172] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 305, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0173] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 306, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0174] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 306, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0175] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 306, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0176] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 307, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0177] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 307, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0178] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 307, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0179] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 308, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0180] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 308, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0181] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 308, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0182] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 309, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0183] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 309, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0184] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 309, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0185] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 310, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0186] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 310, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0187] The antibody or antigen-binding fragment thereof is a) A heavy chain containing or consisting of sequence sequence number 310, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0188] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 311, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0189] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 311, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0190] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 311, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0191] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 312, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 282, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0192] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 312, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 283, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0193] The antibody or antigen-binding fragment thereof is a) a heavy chain comprising or consisting of SEQ ID NO: 312, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and b) a humanized antibody comprising, or consisting of, a light chain comprising, or consisting of, SEQ ID NO: 284, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0194] The constant region domain of an antibody, if present, may be selected in consideration of the proposed function of the antibody molecule, particularly any effector function that may be required. For example, the constant region domain may be a human IgA, IgD, IgE, IgG, or IgM domain. In particular, if the antibody molecule is intended for therapeutic use and antibody effector function is required, the human IgG constant region domain, especially the IgG1 and IgG3 isotypes, may be used. Alternatively, if the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, the IgG2 and IgG4 isotypes may be used. It will be understood that sequence variants of these constant region domains may also be used. It will also be known to those skilled in the art that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody, as well as the cell culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation.

[0195] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes one or more amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0196] The antibody or antigen-binding fragment thereof is It may comprise or consist of an ScFv comprising or consisting of SEQ ID NO: 101, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0197] The antibody or antigen-binding fragment thereof is It may comprise or consist of an ScFv comprising or consisting of SEQ ID NO: 102, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0198] The antibody or antigen-binding fragment thereof is It may comprise or consist of an ScFv comprising or consisting of SEQ ID NO: 103, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0199] The antibody or antigen-binding fragment thereof is It may comprise or consist of an ScFv comprising or consisting of SEQ ID NO: 104, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0200] The antibody or antigen-binding fragment thereof is It may comprise or consist of an ScFv comprising or consisting of SEQ ID NO: 105, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0201] The antibody or antigen-binding fragment thereof is It may comprise or consist of an ScFv comprising or consisting of SEQ ID NO: 106, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0202] In another aspect, an antibody or antigen-binding fragment thereof is provided that binds to an epitope on CD1a comprising or consisting of residues Arg83, Tyr84, His86, Glu87, Gln89, Phe90, Glu91, Asn139, Met140, Lys142, His143, Lys146, Val147, and Gln150 of CD1a (residue numbering according to SEQ ID NO: 252).

[0203] In another aspect, an antibody or antigen-binding fragment thereof is provided that binds to an epitope on CD1a comprising or consisting of residues Glu62, Glu65, Leu66, Thr68, Leu69, Ile72, Asn151, His153, Glu154, Ile157, Asn160, Asp164, Thr165, and Arg168 of CD1a (residue numbering according to SEQ ID NO: 252).

[0204] In another embodiment, the residues Glu79, Arg82, Arg83 of CD1a, Antibodies or antigen-binding fragments thereof that bind to an epitope on CD1a comprising or consisting of His86, Glu87, Gln89, Phe90, Glu91, Tyr92, Val147, and Asn150 (residue numbering according to SEQ ID NO: 252) are provided.

[0205] More specifically, the antibody or its antigen-binding fragment binds to one or more disclosed residues, more specifically, to five or more residues.

[0206] The epitopes can be identified by any suitable epitope mapping method known in the art in combination with any of the antibodies provided herein. Examples of such methods include screening peptides of various lengths derived from the full-length target protein for binding to the antibody or fragment thereof of the present disclosure, and identifying the smallest fragment that can specifically bind to the antibody containing the sequence of the epitope recognized by the antibody. The target peptides can be produced synthetically. The peptides that bind to the antibody can be identified, for example, by mass spectrometry. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitopes to which the antibodies of the present invention bind. Typically, when epitope determination is performed by X-ray crystallography, amino acid residues of the antigen within 4 Å of the CDR are considered to be amino acid residues that are part of the epitope. Once identified, the epitopes can be used to prepare fragments that bind to the antibodies of the present invention and, if necessary, can be used as immunogens to obtain further antibodies that bind to the same epitope.

[0207] Epitopes can be determined using several techniques that are available to and known to those skilled in the art, such as X-ray crystallography.

[0208] Whether an antibody binds to the same epitope as a reference antibody or competes with a reference antibody for binding can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is bound to a protein or peptide under saturating conditions. The ability of the test antibody to bind to the protein or peptide is then evaluated. If the test antibody can bind to the protein or peptide after saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope from the reference antibody. On the other hand, if the test antibody cannot bind to the protein or peptide after saturation binding with the reference antibody, the test antibody can bind to the same epitope as the epitope bound by the reference antibody of the present invention.

[0209] In one embodiment, an antibody or its antigen-binding fragment is provided that competes with the antibody or its antigen-binding fragment for binding to CD1a.

[0210] The term "antibody or antigen-binding fragment thereof that competes with" a reference antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay.

[0211] To determine whether an antibody competes for binding with a reference antibody, the above binding methodology is performed in two ways. In the first way, the reference antibody is allowed to bind to the protein / peptide under saturating conditions, followed by evaluating the binding of the test antibody to the protein / peptide molecule. In the second way, the test antibody is allowed to bind to the protein / peptide under saturating conditions, followed by evaluating the binding of the reference antibody to the protein / peptide. If only the first (saturating) antibody can bind to the protein / peptide in both ways, it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily have to bind to the same epitope as the reference antibody, but can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0212] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if mutations of essentially all amino acids in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if mutations of several amino acids that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0213] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0214] The antibody or antigen-binding fragment thereof of the present invention can be isolated.

[0215] In any embodiment, “antibody or its antigen-binding fragment” may refer to one or more, for example, two, of the listed antibodies or their antigen-binding fragments. For example, in any embodiment, two antibodies or their antigen-binding fragments that bind to CD1a at different binding sites may be considered.

[0216] For example, any combination of antibodies or antigen-binding fragments may be utilized in any of the therapeutic applications disclosed herein and / or in any of the monitoring methods disclosed herein. For example, Abs 116 and 16 may be used in combination. Alternatively, Abs 16 and 110 may be used in combination.

[0217] In another embodiment, Ab 116 may be used in any therapeutic application disclosed herein, and Ab 116 may be used in monitoring the same subject. Alternatively, Ab 16 may be used in any therapeutic application disclosed herein, and Ab 116 may be used in monitoring the same subject.

[0218] Any reference to an antibody or its antigen-binding fragment by its internal name (e.g., Ab 116 or Ab 16) includes any chimeric or humanized versions disclosed herein.

[0219] The term "antibody" as referred to herein refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (V H ) and a heavy chain constant region. Each light chain is composed of a light chain variable region (V L The antibody consists of a heavy chain and a light chain constant region. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including the first component (Clq) of the classical complement system.

[0220] The term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. These antigen-binding fragments may be, but are not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, bivalent, trivalent, or tetravalent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies, and any of the above epitope-binding fragments (Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for preparing and manufacturing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0221] The term "chimeric" antibody refers to an antibody in which the variable domains (or at least a portion thereof) of the heavy and / or light chains are derived from a particular source or species, while the remainder of the heavy and / or light chains (i.e., the constant domains) are derived from a different source or species. (Morrison; PNAS 81, 6851 (1984)). Chimeric antibodies can, for example, contain non-human variable domains and human constant domains. Chimeric antibodies are typically produced using recombinant DNA methods. A subcategory of "chimeric antibodies" is "humanized antibodies."

[0222] The term "humanized" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof comprising amino acid residues from non-human HVRs and human FRs. Typically, the heavy and / or light chains comprise one or more CDRs (optionally including one or more modified CDRs) from a donor antibody (e.g., a non-human antibody such as a mouse or rabbit monoclonal antibody) and are grafted onto the heavy and / or light chain variable region frameworks of an acceptor antibody (human antibody) (see, e.g., Vaughan et al., Nature Biotechnology, 16, 535-539, 1998). The advantage of such humanized antibodies is that they retain the specificity and affinity of the parent non-human antibody while reducing immunogenicity in humans. Rather than transferring the entire CDR, only one or more of the specificity-determining residues from any one of the CDRs described herein above can be transferred to the human antibody framework (see, e.g., Kashmiri et al., 2005, Methods, 36, 25-34). A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0223] The term "framework" or "FR" refers to variable domain residues other than hypervariable region residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in VH (or VL) as the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0224] The antibody or antigen-binding fragment thereof can be a monoclonal antibody, a full-length antibody, a bispecific antibody, a multispecific antibody, an ScFv or other single chain or modified form, a Fab, (Fab')2, an Fv, a dAb, an Fd, a nanobody, a camelid antibody, or a diabody. Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0225] The bispecific antibody may comprise a CD1a targeting moiety comprising an antibody of the invention or an antigen-binding fragment thereof, and a T cell engaging moiety. The T cell engaging moiety may be a CD3 targeting moiety, such as the antibody UCHT1 (SEQ ID NO: 298). The CD1a targeting moiety may comprise the light chain variable region of SEQ ID NO: 314 and the heavy chain variable region of SEQ ID NO: 314, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto. The CD1a targeting moiety may comprise the light chain of SEQ ID NO: 210 and the heavy chain of SEQ ID NO: 299, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0226] The term "full-length antibody" is used herein to refer to an antibody having a heavy chain with a structure substantially similar to that of a native antibody or including an Fc region as defined herein. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH) composed of three constant domains, CH1, CH2, and CH3, or four constant domains, CH1, CH2, CH3, and CH4, depending on the Ig class. The antibody constant region may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. IgG antibodies are examples of full-length antibodies, such as IgG1 or IgG4 antibodies.

[0227] We targeted CD1a and its potential role in inflammatory skin and mucosal diseases and disorders, related systemic diseases or disorders, or systemic inflammatory drug responses by generating effective monoclonal antibodies. Because CD1a is highly expressed in skin and mucosal membranes, the use of such antibodies offers an opportunity to selectively treat inflammatory skin and mucosal diseases and disorders while minimizing off-target effects. CD1a is not expressed in mice but is expressed in other mammals. Human CD1a (UniProtKB / Swiss-Prot:P06126-CD1A_HUMAN) is expressed from dominant alleles worldwide, with variants present in some Chinese populations (18). Targeting CD1a antigen presentation also disrupts inflammatory pathways upstream of other cytokine-directed antibody therapies (e.g., anti-IL17 therapy or other immunotherapies), thus providing a powerful means to modulate pro-inflammatory disorders early in the immune cascade. Furthermore, exploiting the specificity of CD1a for skin may provide a means to deliver additional therapies to the skin, for example, by using bispecific or multispecific antibodies or conjugated antibody technology to specifically target small molecule, drug, nucleic acid, peptide, antibody, or cell conjugate therapies. Furthermore, because CD1a is relatively non-polymorphic, the present invention offers universal potential for the prevention and / or treatment of inflammatory skin and mucosal diseases, such as atopic dermatitis and psoriasis, or CD1a-expressing malignancies, in which the frequency of CD1a-expressing dendritic cell subsets is increased and the migration pattern of LCs is altered. (13-15)

[0228] By altering the number and function of CD1a-expressing cells, the antibodies will have effects beyond lipid responsiveness and affect all roles of CD1a-expressing cells, including antigen presentation to peptide-specific T cells and innate pathways (e.g., neutrophils). The antibodies of the present invention, despite their murine IgG1 nature, are able to deplete Langerhans cells. Such depletion provides a means of controlling a wide range of inflammatory pathways in the absence of complement / ADCC-associated inflammation, which may offer therapeutic benefits. This is shown in the imiquimod model described herein, where antibodies according to the present invention reduce inflammation (e.g., to levels significantly lower than in wild-type mice) and demonstrate significant anti-inflammatory effects on pathways beyond CD1a-expressing cells (including innate pathways such as neutrophils and eosinophils). The antibodies of the present invention also inhibit the production of a variety of cytokines, including IFN-gamma and IL-22, that are associated with a wide range of clinical diseases.

[0229] In another aspect, the present invention provides nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention. Such nucleic acids may be provided by any of SEQ ID NOS: 51-90. Those skilled in the art will appreciate that due to codon redundancy, numerous DNA sequences can be used that can encode the antibodies or antigen-binding fragments thereof of the present invention. Alternatively, codon optimization of nucleotide sequences can be used to improve the efficiency of translation in expression systems for producing the antibodies or antigen-binding fragments thereof of the present invention.

[0230] In another aspect, the present invention provides a vector containing a nucleic acid of the present invention. Suitable vectors can be selected or constructed, including appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences, as needed. The vector can be, for example, a plasmid or viral vector. For further details, see, for example, Sambrook, J., E.F. Fritsch, and T. Maniatis. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Many known techniques and protocols for manipulating nucleic acids, for example, in preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and analyzing proteins, are described in detail in Ausubel et al., Current protocols in molecular biology. New York: Greene Publishing Association; Wiley-Interscience, 1992. The vector can also be an expression vector. The vector or expression vector can also be a plasmid.

[0231] The nucleic acid molecules or vectors of the present invention can be expressed in, for example, suitable host cells or cell-free systems using any suitable expression system.

[0232] In another aspect, the present invention provides a host cell comprising the antibody or antigen-binding fragment thereof, nucleic acid, and / or vector of the present invention. The host cell may be selected from bacterial host cells (prokaryotic systems) such as E. coli, or eukaryotic cells such as yeast, fungi, insect cells, or mammalian cells. Preferably, the host cell of the present invention is capable of producing the antibody or antigen-binding fragment thereof of the present invention. The produced antibody or antigen-binding fragment thereof may be enriched by selection and / or isolation.

[0233] The antibodies or antigen-binding fragments thereof of the present invention can also be produced by chemical synthesis. The resulting antibodies or antigen-binding fragments thereof can be enriched by selection and / or isolation.

[0234] According to a further aspect, the present invention provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, and / or a host cell of the invention, optionally together with one or more pharmaceutically acceptable excipients or diluents.

[0235] The antibodies or antigen-binding fragments thereof, nucleic acids, vectors, or host cells of the present invention can be formulated into pharmaceutical compositions using established preparation methods (Gennaro, AL and Gennaro, AR (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). Pharmaceutically inert inorganic or organic excipients may be used to prepare pharmaceutical compositions. For example, to prepare pills, powders, gelatin capsules, or suppositories, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural oils, and hardened oils are examples of pharmaceutically acceptable excipients that can be used. Suitable excipients for the manufacture of solutions, suspensions, emulsions, aerosol mixtures, or powders for reconstitution into a solution or aerosol mixture before use include water, alcohols, glycerol, polyols, and suitable mixtures thereof, as well as vegetable oils.

[0236] The pharmaceutical compositions of the present invention can be administered via any therapeutically effective parenteral or non-parenteral (enteral) route. Parenteral application methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal, or intravenous injection and infusion techniques (e.g., in the form of an injection solution, infusion solution, or mixture), as well as aerosol placement and inhalation (e.g., in the form of an aerosol mixture, spray, or powder). The pharmaceutical compositions of the present invention can be administered systemically or locally, if desired, in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives, and vehicles. A combination of intravenous and subcutaneous infusion and / or injection may be most convenient for compounds with a relatively short or long serum half-life or requiring a rapid onset of action. Preferably, the pharmaceutical composition is administered subcutaneously or intravenously. The pharmaceutical composition may be an aqueous solution, an oil-in-water emulsion, or a water-in-oil emulsion.

[0237] For intravenous injection or injection at the affected site or other administration site, the active ingredient is in the form of a pyrogen-free parenterally acceptable aqueous solution having suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0238] The composition is preferably administered to an individual in a "therapeutically effective amount," which is sufficient to show benefit to the individual. The optimal dosage depends on the biodistribution of the antibody or antigen-binding fragment thereof, the mode of administration, the severity of the disease / disorder being treated, and the patient's medical condition. If desired, the antibody or antigen-binding fragment thereof can be administered in a sustained-release formulation, such as a liposomal dispersion or hydrogel-based polymer microspheres, e.g., PolyActive™ or OctoDEX™ (see Bos et al., Business Briefing: Pharmatech 2003:1-6). Other available sustained-release formulations include PLGA-based polymers (PR pharmaceuticals), PLA-PEG-based hydrogels (Medincell), and PEA-based polymers (Medivas). Prescribing treatment (e.g., determining dosage) is within the physician's responsibility and typically takes into account the disorder being treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to the physician.

[0239] The pharmaceutical compositions may also contain additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and even solvents or solubilizers or agents for achieving a depot effect, the latter being that the fusion protein may be incorporated into slow- or sustained-release systems or targeted delivery systems such as liposomes and microcapsules.

[0240] In another aspect, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or pharmaceutical composition of the invention may be for use in the treatment or prevention of one or more diseases or disorders in a subject.

[0241] In one aspect, provided is a method for treating or preventing one or more diseases or disorders in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or composition of the invention.

[0242] In one embodiment, the use of the present invention in the manufacture of a pharmaceutical product for the treatment or prevention of one or more diseases or disorders in a subject is provided.

[0243] In any embodiment, the subject can be a mammal. The mammal can express a CD1a ortholog. Preferably, the subject is a human.

[0244] One or more diseases or disorders may be one or more inflammatory skin or mucous membrane disorders or diseases, or one or more related systemic diseases or disorders, or one or more systemic inflammatory drug reactions, or CD1a-expressing malignancies.

[0245] The inflammatory skin or mucosal disease or disorder may be selected from: a) Primarily neutrophilic skin diseases, such as acne, generalized pustular psoriasis, plaque psoriasis, guttate psoriasis, palmoplantar pustulosis, SAPHO syndrome, acute febrile neutrophilic dermatosis (Sweet's syndrome), histiocytic neutrophilic dermatitis, neutrophilic dermatosis of the dorsum of the hands, pyoderma gangrenosum, neutrophilic eccrine hidradenitis, hidradenitis suppurativa, erythema elevatum perforans, Behçet's disease, gut-related dermatitis-arthritis syndrome, other infection-related inflammation, neutrophilic urticarial dermatosis, palisading neutrophilic granulomatous dermatitis, erythema gyratum circumscriptum, neutrophilic annular erythema, acute generalized exanthematous pustulosis (AGEP), vasculitis, etc. b) Autoimmune disorders, such as connective tissue diseases (e.g. lupus, dermatomyositis, scleroderma / systemic sclerosis, Churg-Strauss syndrome), panniculitis, vasculitis, autoimmune blistering conditions (e.g. bullous pemphigoid, pemphigus, linear IgA disease), dermatitis herpetiformis, celiac disease, some autoinflammatory diseases, vitiligo, alopecia areata, alopecia universalis, alopecia totalis, panniculitis, lichen planus, erythema multiforme, lichen sclerosus, other lichenoid and erythema multiforme-like diseases, blistering psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, Guillain-Barré syndrome, thyroiditis, transverse myelitis, neurodegeneration, etc. c) Mast cell disorders and eosinophilic disorders, such as Muckle-Wells syndrome, eosinophilia and systemic symptoms syndrome, urticaria, angioedema, keratoconjunctivitis, food allergies, other allergies or atopy (including atopic dermatitis), rhinitis, conjunctivitis, asthma, eosinophilic esophagitis and other eosinophilic mucosal diseases, contact dermatitis, chronic obstructive airway disease, etc. d) Adverse drug reactions manifesting as inflammatory skin or mucosal diseases or disorders, such as Stevens-Johnson syndrome, toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms syndrome (DRESS) and acute generalized exanthematous pustulosis (AGEP), erythema multiforme, bullous fixed drug eruptions, checkpoint inhibitor-associated dermatitis, and other inflammation. e) Graft-versus-host disease f) Itching and pruritic conditions, including nodular prurigo.

[0246] The CD1a-expressing malignancies referred to herein may be any malignancies in which CD1a expression can be detected. Such malignancies may include Langerhans cell histiocytosis, Langerhans cell sarcoma, a subset of T-cell lymphomas, a subset of thymomas, or rare instances of other malignancies (such as a subset of mastocytosis). Preferably, the CD1a-expressing malignancies are a subset of T-cell lymphomas.

[0247] Preferably, the one or more diseases or disorders comprise or consist of psoriasis, dermatitis, lupus erythematosus, neutrophilic dermatosis, a related systemic disease or disorder, and / or a systemic inflammatory drug reaction, or a CD1a-expressing malignancy.

[0248] As used herein, an associated systemic disease or disorder may refer to the involvement of any non-cutaneous site that may be associated with an inflammatory skin or mucosal disease or disorder as defined herein, which may include non-cutaneous lupus erythematosus.

[0249] The inflammatory drug reaction occurring systemically may be at a non-cutaneous site, such as the spleen. The inflammatory drug reaction occurring systemically may be the result of an associated systemic disease or disorder or an inflammatory response. The inflammatory response may be to a drug, such as Aldara (5% imiquimod cream). The inflammatory response may result in an increase in the number or activity of CD4 T cells, CD8 T cells, neutrophils, or eosinophils, and / or an increase in the levels of IL-23, IL-12, IL-1β, and / or MCP-1, and / or a decrease in IL-10 and / or IL-27.

[0250] Furthermore, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or pharmaceutical compositions of the invention can be administered alone or in combination with one or more other therapeutic agents, either simultaneously, sequentially, or separately, depending on the condition being treated. The one or more other therapeutic agents may be selected from the group comprising cytotoxic agents, immunoactivators (e.g., checkpoint inhibitors or TLR agonists), anti-inflammatory agents (e.g., steroids), CAR-T cells (e.g., regulatory or cytolytic CAR-T cells), or other cells expressing or presenting one or more antibodies or antigen-binding fragments of the invention.

[0251] In another aspect, there is provided a method of monitoring therapeutic efficacy or disease status in a subject diagnosed with a CD1a-expressing malignancy, comprising: i. providing a biological sample obtained from a subject; ii. determining the level of binding of one or more antibodies or antigen-binding fragments of the invention to CD1a-expressing cells in a sample obtained from the subject before treatment, or between treatments, or during a treatment-free period; iii. determining that the treatment is effective or the disease state is improved if the tumor volume or the level of binding of one or more antibodies or antigen-binding fragments of the invention to CD1a-expressing cells is reduced after the treatment, or between treatments, or during a treatment-free period.

[0252] The biological samples referred to herein may be blood or serum samples, tissue biopsies, cerebrospinal fluid, saliva, or urine samples. Preferably, the biological sample may be a blood or serum sample.

[0253] The level of binding of one or more antibodies or antigen-binding fragments of the invention to CD1a-expressing cells in a sample can be determined using any method known to those of skill in the art. One such method can be, for example, flow cytometry or any other technique that utilizes a detectable label to determine the number of CD1a-expressing cells in a sample.

[0254] Tumor volume can be determined by any preferred technique known to those skilled in the art.

[0255] The reduction in tumor volume, or the reduction in the binding level of one or more antibodies or antigen-binding fragments of the present invention to CD1a-expressing cells, may be 10% or more, for example, 25% or more, 50% or more, 75% or more, or 90% or more.

[0256] The interval between treatments or periods without treatment may be two weeks or longer, for example, four weeks or longer, eight weeks or longer, twelve weeks or longer, six months or longer, or twelve months or longer.

[0257] In another embodiment, a method for diagnosing a subject having an inflammatory skin and mucous membrane disease or disorder, or a related systemic disease or disorder, or a systemic inflammatory drug reaction, or a CD1a-expressing malignant tumor, i. providing a biological sample obtained from a subject; ii. Determining the expression level of CD1a in a sample obtained from a subject using one or more antibodies or antigen-binding fragments thereof of the present invention, iii. The expression level of CD1a in the sample obtained from the subject is compared with the expression level of CD1a in the positive or negative reference sample, iv. determining that the subject has an inflammatory skin and mucosal disease or disorder, or an associated systemic disease or disorder, or a systemic inflammatory drug reaction, or a CD1a-expressing malignancy, if the expression level of CD1a in the sample obtained from the subject is higher than the expression level of CD1a in the negative reference sample, or is equal to or higher than the expression level of CD1a in the positive reference sample.

[0258] Alternatively, in step iv, if the expression level of CD1a in the sample obtained from the subject is equal to or lower than the expression level of CD1a in the negative reference sample, or lower than the expression level of CD1a in the positive reference sample, the subject can be determined to not have an inflammatory skin and mucosal disease or disorder, or an associated systemic disease or disorder, or a systemic inflammatory drug reaction, or a CD1a-expressing malignancy.

[0259] A negative reference sample may refer to a biological sample taken from a healthy subject known to be free of inflammatory skin and mucosal diseases or disorders, or related systemic diseases or disorders, or systemic inflammatory drug reactions, or CD1a-expressing malignancies. A positive reference sample may refer to a biological sample taken from a subject already diagnosed with inflammatory skin and mucosal diseases or disorders, or related systemic diseases or disorders, or systemic inflammatory drug reactions, or CD1a-expressing malignancies.

[0260] The expression level of CD1a in diagnostic methods may refer to the level of CD1a molecules expressed on a given cell(s) in a population, or the percentage of cells in a population or sample that are determined to express CD1a.

[0261] Techniques for producing antibodies and antigen-binding fragments thereof are well known in the art. The term "antibody" also includes immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2). Illustrative examples of antibodies or antigen-binding fragments thereof include Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), diabodies, domain antibodies, or bispecific antibodies (Holt LJ et al., Trends Biotechnol. 21(11), 2003, 484-490). Examples also include dAB fragments consisting of a single CH domain or VL domain capable of binding to an antigen alone. Antibodies or antigen-binding fragments thereof may be chimeric, nanobody, single-chain, and / or humanized. Antibodies or antigen-binding fragments thereof may be of the human IgG1 or IgG4 isotype, or other natural or modified isotypes. Antibodies can be monoclonal (mAb) or polyclonal.

[0262] The antibody or antigen-binding fragment thereof can be modified to alter its in vivo stability and / or half-life. The modification can be, for example, PEGylation.

[0263] Antibodies or antigen-binding fragments thereof may also be antibody-like molecules, including those using the CDRs separately or in combination in synthetic molecules such as SMIPs and small antibody mimetics.

[0264] The percent identity of two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for best alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of the two sequences that results in the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides in the sequences (i.e., % identity = number of identical positions / total number of positions × 100).

[0265] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, PNAS, 87(6)2264-8, modified as in Karlin and Altschul, 1993, PNAS, 90(12):5873-5877. The NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol., 215:403-10 incorporate such an algorithm. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, GappedBLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilized for sequence comparison is the Myers and Miller algorithm. The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, incorporates such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, as described in Torellis and Robotti (1994), and FASTA, as described in Pearson and Lipman (1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0266] The antibodies or antigen-binding fragments thereof of the invention may contain one or more mutated amino acid residues. With respect to the nucleic acids or antibodies or antigen-binding fragments thereof of the invention, the terms "mutated," "mutant," and "mutation" refer to a substitution, deletion, or insertion of one or more nucleotides or amino acids, respectively, compared to a reference sequence that can be taken to define a "naturally" occurring nucleic acid or polypeptide, i.e., wild-type.

[0267] The amino acid variations in the CDR sequences may be conservative amino acid substitutions.

[0268] The mutation may be a conservative substitution. Conservative substitutions are generally the following (listed according to the amino acid to be mutated, each followed by one or more substitutions that may be considered conservative): Ala → Gly, Ser, Val; Arg → Lys; Asn → Gln, His; Asp → Glu; Cys → Ser; Gln → Asn; Glu → Asp; Gly → Ala; His → Arg, Asn, Gln; Ile → Leu, Val; Leu → Ile, Val; Lys → Arg, Gln, Glu; Met → Leu, Tyr, He; Phe → Met, Leu, Tyr; Ser → Thr; Thr → Ser; Trp → Tyr; Tyr → Trp, Phe; Val → He, Leu. Other substitutions are also permissible and can be determined empirically or according to other known conservative or non-conservative substitutions.

[0269] One, two, or three conservative substitutions may be made in the CDRs of an antibody or antigen-binding fragment thereof of the present invention.

[0270] Methods for producing antibodies or antigen-binding fragments thereof are well known in the art. For example, those skilled in the art may use hybridoma technology or recombinant DNA technology to clone each antibody sequence into a vector, such as an expression vector. Methods for producing bispecific antibody molecules are also known in the art, including recombinant DNA technology, chemical conjugation of two different monoclonal antibodies, or chemical linkage of two antibody fragments (e.g., two Fab fragments). Alternatively, bispecific antibody molecules can be produced by quadroma technology, which fuses hybridomas producing parent antibodies. Because heavy and light chains are randomly assorted, a mixture of 10 different possible antibody structures is produced, of which only one has the desired binding specificity. The bispecific antibody molecules of the present invention can act as monoclonal antibodies (mAbs) against each target. The antibodies or antigen-binding fragments thereof may be chimeric, humanized, or fully human. The antibodies or antigen-binding fragments thereof may be of the human IgG1 or IgG4 isotype, or other natural or modified isotypes. The bispecific antibody molecule or multispecific antibody can be, for example, a bispecific tandem single chain Fv, a bispecific Fab2, or a bispecific diabody.

[0271] References to "OX16," "OX116," "OX110," "OX111," "OX77a," or "OX25" refer to antibodies 16, 116, 110, 111, 77a, or 25, respectively (as defined in Table 11).

[0272] All features disclosed in this specification may be combined in any combination, including in any aspect or embodiment. [Brief explanation of the drawings]

[0273] [Figure 1]This study demonstrates the inhibition of polyclonal T cell responses by a panel of anti-CD1a antibodies. A. Dose titration curves of polyclonal T cell IFNγ responses as anti-CD1a antibody concentrations increase (0.01–10 μg / ml) (n=6 donors). B. Calculated IC50 values ​​for a panel of newly generated anti-CD1a antibodies and commercially available antibodies (OKT6, HI149, and SK9, n=6 donors). [Figure 2-1] Figure 1 shows inhibition of CD1a-restricted enriched T cell line responses by a panel of anti-CD1a antibodies. A-B. Cytokine secretion responses of CD1a-restricted enriched T cell lines induced by empty vector (EV) or CD1a-transfected K562 presenting endogenous ligand. Inhibition of IFNγ (A) or IL-22 (B) was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. C. IFNγ secretion responses of CD1a-restricted enriched T cell lines induced by CD1a-coated beads presenting endogenous ligand. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. (N = 4 19 enriched T cell lines, two-way ANOVA with Tukey's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (* indicates significance compared to "CD1a"). [Figure 2-2]Figure 1 shows inhibition of CD1a-restricted enriched T cell line responses by a panel of anti-CD1a antibodies. A-B. Cytokine secretion responses of CD1a-restricted enriched T cell lines induced by empty vector (EV) or CD1a-transfected K562 presenting endogenous ligand. Inhibition of IFNγ (A) or IL-22 (B) was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. C. IFNγ secretion responses of CD1a-restricted enriched T cell lines induced by CD1a-coated beads presenting endogenous ligand. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. (N = 4 19 enriched T cell lines, two-way ANOVA with Tukey's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (* indicates significance compared to "CD1a"). [Figure 3-1]Characterization of CD1a transgenic mice. Representative flow cytometry plots (A) and graphical summary (B) of CD1a protein expression by cells from wild-type (WT) and CD1a transgenic (CD1a) mice. CD1a protein expression was assessed (from left to right) on total live ear skin cells, CD45+ skin cells, dermal dendritic cells (dDCs, CD45+ / CD11c+ / Langerin-), and Langerhans cells (LCs, CD45+ / CD11c+ / Langerin+). C. CD1a protein expression in the ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice visualized by immunofluorescence. Cryosections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars (from left to right): 50 μm, 50 μm, and 10 μm. D. Exemplary PCR genotyping of CD1a transgenic mouse strain littermates using CD1a forward and reverse primers and tail genomic DNA (lanes A-F). The expected CD1a band is 655 bp. Lane G: Positive control genomic DNA from a founder mouse. Lane H: Negative control lacking DNA template. E. Representative flow cytometry plots of thymic CD1a protein expression by wild-type mice (WT) and CD1a transgenic mice (CD1a). [Figure 3-2]Characterization of CD1a transgenic mice. Representative flow cytometry plots (A) and graphical summary (B) of CD1a protein expression by cells from wild-type (WT) and CD1a transgenic (CD1a) mice. CD1a protein expression was assessed (from left to right) on total live ear skin cells, CD45+ skin cells, dermal dendritic cells (dDCs, CD45+ / CD11c+ / Langerin-), and Langerhans cells (LCs, CD45+ / CD11c+ / Langerin+). C. CD1a protein expression in the ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice visualized by immunofluorescence. Cryosections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars (from left to right): 50 μm, 50 μm, and 10 μm. D. Exemplary PCR genotyping of CD1a transgenic mouse strain littermates using CD1a forward and reverse primers and tail genomic DNA (lanes A-F). The expected CD1a band is 655 bp. Lane G: Positive control genomic DNA from a founder mouse. Lane H: Negative control lacking DNA template. E. Representative flow cytometry plots of thymic CD1a protein expression by wild-type mice (WT) and CD1a transgenic mice (CD1a). [Figure 4] Characterization of anti-CD1a antibodies in vivo. A. Schematic diagram of imiquimod-induced dermatitis and prophylactic administration of anti-CD1a. B. Daily measurements of ear swelling induced by imiquimod treatment in wild-type mice (WT) and CD1a transgenic mice (CD1a) injected intraperitoneally with mouse IgG1 isotype control, and in CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies, as shown in panel A of the schematic diagram. (N=6, two-way ANOVA with Dunnett's test, **, P<0.01; ****, P<0.0001 indicates significance compared to "CD1a" at day 6 or the indicated time point). [Figure 5]This study demonstrates the effect of anti-CD1a on imiquimod-induced cutaneous immune responses. A-C. Flow cytometry analysis of ear skin from mouse IgG1 isotype-treated wild-type mice (WT) and CD1a transgenic mice (CD1a), as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to a prophylactic model of administration. Skin T cells were counted (A), cell surface CD69 expression was assessed (B), and the frequencies of skin neutrophils (C) and eosinophils (D) were determined. (N=4, one-way ANOVA with Dunnett's test, *,P<0.05;**,P<0.01;***,P<0.001). [Figure 6] This study demonstrates the effects of anti-CD1a on imiquimod-induced Langerhans cell cutaneous and lymph node responses. Flow cytometry analysis was performed on ear skin (A-B) and inflow area cervical lymph nodes (C-D) of mouse IgG1 isotype-treated (wild-type WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to a prophylactic model of administration. Skin LCs were counted (A) and cell surface CD1a expression was evaluated (B). Lymph node LCs were counted (C) and cell surface CD1a expression was evaluated (D). (N=4, one-way ANOVA with Dunnett's test, *,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001). [Figure 7-1]Antibody-dependent depletion (phenotypic changes) is shown. A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with the indicated EV or CD1a-K562 for 48 hours. The percentage of antibody-induced depletion relative to the reference population of untreated K562 cells was calculated and normalized to the EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion with increasing concentrations of anti-CD1a antibody (0.625 to 5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days, as indicated. Antibodies and cytokines were added on day 0 or day 2. The antibody-induced reduction (%) relative to the isotype control, as measured by percentage confluence, was calculated using Incucyte live cell imaging (N = 4, two-way ANOVA with Tukey's test) (C), and representative images of MoLCs (D). E. K562-CD1a or K562-EV empty vector were incubated with anti-CD1a antibody for 24 hours, stained for Annexin V, and analyzed by flow cytometry (N = 3-4, one-way ANOVA with Tukey's test). F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody for 3 hours at 37°C. Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were cocultured with PBMCs at a 1:50 ratio for 5 hours at 37°C in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody.Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=4-6, one-way ANOVA with Tukey's test). H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells, and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14. Measurement of tumor volume over time. (N=6-15, two-way ANOVA with Tukey's test; asterisks indicate significance compared to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-2]Antibody-dependent depletion (phenotypic changes) is shown. A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with the indicated EV or CD1a-K562 for 48 hours. The percentage of antibody-induced depletion relative to the reference population of untreated K562 cells was calculated and normalized to the EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion with increasing concentrations of anti-CD1a antibody (0.625 to 5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days, as indicated. Antibodies and cytokines were added on day 0 or day 2. The antibody-induced reduction (%) relative to the isotype control, as measured by percentage confluence, was calculated using Incucyte live cell imaging (N = 4, two-way ANOVA with Tukey's test) (C), and representative images of MoLCs (D). E. K562-CD1a or K562-EV empty vector were incubated with anti-CD1a antibody for 24 hours, stained for Annexin V, and analyzed by flow cytometry (N = 3-4, one-way ANOVA with Tukey's test). F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody for 3 hours at 37°C. Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were cocultured with PBMCs at a 1:50 ratio for 5 hours at 37°C in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody.Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=4-6, one-way ANOVA with Tukey's test). H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells, and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14. Measurement of tumor volume over time. (N=6-15, two-way ANOVA with Tukey's test; asterisks indicate significance compared to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-3]Antibody-dependent depletion (phenotypic changes) is shown. A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with the indicated EV or CD1a-K562 for 48 hours. The percentage of antibody-induced depletion relative to the reference population of untreated K562 cells was calculated and normalized to the EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion with increasing concentrations of anti-CD1a antibody (0.625 to 5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days, as indicated. Antibodies and cytokines were added on day 0 or day 2. The antibody-induced reduction (%) relative to the isotype control, as measured by percentage confluence, was calculated using Incucyte live cell imaging (N = 4, two-way ANOVA with Tukey's test) (C), and representative images of MoLCs (D). E. K562-CD1a or K562-EV empty vector were incubated with anti-CD1a antibody for 24 hours, stained for Annexin V, and analyzed by flow cytometry (N = 3-4, one-way ANOVA with Tukey's test). F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody for 3 hours at 37°C. Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were cocultured with PBMCs at a 1:50 ratio for 5 hours at 37°C in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody.Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=4-6, one-way ANOVA with Tukey's test). H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells, and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14. Measurement of tumor volume over time. (N=6-15, two-way ANOVA with Tukey's test; asterisks indicate significance compared to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-4]Antibody-dependent depletion (phenotypic changes) is shown. A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with the indicated EV or CD1a-K562 for 48 hours. The percentage of antibody-induced depletion relative to the reference population of untreated K562 cells was calculated and normalized to the EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion with increasing concentrations of anti-CD1a antibody (0.625 to 5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days, as indicated. Antibodies and cytokines were added on day 0 or day 2. The antibody-induced reduction (%) relative to the isotype control, as measured by percentage confluence, was calculated using Incucyte live cell imaging (N = 4, two-way ANOVA with Tukey's test) (C), and representative images of MoLCs (D). E. K562-CD1a or K562-EV empty vector were incubated with anti-CD1a antibody for 24 hours, stained for Annexin V, and analyzed by flow cytometry (N = 3-4, one-way ANOVA with Tukey's test). F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody for 3 hours at 37°C. Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were cocultured with PBMCs at a 1:50 ratio for 5 hours at 37°C in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody.Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=4-6, one-way ANOVA with Tukey's test). H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells, and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14. Measurement of tumor volume over time. (N=6-15, two-way ANOVA with Tukey's test; asterisks indicate significance compared to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-5]Antibody-dependent depletion (phenotypic changes) is shown. A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with the indicated EV or CD1a-K562 for 48 hours. The percentage of antibody-induced depletion relative to the reference population of untreated K562 cells was calculated and normalized to the EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion with increasing concentrations of anti-CD1a antibody (0.625 to 5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days, as indicated. Antibodies and cytokines were added on day 0 or day 2. The antibody-induced reduction (%) relative to the isotype control, as measured by percentage confluence, was calculated using Incucyte live cell imaging (N = 4, two-way ANOVA with Tukey's test) (C), and representative images of MoLCs (D). E. K562-CD1a or K562-EV empty vector were incubated with anti-CD1a antibody for 24 hours, stained for Annexin V, and analyzed by flow cytometry (N = 3-4, one-way ANOVA with Tukey's test). F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody for 3 hours at 37°C. Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were cocultured with PBMCs at a 1:50 ratio for 5 hours at 37°C in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody.Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=4-6, one-way ANOVA with Tukey's test). H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells, and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14. Measurement of tumor volume over time. (N=6-15, two-way ANOVA with Tukey's test; asterisks indicate significance compared to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-6]Antibody-dependent depletion (phenotypic changes) is shown. A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with the indicated EV or CD1a-K562 for 48 hours. The percentage of antibody-induced depletion relative to the reference population of untreated K562 cells was calculated and normalized to the EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion with increasing concentrations of anti-CD1a antibody (0.625 to 5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days, as indicated. Antibodies and cytokines were added on day 0 or day 2. The antibody-induced reduction (%) relative to the isotype control, as measured by percentage confluence, was calculated using Incucyte live cell imaging (N = 4, two-way ANOVA with Tukey's test) (C), and representative images of MoLCs (D). E. K562-CD1a or K562-EV empty vector were incubated with anti-CD1a antibody for 24 hours, stained for Annexin V, and analyzed by flow cytometry (N = 3-4, one-way ANOVA with Tukey's test). F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody for 3 hours at 37°C. Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were cocultured with PBMCs at a 1:50 ratio for 5 hours at 37°C in the presence of 5 μg / ml of either an isotype control antibody or the indicated antibody.Percent cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells. (N=4-6, one-way ANOVA with Tukey's test). H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells, and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14. Measurement of tumor volume over time. (N=6-15, two-way ANOVA with Tukey's test; asterisks indicate significance compared to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 8-1] (A) Heatmap from CD1a epitope analysis. Matrix heatmap representation of CD1a antibody binding by flow cytometry, measured by CD1a-AF647 mean fluorescence intensity (MFI). The relevant purified antibody was incubated with cells prior to staining CD1a-K652 with an anti-CD1a antibody conjugated to the fluorophore AF647 to assess interference of the AF647-conjugated antibody with CD1a binding. The gray scale indicates the degree of interference, with the upper shade (-) indicating no interference. (B) Results of an in vivo CD1a antibody epitope competition assay. Flow cytometry plots of CD1a expression measured by staining with anti-CD1a antibody SK9 (left panel) or HI149 (right panel). Anti-CD1a antibody 116 (100 μg i.p.) was administered on days 0, 2, and 4, and ear skin tissue was collected, processed, and stained for CD1a on day 5. [Figure 8-2](A) Heatmap from CD1a epitope analysis. Matrix heatmap representation of CD1a antibody binding by flow cytometry, measured by CD1a-AF647 mean fluorescence intensity (MFI). The relevant purified antibody was incubated with cells prior to staining CD1a-K652 with an anti-CD1a antibody conjugated to the fluorophore AF647 to assess interference of the AF647-conjugated antibody with CD1a binding. The gray scale indicates the degree of interference, with the upper shade (-) indicating no interference. (B) Results of an in vivo CD1a antibody epitope competition assay. Flow cytometry plots of CD1a expression measured by staining with anti-CD1a antibody SK9 (left panel) or HI149 (right panel). Anti-CD1a antibody 116 (100 μg i.p.) was administered on days 0, 2, and 4, and ear skin tissue was collected, processed, and stained for CD1a on day 5. [Figure 9-1]This figure demonstrates the effectiveness of anti-CD1a antibody treatment in treating imiquimod-induced inflammation. A. Schematic of the imiquimod-induced inflammation model with therapeutic anti-CD1a administration. B. Daily measurements of ear swelling. C. Representative images of inflammation (day 8) induced by imiquimod treatment of wild-type (WT) and CD1a transgenic (CD1a) mice as in panel A of the schematic, followed by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies into CD1a transgenic mice (arrows on day 3) (N = 2-10, two-way ANOVA with Dunnett's test; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or the indicated time point). D. Thickness of the ear and epidermis of wild-type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) mice, as well as CD1a protein expression in the ear skin, visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, 10 µm (upper panel) and 100 µm (lower panel). E–G. Flow cytometry analysis of ear skin from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the therapeutic model of administration. Cutaneous T cells were enumerated and assessed for cell surface CD11a expression (E), and the frequencies of neutrophils (F) and eosinophils (G) were determined. (N=7–9, one-way ANOVA with Dunnett's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001). [Figure 9-2]This figure demonstrates the effectiveness of anti-CD1a antibody treatment in treating imiquimod-induced inflammation. A. Schematic of the imiquimod-induced inflammation model with therapeutic anti-CD1a administration. B. Daily measurements of ear swelling. C. Representative images of inflammation (day 8) induced by imiquimod treatment of wild-type (WT) and CD1a transgenic (CD1a) mice as in panel A of the schematic, followed by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies into CD1a transgenic mice (arrows on day 3) (N = 2-10, two-way ANOVA with Dunnett's test; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or the indicated time point). D. Thickness of the ear and epidermis of wild-type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) mice, as well as CD1a protein expression in the ear skin, visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, 10 µm (upper panel) and 100 µm (lower panel). E–G. Flow cytometry analysis of ear skin from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the therapeutic model of administration. Cutaneous T cells were enumerated and assessed for cell surface CD11a expression (E), and the frequencies of neutrophils (F) and eosinophils (G) were determined. (N=7–9, one-way ANOVA with Dunnett's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001). [Figure 9-3]This figure demonstrates the effectiveness of anti-CD1a antibody treatment in treating imiquimod-induced inflammation. A. Schematic of the imiquimod-induced inflammation model with therapeutic anti-CD1a administration. B. Daily measurements of ear swelling. C. Representative images of inflammation (day 8) induced by imiquimod treatment of wild-type (WT) and CD1a transgenic (CD1a) mice as in panel A of the schematic, followed by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies into CD1a transgenic mice (arrows on day 3) (N = 2-10, two-way ANOVA with Dunnett's test; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or the indicated time point). D. Thickness of the ear and epidermis of wild-type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) mice, as well as CD1a protein expression in the ear skin, visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, 10 µm (upper panel) and 100 µm (lower panel). E–G. Flow cytometry analysis of ear skin from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the therapeutic model of administration. Cutaneous T cells were enumerated and assessed for cell surface CD11a expression (E), and the frequencies of neutrophils (F) and eosinophils (G) were determined. (N=7–9, one-way ANOVA with Dunnett's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001). [Figure 10-1]The CD1a dependency of the systemic effect of imiquimod application is shown. A. Schematic diagram showing spleen weight (mg) measurements and representative images on day 8 after imiquimod treatment of wild-type (WT) and CD1a transgenic (CD1a) mice, followed by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies into CD1a transgenic mice (Figure 9A). B-E. Blood cell analysis of blood from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, and CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the therapeutic model of administration. Splenic CD4 (B) and CD8 (C) T cell CD69 expression was assessed, and neutrophils (D) and eosinophils (E) were enumerated. (N=7-9, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). F. Plasma cytokine levels in the blood of mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice injected with anti-CD1a antibodies according to the therapeutic model of administration (N=7-9, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 10-2]The CD1a dependency of the systemic effect of imiquimod application is shown. A. Schematic diagram showing spleen weight (mg) measurements and representative images on day 8 after imiquimod treatment of wild-type (WT) and CD1a transgenic (CD1a) mice, followed by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies into CD1a transgenic mice (Figure 9A). B-E. Blood cell analysis of blood from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, and CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the therapeutic model of administration. Splenic CD4 (B) and CD8 (C) T cell CD69 expression was assessed, and neutrophils (D) and eosinophils (E) were enumerated. (N=7-9, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). F. Plasma cytokine levels in the blood of mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice injected with anti-CD1a antibodies according to the therapeutic model of administration (N=7-9, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 11] The CD1a dependency of the systemic effect of imiquimod application is shown. A-E. Blood cell analysis of blood from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice injected with a refined panel of anti-CD1a antibodies according to the therapeutic model. Circulating T cells (A), CD4+ (B), and CD8+ (C), neutrophils (D), and eosinophils (E) were enumerated. (N = 5-7, one-way ANOVA with Dunnett's test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). [Figure 12]This demonstrates that imiquimod does not constitute a CD1a ligand. Isoelectric focusing (IEF) gel pH 3-7 shows isoelectric point-dependent transfer of mock and imiquimod-loaded CD1a proteins. Mock: Vehicle control TBS 2% CHAPS 7% DMSO. [Figure 13-1] Efficacy of anti-CD1a antibody application in sustained control of imiquimod-induced inflammation. A. Schematic of the imiquimod rechallenge model without subsequent anti-CD1a administration. B. Daily measurements of ear swelling induced by imiquimod treatment in wild-type mice (WT) and CD1a transgenic mice (CD1a) injected intraperitoneally with a mouse IgG1 isotype control, and in CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies as in panel 13A of the schematic (two-way ANOVA with Dunnett's test; *, P<0.05; **, P<0.01 indicates significance compared to the "CD1a" isotype on day 7 of imiquimod reapplication). [Figure 13-2] Efficacy of anti-CD1a antibody application in sustained control of imiquimod-induced inflammation. A. Schematic of the imiquimod rechallenge model without subsequent anti-CD1a administration. B. Daily measurements of ear swelling induced by imiquimod treatment in wild-type mice (WT) and CD1a transgenic mice (CD1a) injected intraperitoneally with a mouse IgG1 isotype control, and in CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies as in panel 13A of the schematic (two-way ANOVA with Dunnett's test; *, P<0.05; **, P<0.01 indicates significance compared to the "CD1a" isotype on day 7 of imiquimod reapplication). [Figure 14]Efficacy of anti-CD1a antibody application in treating imiquimod-induced inflammation compared to standard treatment. As shown in the schematic panel of Figure 9A, daily measurements of ear swelling induced by imiquimod treatment of wild-type mice (WT) and CD1a transgenic mice (CD1a), followed by intraperitoneal treatment with a mouse IgG1 isotype control (CD1a) or injection of a refined panel of anti-CD1a antibodies and anti-IL-17A into CD1a transgenic mice. dx = number of days in the model at which significance was reached compared to ear thickness in CD1a transgenic mice. [Figure 15-1] Comparative analysis of the effectiveness of anti-CD1a antibody administration in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with therapeutic anti-CD1a administration. B. Schematic panel A shows imiquimod treatment of wild-type (WT) and CD1a transgenic mice (CD1a), followed by daily measurement of ear swelling induced by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies or CR2113 into CD1a transgenic mice. N = 2-7, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B) corrected for WT. D. Schematic of MC903-induced inflammation followed by preventive MC903-induced inflammation. E. Daily measurements of ear swelling induced by MC903 treatment of wild-type (WT) and CD1a transgenic (CD1a) mice after intraperitoneal treatment with a mouse IgG1 isotype control, as in panel D of the schematic, or by injection of 16, 110, or 116 anti-CD1a antibodies or CR2113 into CD1a transgenic mice. Corrected for WT. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05 indicates significance compared to "CD1a" on day 7. F. Percentage of cutaneous T cells and eosinophil counts measured by flow cytometry. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 15-2] Comparative analysis of the effectiveness of anti-CD1a antibody administration in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with therapeutic anti-CD1a administration. B. Schematic panel A shows imiquimod treatment of wild-type (WT) and CD1a transgenic mice (CD1a), followed by daily measurement of ear swelling induced by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies or CR2113 into CD1a transgenic mice. N = 2-7, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B) corrected for WT. D. Schematic of MC903-induced inflammation followed by preventive MC903-induced inflammation. E. Daily measurements of ear swelling induced by MC903 treatment of wild-type (WT) and CD1a transgenic (CD1a) mice after intraperitoneal treatment with a mouse IgG1 isotype control, as in panel D of the schematic, or by injection of 16, 110, or 116 anti-CD1a antibodies or CR2113 into CD1a transgenic mice. Corrected for WT. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05 indicates significance compared to "CD1a" on day 7. F. Percentage of cutaneous T cells and eosinophil counts measured by flow cytometry. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 15-3]Comparative analysis of the effectiveness of anti-CD1a antibody administration in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with therapeutic anti-CD1a administration. B. Schematic panel A shows imiquimod treatment of wild-type (WT) and CD1a transgenic mice (CD1a), followed by daily measurement of ear swelling induced by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies or CR2113 into CD1a transgenic mice. N = 2-7, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B) corrected for WT. D. Schematic of MC903-induced inflammation followed by preventive MC903-induced inflammation. E. Daily measurements of ear swelling induced by MC903 treatment of wild-type (WT) and CD1a transgenic (CD1a) mice after intraperitoneal treatment with a mouse IgG1 isotype control, as in panel D of the schematic, or by injection of 16, 110, or 116 anti-CD1a antibodies or CR2113 into CD1a transgenic mice. Corrected for WT. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05 indicates significance compared to "CD1a" on day 7. F. Percentage of cutaneous T cells and eosinophil counts measured by flow cytometry. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 15-4]Comparative analysis of the effectiveness of anti-CD1a antibody administration in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with therapeutic anti-CD1a administration. B. Schematic panel A shows imiquimod treatment of wild-type (WT) and CD1a transgenic mice (CD1a), followed by daily measurement of ear swelling induced by intraperitoneal treatment with a mouse IgG1 isotype control or injection of a refined panel of anti-CD1a antibodies or CR2113 into CD1a transgenic mice. N = 2-7, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 indicate significance compared to "CD1a" on day 8 or OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B) corrected for WT. D. Schematic of MC903-induced inflammation followed by preventive MC903-induced inflammation. E. Daily measurements of ear swelling induced by MC903 treatment of wild-type (WT) and CD1a transgenic (CD1a) mice after intraperitoneal treatment with a mouse IgG1 isotype control, as in panel D of the schematic, or by injection of 16, 110, or 116 anti-CD1a antibodies or CR2113 into CD1a transgenic mice. Corrected for WT. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05 indicates significance compared to "CD1a" on day 7. F. Percentage of cutaneous T cells and eosinophil counts measured by flow cytometry. N = 3-4, two-way ANOVA with Dunnett's test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. [Figure 16-1]Comparative analysis of the effects of anti-CD1a antibodies on cutaneous and systemic immune responses associated with imiquimod-induced inflammation. Ear skin, inflow area cervical lymph node, and plasma samples were analyzed from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the administration therapy model shown in the schematic diagram in Figure 15A. A. IL-17A expression on cutaneous T cells was analyzed using intracellular cytokine expression directly detectable ex vivo by flow cytometry (left panel), and eosinophils in cervical lymph nodes were counted (right panel). B-C. Cytokine levels in plasma (B) and cutaneous digest (C) were measured by ELISA (N=2-7, one-way ANOVA with Dunnett's test, *,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001). [Figure 16-2] Comparative analysis of the effects of anti-CD1a antibodies on cutaneous and systemic immune responses associated with imiquimod-induced inflammation. Ear skin, inflow area cervical lymph node, and plasma samples were analyzed from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the administration therapy model shown in the schematic diagram in Figure 15A. A. IL-17A expression on cutaneous T cells was analyzed using intracellular cytokine expression directly detectable ex vivo by flow cytometry (left panel), and eosinophils in cervical lymph nodes were counted (right panel). B-C. Cytokine levels in plasma (B) and cutaneous digest (C) were measured by ELISA (N=2-7, one-way ANOVA with Dunnett's test, *,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001). [Figure 16-3]Comparative analysis of the effects of anti-CD1a antibodies on cutaneous and systemic immune responses associated with imiquimod-induced inflammation. Ear skin, inflow area cervical lymph node, and plasma samples were analyzed from mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) mice, as well as CD1a transgenic mice injected with a refined panel of anti-CD1a antibodies according to the administration therapy model shown in the schematic diagram in Figure 15A. A. IL-17A expression on cutaneous T cells was analyzed using intracellular cytokine expression directly detectable ex vivo by flow cytometry (left panel), and eosinophils in cervical lymph nodes were counted (right panel). B-C. Cytokine levels in plasma (B) and cutaneous digest (C) were measured by ELISA (N=2-7, one-way ANOVA with Dunnett's test, *,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001). [Figure 17] Crystal structure analysis of OX16, OX110, and OX116. Overview of the crystal structures corresponding to human CD1a bound to three different antibody fragments. Left panel: The heavy chain of CD1a is shown in gray, β2-microglobulin in blue, and the corresponding scFv molecules in green for OX16, yellow for OX110, and pink for OX116. Segments of each scFv fragment corresponding to the VH domain are shown in darker colors, and the VL is shown in lighter colors. Right panel: Surface representation of the CD1a (gray) region recognized by each antibody fragment. CD1a residues in contact with each scFv molecule are shown in color as illustrated. Contact residues are thought to be within 3.5 Å of the interacting chain. [Figure 18]Effect of lipids on the binding of OX16 and OX116 to CD1a. The interaction between OX16 or OX116 scFv and CD1a bearing different lipid ligands was measured using surface plasmon resonance (SPR). Binding curves were calculated by fitting the response units measured upon injection of CD1a-endogenous lipids (red), CD1a-sphingomyelin (blue), CD1a-lysophosphatidylcholine (green), or CD1a-GD3 ganglioside (brown) to flow cells containing OX16-scFv (top) or OX116-scFv (bottom). Bmax values ​​are in relative response units, and KD values ​​are provided in nM. [Figure 19-1] Effect of blocking polyclonal and clonal T cell function by anti-CD1a antibodies. A-B. Determination of the ability of anti-CD1a antibody variants to inhibit CD1a-dependent activation of polyclonal T cell IFNγ (A) and IL-22 (B) production. T cells were isolated from donor PBMCs by CD3 microbead separation. T cells were cocultured overnight with CD1a-K562 or EV-K562, and IFNγ or IL-22 production was detected by ELISpot in the presence of 10 μg / ml anti-CD1a antibody. Antibody formats were compared to a mouse IgG1 isotype control (top statistic) or to the respective isotype (bottom statistic). % blockade was calculated comparing antibody treatment and isotype control after subtracting EV background levels in the cytokine spots. (N = 4 donors, one-way ANOVA with Sidak's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, mean ± SD). C. Cytokine secretion responses of CD1a-restricted T cell clones elicited by empty vector (EV) or CD1a-transfected K562-presenting endogenous ligand. Inhibition of IFNγ was assessed with anti-CD1a antibodies by flow cytometry and normalized to the isotype response (N = 4–8 T cell clones, one-way ANOVA with Tukey's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). [Figure 19-2] Effect of blocking polyclonal and clonal T cell function by anti-CD1a antibodies. A-B. Determination of the ability of anti-CD1a antibody variants to inhibit CD1a-dependent activation of polyclonal T cell IFNγ (A) and IL-22 (B) production. T cells were isolated from donor PBMCs by CD3 microbead separation. T cells were cocultured overnight with CD1a-K562 or EV-K562, and IFNγ or IL-22 production was detected by ELISpot in the presence of 10 μg / ml anti-CD1a antibody. Antibody formats were compared to a mouse IgG1 isotype control (top statistic) or to the respective isotype (bottom statistic). % blockade was calculated comparing antibody treatment and isotype control after subtracting EV background levels in the cytokine spots. (N = 4 donors, one-way ANOVA with Sidak's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, mean ± SD). C. Cytokine secretion responses of CD1a-restricted T cell clones elicited by empty vector (EV) or CD1a-transfected K562-presenting endogenous ligand. Inhibition of IFNγ was assessed with anti-CD1a antibodies by flow cytometry and normalized to the isotype response (N = 4–8 T cell clones, one-way ANOVA with Tukey's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). [Figure 20]Complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-CD1a antibodies A. Flow cytometry analysis of complement-dependent cell-mediated cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum at 37°C for 3 hours in the presence of either 5 μg / ml of isotype control antibody or the indicated antibody. The percentage of cytotoxicity (%) was calculated against a reference population of untreated K562 cells and normalized to isotype control-treated cells. B. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were incubated with PBMCs at a 50:1 effector / target ratio at 37°C for 5 hours in the presence of either 5 μg / ml of isotype control antibody or the indicated antibody, as well as 0.5% FCS and 100 U / ml of IL-2. The percentage of cytotoxicity (%) was calculated against a reference population of untreated K562 cells and normalized to isotype control-treated cells. (N=4~6, one-way ANOVA with Tukey's test). *P<0.05, **,P<0.01; ***,P<0.001, ****,P<0.0001. [Figure 21] Inhibition of TCR binding to CD1a by OX116. A. Schematic of the experimental setup to determine the ability of OX116 to inhibit TCR binding to CD1a. B. Binding curves showing the interaction of CD1a-restricted TCRs (CO3 γδTCR - red; BK6 αβTCR - blue; CD22 γδTCR - green) with CD1a bound to the OX116 Ab fragment. [Figure 22]Clone OX25 CD1a Binding Characteristics A. CD1a transfected cells and recombinant proteins (and controls) were investigated for binding by the antibody OX25 by flow cytometry (geometric mean fluorescence intensity) and ELISA (optical density). Target cells and proteins included major and minor variants of CD1a, as well as cynomolgus monkey CD1a and cells that naturally express CD1a (MOLT4). B. ELISA (optical density) of full-length CD1a compared to chimeric CD1a having human alpha-1 and alpha-2 domains fused to the mouse alpha-3 domain of CD1d, using size exclusion pool B. C. Surface plasmon resonance of OX25 interaction with CD1a. D. Heavy and light chain CDR3 regions of OX25. [Figure 23] Humanized antibodies can deplete CD1a expression transfectants. Modified human IgG1 anti-CD1a antibody or isotype control (5 μg / ml) was incubated with K562-CD1a for 48 hours as shown. The percentage of antibody-induced reduction (%) compared to the isotype control, measured by percentage confluence, was calculated using Incucyte live-cell imaging (N=3, two-way ANOVA with Dunnett's test; **, P<0.01; ****, P<0.0001, where * indicates significance compared to "CD1a"). [Figure 24] Humanized antibodies can inhibit CD1a autoreactive T cells. The cytokine secretion response of CD1a-restricted enriched T cell lines induced by an empty vector (EV) or a K562-presenting endogenous ligand transfected with CD1a was investigated. IFNγ inhibition was evaluated by flow cytometry in a panel of modified human IgG1 anti-CD1a antibodies and isotype controls (5 μg / ml). (N=5-6 enriched T cell lines, one-way ANOVA with Dunnett's test, *,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001, where * indicates significance compared to "CD1a"). [Figure 25]OX25 binds to the alpha-3 domain of CD1a. Characterization of anti-CD1a antibody OX25. A. ELISpot analysis of OX25 and commercial comparators (SK9 - non-blocking, OKT6, and HI148 - blocking antibodies). Polyclonal T cell IFNγ responses to overnight co-culture with CD1a-transfected (CD1a) or empty vector (EV) K562 model antigen-presenting cells. The effect of anti-CD1a antibody (10 μg / ml) on T cell activation was measured by IFNγ (IFNγ spot) ELISpot (n=8 T cell donors). B. Matrix heatmap representation of CD1a antibody binding by flow cytometry as measured by CD1a-AF647 mean fluorescence intensity (MFI). The relevant purified antibodies were incubated with K562 cells prior to staining CD1a-K652 or empty vector (EV) K562 cells with anti-CD1a antibodies conjugated to the fluorophore AF647 to assess interference of AF647-conjugated antibodies with CD1a binding. The gray scale indicates the degree of interference, with the top (-) shade indicating no interference and 100% binding. [Figure 26] Figure 1 shows the efficacy of OX116 in treating CD1a- and checkpoint inhibition-dependent skin inflammation. A. Schematic of imiquimod-induced skin inflammation and administration of anti-CD1a (clone OX116) and anti-PD1 (clone J43) or isotype control. B. Daily measurements of ear swelling induced by imiquimod treatment of wild-type (WT) and CD1a transgenic (CD1a) mice intraperitoneally injected with mouse IgG1 isotype control, anti-PD1 (J43), or anti-CD1a OX116 as in panel A of the schematic (N = 4, two-way ANOVA with Dunnett's test; *, P < 0.05; **, P < 0.01; indicates significance on day 6, as indicated). C. Intracellular flow cytometry analysis of wild-type (WT) and CD1a transgenic (CD1a) ear skin cells injected intraperitoneally with mouse IgG1 isotype control, anti-PD1 (J43), or anti-CD1a (OX116) with or without imiquimod treatment. Cutaneous T cells were identified by CD3+ surface staining, and IL-17 production was analyzed ex vivo by intracellular staining with anti-IL17 antibody. [Figure 27-1] A. Pruritus and pruritic cytokines are significantly reduced after administration of anti-CD1a antibodies. Ear tissues from hCD1a and WT mice were topically treated with 1 nmol of MC903 / EtOH on days 0, 2, and 5. 100 μg / 100 μl of OX116, OX16, and isotype control were administered intraperitoneally on days -2, 0, 2, and 4. (A) Pruritus frequency was assessed at the endpoint. Each data plot represents an individual ear. Plots show mean ± SEM. Statistics were calculated using one-way ANOVA with Tukey's post-hoc test. *P<0.05, ***P<0.001, NS=not significant. B. Epidermal cytokines are reduced in skin tissues after administration of anti-CD1a antibodies. Ear tissues from hCD1a and WT mice were topically treated with 1 nmol of MC903 / EtOH on days 0, 2, and 5. 100ug / 100ul of OX116, OX16, and isotype control were administered intraperitoneally on days -2, 0, 2, and 4. TSLP levels (left panel) and IL-33 levels (right panel) (pg / ml / ear) were assessed at endpoint by Legendplex™. Each data plot represents an individual ear. Plots show mean ± SEM. Statistics were calculated using one-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, NS=not significant. [Figure 27-2]A. Pruritus and pruritic cytokines are significantly reduced after administration of anti-CD1a antibodies. Ear tissues from hCD1a and WT mice were topically treated with 1 nmol of MC903 / EtOH on days 0, 2, and 5. 100 μg / 100 μl of OX116, OX16, and isotype control were administered intraperitoneally on days -2, 0, 2, and 4. (A) Pruritus frequency was assessed at the endpoint. Each data plot represents an individual ear. Plots show mean ± SEM. Statistics were calculated using one-way ANOVA with Tukey's post-hoc test. *P<0.05, ***P<0.001, NS=not significant. B. Epidermal cytokines are reduced in skin tissues after administration of anti-CD1a antibodies. Ear tissues from hCD1a and WT mice were topically treated with 1 nmol of MC903 / EtOH on days 0, 2, and 5. 100ug / 100ul of OX116, OX16, and isotype control were administered intraperitoneally on days -2, 0, 2, and 4. TSLP levels (left panel) and IL-33 levels (right panel) (pg / ml / ear) were assessed at endpoint by Legendplex™. Each data plot represents an individual ear. Plots show mean ± SEM. Statistics were calculated using one-way ANOVA with Tukey's post-hoc test. *P<0.05, **P<0.01, NS=not significant. [Figure 28]This study demonstrates that bispecific CD1a Ab controls CD1a-expressing target cells in vitro and in vivo. A. Left panel: K562 cells expressing CD1a were co-cultured in vitro with Jurkat cells (CD1a-KO) expressing the activation reporter gene (NFAT-GFP) (effector:target 1:1, 50,000 cells each). WIMM-3 bispecific antibody was added, and the cells were incubated at 37°C and 5% CO2 for 18 hours. Cells were stained with viability dye and anti-CD1a antibody to identify viable CD1a-negative Jurkat T cells. Right panel: K562-CD1a-GFP and K562-empty vector-mCherry cells were mixed in equal numbers (25,000 cells each) and co-cultured with 125,000 human CD8+ T cells that had been isolated from PBMCs, stimulated with anti-CD3 and anti-CD28 beads, and rested for 12 days. The co-culture was incubated for 48 hours. B. 500,000 K562 cells expressing CD1a were subcutaneously injected into the flanks of anesthetized NSG mice (n=7 / group). Four days later, the mice were intravenously injected with human CD8+ T cells isolated from PBMCs and stimulated with anti-CD3 / anti-CD28 beads 14 days prior. The mice were also injected with 100 μl of bispecific antibody WIMM-3 (0.5 mg / Kg) or PBS. The mice underwent a second treatment with human CD8+ T cells and WIMM-3 or PBS 14 days after the first injection. The left panel shows the mean tumor size, the right panel shows data for individual mice, and the bottom panel shows survival rate. Student's t-test was used at each time point. P values ​​<0.0001***, <0.001***, <0.005**, <0.05*. DETAILED DESCRIPTION OF THE INVENTION

[0274] Materials and Methods mouse All mice were housed in a specific pathogen-free facility. For each experiment, mice were matched for age, sex, and background strain, with wild-type littermates used as matched controls. All experiments performed in this study were approved by the UK Home Office.

[0275] Generation of CD1a transgenic mice Mice were generated by the Wellcome Trust Centre for Human Genetics, Oxford. A 5.7-kb genomic fragment encompassing the entire CD1A gene, including 0.8 kb of upstream and 0.8 kb of downstream sequences, was amplified from human genomic DNA by PCR using primers 5'-ATGGTACCAAGAGGAATGTAAATGTGTCCGGC-3' and 5'-AAGCGGCCGCGATCATGTTAACCAAGGTCAGGAA-3' and subcloned into the Litmus28 vector (NEB) via the KpnI and NotI sites incorporated into these PCR primers. After sequence verification of the coding exons, the transgene fragment was excised from the vector backbone, purified, resuspended at 2 ng / µL in microinjection buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA), and microinjected into the pronuclei of fertilized zygotes prepared from C57BL / 6J mice. After overnight culture, the resulting 2-cell embryos were surgically transferred into the oviducts of pseudopregnant CD1 foster mothers and raised to term. Transgenic offspring were identified by PCR using transgene-specific primers and bred as individual strains with wild-type C57BL / 6J mice.

[0276] CD1a genotyping Crude genomic DNA was prepared using ear notch samples from CD1a transgenic mice. 100 μl of DirectPCR ear lysis buffer (Viagen) supplemented with 0.4 mg / ml of proteinase K (Sigma) was added to the ear notch and incubated overnight at 55°C. The enzyme was then thermally inactivated at 85°C for 1 hour. The samples were centrifuged to pellet the debris, and the lysate was transferred to a clean tube. 1 μl of the lysate was used as a template for genotyping. The following PCR reaction was used for genotyping. The PCR product was loaded onto a 1% TAE agarose gel using SyberSafe, electrophoresis was performed, and the gel was imaged under UV light. If a 655 bp band was detected as expected, the mouse was considered positive for the CD1a transgene.

[0277] [Table 1]

[0278] [Table 2]

[0279] cell line K562 (EV-K562) cells transfected with an empty vector and K562 (CD1a-K562) cells transfected with CD1a (donated by B. Moody, Brigham and Women's Hospital, Harvard Medical School, Boston, MA) were maintained in RPMI1640 medium supplemented with 10% FCS, 100 IU / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 1 × non-essential amino acids (NEAA) (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 500 μM 2-mercaptoethanol (Gibco), and 200 μg / ml G418 antibiotic (Thermo Fisher Scientific).

[0280] ELISpot analysis Activation-induced cytokine secretion from polyclonal T cells was detected using an ELISpot assay (IFNγ ELISpot kit, Mabtech, AB) upon co-culture with model CD1a-expressing antigen-presenting cells. PBMCs from healthy donor blood were isolated by density gradient (Lymphoprep), and T cells were purified using anti-CD3 magnetic bead sorting according to the manufacturer's protocol (MACS, Miltenyi). All study participants provided informed written consent (National Health Service (NHS) National Research Ethics Service (NRES) research ethics committee 14 / SC / 0106). T cells were then expanded by culture with IL-2 (200 U / ml) for 3 days, followed by overnight co-culture with K562 cells transfected with unpulsed / endogenous lipid-bound CD1a (CD1a-K562) or K562 cells transfected with a control empty vector (EV-K562). To assess the functionality of anti-CD1a antibodies, K562 cells were incubated with 10 μg / ml anti-CD1a antibodies 1 hour before and during coculture with polyclonal T cells on anti-IFNγ or anti-IL-22 capture antibody-coated ELISpot plates (Millipore Corp., MA). IFNγ and IL-22 secretion was detected with biotinylated anti-cytokine detection antibodies and visualized with streptavidin-alkaline phosphatase chromogenic assays. The resulting spots, representing cytokine-producing T cells, were counted using an automated ELISpot reader (Autimmun Diagnostika GmbH ELISpot Reader Classic). After subtracting the EV background level of cytokine-producing spots, percent blockade was calculated by comparing antibody-treated and untreated groups. The contribution of EV-K562 was subtracted from the CD1a IFNγ / IL-22 spot counts. The adjusted spot counts for the CD1a-K562 antibody treated group were then divided by the CD1a group without antibody and used to calculate % blocking.

[0281] Analysis of CD1a-reactive T cell generation and activation: CD1a-restricted T cells were isolated by fluorescence-activated cell sorting. T cells were cocultured with CD1a-K562 and EV-K562, and cytokine-producing responder T cells were detected using the Miltenyi MACS cytokine secretion assay according to the manufacturer's instructions. Briefly, to detect CD1a-dependent autocrine cytokine production, T cells were cultured with CD1a-K562 for 6 hours and then coated with anti-cytokine (IL-22 or IFNγ) antibodies. Live responder cells were then sorted onto culture plates. CD1a-restricted T cells were expanded in a mixed lymphocyte reaction, and purity and CD1a responsiveness were assessed using an analytical flow cytometer with the FACS-based cytokine secretion assay described above. CD1a-restricted T cell activation was analyzed as follows: 2 × 10 5 1-5 × 10 K562 cells 5 The cells were co-cultured with CD1a autoreactive T cell clones for 4 hours. Helper cytokines were added to the co-cultures to support CD1a-dependent cytokine production. IFNγ-producing T cell cultures were supplemented with IL-12 (1 ng / mL, BioLegend), IL-18 (1 ng / mL, BioLegend), and IL-2 (25 U / mL, BioLegend). T cell activation was assessed by T cell cytokine production using a cytokine secretion assay (Miltenyi Biotec) according to the manufacturer's instructions.

[0282] Imiquimod administration to mice Mice were anesthetized with isoflurane, and 15 mg of Aldara cream containing 5% imiquimod was applied to the dorsal and ventral ear pinnae on days 0, 1, 2, 3, 4, and 5 for the preventive model (Figure 4A) or days 0, 1, 2, 4, 5, 6, and 7 for the therapeutic model (Figure 9A). 100 μg of anti-CD1a antibody or mouse IgG1 isotype control was administered intraperitoneally on days -5, -3, -1, 1, 3, and 5 for the preventive model (Figure 4A) or days 3, 5, and 7 for the therapeutic model (Figure 9A). Ear thickness was measured daily using a micrometer (Mitutoyo) throughout the Aldara application period, from days 0 to 6 for the preventive model (Figure 4A) or days 0 to 8 for the therapeutic model (Figure 9A). Mice were sacrificed, and tissues were collected 24 h after challenge.

[0283] MC903 administration to mice Mice were lightly anesthetized with isoflurane and MC903 was applied to the ventral and dorsal sides of the ear at a dose of 2 nmol (10 microliters per side of the ear) daily for 7 days. 100 μg of anti-CD1a antibody or mouse IgG1 isotype control was administered intraperitoneally as shown in Figure 15D. Ear thickness measurements were taken daily using a micrometer (Mitutoyo).

[0284] Tissue processing Twenty-four hours after the final imiquimod challenge, mice were sacrificed and tissues were harvested. Ears, cervical lymph nodes (cLNs), and spleens were harvested for immunophenotyping or imaging. Cell suspensions of spleens and cLNs were obtained by passing the tissues through a 70-μm strainer and washing with RPMI containing 10% FCS. Erythrocytes in the spleen cell suspension were removed by incubation with RBC lysis solution (eBioscience).

[0285] Ear skin tissue was washed with HBSS to remove excess imiquimod, split ventrally, diced into <0.5 mm pieces, and digested with 1 mg / mL collagenase P (Roche) and 0.1 mg / mL DNase I (Sigma-Aldrich) in DMEM for 3 × 30 min with agitation, with 5 mg / mL dispase added for the final 30 min of the digestion step. Single-cell suspensions were obtained by washing through a 70 μm filter in DMEM containing 10% FCS prior to analysis by flow cytometry.

[0286] Flow cytometry For FACS surface staining, cells were labeled with the following anti-mouse antibodies (procured from Biolegend unless otherwise stated): CD3 (500A2, BUV495:741064 BD Pharmingen), CD11b (M1 / 70, BUV395:563553 BD Pharmingen), CD11c (N418, BV711:117349), CD8 (53-6.7, BUV805:612898 BD Pharmingen), CD4(GK1.5,AF700:100430),CD45(2D1,FITC:368507),CD11a(I21 / 7,PE Cy7:153108), CD69(H1.2F3,BV650:104541), Langerin(4C7,PE:144204), Ly6C(RB6-8C 5, BV605:108440), Ly6G (1A8, PETxRed:127648), MHCII (M5 / 114.15.2, BV785:107645), SiglecF (S17007L, BV421:155509), IL-17A (TC11-18H10.1, PECy7:506922), Live / Dead Aqua (Invitrogen), and anti-human CD1a (APC or purified SK9, HI149, OKT6, NA1 / 34).

[0287] Flow cytometry: epitope competition assay CD1a-K562 cells were incubated with purified, primary, freshly produced, commercially available anti-CD1a antibodies (25 μg / ml) on ice for 30 minutes. Unbound antibodies were then washed away, and Alexa-Fluor-647 conjugated forms of the different antibodies were then incubated with the cells in a matrix configuration for 30 minutes on ice (10 μg / ml). The mean fluorescence intensity (MFI) was used to assess the extent of binding of the fluorophore-conjugated antibodies.

[0288] Confocal imaging Mouse ear skin was frozen in optimal cutting temperature embedding compound and stored at -80°C. 10 μm frozen sections were cut using a Leica cryostat, collected onto Superfrost Plus slides, air-dried for 30 minutes, and then stored at -80°C. Slides were rehydrated in PBS for 10 minutes before staining. Endogenous peroxidase activity in the samples was quenched by adding 0.15% hydrogen peroxide solution for 5 minutes at room temperature. Endogenous biotin was blocked with an avidin / biotin blocking kit (Vector Laboratories Ltd), and 10% goat serum was used to reduce nonspecific antibody binding. Anti-CD1a antibody (1:100, OKT6; in-house production, conjugated to biotin) was used for confocal microscopy. Signal enhancement was achieved using the Alexa Fluor 594 Tyramide SuperBoost Kit (streptavidin; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, slides were incubated with primary antibodies overnight at 4°C. After washing, HRP-conjugated streptavidin was added to the sections and incubated overnight at 4°C. Excess streptavidin-HRP was washed away, and the tissues were incubated with tyramide working solution for 8 minutes at room temperature. The reaction was then stopped with a stop reagent. After staining, slides were mounted using antifade mounting medium containing DAPI (Vector Laboratories Ltd), coverslipped, and stored refrigerated in the dark before analysis under a confocal microscope (Zeiss LSM 780 confocal microscope - inverted; 25x / 0.8 Imm Korr DIC M27; room temperature; Axiocam camera; Zen software). Images were processed using Fiji.

[0289] Cell phenotyping and cytotoxicity assays: Anti-CD1a antibody (5 μg / ml) and / or commercial comparator NA1 / 34 (5 μg / ml) were incubated with CD1a-expressing K562 or EV control K562 for 48 hours, and cell depletion was assessed by flow cytometry. To measure direct antibody-induced cell depletion, K562 were fluorescently labeled with CellTraceViolet and then incubated with anti-CD1a antibody for 48 hours. Prior to evaluation of depletion by flow cytometry, a reference population of untreated CFSE-labeled K562 was added to the antibody-treated K562 at a 1:1 ratio. The percentage of induced depletion was then calculated by comparing the frequency of viable cells in the different populations analyzed, antibody-treated and untreated reference CD1a+ and EV K562, using the following formula: % reduction = 100 - ((% viable cells of antibody-treated CD1a-K562 / % viable cells of reference CFSE-labeled K562) / (% viable cells of untreated CD1a-K562 / % viable cells of reference CFSE-labeled K562) × 100). To examine the effect of anti-CD1a antibodies on apoptosis of CD1a-expressing cells, K562-CD1a or K562-EV were incubated with either an isotype control or anti-CD1a antibody (5 μg / ml) and stained for Annexin-V (Biolegend) after 24 h of incubation.

[0290] Complement-mediated lysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays: For the CDC assay, K562-CD1a cells (5 × 10 per well) 4 PBMCs were used for the ADCC assay. K562-CD1a cells (5 × 10 per well) were pretreated with 5 μg / ml of either an isotype control antibody or the indicated antibody for 30 minutes and then incubated with 10% normal human serum at 37°C in 5% CO2 for 3 hours. 3 cells) were incubated with PBMCs (2.5 × 10 cells per well) for 5 h at 37°C in 5% CO with IL-2 (100 U / ml) in combination with 5 μg / ml of either an isotype control antibody or the indicated antibody. 5Cells) were co-cultured with K562-CD1a cells (effector / target ratio of 50:1). Cytotoxicity was determined by calculating the percentage of viable target K562-CD1a cells using the following formula: % cytotoxicity = 100 - ((% viable CD1a-K562 cells treated with CD1a antibody / % viable reference K562 cells) / (% viable isotype antibody-treated CD1a-K562 cells / % viable reference K562 cells) × 100).

[0291] In vivo depletion of CD1a+ cells: "NSG" NOD-scid IL2R gamma ヌル Mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells in ECM gel (Merck) suspension (vol = 100 μl), and tumors were allowed to develop for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibody on days 6, 10, and 14, and tumor size was measured.

[0292] Isoelectric focusing assay (IEF): Lipid loading was evaluated by incubating 10 μg of CD1a with Tris-buffered saline and 100 × molar excess imiquimod (Invivogen) solubilized in 2% CHAPS, 7% DMSO, or vehicle alone (mock) at 37°C for 2 hours and overnight at room temperature. CD1a samples were separated by isoelectric focusing (IEF). Briefly, CD1a-imiquimod and CD1a-mock proteins were loaded onto IEF pH 3-7 gels (Novex), and then electrophoresed at 100 V for 1 hour, 200 V for 1 hour, and finally at 500 V for 30 minutes. The gels were then fixed with 12% TCA, stained with SimplyBlue SafeStain for 7 minutes, and destained overnight with DI water.

[0293] Statistical analysis: One-way and two-way ANOVA tests were performed using GraphPad Prism version 6.00 (GraphPad Software). Error bars represent standard deviations as indicated.

[0294] Generation and selection of therapeutic anti-CD1a antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) Numerous animals across different species (including mice and rabbits) were immunized. Mice were immunized with NIH3T3 cells transfected with human CD1a and mouse B2M. Rabbits were immunized with Rab9 cells transfected with human CD1a and rabbit B2M. After 3–5 injections, the animals were sacrificed and PBMCs, spleen, bone marrow, and lymph nodes were collected. Serum was monitored for binding to HEK-293 cells expressing human CD1a and human B2M via flow cytometry.

[0295] Memory B cell cultures (related to 77A (VR11851), 110 (VR12112), 111 (VR12113), and 116 (VR12117)) were set up, and the supernatant was first screened for its ability to bind to human CD1a transiently transfected HEK-293 cells using a bead-based assay on the TTP Labtech Mirrorball system. This was a multiplex assay that counterscreened HEK-293 cells expressing human CD1a and human B2M stained with cell dye against HEK-293 cells expressing CD1b, CD1c, or CD1d counterstained with human B2M, using goat antibiotic Fc-FITC conjugate as the detection agent.

[0296] Approximately 3500 CD1a-specific positive hits were identified in the primary Mirrorball screen from a total of 10 x 200 plate B culture experiments. Positive supernatants from this assay were then carried forward for further characterization by: ELISA to confirm binding to human CD1a protein (details below) ELISA to confirm binding to the CD1a lipid-binding domain (human lipid-binding domain of CD1a, mouse Ig domain of CD1d) on the chimeric CD1a protein (details below) Flow cytometry to confirm binding to human CD1a expressed on HEK-293 cells (co-expressed with human β2M) (details below)

[0297] Wells that showed binding in the above assay were advanced for recovery of V regions using fluorescent focus techniques.

[0298] Furthermore, bone marrow-derived plasma cells were directly screened for their ability to bind to human CD1a using fluorescence focusing (related to 16(VR11834)). Here, B cells secreting CD1a-specific antibodies were picked up on biotinylated human CD1a immobilized on streptavidin beads using a goat antibiotic Fc-FITC conjugate detection reagent. Approximately 300 direct foci were picked up.

[0299] After reverse transcription (RT) and PCR of the harvested cells, "transcriptionally activated PCR" (TAP) products encoding the antibody V-regions were generated and used to transiently transfect HEK-293 cells. The resulting TAP supernatants containing the recombinant antibodies were further characterized by: ELISA to confirm binding to human CD1a protein and chimeric CD1a protein (human lipid-binding domain of CD1a, mouse Ig domain of CD1d) (details below) Flow cytometry to confirm binding to human CD1a expressed on HEK-293 cells (co-expressed with human β2M) and counter-screening for cross-binding to related homologous proteins: CD1b, CD1c, or CD1d, also expressed on HEK-293 cells (co-expressed with human β2M). (See below for details.)

[0300] Next, heavy and light chain variable region gene pairs from the target TAP product were cloned as full-length antibodies in either rabbit or mouse and re-expressed in a HEK-293 transient expression system. A total of 119 V regions were cloned and registered. The recombinant cloned antibodies were then further characterized as follows: • Repeat the above flow cytometry and ELISA assays. Flow cytometry to assess binding to CD1a expressed on multiple cell lines. This provided the first indication that binding was lipid independent. Supernatants were screened for binding to: - Stably transduced C1R cells expressing CD1a or empty vector (co-expressed with human β2M), these are related to 110 (VR12112), 111 (VR12113), and 116 (VR12117) (see below for details). - MOLT4 cells endogenously expressing CD1a, CD1b, CD1c, CD1d, and β2M. These are associated with 110 (VR12112), 111 (VR12113), and 116 (VR12117). (Details below) • Profiling in BIAcore to estimate dissociation rate and affinity (details below)

[0301] Antibodies that showed binding and affinity <100 nM in the above assay were selected for purification. Cell culture supernatant was purified using protein A affinity purification. The purified samples were buffered with 10 mM PBS (pH 7.4), and their recovery and purity were analyzed using UV spectroscopy, analytical size exclusion chromatography, SDS-Page electrophoresis, and LAL endotoxin assay, respectively. Where necessary, the samples were subjected to a second round of purification to increase monomer levels. The final samples were sterile filtered and stored in 10 mM PBS (pH 7.4).

[0302] After purification, all five antibodies were further characterized as follows: • Repeat the above flow cytometry, ELISA, and BIAcore assays. • ELISA to evaluate the binding of cynomolgus monkey CD1a protein and human CD1a protein to variants common in China (18) (details below) • Flow cytometry to evaluate binding to transiently transfected HEK-293 cells (details below) - Co-transfection of cynomolgus macaque CD1a with cynomolgus macaque β2M - Co-transfection of human CD1a common variants in China with human β2M

[0303] Antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) demonstrated the ability to bind to all forms of recombinant and cell-expressed CD1a protein tested during their respective stages of antibody discovery (Tables 1-9). The only exception was 116 (VR12117), which showed no binding to recombinant or cell-expressed cynomolgus monkey CD1a (Tables 4 and 9). While including antibody 116 in subsequent in vitro and in vivo analyses was not considered obvious, it was nevertheless a deliberate step to focus on epitope-binding regions where the lipid-binding domain differs from that of human and cynomolgus monkeys and may have different functional effects. None of the antibodies showed binding to CD1b, CD1c, or CD1d expressed on HEK-293 cells (Table 5). This indicates that these antibodies are CD1a-specific. Expression of CD1a, CD1b, CD1c, and CD1d on HEK-293 cells was confirmed using commercially available antibodies, supporting this conclusion (data not shown). Binding to CD1a expressed on multiple cell types (HEK, C1R, and MOLT4) provided the first indication that antibody binding may be lipid-independent, as CD1a is likely loaded from different pools of lipids in each cell line.

[0304] After the antibody was discovered, its in vitro function was evaluated using a T-cell assay as follows.

[0305] DNA encoding the heavy and light chain V regions of 77A (VR11851), 110 (VR12112), 111 (VR12113), and 116 (VR12117) on a murine IgG1 backbone was synthesized at ATUM and expressed in an in-house HEK-293 transient expression system. The antibodies were then purified and endotoxin-removed and tested in in vivo assays as follows.

[0306] Affinity of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) for human CD1a The affinity of purified antibodies for human CD1a was assessed using a Biacore T200 instrument (GE Healthcare) by capturing the antibodies against immobilized anti-species IgG F(ab')2, followed by titration with human CD1a. Affinipure goat anti-species IgG-F(ab')2 fragment specific (Jackson ImmunoResearch) was immobilized onto a CM5 sensor chip (GE Healthcare) via amine coupling chemistry to a capture level of approximately 5000 response units (RU). HBS-EP+ buffer (10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare) was used as the running buffer at a flow rate of 10 μL / min. Capture with immobilized goat anti-species Fab was performed using a 10 μL injection of 0.5 μg / mL of test antibody. Human CD1a was titrated against the capture antibody (0 nM, 0.6 nM, 1.8 nM, 5.5 nM, 16.6 nM, and 50 nM, diluted in running buffer) at a flow rate of 30 μL / min to assess affinity.

[0307] The surface was regenerated between cycles by two 10 μL injections of 40 mM HCl, interspersed with 10 μL injections of 5 mM NaOH at a flow rate of 10 μL / min. Background-subtracted binding curves were analyzed using Biacore T200 evaluation software according to standard procedures. Kinetic parameters were determined from fitting algorithms. The assay was performed at the clone supernatant and purified antibody stages. Table 10 shows the kinetic parameters of antibody binding to human CD1a.

[0308] Binding of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) assessed by ELISA CD1a-specific antibodies were identified by ELISA. ELISA plates were coated with 2 μg / mL of the protein of interest (human CD1a pool B, chimeric CD1a pool B [human lipid-binding domain and mouse CD1d Ig domain], Chinese variant CD1a, or cynomolgus monkey CD1a) (20 μL / well) overnight at 4°C and then washed with wash buffer (0.2% (v / v) Tween-20 in PBS (pH 7.4)). The plates were then blocked with 80 μL / well of blocking buffer (1% (w / v) bovine serum albumin) at room temperature for 1 hour and then washed with wash buffer. 20 μL of diluted antibody samples (B cell culture supernatants, TAP supernatants, clone supernatants, purified antibody solutions) were transferred to the ELISA plates, incubated at room temperature for 1 hour, and subsequently washed with wash buffer. 20 μl / well of peroxidase-conjugated goat anti-species IgG Fc-specific F(ab')2 fragment (Jackson ImmunoResearch) diluted to 5:5000 in blocking buffer was added and incubated for 1 hour at room temperature, followed by washing with wash buffer. Binding was visualized by adding 20 μL / well of TMB substrate (EMD Millipore). The reaction was incubated at room temperature for 5 minutes, and then the optical density was measured at 630 nM using a microplate reader. This assay was performed on B cell supernatants (human CD1a pool B), TAP supernatants (human CD1a pool B, chimeric CD1a pool B), clone supernatants (human CD1a pool B, chimeric CD1a pool B), and purified antibodies (human CD1a pool B, chimeric CD1a pool B, Chinese variant CD1a, and cynomolgus CD1a). Tables 1-4 show the data for purified antibodies.

[0309] The coupling of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) was evaluated by flow cytometry. CD1a-specific antibodies were identified by flow cytometry. Binding to proteins expressed on HEK, C1R, and MOLT4 cell lines was assessed. HEK-293 cells were transfected with the protein of interest (CD1a, CD1b, CD1c, CD1d, Chinese variant CD1a, or cynomolgus monkey CD1a) and species-specific β2M (as described above). Transfections were performed using the Expifectamine 293 kit (Gibco) and incubated overnight. C1R-CD1a, C1R-empty vector, and MOLT4 cell lines were washed with 1x PBS on the required day. All cell lines were counted, resuspended in 1x PBS, and then stained for 30 minutes at 37°C using DiI or DiO cell stain (Invitrogen). After washing the cells with flow cytometry buffer (1% bovine serum albumin, 2 mM EDTA, and 0.1% sodium azide in PBS), the two DiI-stained and DiO-stained populations were mixed together. The cells (20 μl / well) were then added to a dilution of antibody samples (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) (20 μl / well) and incubated for 1 hour at 4°C in a flow cytometry assay plate, followed by washing with flow cytometry buffer. 10 μl / well of Alexafluor 647-conjugated goat anti-species IgG Fc-specific F(ab')2 fragment (Jackson ImmunoResearch) diluted 1:2500 in flow cytometry buffer was added and incubated for 30 minutes at 4°C, followed by washing with wash buffer. Fluorescence intensity was then measured using an iQue Screener PLUS. This assay was performed on B cell supernatants (HEK-293 cells expressing human CD1a), TAP supernatants (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d), clone supernatants (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d; C1R cells expressing human CD1a or an empty vector; and MOLT4 cell line), and purified antibodies (HEK-293 cells expressing human CD1a, CD1b, CD1c, CD1d, Chinese variant CD1a, or cynomolgus CD1a; C1R cells expressing human CD1a or an empty vector; and MOLT4 cells).Tables 5 to 9 show data on purified antibodies.

[0310] Table 1. Antibodies that bind to human CD1a pool B protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind to human CD1a protein in an ELISA. The antibodies were titrated through a dilution series and compared to a control rabbit IgG antibody. All five antibodies bound to human CD1a pool B protein. Data for purified antibodies is shown.

[0311] [Table 3]

[0312] Table 2. Antibodies that bind to chimeric CD1a Pool B protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind to chimeric CD1a [human CD1a lipid-binding domain, mouse CD1d Ig domain] protein in an ELISA. Antibodies were titrated through a dilution series and compared to a control rabbit IgG antibody. All five antibodies bound to chimeric CD1a Pool B protein. Data for purified antibodies is shown.

[0313] [Table 4]

[0314] Table 3. Antibodies that bind to the Chinese variant CD1a protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind to the Chinese variant CD1a protein in an ELISA. The antibodies were titrated through a dilution series and compared to a control rabbit IgG antibody. All five antibodies bound to the Chinese variant CD1a protein. Data for purified antibodies is shown.

[0315] [Table 5]

[0316] Table 4. Antibody binding to cynomolgus monkey CD1a protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind to cynomolgus monkey CD1a protein in an ELISA. The antibodies were titrated through a dilution series and compared to a control rabbit IgG antibody. All five antibodies except 116 (VR12117) bound to cynomolgus monkey CD1a protein. Data is shown for purified antibodies.

[0317] [Table 6]

[0318] Table 5. Antibodies that bind to human CD1a, CD1b, CD1c, or CD1d expressed on HEK-293 cells. HEK-293 cells were transiently transfected with human CD1a, CD1b, CD1c, or CD1d and cotransfected with human β2M. Antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through a dilution series and tested for binding to the transfected protein. Binding was quantified as the fold change in geometric mean fluorescence intensity over background, assessed by flow cytometry. All five antibodies bound to human CD1a expressed on HEK-293 cells. No binding was observed to CD1b, CD1c, or CD1d expressed on HEK-293 cells. Data for purified antibodies is shown.

[0319] [Table 7]

[0320] Table 6. Antibodies that bind to human CD1a, CD1b, CD1c, or CD1d expressed on C1R cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through a dilution series and tested for binding to C1R cells stably transduced with human CD1a or empty vector and human β2M. Binding was quantified as the fold change in geometric mean fluorescence intensity over background, assessed by flow cytometry. All five antibodies bound to human CD1a expressed on C1R cells. No binding was observed to C1R cells expressing empty vector. Data is shown for purified antibodies.

[0321] [Table 8]

[0322] Table 7. Antibody binding to MOLT4 cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through a dilution series and tested for binding to MOLT4 cells, which endogenously express CD1a, CD1b, CD1c, CD1d, and β2M. Binding was quantified as the fold change in geometric mean fluorescence intensity over background, assessed by flow cytometry. All five antibodies bound to MOLT4 cell surface proteins, most likely CD1a. Data shown is for purified antibodies.

[0323] [Table 9]

[0324] Table 8. Antibodies binding to common Chinese variant CD1a expressed on HEK-293 cells. Antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through a dilution series and tested for binding to HEK-293 cells transiently transfected with common Chinese variant CD1a (18) and human β2M. Binding was quantified as the fold change in geometric mean fluorescence intensity over background, assessed by flow cytometry. All five antibodies bound to Chinese variant CD1a expressed on HEK-293 cells. Data for purified antibodies is shown.

[0325] [Table 10]

[0326] Table 9. Antibody binding to cynomolgus monkey CD1a expressed on HEK-293 cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through a dilution series and tested for binding to HEK-293 cells transiently transfected with cynomolgus monkey CD1a and cynomolgus monkey β2M. Binding was quantified as the fold change in geometric mean fluorescence intensity over background, assessed by flow cytometry. All five antibodies except 116 (VR12117) bound to cynomolgus monkey CD1a expressed on HEK-293 cells. Data for purified antibodies is shown.

[0327] [Table 11]

[0328] Table 10. Antibody affinity to human CD1a. The affinity of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) to human CD1a was assessed using biacore. A 1:1 binding model was used to fit the data in all cases except for 16 (VR11834), which required a heterologous ligand binding model. To allow for progression, affinities <100 nM were required. Data shown for purified antibodies.

[0329] [Table 12]

[0330] [Table 13]

[0331] antibody production For surface plasmon resonance and crystallization studies, single-chain variable regions (ScFv) were generated for OX16, OX110, and OX116 using a flexible glycine-serine linker between the heavy and light chains. For in vitro functional assays, several constructs containing existing mouse or rabbit variable regions on a human IgG1 Fc region were generated, including wild-type; Leu234Ala, Leu235Ala, and Gly237Ala ("LALAGA"); afucosylated; and Fab versions of existing mouse or rabbit variable regions on a human IgG1 Fc region, as previously described. These were established for OX16, OX110, OX116, and the comparative antibodies CR2113 and mAB571 (U.S. Patent No. 10,844,118 and WO 2022 / 077021). All were expressed in an in-house HEK-293 transient expression system, and the antibodies were then subjected to purification and endotoxin removal.

[0332] Crystallization Human CD1a / β2m heterodimers containing a fos-jun zipper were expressed in HEK293S cells and purified by nickel affinity and size-exclusion chromatography. CD1a was deglycosylated using EndoH (NEB), and the fos-jun zipper, BirA tag, and His tag were cleaved using thrombin overnight at room temperature. For OX scFvs, the BirA and His tags were cleaved using 3C protease overnight at 4°C. The CD1a protein used in crystallization studies contained a heterogeneous mixture of lipids derived from the expression system (CD1a-endo). Monoclonal antibody fragments were expressed as scFv constructs in suspension HEK293F cells and purified by nickel affinity and size-exclusion chromatography. CD1a and antibody fragments were mixed at a 1:1 molar ratio and incubated overnight at 4°C. Sitting-drop crystallization studies were performed at the Monash Macromolecular Crystallization Facility, and the sample concentrations used in each case ranged from 5 to 10 mg / ml. Initial hits were further optimized in hand trays by the hanging-drop method. Crystals of OX16-CD1a appeared in 0.2 M sodium malonate, 20% PEG 3350 and diffracted to 3.2 Å. Crystals of OX110-CD1a were obtained in 0.1 M MES (pH 6), 20% PEG 8000, and 0.2 M sodium acetate and diffracted to 3.4 Å. Crystals of OX116-CD1a were grown in 1.5 M ammonium sulfate, 0.1 M Bis-Tris (pH 6) and diffracted to 2.7 Å. In each case, the structures were solved by molecular replacement using the CD1a binary structure (PDB: 6NUX) and an Alphafold-generated model of the corresponding antigen fragment. Next, the structure was refined through a cycle of manual refinement in Coot, followed by automated refinement in Phenix.

[0333] Surface plasmon resonance SPR experiments were performed on a Biacore 3000 using streptavidin-coated chips (Cytiva). Antibody fragments were expressed with a biotinylation tag at their C-terminus and biotinylated overnight using BirA ligase. Biotinylated scFv molecules were coupled to the chip surface until a total of 150 or 1000 response units per flow cell was achieved, depending on the experiment. To assess the effect of lipid antigen head groups on binding to the OX16 and OX116 antibodies, increasing concentrations of deglycosylated CD1a-endo or CD1a loaded with specific lipids were injected over each flow cell. Lipid loading of CD1a was performed as previously described (Cotton et al., J Exp Med. 5;218:e20202699 (2021)). Briefly, the lipids used were sphingomyelin (Avanti 860593), lysophosphatidylcholine (Avanti 845875), GD3 ganglioside (Avanti 860060), egg PG (Avanti 841138), sulfatide (Avanti 131305), and phosphatidylcholine (Avanti 850375). Each lipid was solubilized to 5–10 mM in 20 mM Tris (pH 8), 150 mM NaCl, and 0.5% CHAPS. CD1a-endo was incubated with a 15–40-fold molar excess of lipid overnight at room temperature. The mixture was then purified by anion exchange chromatography using a MonoQ column (GE Healthcare). Fractions corresponding to lipid-loaded CD1a were pooled together and concentrated to 50 μM. Serial dilutions of CD1a up to a maximum concentration of 10 μM were injected for 60 s in 20 mM Tris (pH 8), 150 mM NaCl buffer at 25°C. Dissociation times between injections ranged from 5 min to a maximum of 1 h. For TCR binding to CD1a-Ab complexes, 1000 response units of biotinylated OX116 were coupled onto the SA chip. Each injection cycle consisted of a 60 s injection of 1 μM CD1a alone, followed immediately by injections of increasing concentrations of TCR (0–50 μM) supplemented with 100 nM CD1a to prevent further dissociation of CD1a from the coupled Ab fragment.Electrophoresis was performed in 20 mM Tris (pH 8), 150 mM NaCl, and 0.5% BSA buffer at 25°C. In all SPR experiments, the relative binding response was calculated by subtracting the nonspecific response on the reference cell coupled with an unrelated protein. Binding curves were obtained by fitting the measured responses to the 1:1 specific binding model in GraphPad.

[0334] Antibody binding detected by ELISA The ELISA plate was coated overnight at 4°C with 2 μg / mL of the target protein (human CD1a pool B, chimeric CD1a pool B [human lipid-binding alpha-1 / 2 domain and mouse CD1d alpha-3 Ig domain], minor variant CD1a, or cynomolgus monkey CD1a) (20 μL / well), and then washed with wash buffer (0.2% (v / v) Tween-20 in PBS (pH 7.4)). The plate was then blocked at room temperature for 1 hour with 80 μl / well of blocking buffer (1% (w / v) bovine serum albumin), and then washed with wash buffer. 20 μL of antibody sample (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) dilution was transferred to the ELISA plate, incubated at room temperature for 1 hour, and then washed with wash buffer. 20 μl / well of peroxidase-conjugated goat antibody IgG Fc-specific F(ab')2 fragment (Jackson ImmunoResearch), diluted to 5000 with blocking buffer, was added and incubated at room temperature for 1 hour, followed by washing with wash buffer. TMB substrate (EMD Millipore) was added (20 μL / well) to visualize binding, and the reaction was incubated at room temperature for 5 minutes. Optical density was then measured at 630 nM using a microplate reader.

[0335] Antibody binding detected by flow cytometry CD1a-specific antibodies were identified by flow cytometry. Binding to proteins expressed on HEK, C1R, and MOLT4 cell lines was assessed. HEK-293 cells were transfected with the protein of interest (CD1a, CD1b, CD1c, CD1d, Chinese variant CD1a, or cynomolgus monkey CD1a) and species-specific β2M (as described above). Transfections were performed using the Expifectamine 293 kit (Gibco) and incubated overnight. C1R-CD1a, C1R-empty vector, and MOLT4 cell lines were washed with 1x PBS on the required day. All cell lines were counted, resuspended in 1x PBS, and then stained for 30 minutes at 37°C using DiI or DiO cell stain (Invitrogen). After washing the cells with flow cytometry buffer (1% bovine serum albumin, 2 mM EDTA, and 0.1% sodium azide in PBS), the two DiI-stained and DiO-stained populations were mixed together. The cells (20 μl / well) were then added to a dilution of antibody samples (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) (20 μl / well) and incubated for 1 hour at 4°C in a flow cytometry assay plate, followed by washing with flow cytometry buffer. 10 μl / well of Alexafluor 647-conjugated goat anti-species IgG Fc-specific F(ab')2 fragment (Jackson ImmunoResearch) diluted 1:2500 in flow cytometry buffer was added and incubated for 30 minutes at 4°C, followed by washing with wash buffer. Fluorescence intensity was then measured using an iQue Screener PLUS. This assay was performed on B cell supernatants (HEK-293 cells expressing human CD1a), TAP supernatants (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d), clone supernatants (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d; C1R cells expressing human CD1a or an empty vector; and MOLT4 cell line), and purified antibodies (HEK-293 cells expressing human CD1a, CD1b, CD1c, CD1d, Chinese variant CD1a, or cynomolgus CD1a; C1R cells expressing human CD1a or an empty vector; and MOLT4 cells).

[0336] Humanization method for antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834). Antibodies were humanized by transplanting CDRs derived from the V regions of rabbit and mouse antibodies onto a human germline antibody V region framework. To restore antibody activity, several framework residues from the rabbit and mouse V regions were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanised antibodies. International Publication No. 91 / 09967). The CDRs transplanted from the donor to the acceptor sequence are as defined by Kabat (Kabat et al., 1987), with the exception of CDRH1, where a Chothia / Kabat definition combination is used (see Adair et al., 1991, Humanised antibodies. International Publication No. 91 / 09967). Generally, the VH genes of rabbit antibodies are shorter than the selected human VH acceptor genes. When aligned with the human acceptor sequence, framework 1 of the VH region of rabbit antibodies typically lacks the N-terminal residue retained in the humanized antibody. Framework 3 of rabbit antibody VH regions also typically lacks one or two residues (75, or 75 and 76) in the loop between beta sheet strands D and E; in humanized antibodies, the gap is filled with the corresponding residues from the selected human acceptor sequence.

[0337] Antibody 77A The human V region IGKV1-5 + IGKJ4 J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the antibody 11851 light chain CDRs. In addition to the CDRs, 0, 1, 2, 3, or 4 of the following framework residues (donor residues) from the 11851 VK gene can be retained at positions 1, 2, 3, and 71 (Kabat numbering): alanine (A1), valine (V2), glutamic acid (E3), and tyrosine (Y71), respectively. In some cases, CDRL3 can be mutated to remove the unpaired cysteine ​​residue at position 90 (Kabat numbering) (C90, CDRL3 variant, SEQ ID NOS: X-Y).

[0338] The human V region IGHV3-23+IGHJ5 J region (IMGT, http: / / www.imgt.org / ) The heavy chain CDRs of antibody 11851 were selected as acceptors. In addition to the CDRs, 0, 1, 2, 3, 4, 5, 6, or 7 of the following framework residues (donor residues) from the 11851 VH gene can be retained at positions 24, 48, 49, 71, 73, 78, and 94 (Kabat numbering): valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), valine (V78), and arginine (R94), respectively.

[0339] antibody 110 The human V region IGKV1-D13 + IGKJ4 J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the antibody 12112 light chain CDRs. In addition to the CDRs, 0, 1, 2, or 3 of the following framework residues (donor residues) from the 12112 VK gene can be retained at positions 2, 3, and 70 (Kabat numbering): glutamine (Q2), valine (V3), and glutamine (Q70), respectively. In some cases, CDRL3 can be mutated to eliminate the disulfide bond between cysteine ​​residues at positions 94 and 95d (Kabat numbering) (C94 and C95d, CDRL3 variants, SEQ ID NOS: X-Y).

[0340] Human V region IGHV3-48 + IGHJ2 J region (IMGT, http: / / www.imgt.org / ) The heavy chain CDR of antibody 12112 was selected as the acceptor. In addition to the CDR, 0, 1, 2, 3, 4, 5, or 6 of the following framework residues (donor residues) from the 12112 VH gene may be retained at positions 24, 48, 49, 71, 73, and 78 (Kabat numbering): valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), and valine (V78), respectively. In some cases, CDRH2 may be mutated to remove a potential N-linked glycosylation site (CDRH2 variants, SEQ ID NOs. X-Y). In some cases, CDRH3 may be mutated to modify a potential aspartate-proline hydrolysis site (CDRH3 variants, SEQ ID NOs. X-Y).

[0341] Antibody 111 The human V region IGKV1-5 + IGKJ4 J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the antibody 12113 light chain CDR. In addition to the CDR, 0, 1, 2, 3, or 4 of the following framework residues (donor residues) from the 12113 VK gene may be retained at positions 1, 2, 3, and 71 (Kabat numbering): alanine (A1), valine (V2), glutamic acid (E3), and tyrosine (Y71), respectively. In some cases, CDRL3 may be mutated to remove an unpaired cysteine ​​residue at position 90 (Kabat numbering) (C90, CDRL3 variant, SEQ ID NOs. X-Y).

[0342] Human V region IGHV3-23+IGHJ2 J region (IMGT, http: / / www.imgt.org / ) It was chosen as the acceptor for the heavy chain CDRs of antibody 12113. In addition to the CDRs, 0, 1, 2, 3, 4, 5, or 6 of the following framework residues (donor residues) from the 12113 VH gene can be retained at positions 48, 49, 71, 73, 78, and 94 (Kabat numbering): isoleucine (I48), glycine (G49), lysine (K71), serine (S73), valine (V78), and arginine (R94), respectively.

[0343] antibody 116 The human V region IGKV1-D13 + IGKJ4 J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the antibody 12117 light chain CDRs. In addition to the CDRs, 0, 1, 2, or 3 of the following framework residues (donor residues) from the 12117 VK gene could be retained at positions 2, 3, and 70 (Kabat numbering): glutamine (Q2), valine (V3), and glutamine (Q70), respectively. In some cases, CDRL1 could be mutated to modify a potential deamidation site (CDRL1 variants, SEQ ID NOS: X-Y). In some cases, CDRL3 could be mutated to eliminate the disulfide bond between cysteine ​​residues at positions 94 and 95d (Kabat numbering) (C94 and C95d, CDRL3 variants, SEQ ID NOS: X-Y).

[0344] Human V region IGHV3-66 + IGHJ4 J region (IMGT, http: / / www.imgt.org / ) The heavy chain CDRs of antibody 12117 were selected as acceptors. In addition to the CDRs, 0, 1, 2, 3, 4, 5, or 6 of the following framework residues (donor residues) from the 12117 VH gene can be retained at positions 24, 48, 49, 71, 73, and 78 (Kabat numbering): valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), and valine (V78), respectively.

[0345] antibody 16 The human V region IGKV1-39+IGKJ1 J region (IMGT, http: / / www.imgt.org / ) was selected as the acceptor for the antibody 11834 light chain CDRs. In addition to the CDRs, 0, 1, 2, 3, or 4 of the following framework residues (donor residues) from the 11834 VK gene can be retained at positions 48, 70, 71, and 85 (Kabat numbering): valine (V48), glutamine (Q70), tyrosine (Y71), and arginine (R85), respectively. In some cases, CDRL2 can be mutated to remove a potential aspartate isomerization site (CDRL2 variant, SEQ ID NO: X).

[0346] Human V region IGHV3-23 + IGHJ4 J region (IMGT, http: / / www.imgt.org / ) The heavy chain CDRs of antibody 11834 were selected as acceptors. In addition to the CDRs, 0, 1, 2, or 3 of the following framework residues (donor residues) from the 11834 VH gene can be retained at positions 44, 49, and 94 (Kabat numbering): arginine (R44), alanine (A49), and arginine (R94), respectively. In some cases, CDRH2 can be mutated to modify two potential asparagine deamidation sites (CDRH2 variants, SEQ ID NOS: X-Y). [Example]

[0347] Example 1 - Improvement of the anti-CD1a panel: Functional evaluation of anti-CD1a antibodies Following CD1a binding evaluation, we screened a large panel of generated anti-CD1a antibodies for inhibitory function. T cell cytokine production was measured by EliSpot in an in vitro antigen-presentation model. Figure 1 summarizes these data. Several newly generated antibodies proved more potent at inhibiting CD1a T cell responses than the commercially available anti-CD1a antibodies OKT6, HI149, and SK9. Notably, antibodies 16, 22, 39, 46, 77, 87, 110, and 116 all had IC50s at least logarithmically lower than OKT6. This represents an improvement over antibodies described in the prior art, despite the use of polyclonal T cells, which are expected to be less sensitive than transduced, clonal, immortalized T cells (Figure 1B).

[0348] Example 2 - Anti-CD1a panel improvement: inhibition of responses of CD1a-restricted enriched T cell lines To help narrow down antibody candidates for in vivo analysis, we took a different approach to assess CD1a T cell responses: we isolated and expanded CD1a-restricted enriched T cell lines to analyze CD1a responses alone, rather than against a mixed polyclonal T cell background where a low signal-to-noise ratio may partially mask potential inhibitory antibodies.

[0349] In these assays, antibodies 116 and 16 stood out as potent inhibitors, with 16 uniquely inhibiting autoreactive / endogenous production of IL-22 (Figures 2A and 2B). This improvement suggests that the antibodies may be useful in conditions where IL-22 plays a pathogenic role, in addition to those where IFNγ plays a role. The differential effects observed for different cytokines were surprising. Furthermore, an APC-free system was used to assess antibody-dependent inhibition of CD1a-restricted T cell activation. CD1a-coated beads were used instead of APCs, and IFNγ production by the resulting T cells was measured by flow cytometry. This assay revealed significant inhibition of CD1a-dependent cytokine responses with all antibodies, particularly 77a, 87, 110, 111, and 116 (Figure 2C).

[0350] Example 3 - In vivo evaluation of inhibitory antibodies in skin inflammation The goal of this study was to generate antibodies for clinical use in the treatment of human diseases and disorders; therefore, it was essential to confirm their efficacy in a complex immune system similar to that of human disease. A highly refined panel of the best newly generated antibodies was selected from the analysis of the above data (antibodies 16, 77a, 110, 111, and 116) to determine their potential in in vivo models of psoriasis, dermatitis, and lupus, and as models of inflammatory skin or mucosal diseases or disorders, or related systemic diseases or disorders, or drug reactions manifesting as one or more inflammatory drug reactions manifested systemically. Experimental psoriasis and dermatitis have been shown to be exacerbated in CD1a transgenic mice compared to wild-type mice, and CD1a-dependent inflammation can be ameliorated by administration of anti-CD1a antibodies (Kim et al., 2016). Also of note, some individuals develop cutaneous / mucosal inflammatory drug reactions to imiquimod, which is used topically for several skin disorders. Such drug reactions include psoriatic reactions, dermatitis reactions, bullous disease, alopecia, blister formation, lichenoid reactions, neutrophilic disease, lupus erythematosus, erythema multiforme, oral erosions, and severe drug reactions such as DRESS, AGEP, Stevens-Johnson syndrome, and toxic epidermal necrolysis (19-29).

[0351] Generation of CD1a transgenic mice To evaluate the possible role of CD1a in skin and associated systemic inflammation, we generated CD1a transgenic mice. CD1a is not present in the mouse genome; therefore, we cloned the human CD1a locus with 0.8 kb of 5'-flanking region and 0.8 kb of 3'-flanking region, including the promoter element, and inserted the transgene by microinjection. This is similar to a published CD1a transgenic model but requires additional transgene fragment stitching (Illing et al., Nature 486, 554-558 (2012)). Genotype-positive founder mice were bred and lines were screened for CD1a transgene expression. We then proceeded to phenotype the mice to determine whether CD1a protein expression followed the expected profile and represented cellular expression of human CD1a. Ear skin from wild-type and CD1a transgenic (CD1aTg) mice was harvested and enzymatically processed to allow analysis of the skin cellular environment by flow cytometry (Figure 3A). CD1a expression was detected in the skin, constituting 4.2% (+ / - 1.79) of total skin cells and 23.6% (+ / - 6.68) of CD45+ cells. To assess cellular regulation of expression, dermal DCs (dDCs) and Langerhans cells (LCs) were assessed for CD1a protein. A subset of dermal DCs has been reported to express CD1a, and Langerhans cells characteristically express constitutive CD1a. 高 CD1a was found to be expressed by 41.5% (±20.38) of dDCs and 88% (±4.606) of LCs (Figures 3A-B). CD1a protein expression in the skin was further characterized by immunofluorescence, revealing cells with a characteristic epidermal location and dendrites typical of LCs (Figure 3C). CD1a genotype was confirmed (Figure 3D), and CD1a expression in the thymus was observed primarily by the percentage of CD4+CD8+ double-positive thymocytes (Figure 3E). CD1a Tg mice did not exhibit abnormal skin inflammation at steady state. In summary, we generated CD1a transgenic mice that exhibit CD1a expression in a manner phenotypically similar to that in human tissues.

[0352] This model was used to test anti-CD1a antibodies for the prevention of inflammatory skin diseases and disorders (Figure 4A). Application of Aldara cream containing 5% imiquimod (a TLR7 / 8 agonist) is an established model that induces psoriasis-, dermatitis-, and lupus-like skin inflammation characterized by skin thickening, scaling, and redness (30, 31). Ear inflammation in CD1a transgenic mice was found to be significantly higher in response to Aldara than in their WT counterparts. Furthermore, while all anti-CD1a antibodies administered prior to imiquimod reduced subsequent ear thickening, antibodies 116 and 16 abolished CD1a-dependent inflammation, at least to WT levels (Figure 4B). By the end of the experiment, CD1a transgenic (-Tg) mice treated with antibodies 16 and 110 showed reduced inflammation, down to WT levels of ear thickening. Surprisingly and unexpectedly, antibody 116 treatment reduced the level of ear skin inflammation in CD1a-Tg individuals, which was significantly lower than that in wild-type individuals (Figure 4B).

[0353] Example 4 - In vivo effect of inhibitory antibodies on skin immune response. We attempted to analyze the contribution of the skin immune population to imiquimod-induced CD1a-dependent otoitis.

[0354] We found that cutaneous T cell infiltration was elevated in CD1a transgenic mice, and the frequency of this population was reduced by anti-CD1a antibodies, particularly antibodies 116, 16, and 110, in a preventive model (Figure 5A). Notably, 16 and 116 were able to reduce cutaneous T cell infiltration to levels lower than those of wild-type mice. This suggested a significant improved effect on inflammation in vivo. Furthermore, the activation marker CD69 was increased on the surface of cutaneous T cells in CD1a transgenic mice and was inhibited by some anti-CD1a antibodies, particularly 116 and 16, in a preventive model (Figure 5B). Neutrophils are known to be key cells in several inflammatory disorders, including the psoriatic response and the murine imiquimod model. Here, we found that neutrophil frequency in the skin was elevated upon imiquimod treatment and further increased in CD1a transgenic mice, which was reduced to below WT levels using anti-CD1a antibodies 116 and 16 in a preventive model (Figure 5C). Furthermore, a decrease in cutaneous eosinophils in response to the antibody was observed. This is interesting considering the known role of eosinophils in many forms of drug responsiveness (Figure 5D). This unexpected finding represents an improvement, as effects on eosinophils had not been observed previously.

[0355] Langerhans cells (defined herein as CD11c+ Langerin+) also increased in the skin of CD1a transgenic mice upon imiquimod loading compared to wild-type mice, as observed in human skin inflammatory disorders. Administration of antibodies 16, 116, 111, and, though not statistically significant, 110 reduced the number of skin LCs in the prophylactic model (Figure 6A). In particular, antibody 116 reduced the number of skin LCs more significantly than in wild-type skin, showing a remarkable level of improvement. The effect of antibodies on CD1a expression in LCs was evaluated as the primary CD1a-expressing population. Notably, staining was reduced with antibodies 110 and 116, which was due to interference of the HI149 detection antibody on the binding of antibodies 110 / 116 (Figure 6B). This indicates that the antibodies bind persistently in vivo, which is a remarkable effect and is associated with therapeutic benefits. This finding also increases the potential for the use of antibodies for diagnostic or prognostic purposes, or for monitoring CD1a-expressing cells before and during treatment. This observation was not seen with non-competitive SK9 detection antibodies, as shown below. The observed decrease in LCs may be due to antibody-dependent LC death or migration or phenotypic changes. Therefore, cervical lymph nodes were analyzed for the presence of CD11c+Langerin+LCs. Compared to WT mice, an increase in the number of LCs in the lymph nodes of CD1a transgenic mice was found, but migration to the LN did not appear to explain the decrease in cutaneous LCs in mice treated with antibodies 110 and 116 (Figure 6C). In particular, antibody 116 resulted in an immunological improvement similar to that in wild-type skin, showing a surprisingly high level of improvement. Interestingly, the expression level of CD1a on lymph node-derived LCs followed a similar pattern to that of skin LCs, in that LC staining was reduced. This was attributed to the interference of antibodies 110 / 116 with binding by the HI149 detection antibody, as further explained below (Figure 6D). This was not seen with non-competitive SK9 detection antibodies. Notably, lymph node-derived LCs express less CD1a per cell than those from the skin, which may be a regulatory mechanism to prevent systemic inflammation.Thus, the effect of antibodies against LC in vivo is maintained in the skin and after migration to lymph nodes, an important enhancement because it leads to a longer-lasting clinical effect.

[0356] Example 5 - Anti-CD1a antibodies exhibit cytotoxicity in terms of their effect on the phenotype of CD1a-expressing cells Given that enhanced migration alone did not fully explain the reduction in skin LCs, we investigated the possibility of antibody-induced changes in the phenotype of CD1a+ cells, despite their murine IgG1 nature.

[0357] All anti-CD1a antibodies, particularly 110 and 116, were able to reduce the number of CD1a K562 cells lacking MHC class I and II in vitro, demonstrating comparable responses (Figure 7A). More detailed testing of antibodies 110 and 116 demonstrated a dose-dependent reduction (Figure 7B). This was an improvement and surprising given their murine IgG1 isotype. This was distinct from the published CR2113 antibody (16, 18) (U.S. Patent No. 10,844,118 (B2) and Canadian Patent Application Publication No. 2,924,882 (A1)), which is believed to require complement and / or antibody-dependent cellular cytotoxicity. Specifically, it has been stated that "CR2113 does not directly induce apoptosis" (17), and it has been noted that NA1 / 34 does not induce direct killing. However, because different Fc regions affect effector function, the comparative effects of CR2113 on a murine IgG1 background will be discussed immediately below. We sought to evaluate the ability of antibodies to induce direct depletion of primary human CD1a-expressing cells. DC- and LC-like cells were generated through 5-day in vitro differentiation of monocytes using the cytokines IL-4 / GM-CSF and IL-4 / GM-CSF / TGF-β, respectively, and anti-CD1a antibodies were added on day 0 or day 2 of culture. We observed that antibodies 110 and 116 depleted LCs in vitro and, to a lesser extent, DCs (Figure 7C, upper and lower panels, respectively). In exploring the mechanism underlying this depletion, we found that this depletion was associated with a culture phenotype of pronounced cell clustering (Figure 7D). While the reduction in numbers may be partially explained by this clustering, we further tested whether the antibodies could induce apoptosis of CD1a-expressing target cells and compared them to CR2113 (in a murine IgG1 background). Figure 7E shows that 110 and 116 (but not 16), as well as CR2113 (in a mouse IgG1 background), induce annexin V expression by CD1a-expressing K562 cells even in the absence of complement or ADCC, suggesting that 110, 116, and CR2113 antibodies may mediate K562 cell death to some extent.To investigate the role of complement-mediated lysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC), K562-CD1a was incubated with complement (Figure 7F) and / or human PBMCs (Figure 7G). Despite the mouse IgG1 nature of the antibody, there was evidence of complement-mediated lysis and ADCC. The effects of antibodies against the human IgG1 Fc region are investigated below. To further explore the in vivo mechanism, a new model was established using K562-CD1a subcutaneous tumors in an immunodeficient NSG model, which has a widespread lack of lymphocyte responses and other effects. Data showed that all three antibodies reduced lymphoid cell tumor size by day 10 (a 25% or greater reduction in CD1a-expressing tumor cell volume), an effect that persisted through days 15–20 for 16 and 116, but was lost for CR2113 (Figure 7H). The differences between in vitro and in vivo responses may be explained by other cofactors present in vivo, such as complement, numerous innate cell subsets with FcRs with different Fc specificities, differential target cell density, reduced in vivo antibody half-life, and altered tissue access. Such direct changes in the phenotype of CD1a-expressing target cells may promote a less inflammatory response of CD1a-expressing cells. Thus, the reduction in LCs in the skin of CD1a-Tg mice treated with 110 and 116 may be partially explained by direct, antibody-dependent changes in the phenotype of CD1a+LCs, contributing to clinical efficacy, e.g., 116 reducing inflammation to a lower level than wild-type mice. This data also suggests that the antibody may be useful in treating Langerhans cell histiocytosis and CD1a-expressing malignancies, including some forms of T-cell lymphoma and some forms of thymoma. However, the CD1a-bead assay (Fig. 2C) was not affected by any depletion effect, so a change in target cell phenotype cannot explain the reduced T cell functional response shown in Fig. 2 .

[0358] Example 6 - Epitope binding analysis of CD1a antibody The data presented herein demonstrate that the five newly generated anti-CD1a antibodies have diverse functionalities, and we sought to determine whether the antibodies have overlapping binding sites using flow cytometry cross-blocking assays. Furthermore, we assessed epitope overlap using commercially available antibodies OKT6, HI149, SK9, and NA1 / 34 (which, as noted above, have binding sites known to overlap with CR2113).

[0359] CD1a-K562 cells were incubated with purified primary anti-CD1a antibodies (Y-axis, Figure 8A, 25 μg / ml), followed by washing away unbound antibodies. Different antibodies in Alexa-Fluor-647 conjugated form were then incubated with the cells in the matrix configuration shown in Figure 8A (X-axis, 10 μg / ml). Mean fluorescence intensity (MFI) was used to assess the extent of binding of the fluorophore-conjugated antibodies. Any steric hindrance caused by binding of the primary purified antibodies would be represented by a decrease in MFI. The results indicated that antibodies HI149, OKT6, 110, and 116 may have overlapping or closely related epitopes, and that a second group, including antibodies NA1 / 34, 77a, 111, and 16, may have closely related binding sites. This suggests that the observed decrease in CD1a expression in vivo (Figures 6B and D) was due to interference of the 110 / 116 antibodies with binding by the HI / 149 detection antibody. Indeed, this effect was not seen with the non-competing SK9 detection antibody (Figure 8B). Importantly and unexpectedly, the antibodies thus maintained their presence on LCs even after in vivo migration to lymph nodes in the skin and after enzymatic digestion of skin tissue. This is likely to be associated with substantial longer-term clinical benefit. Because the antibodies are classified into two major non-competing groups, Figure 8 (A and B) shows that combinations of antibody members selected from each group can be used together, for example, for treatment / monitoring or combination therapy. One such combination is 116 and 16.

[0360] Example 7 - Demonstration of the efficacy of the antibody of the present invention for the treatment of imiquimod-induced inflammation and systemic related inflammation. Although the existence of circulating CD1a-reactive T cells has been demonstrated (11), given the skin-dominant expression of CD1a, most studies have focused on skin-specific functional effects. The role of CD1a in inflammation in tissues other than the skin has not been extensively investigated. Furthermore, although CD1a is known to amplify imiquimod cutaneous responses (16), no studies have been conducted on associated systemic sequelae. We generated novel CD1a transgenic mice and CD1a-reactive T cells and characterized anti-CD1a antibodies for in vitro and in vivo functionality using human and mouse assays, respectively. This finding confirms that CD1a-dependent effects extend to systemic effects and has implications for the treatment of systemic involvement in skin diseases, including adverse inflammatory drug responses.

[0361] Therapeutic potential of anti-CD1a antibodies To further evaluate the therapeutic potential of the newly generated anti-CD1a antibodies, we tested the three most clinically effective antibodies, 16, 110, and 116, in an imiquimod treatment model, in which the anti-CD1a antibodies were introduced after imiquimod-induced inflammation was established (Figure 9A). All three antibodies rapidly improved the clinical response after initiation, even during imiquimod administration (Figures 9B-C). This response was most pronounced for 116, which reduced ear thickness (Figure 9B). Thickening of the whole skin (upper panel) and epidermis (lower panel) was visualized by confocal microscopy (Figure 9D), confirming the micrometer assessment (Figure 9B). CD1a protein expression was assessed in the epidermis of CD1a transgenic mice (anti-CD1a OKT6 AF-594, red). Decreased expression due to cell death and epitope competition was observed in skin treated with 110 and 116 (Figures 8A and 9D). Analysis of skin cellular immune responses according to the imiquimod treatment model revealed a decrease in the number and activation of cutaneous T cells, a decrease in cutaneous LCs, and a decrease in cutaneous neutrophils after antibody administration (Figures 9E-G).

[0362] CD1a is involved in the systemic immune response to imiquimod. The effects of imiquimod treatment in humans may extend beyond the skin, and in mouse models, it has been shown to induce splenomegaly. We evaluated the contribution of CD1a to this pathway. Surprisingly, imiquimod-treated CD1a Tg mice had increased spleen weight compared to wild-type mice, and antibody treatment reduced both spleen size and weight. This was consistent with the systemic effects beyond the skin (Figure 10A). Furthermore, the antibody reduced CD4 and CD8 T cell activation, as determined by CD69 expression (116 and 110, Figures 10B-C), splenic neutrophils (no significant trend), and eosinophil frequencies (16, 110, 116) (Figures 10D and 10E, respectively). Plasma cytokine levels were assessed on day 8. Significant increases in IL-23, IL-12p70, IL-1β, IL-1α, and MCP-1 were observed in imiquimod-treated CD1a transgenic mice, and decreased in some or all of the 16, 110, and 116-treated groups (Figure 10F). Plasma immunomodulatory cytokines IL-10 and IL-27 increased (and others also tended to increase) in the presence of antibodies 16 and 116, respectively. Next, the effects on circulating immune cells were examined. As with the spleen, serum CD4 and CD8 T cell counts, neutrophilia, and eosinophilia increased in the imiquimod-treated CD1a transgenic group. This increase was significantly blocked after treatment with 16, 110, or 116 (Figures 11A-E). Finally, the inventors investigated whether imiquimod itself could be a CD1a ligand and showed that this was not the case, suggesting broader autoimmune and autoinflammatory effects of the CD1a pathway (Figure 12). Therefore, it may be suggested that a wide range of systemic inflammatory immune responses are primed or influenced by CD1a in the skin.

[0363] To investigate whether anti-CD1a antibodies could produce a sustained reset of skin inflammation after imiquimod application, we attempted the model shown in Figure 13A, in which imiquimod rechallenge was used in the absence of anti-CD1a antibody rechallenge (Figure 13B). Surprisingly, 16, 110, and 116 all produced sustained improvements in ear thickness in the absence of repeated antibody administration, consistent with sustained immunological effects. The immunological responses were also sustained, with significant improvements in skin T cell frequency (110, 116), skin T cell activation (16, 110, 116), skin eosinophils (116), and skin neutrophils (16, 110, 116), lymph node T cell frequency (110, 116), lymph node T cell activation (16, 116), lymph node Langerhans cells (116), and lymph node eosinophils (11). 6), lymph node neutrophils (116), blood T cell frequency (110, 116), blood T cell activation (116), blood eosinophils (110, 116), plasma IL-1α (116), IFNγ (16, 110, 116), IL-1β (16, 110, 116), IL-6 (16, 116), and IL-17A (16, 110, 116) were significantly reduced.

[0364] To compare the performance of antibodies in managing moderate-to-severe psoriasis with the current standard of care, the imiquimod treatment model (Figure 9A) was repeated with anti-IL-17A (IgG1 isotype), administered at the same time and dose (100 μg) as the anti-CD1a antibody (Figure 14). All anti-CD1a antibodies again demonstrated significant improvement in ear thickness outcomes, and all produced significant improvement earlier than anti-IL-17A. In contrast to the different anti-CD1a antibodies, it was noted that anti-IL-17A did not significantly reduce the frequencies of cutaneous T cells, cutaneous Langerhans cells, cutaneous eosinophils, lymph node T cells, lymph node neutrophils, lymph node eosinophils, plasma IL-23, MCP-1, or IL-6.

[0365] To directly compare cutaneous and systemic inflammatory outcomes between the antibodies described herein and CR2113, an imiquimod skin treatment model was attempted (Figure 15A). All anti-CD1a antibodies had a beneficial effect on ear thickness, but antibody 116 demonstrated a significant improvement over CR2113 (Figures 15B-C). To extend the investigation of the improvement of anti-CD1a antibodies 16, 110, and 116 relative to CR2113, a comparison was performed in an additional model of cutaneous inflammation (i.e., MC903-induced inflammation) (Figure 15D). Significant benefits were observed for antibodies 16, 110, and 116, thus demonstrating improvement, but not for CR2113 (Figure 15E). It was noted that 16 and 116 demonstrated significant reductions in the percentage of cutaneous T cells and skin eosinophil counts, whereas CR2113 did not (Figure 15F). Skin-extracted cytokines were significantly reduced for IL-5(16, 110, 116), IL-6(16, 110, 116), IL-9(16), IL-23(116), and IL-17F(16, 110, 116), but CR2113 did not show a significant reduction.

[0366] It was further observed that antibody 116 demonstrated consistent improvement over CR2113 in reducing the inflammatory response to imiquimod in the skin, lymph nodes, and plasma (Figure 16). For several outcomes, antibody 16 was also significantly improved over CR2113 (Figure 16). Specifically, antibody 116 demonstrated improvement over CR2113 in reducing IL-17A expression by skin T cells and lymph node eosinophil influx frequency. 116 also demonstrated improvement over CR2113 in reducing plasma IFNγ, IL-1α, IL-1β, IL-5, IL-9, IL-17A, IL-17F, and IL-22, as well as skin digestive IL-1α, IL-22, and TNFα. 16 was better than CR2113 in reducing lymph node eosinophils, plasma IL-1β, IL-22, IL-9, and IL-5, and skin digesta IL-1α (with a strong trend for IL-17A). Overall, the data confirm that the antibodies described herein can inhibit cutaneous and systemic inflammatory responses to imiquimod and MC903.

[0367] Example 8 - Anti-CD1a crystal structure The crystal structures of CD1a bound to single-chain variable constructs of the OX16, OX110, and OX116 antibodies were solved at 3.3 Å, 3.5 Å, and 2.7 Å resolution, respectively ( FIG. 17 ). Electron density maps of the CD1a, β2m, and scFv chains were of excellent quality, allowing for detailed molecular analysis of the interactions. Molecular details of the complexes are as follows: OX16-CD1a: OX16 binds directly to CD1a, and OX16 extends across the entire A' roof, docking to both the α1 and α2 helices. The total buried surface area (BSA) of the interface is 1528 Å (781 Å for OX16 and 747 Å for CD1a). CD1a residues contributing to the interaction are Glu62, Glu65, Leu66, Thr68, Leu69, Ile72 on the α1 helix, and Asn151, His153, Glu154, Ile157, Asn160, Asp164, Thr165, and Arg168 on the α2 helix. The heavy chain provides 70% of the interaction, and 30% corresponds to the light chain of the antibody. The antibody variable loops involved in the interaction are: heavy chain: H1 (Tyr34) H3 (Arg100-Trp106; Arg100, Tyr103, Tyr104, Tyr106); light chain: L1 (Tyr169), L2 (Tyr186), L3 (Tyr229, Trp233). The CDR3 loop of the heavy chain is central to the interaction, as it comprises 60% of the total buried surface area.

[0368] The blocking ability of OX16 appears evident given that its epitope largely overlaps with that of self-reactive TCRBK6, the only αβTCR with a known crystalline structure that binds to CD1a. Most of the CD1a residues recognized by BK6 overlap with those central to the OX16-CD1a interaction (Glu62, Glu65, Ile157, Asn160, Asp164, Thr165, Arg168 (Birkinshaw et al., Nature Immunology 2015)), and therefore, OX16 binding is incompatible with BK6. Although Ab does not occlude the F' portal, the L1 loop is located almost directly above it, leaving only a limited amount of space for the protruding head group. This prompted SPR experiments to investigate whether the size and "bulkiness" of the head group may influence CD1a recognition by OX16.

[0369] OX110-CD1a. The OX110 antibody binds to CD1a at the end of the α1 domain of CD1a, immediately adjacent to the F' pocket, reminiscent of the binding shown by the recently published γδ T cell receptor CO3 (Wegrecki et al, Nat comm 2022). In this crystal structure, four CD1a-antibody complexes are observed asymmetrically, and surprisingly, while there are only slight differences between them in terms of the interacting side chains, the overall docking pattern remains nearly identical. For example, the CD1a loop Tyr19-Trp23 can take on various conformations, with two copies of the complex interacting with the antibody, while the other two do not. This indicates that there is some flexibility in the recognition of CD1a by OX110, and therefore demonstrates that the interaction is very robust. From a functional standpoint, this may be important. This is because, even if CD1a undergoes slight conformational changes upon binding to specific lipid ligands on the cell surface, these changes are unlikely to affect OX110's recognition of CD1a. To clarify, the analysis focused on the complex with the best electron density map within the asymmetric unit. The total buried area at complex formation was 1404 Å, of which 680 Å corresponds to OX110 Å and 724 Å corresponds to CD1a. The residues from the α1 helix of CD1a that come into contact with the antibody are Glu79, Arg82, Arg83, His86, Glu87, Gln89, Phe90, Glu91, Tyr92, and Val147 and Asn150 from the α2 domain. His86 is considered central to this interaction as it establishes H bonds and salt bridges with three residues from the heavy chain of OX110. This explains why the point mutant CD1a[H86A] completely abolishes binding of OX110 to CD1a, as seen in epitope mapping experiments: antibody contributions are 68% and 32% for the heavy and light chains, respectively.The variable loops that interact with CD1a are H1 (Ser31, Ser32), H2 (Asn53, Ser54, Ser55), H3 (Asp97, Tyr99, Tyr101, Tyr103, Gly104, Trp105), L1 (Phe165, Asn166), and L3 (Glu228, Phe229, Ser230, Cys231). Most of the antibody contribution comes from the H3 loop, which again provides 30% of the total buried surface area. Interestingly, L3 contains an intraloop disulfide bond between Cys231 and Cys236, which is common in single-chain antibodies to stabilize the long CDR3 loop. Here, L3 plays no apparent role because it only contributes slightly to the interaction interface.

[0370] OX116-CD1a. The OX116 antibody also binds to the lateral side of CD1a, next to the F' pocket. The epitopes partially overlap with those of OX110, but OX116 spans both the α1 and α2 domains of CD1a. The embedded region of the assembly is 1526 Å (CD1a provides 793 Å and OX116 provides 733 Å). CD1a residues that interact with the antibody include Arg83, Tyr84, His86, Glu87, Gln89, Phe90, Glu91 on the α1 domain, and Asn139, Met140, Lys142, His143, Lys146, Val147, Gln150 on the α2 domain. In this case, His86 contacts the antibody, but these contacts do not involve H-bonding and consist only of weak van der Waals contact, which explains why the CD1a[H86A] mutant did not affect the interaction in epitope binding experiments. The OX116 residues involved in complex formation belong to H1 (Ser31, Asn32, Ala34), H2 (Tyr53, Thr54, Thr55, Gly56, Phe57, Tyr59), H3 (Ala99, Thr100, Tyr101, Val102, Pro104), L1 (Tyr166, Asn167), and L3 (Glu229, Phe230, Ser231, Cys232). As with the other two complexes, here the VH domain constitutes 75% of the assembly interface, and the VL domain provides the remaining 25%. However, the H3 loop governing the interaction in OX16-CD1a and OX110-CD1a provides only 25% of the total interaction area here. Surprisingly, 35% of the BSA originates from the germline-encoded H2 loop, which has only a small contribution (16% of the BSA) in OX110-CD1a and no contribution in OX16-CD1a. Here again, an intraloop disulfide bond is present within L3. In fact, the sequence of loop L3 is nearly identical between OX110 (GEFSCSSTDCVTF) and OX116 (GEFSCSSVDCATF), and in each case, the same residue from L3 is in contact with the same segment (Gln89) of CD1a, but the angle of interaction is different, and the heavy chain docks to different epitopes.

[0371] Although both OX110 and OX116 bind to the F' pocket side of CD1a, their binding modes differ. OX110 binding around His86 induces a conformational change in that portion of the α1 helix, which adopts a different conformation from any other structure of CD1a. Furthermore, it affects the amino-terminal portion of the α1 helix in the A' roof region, slightly altering how the α1 and α2 helices interact to form the A' roof. A similar effect was not observed upon binding of OX116 to the α1-α2 interface on the F' side of the cleft. In summary, the shape of the CD1a binding cleft appears unaffected by association with OX16 or OX116, but OX110 binding affects its association with the α1 and α2 helices.

[0372] Example 9 - Effect of lipids on the binding of OX16 and OX116 to CD1a CD1a cells were loaded with different lipids known to be permissive (endogenous "endo," lysophosphatidylcholine 18:1 (LPC)) or nonpermissive (sphingomyelin 24:1 (SM24:1)) or with a large headgroup lipid control (ganglioside GD3). The lipid-loaded CD1a cells were then tested for binding of the OX16 (Figure 18A) and OX116 (Figure 18B) antibodies using surface plasmon resonance. OX16 bound to CD1a, including all lipids, with some enhanced binding to permissive ligands, consistent with a degree of selectivity for lipids that may promote autoreactive T cell responses. Lipid antigens (sphingomyelin, GD3) that protrude significantly through the F' portal appear to negatively affect recognition by OX16. As described in Example 8, this may be explained by the OX16 overhang on the F' portal, which may limit binding to nonpermissive lipids with large protruding headgroup antigens. Thus, binding of OX116 to CD1a precisely follows the molecular pattern previously described for the autoreactive αβ T cell receptor. OX116 exhibited binding to all lipids tested, including endogenous lipid ("endo"), SM24:1, LPC, GD3, sulfatide, and phosphatidylcholine, without preferential binding to known permissive or nonpermissive ligand classes. This was unexpected, given the proximity of OX116 to the F' portal. Furthermore, we demonstrate that recognition of CD1a by OX116 is robust and unlikely to be affected by the identity of the antigenic lipid present in the CD1a cleft at the cellular level. Overall, these data demonstrate that OX16 and OX116 can bind to a wide range of lipid-loaded CD1a.

[0373] Example 10 - Effect of blocking polyclonal and clonal T cell function with anti-CD1a antibodies K562 cells expressing CD1a or an empty vector control (EV) were incubated overnight with different anti-CD1a antibodies and polyclonal T cells isolated from healthy adult donors. The number of cells expressing IFNg or IL-22 was measured using ELISpot, and percentage inhibition was compared to isotype controls (Figures 19A-19B). Wild-type human IgG1 Fc showed a significant decrease in IL-22 production with all antibodies, but decreased IFNg production only with antibodies OX16, OX110, and OX116. Afucosylated IgG1 showed a significant decrease in IL-22 with OX16, OX110, OX116, CR2113, and mAb571. Afucosylated IgG1 showed increased IFNg production with OX16, OX110, CR2113, and mAb571, and the known enhanced Fc effect of afucosylated IgG1 This was consistent with the effector function. Notably, despite the enhancement of the effector function of afucosylated IgG1, antibody OX116 did not show a significant increase in IFNg induction. Possible mechanisms were investigated below. Overall, these data demonstrate that the Fab versions of the antibodies inhibit polyclonal CD1a-dependent T cell reactivity, and provide evidence that OX16, OX110, and OX116 are improved compared to CR2113 and mAb571. These were highlighted by using comparison targets for different IgG1 Fc regions. Next, the ability of antibodies to modulate IFNg production by CD1a-responsive T cell clones was investigated (Figure 19C). All antibodies, whether on the wild-type human IgG1 Fc region or as Fab variants, showed the ability to inhibit IFNg production by CD1a-responsive T cell clones.

[0374] Example 11 - CDC and ADCC effects of anti-CD1a antibody Given the findings in Figure 7 using mouse Fc regions, we investigated the ability of antibodies on different human Fc backgrounds to induce complement-mediated cytotoxicity compared with CR2113 and mAb571 (Figure 20A). While OX16 and OX110 did not induce CDC, OX116 induced significant killing in the presence of complement, even when placed in a human IgG1 background of all variants. However, when the Fab version of OX116 was used, CDC was not observed, suggesting an Fc-dependent effect. Antibody OX116, when placed in a different IgG1 Fc region, showed improvement over the published antibodies CR2113 and mAb571, relevant for the use of antibodies for specific indications, such as when cytotoxicity of CD1a-expressing cells may benefit patients, such as in the setting of CD1a-expressing malignancies. Next, anti-CD1a antibodies on different human Fc backgrounds were tested for their ability to induce antibody-dependent cellular cytotoxicity (ADCC). For all anti-CD1a antibodies, human IgG1 and α-fucosylated IgG1 showed evidence of ADCC of CD1a-expressing target cells (Figure 20B). No significant ADCC was observed for the Fab versions of the antibodies. Notably, ADCC could not solely explain the findings in Figure 19 because, in the latter, the effector population consisted of T cells, not NK cells, and a different effector:target ratio was used. Furthermore, the inhibitory response in Figure 19 was observed using the Fab form of the antibody. Thus, anti-CD1a antibodies exhibit T cell-blocking function, and some Fc forms of the antibodies exhibit ADCC. Furthermore, as summarized above, OX116 can also induce direct killing of CD1a-expressing cells.

[0375] Example 12 - Inhibition of TCR binding to CD1a by OX116 We next investigated whether OX116 could inhibit the binding of known CD1a-reactive TCRs. The biotinylated ScFv of OX116 was captured on a streptavidin chip (Figure 21A), followed by injection of CD1a and three different TCRs (CO22, CO3, and BK6) (Birkinshaw RW et al., Nat Immunol. 16:258-66 (2015); Wegrecki M et al., Nat Commun. 13:3872 (2022)). The αβ TCR of BK6 binds to the A' roof of CD1a, while the γδ TCR of CO3 recognizes the α1 domain of CD1a within a region overlapping with the epitopes of the OX110 and OX116 antibodies. The CO22 binding site has not been established, but is independent of the A' roof and instead requires the α3 domain of CD1a. As expected, the TCR of CO22 bound to the CD1a-OX116 complex (Figure 21B, green curve). To explore whether OX116 binding to the lateral surface of CD1a might have an indirect, distal effect on the shape of the A' roof of CD1a and its recognition by autoreactive TCRs, we used the TCR of BK6. However, in this case, binding was still detectable (Figure 21B, blue curve). This suggests that OX116 did not interfere with A' roof recognition. We used scFv fragments in the SPR experiments. Therefore, binding of full-length antibodies may result in more significant steric hindrance, with an inhibitory effect on autoreactive TCRs. As expected, the interaction of the TCR of CO3 with CD1a, which is known to bind near the F' portal, was completely abolished by OX116 (Figure 21B, red curve), confirming that TCR and antibody binding are mutually exclusive due to the proximity of epitopes on the surface of CD1a. Overall, these data indicate that OX116 may inhibit TCR engagement with CD1a.

[0376] Example 13 - Generation of the OX25 antibody that binds to the alpha3 domain of CD1a Having established that the footprints of the OX16, OX110, and OX116 anti-CD1a antibodies are directed against the alpha1 and alpha2 domains, we sought to discover an antibody directed against the alpha3 domain. Ab25 was generated and selected according to the Materials and Methods section under "Generation and Selection of Therapeutic Anti-CD1a Antibodies" (Figure 22).

[0377] Example 14 - Humanized antibodies can deplete CD1a expression transfectants and inhibit CD1a autoreactive T cells. Humanized variants of the antibodies were generated (Ab1-51) and tested for binding to K562-CD1a transfectants. Ab1-15 was derived from OX116, and Ab16-51 was derived from OX16. All 51 variant antibodies showed evidence of binding to CD1a expressed by the transfectants. However, it was noted that the mean fluorescence intensity (MFI) varied, and the top antibodies were advanced for functional analysis. Figure 23 shows depletion of K562-CD1a transfectants in the presence of anti-CD1a antibodies. All humanized antibodies derived from OX116 (1, 2, 3, 4, 5) retained depletion ability, and none of the humanized antibodies derived from OX16 (16, 17, 21, 22, 28, 31, 34, 36, 38, 39, 41, 42, 46, 47, 48, 51) possessed intrinsic depletion activity, except for variants 28 and 51, which showed significant depletion ability. The variants were then tested for their ability to block CD1a-reactive T cell clones (Figure 24). Most showed a significant ability to block IFNg production, with antibody 51 exhibiting the most potent performance. The humanized antibodies may have direct diagnostic / monitoring / therapeutic utility or may be included as part of other approaches, including bispecific or multispecific molecules, or as part of cellular therapeutics.

[0378] Example 15 - Antibody 25 Activity The antibody OX25 has been established to bind to the alpha-3 domain of CD1a (Figure 22), and its ability to block CD1a-reactive T cells was then tested (Figure 25). This confirmed that IFNg-induced polyclonal CD1a autoreactive T cell production was not inhibited by OX25, as expected. Furthermore, OX25 was found to compete with SK9 for CD1a binding but not with antibodies that bind to the alpha-1 and alpha-2 domains of CD1a. These data confirm the membrane-proximal binding site of OX25, which may offer diagnostic, monitoring, and / or therapeutic utility when non-competitive membrane-proximal domains are advantageous, as observed with certain checkpoint agonists.

[0379] Example 16 - CD1a antibodies can treat skin inflammation While checkpoint inhibitors are increasingly used to manage malignancies, their use can be limited by side effects, including various inflammatory skin reactions such as psoriasis-like inflammation. This can sometimes necessitate discontinuation of checkpoint inhibitors, resulting in the management of the underlying malignancy, or the use of broad immunosuppressants, which also have potential effects on the immunological control of the underlying malignancy. Approaches to treat tissue inflammation that enable the continued use of checkpoint inhibitors would have therapeutic utility. In an imiquimod model of skin inflammation, we investigated whether the CD1a and Langerhans cell pathways could be involved by using OX116 and anti-PD-1 antibodies alone or in combination. Figure 26A shows a schematic diagram of an approach that established a significant reduction in ear thickness (Figure 26B) and a decrease in the percentage of cutaneous IL-17A+ T cells (Figure 26C) in PD-1-related inflammation in the presence of OX116. The degree of reduction in ear thickness was surprising and dramatic, revealing the involvement of these pathways.

[0380] Example 17 - CD1a antibodies can treat pruritus A major impact of inflammatory skin diseases and other conditions on quality of life is pruritus. CD1a has not previously been associated with pruritus, and therefore establishing the pathway's relevance would represent a new area of ​​biology. Next, the role of CD1a and Langerhans cells in pruritus was evaluated using the MC903 model of skin inflammation. Figure 27A shows that both OX116 and OX16 significantly reduced pruritus, accompanied by a reduction in alarmin cytokines known to play a role in pruritus (Figure 27B). These data are surprising, novel, and inventive, as the CD1a pathway has not previously been implicated in pruritus. This finding would support the use of anti-CD1a antibodies in the prevention and treatment of pruritus and related disorders.

[0381] Example 18 - The efficacy of the CD1a antibody can be enhanced by coupling it with other antibodies. Anti-CD1a antibodies can serve as an approach for targeting bispecific modalities involving T cell engagement. OX16 was coupled to a humanized version of UCHT-1 (anti-CD3 (Shalaby MY, Journal Experimental Medicine 1992)) and tested for its ability to activate reporter T cells when cocultured with CD1a-expressing transfectants. Table B shows the sequence of WIMM-3 utilized. Figure 28A (left panel) shows that the WIMM-3 T cell engager correctly activates reporter T cells in vitro. Figure 28A (right panel) shows that WIMM3 promotes effective CD8+ T cell-mediated cytotoxicity of CD1a-expressing target cells. Figure 28B shows this is also reflected in an immunodeficient xenograft model in which CD1a-expressing target cells were administered together with human CD8+ T cells. The WIMM-3 CD1a-T cell engager effectively inhibited tumor growth, which was associated with a significant improvement in survival. These data demonstrate that anti-CD1a antibodies can effectively target bispecific molecules to CD1a-expressing cells, which has many potential applications, including the treatment of CD1a-expressing malignancies, as well as the principle of targeting other functional or binding modes to CD1a-expressing cells.

[0382] Consideration Skin inflammation, such as dermatitis, psoriasis, and lupus, is a common disorder with significant associated physical and psychological morbidity. Adverse cutaneous reactions to drugs are also common, with incidence rates ranging from 1.8 to 7 per 1,000 hospitalized patients. Severe adverse cutaneous reactions with widespread and systemic effects, such as SJS / TEN, AGEP, and DRESS, are less common. For example, SJS / TEN has an incidence rate of approximately 1 to 6 cases per million individuals per year (M. Mockenhaupt, Allergol Select 1, 96-108 (2017)). In the 1960s, Gell and Coombs defined a classification of hypersensitivity disorders, in which delayed type IV hypersensitivity requires the role of effector T cells (R.R.A. Coombs, Gell, P.G.H., Classification of allergic reactions responsible for drug hypersensitivity reactions. In Clinical Aspects of Immunology. (Davis, Philadelphia, ed. second, 1968)). There is growing recognition that this classification cannot explain all aspects of drug hypersensitivity, but the primary focus remains on altered recognition of covalently bound haptens or non-covalently modified peptides / MHC molecules. However, current models cannot explain the predominance of skin and mucosal involvement in drug hypersensitivity (M. Mockenhaupt, Allergol Select 1, 96-108 (2017)).

[0383] Through the generation of CD1a transgenic mice and autoreactive human CD1a-restricted enriched T cell lines, and the characterization of functional anti-CD1a antibodies, the data presented herein demonstrate the induction of CD1a presentation by endogenous lipid ligands. This results in autoreactive T cell-mediated cutaneous and systemic inflammation. Anti-CD1a antibodies had clinical and immunological effects whether they blocked or blocked / modulated CD1a. This suggests that CD1a lipid presentation to T cells is important. It is interesting that TLR7 can recognize single-stranded RNA, and therefore its reactivity to viral infections may mimic the clinical phenotypes of various severe forms of cutaneous inflammation, including psoriasis, dermatitis, lupus, and adverse inflammatory responses to drugs, including SJS and TEN. Such shared, ultimate common clinical symptoms may indicate that several triggers can promote CD1a autoreactivity and autoinflammation. This model may also help explain the increased risk of autoimmunity associated with certain drug responses, including lupus erythematosus and DRESS syndrome. Furthermore, this finding suggests CD1a autoreactivity in the disruption of broader T cell tolerance.

[0384] In addition to the effects on T-cell responses to the imiquimod-containing drug Aldara, increased neutrophil and eosinophil responses in the skin, aspiration area lymph nodes, and spleen were observed in CD1a transgenic mice. These effects were inhibited by administration of the antibodies of the present invention, particularly 16, 110, and 116. This suggests a CD1a-dependent immune cascade that is more extensive than initially anticipated. Neutrophil depletion has been shown to improve the severity of imiquimod-induced inflammation (H. Sumida et al., Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod-induced psoriasis. J Immunol 192, 4361-4369 (2014)).

[0385] Application of Aldara / imiquimod mimics key aspects of various forms of skin inflammation and related systemic diseases and disorders, including psoriasis, dermatitis, lupus, and severe cutaneous hypersensitivity reactions involving T cell and neutrophil infiltration, as discussed above. The data demonstrated herein show that imiquimod-dependent eosinophil infiltration of the skin, lymph nodes, and spleen was enhanced in CD1a transgenic mice and reduced by administration of the antibodies of the invention, particularly 16, 110, and 116.

[0386] Furthermore, LC counts have been reported to increase in lesional skin compared to non-lesional skin in patients with various forms of inflammatory skin diseases or disorders, including psoriasis, dermatitis, lupus, and maculopapular drug rash, and to decrease to non-lesional levels as the rash resolves (DIDascalu, Y. Kletter, M. Baratz, S. Brenner, Acta Derm Venereol 72, 175-177 (1992)). Interestingly, psoriasis is associated with altered LC migration, suggesting that imiquimod application, while a well-studied and effective mouse model for psoriasis, lupus, and dermatitis, also has applicability to inflammatory drug responses to harmful drugs. Here, we have shown that CD1a antibody-dependent regulation of LCs is associated with a reduction in skin inflammation upon administration of the antibodies of the present invention (particularly 110 and 116), which may be therapeutically important for the treatment of psoriasis, dermatitis, lupus, inflammatory drug responses, and other conditions. Epitope analysis highlights the potential therapeutic importance of epitope binding sites. Anti-CD1a antibodies were classified into two groups based on their binding sites and resulting effector function. Epitope sites can facilitate the clustering and alteration of phenotypic effects observed with antibodies 110 and 116, but not with 77a, 111, and 16, which are primarily blocking antibodies. Clustering may indeed result in cross-linked / aggregated cell morphologies. This may also explain the reduction in CD1a-transfected K562 and monocyte-derived LC cell types, as both express high levels of CD1a (higher than monocyte-derived DCs). The different antibody binding sites in these two groups do not compete. Therefore, combinations selected from each of the two groups may be useful, for example, in treatment / monitoring or combination therapy.

[0387] The role of CD1a in the development of skin inflammation and related systemic diseases suggests its role in many conditions, including psoriasis, dermatitis, lupus erythematosus, and drug hypersensitivity. Furthermore, characterizing CD1a blockade and regulatory antibodies offers new potential pathways for the prophylactic and therapeutic development of skin inflammation and CD1a-expressing malignancies.

[0388] The data presented herein define CD1a contact points for the anti-CD1a antibodies OX16, OX110, and OX116. The binding sites of OX110 and OX116 are located near the F' portal, distinct from the structures of OX16 and other published anti-CD1a antibodies (U.S. Pat. No. 10,844,118 and WO 2022 / 077021), which bind on the A' roof. OX16 and OX116 were able to bind to CD1a loaded with a variety of lipids, including permissive and non-permissive ligands. This was surprising given the proximity of OX116 binding to the F' portal of CD1a, but suggests that OX116 may have broad utility in CD1a binding and / or CD1a blocking. OX16 binding was negatively affected by lipid antigens with bulky, protruding head groups that mimic the behavior of autoreactive T cells, raising the possibility that OX16 could be used as a CD1a blocker with only small lipids that are autoreactive and / or permissive, but not lipids with head group co-recognition properties predicted to elicit a desirable immune response, for example, in response to Mtb lipids. These findings were consistent with the ability of the OX16, OX110, and OX116 antibodies to inhibit polyclonal CD1a-dependent responses across a broad range of contexts, including IFNγ and IL-22 responses. Functional inhibition of polyclonal T cell responses was compared with the published antibodies CR2113 and mAb571 (U.S. Patent No. 10,844,118 and WO 2022 / 077021). The antibodies demonstrated broad and significant ability to reduce CD1a-dependent autoreactivity, with OX16, OX110, and OX116 demonstrating significant improvements over their published antibody counterparts.

[0389] The use of different human IgG1 Fc variants altered the functional effects of OX16, OX110, and OX116 on CD1a-expressing target cells. While all three antibodies showed clear improvements over the published CR2113 and mAb571 antibodies, it was noted that OX116 produced the highest CDC and loss of confluence of CD1a-expressing target cells. This suggests the potential utility of OX116 in situations where depletion of CD1a-expressing cells may be advantageous, such as CD1a-expressing malignancies. The use of the Fab version of the antibody did not induce CDC or loss of confluence, suggesting that the Fc region and / or dimerization are required for such effector function.

[0390] Therefore, in summary, a family of antibodies having binding sites across the entire CD1a spectrum is described. As shown in Examples 2-7 above, the presence of various binding sites is useful when detecting CD1a or when regulating CD1a function in different ways, either individually or in combination.

[0391] The antibody OX25 has been demonstrated to bind to the alpha3 domain of CD1a. The effects of the antibody on IFNg and IL-22 production were investigated. These cytokines are widely associated with inflammatory skin diseases and related systemic disorders. For example, IFNg is known to promote T cell and neutrophil responses, IgG class switching, and MHC class I and II induction, thereby amplifying innate and adaptive immune responses. IL-22 has broad effects on epithelial and stromal cells, promoting cell proliferation, antimicrobial peptide expression, and skin and systemic inflammation. IL-22 is associated with many inflammatory diseases, including systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, and psoriasis (Dudakov JA et al., Ann Review Immunol 33:747-85 (2015)).

[0392] Antibodies OX16, OX110, OX116, and OX25 have different binding footprints and distinct associated functions. Their ability to bind to the alpha-1, alpha-2, or alpha-3 domains of CD1a provides opportunities to identify CD1a and modulate CD1a function, either alone or in combination. This may be achieved by using anti-CD1a antibodies in a linked form, separately, or as part of other bispecific constructs (or other conjugates) or cell-based therapeutic approaches. The different binding sites also offer the possibility of utilizing combinations in diagnostic or therapeutic monitoring.

[0393] summary In summary, the inventors have generated a refined panel of anti-CD1a antibodies with therapeutic potential in the prevention and / or treatment of inflammatory skin and mucosal disorders. Antibodies 16, 77a, 110, 111, and 116 were shown to be potent inhibitors of human CD1a antigen presentation in vitro and demonstrated efficacy in drug reactions manifesting as psoriasis, dermatitis, systemic lupus erythematosus, and inflammatory skin or mucosal diseases or disorders, as well as in typical inflammatory skin disease models with the characteristics of systemic (non-cutaneous) inflammatory skin diseases or disorders, and in xenograft tumor models. The success of the antibody discovery process in identifying improved antibodies may be due to a combination of the following: a) screening of a large number of hits (3500); b) use of novel chimeric immunogens (in which the lipid-binding domain of human CD1a is fused to the CD1d Ig domain of the host organism, thereby targeting antibody production to the lipid-binding domain which may have the potential for functional inhibition); and c) analysis of various polyclonal and enriched T cells to examine different functional outcomes.

[0394] In vitro human functional assays, as measured by IC50 evaluation of inhibition of primary polyclonal T cell responses, demonstrated antibodies that were more potent than commercially available antibodies. Furthermore, using a sensitive human CD1a-restricted T cell clonal assay, we found that anti-CD1a antibodies 16 and 116 could block the production of IL-22, a key regulator of inflammatory skin and mucosal diseases. Such activity was an improvement and a surprise, as it had not been demonstrated in existing publications or patents for anti-CD1a CR2113 ((16, 17), U.S. Pat. No. 10,844,118 (B2), and Canadian Patent Application Publication No. 2,924,882 (A1)), in which IL-17 or IFNγ production was induced and inhibited in mouse systems. Because IL-22 is a key regulator of skin and mucosal diseases, inhibition of IL-22 is a key advantage of the antibodies.

[0395] Parallel analysis of human and in vivo mouse models provides a powerful tool for evaluating the therapeutic benefit of newly generated antibodies. In vivo, imiquimod has been utilized to induce psoriasis-like, dermatitis-like, lupus-like, and drug-response-like phenotypes, providing a model skin inflammatory system that may be more broadly applicable to numerous inflammatory diseases and disorders, as well as related systemic diseases or disorders and inflammatory drug reactions manifesting throughout the body. Here, antibodies 110, 116, and 16 were shown to significantly reduce imiquimod-induced CD1a-dependent inflammation, demonstrating improvement over standard treatment (anti-IL-17A) and a comparable anti-CD1a antibody (CR2113) in the same mouse IgG1 background. Importantly and unexpectedly, antibody 116 reduced skin inflammation below that of wild-type imiquimod-treated mice and normalized many skin and systemic immunological markers to those of wild-type mice. This suggests a mechanism by which anti-CD1a 116 exerts effects beyond inhibition of CD1a-TCR signaling. Skin immunophenotyping was performed and a reduction in T cell numbers and activation was observed. Similarly, a reduction in neutrophil infiltration to WT levels was observed with administration of antibodies 110, 116, and 16. The observation that neutrophilia was reduced to WT levels is an unexpected improvement over the published anti-CD1a antibody CR2113 and highlights the potential of antibodies 110, 116, and 16.

[0396] Importantly, when analyzing the LC population within the skin, a significant reduction in CD11c+Langerin+LCs was observed after administration of antibodies 110 and 116. This reduction could not be explained by enhanced migration to draining lymph nodes. However, it is possible that antibodies 110 and (to a greater extent) 116 may directly deplete CD1a+ cells in vivo, explaining the reduction in skin LCs in vivo, as evidenced by the significant reduction in human CD1a+ cells in vitro. This is a surprising result considering the mouse IgG1 isotype of the antibodies, where the mouse IgG2a isotype is more likely to mediate cytotoxicity via complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity. Furthermore, although the patented and published anti-CD1a CR2113 has been reported to be unable to directly deplete cells (17), we have shown here that apoptosis of CD1a-expressing cells can also be induced by CR2113 in a mouse IgG1 background. The regulatory capabilities of these antibodies may help explain the lower reduction in imiquimod-induced inflammation compared to that of WT isotype-treated mice. Antibody 116 not only blocks the interaction between CD1a and the TCR, but also modifies LCs, reducing / resetting the skin's inflammatory potential and normalizing many of the skin and systemic immunological markers to those of WT mice. This may explain the improvement over CD1a-dependent responses in these mice compared to wild-type mice, but not in anti-CD1a CR2113.

[0397] Furthermore, the data suggest that the 16, 110, and / or 116 antibodies presented herein have utility in the treatment of CD1a-expressing malignancies, such as Langerhans cell histiocytosis or some forms of T-cell lymphoma and thymoma. This may be by direct effect or the anti-CD1a antibody is coupled or associated with one or more other therapeutic agents selected from the group including cytotoxic agents, anti-inflammatory agents (e.g., steroids), and CAR-T cells (e.g., regulatory or cytolytic CAR-T cells), or other cells that express or present the antibody or antigen-binding fragment thereof.

[0398] This study demonstrates that antibody 16 is a highly effective blocking antibody that eliminates CD1a-dependent inflammation in vivo without directly inducing apoptosis, 110 modifies LC phenotype and function and significantly reduces CD1a-dependent inflammation in vivo, and 116 is a highly effective blocking and modifying antibody that reduces inflammation to below WT levels and normalizes many skin and systemic immunological markers to WT levels. This grouping of antibodies is consistent with basic epitope analysis, with the directly modifying antibodies 110 and 116 clustering, and the blocking antibodies 77a, 111, and 16 clustering. Epitope analysis also revealed groups 77a, 111, and 16 overlapping with epitopes recognized by non-depleted NA1 / 34. This is significant because NA1 / 34 has been shown to cross-block the binding of anti-CD1a CR2113. Antibodies 110 and 116 do not cross-block NA1 / 34 and therefore likely represent different epitope regions. The antibodies remain present on LC in vivo in the skin and even after migration to lymph nodes. This is a significant enhancement as it leads to a longer-lasting clinical effect.

[0399] These data demonstrate the potential of this refined panel of improved anti-CD1a antibodies for use in the prevention and treatment of inflammatory skin and mucous membrane conditions, including but not limited to psoriasis, dermatitis, and lupus, and in the treatment and / or prevention of one or more related systemic diseases or disorders, or one or more systemic inflammatory drug reactions. The effects on the broad cascade of inflammation, including LCs, T cells, and neutrophils, particularly the effects of antibodies 110, 116, and 16, will have broad effects in inflammatory skin and mucous membrane diseases or disorders, or in inflammatory skin and mucous membrane disorders, including psoriasis, dermatitis, lupus, and drug reactions, which may manifest as CD1a-expressing malignancies.

[0400] Here, we define the structural basis of antibodies binding to CD1a and examine the lipid dependence of binding as well as the functional effects of different human IgG1 variants on in vitro function. We show that OX16 and OX116 bind at different sites, and that OX116 binding is completely lipid antigen-independent despite its proximity to the F' portal, explaining its broad effect on CD1a blockade. However, the interaction between CD1a and OX16 is moderately influenced by the protruding lipid head group. This suggests a possible mechanism for selective autoreactive recognition of CD1a molecules bearing only certain species of smaller, permissive self-lipids, but not those with larger head groups that may be required for immunization. We generated the anti-CD1a antibody OX25, which has a binding site on the alpha3 domain of CD1a. Collectively, the data suggest various antibodies with CD1a-binding sites with distinct, relevant functions. We also demonstrate improvements over other published antibodies, CR2113 and mAb571, consistent with a role for antibodies in the diagnosis, monitoring, prevention, and treatment of CD1a-dependent diseases.

[0401] In conclusion, we demonstrate improved anti-CD1a antibodies 16, 77a, 110, 111, and 116 as a method for preventing and treating inflammatory skin and mucosal diseases or disorders, or related systemic diseases or disorders, or systemic inflammatory drug reactions, or CD1a-expressing malignancies, by blocking CD1a and / or modifying the phenotype / function of CD1a+ cells.

[0402] References 1. GFMurphy, AKBhan, S. Sato, MC Mihm, Jr., TJ Harrist, A new immunologic marker for human Langerhans cells. N Engl J Med 304, 791-792 (1981). 2. Y.L.Chen et al.,Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation.J Exp Med 217,(2020)。 3. S.G.Turville et al.,Diversity of receptors binding HIV on dendritic cell subsets.Nat Immunol 3,975-983(2002)。 4. M.Alcantara-Hernandez et al.,High-Dimensional Phenotypic Mapping of Human Dendritic Cells R...

Claims

1. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof: a) CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or b) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or c) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 14, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or d) CDR1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 20, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 22, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or e) CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or g) CDR1 of SEQ ID NO: 91, CDR2 of SEQ ID NO: 92, and CDR3 of SEQ ID NO: 93, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 94, CDR2 of SEQ ID NO: 95, and CDR3 of SEQ ID NO: 96, or a chimeric antibody or antigen-binding fragment thereof, comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

2. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof: (a) a heavy chain comprising or consisting of SEQ ID NO:219, SEQ ID NO:220, or SEQ ID NO:221, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or SEQ ID NO: 218, or a light chain comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (b) a heavy chain comprising or consisting of SEQ ID NO:215, SEQ ID NO:216, or SEQ ID NO:217, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or SEQ ID NO: 214, or a light chain comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (c) a heavy chain comprising or consisting of SEQ ID NO:211, SEQ ID NO:212, or SEQ ID NO:213, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or SEQ ID NO: 210, or a light chain comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (d) a heavy chain comprising or consisting of SEQ ID NO:254, SEQ ID NO:255, or SEQ ID NO:256, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or SEQ ID NO: 253, An antibody or antigen-binding fragment thereof, which includes or comprises a light chain having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with such a sequence, or is a chimeric antibody or antigen-binding fragment thereof.

3. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof: (a) CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 36, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 13, or SEQ ID NO: 139, CDR2 of SEQ ID NO: 37, and CDR3 of sequence number 38, sequence number 123, sequence number 124, sequence number 125, sequence number 126, sequence number 127, sequence number 128, sequence number 129, sequence number 130, or sequence number 131, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (b) CDR1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134, and CDR3 of sequence number 19 or sequence number 110, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 20, CDR2 of SEQ ID NO: 21, and CDR3 of sequence number 22, sequence number 111, sequence number 112, sequence number 113, sequence number 114, sequence number 115, sequence number 116, sequence number 117, sequence number 118, or sequence number 119, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (c) CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of sequence number 30, sequence number 120, sequence number 121, or sequence number 122, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (d) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 14, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109; or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (e) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO:2, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, or SEQ ID NO:251, and CDR3 of SEQ ID NO: 3, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO:5, SEQ ID NO:140, or SEQ ID NO:141, and CDR3 of SEQ ID NO: 6, or a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (f) CDR1 of SEQ ID NO: 91, CDR2 of SEQ ID NO:92, SEQ ID NO:257, SEQ ID NO:258, or SEQ ID NO:259, and CDR3 of SEQ ID NO: 93, or a heavy chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or c) CDR1 of SEQ ID NO: 94, CDR2 of SEQ ID NO: 95, and CDR3 of SEQ ID NO: 96, or a humanized antibody or antigen-binding fragment thereof, comprising or consisting of a light chain variable region comprising a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

4. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof: (a) a heavy chain variable region comprising or consisting of SEQ ID NO: 39 or SEQ ID NO: 194, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or Sequence ID 40, Sequence ID 179, Sequence ID 180, Sequence ID 181, Sequence ID 182, Sequence ID 183, Sequence ID 184, Sequence ID 185, Sequence ID 186, Sequence ID 187, Sequence ID 188, Sequence ID 189, Sequence ID 190, Sequence ID 191, Sequence ID 192, or Sequence ID 193, or a light chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (b) Heavy chain variable regions comprising or consisting of sequences that are at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto, and / or SEQ ID NO:24, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, or SEQ ID NO:167, or a light chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (c) a heavy chain variable region comprising or consisting of SEQ ID NO: 31 or SEQ ID NO: 178, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or Sequence ID 32, Sequence ID 174, Sequence ID 175, Sequence ID 176, or Sequence ID 177, or a light chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (d) a heavy chain variable region comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 157, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or Sequence ID 16, Sequence ID 153, Sequence ID 154, Sequence ID 155, or Sequence ID 156, or a light chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (e) Heavy chain variable regions comprising or consisting of sequences such as SEQ ID NO: 7, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, or SEQ ID NO: 209, or sequences having at least 80%, 90%, 95%, 98%, 99%, or 100% identity therewith, and / or Sequence ID 8, Sequence ID 195, Sequence ID 196, or Sequence ID 197, or a light chain variable region comprising or consisting of a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (f) a heavy chain variable region comprising or consisting of SEQ ID NO:97, SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:263, or SEQ ID NO:264, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or An antibody or antigen-binding fragment thereof that includes or comprises a light chain variable region having at least 80%, 90%, 95%, 98%, 99%, or 100% identity with sequence number 98 or sequence number 260, or a humanized antibody or antigen-binding fragment thereof.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a full-length antibody.

6. The antibody according to claim 5, wherein the antibody is an IgG1 antibody or an IgG1 antibody having one or more substitutions in its constant region.

7. An antibody or its antigen-binding fragment, (a) an ScFv comprising or consisting of SEQ ID NO: 104, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (b) an ScFv comprising or consisting of SEQ ID NO: 105, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or (c) An antibody or antigen-binding fragment thereof comprising, or consisting of, an ScFv comprising, or consisting of, SEQ ID NO: 106, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

8. an antibody or antigen-binding fragment thereof that binds to an epitope on CD1a or competes with the antibody or antigen-binding fragment thereof for binding to CD1a, wherein the epitope on CD1a is (a) residues Arg83, Tyr84, His86, Glu87, Gln89, Phe90, Glu91, Asn139, Met140, Lys142, His143, Lys146, Val147, and Gln150 of CD1a (residue numbering according to SEQ ID NO: 252); or (b) residues Glu62, Glu65, Leu66, Thr68, Leu69, Ile72, Asn151, His153, Glu154, Ile157, Asn160, Asp164, Thr165, and Arg168 of CD1a (residue numbering according to SEQ ID NO: 252); or (c) An antibody or antigen-binding fragment thereof comprising or consisting of residues Glu79, Arg82, Arg83, His86, Glu87, Gln89, Phe90, Glu91, Tyr92, Val147, and Asn150 of CD1 (residue numbering according to SEQ ID NO: 252).

9. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid described in claim 9.

11. The vector according to claim 10, wherein the vector is an expression vector, a plasmid, or a viral vector.

12. A host cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid according to claim 9, and / or the vector according to claim 10 or claim 11.

13. The host cell according to claim 12, wherein the host cell is a bacterial cell or a mammalian cell.

14. A pharmaceutical composition comprising one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 8, a nucleic acid according to claim 9, a vector according to claim 10 or claim 11, and / or a host cell according to claim 12 or claim 13.

15. 15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid according to claim 9, the vector according to claim 10 or claim 11, the host cell according to claim 12 or claim 13, or the pharmaceutical composition according to claim 14, for use in medicine.

16. For use in the treatment or prevention of one or more inflammatory skin or mucosal diseases or disorders, or one or more related systemic diseases or disorders, or one or more systemic inflammatory drug reactions, or CD1a-expressing malignancies, one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 8, one or more nucleic acids according to claim 9, one or more vectors according to claim 10 or claim 11, one or more host cells according to claim 12 or claim 13, or one or more pharmaceutical compositions according to claim 14.

17. (a) the one or more inflammatory skin or mucosal diseases or disorders are (i) Primarily neutrophilic skin diseases, such as acne, generalized pustular psoriasis, psoriasis vulgaris, guttate psoriasis, palmoplantar pustulosis, SAPHO syndrome, acute febrile neutrophilic dermatosis (Sweet's syndrome), histiocytic neutrophilic dermatitis, neutrophilic dermatosis of the back of the hand, pyoderma gangrenosum, neutrophilic eccrine hidradenitis, hidradenitis suppurativa, erythema elevata, Behçet's disease, intestinal dermatitis-arthritis syndrome, other infection-related inflammations, neutrophilic urticarial dermatosis, palisade neutrophilic granulomatous dermatitis, creeping erythema circumferential, neutrophilic erythema annulare, acute generalized exanthematous pustulosis (AGEP), vasculitis, etc. (ii) autoimmune disorders, such as connective tissue diseases (e.g. lupus, dermatomyositis, scleroderma / systemic sclerosis, Churg-Strauss syndrome), panniculitis, vasculitis, autoimmune blistering conditions (e.g. bullous pemphigoid, pemphigus, linear IgA disease), dermatitis herpetiformis, celiac disease, some autoinflammatory diseases, vitiligo, alopecia areata, alopecia universalis, alopecia totalis, panniculitis, lichen planus, erythema multiforme, lichen sclerosus, other lichenoid and erythema multiforme-like diseases, blistering psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, Guillain-Barré syndrome, thyroiditis, transverse myelitis, neurodegeneration, etc. (iii) mast cell disorders and eosinophilic disorders, such as Muckle-Wells syndrome, eosinophilia and systemic symptom syndrome, urticaria, angioedema, keratoconjunctivitis, food allergies, other allergies or atopy (including atopic dermatitis), rhinitis, conjunctivitis, asthma, eosinophilic esophagitis and other eosinophilic mucosal diseases, contact dermatitis, chronic obstructive airway disease, etc. (iv) adverse drug reactions manifesting as inflammatory skin or mucosal diseases or disorders, such as Stevens-Johnson syndrome, toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms syndrome (DRESS) and acute generalized exanthematous pustulosis (AGEP), erythema multiforme, bullous fixed drug eruptions, checkpoint inhibitor-associated dermatitis and other inflammation, (v) Graft-versus-host disease, (vi) pruritus and pruritic conditions, including prurigo nodularis; (b) Whether the one or more related systemic diseases or disorders, or one or more systemic inflammatory drug reactions, is an inflammatory reaction to Aldara (imiquimod), or (c) one or more antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or pharmaceutical compositions for use according to claim 16, wherein the CD1a-expressing malignant tumor is one or more of Langerhans cell histiocytosis, Langerhans cell sarcoma, a subset of T-cell lymphoma, a subset of thymoma, or a rare example of other malignant tumor (such as a subset of mastocytosis).

18. 18. The one or more antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or pharmaceutical compositions for use according to claim 17, wherein the one or more inflammatory skin or mucosal diseases or disorders are one or more of psoriasis, dermatitis, lupus erythematosus, or a drug reaction that manifests as an inflammatory skin or mucosal disease or disorder.

19. 19. The one or more antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or pharmaceutical compositions for use according to any one of claims 15 to 18, wherein the antigen-binding fragments thereof, nucleic acids, vectors, host cells, or pharmaceutical compositions are intended to be administered alone or in combination with one or more other therapeutic agents.

20. 20. The one or more antibodies or antigen-binding fragments thereof, nucleic acid, vector, host cell, or pharmaceutical composition for use according to claim 19, wherein the one or more other therapeutic agents are selected from the group comprising cytotoxic agents, anti-inflammatory agents (e.g., steroids), and CAR-T cells (e.g., regulatory or cytolytic CAR-T cells), or other cells that express or present one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 8.

21. Use of one or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 8, a nucleic acid according to claim 9, a vector according to claim 10 or claim 11, a host cell according to claim 12 or claim 13, or a pharmaceutical composition according to claim 14 in the manufacture of a medicament for the treatment or prevention of one or more inflammatory skin or mucosal diseases or disorders, or one or more related systemic diseases or disorders, or one or more inflammatory drug reactions that manifest systemically, or one or more CD1a-expressing malignancies.

22. 15. A method for treating one or more inflammatory skin or mucosal diseases or disorders, or one or more related systemic diseases or disorders, or one or more inflammatory drug reactions manifesting systemically, or one or more CD1a-expressing malignancies in a subject, the method comprising administering to the subject an effective amount of one or more antibodies or antigen-binding fragments thereof described in any one of claims 1 to 8, a nucleic acid described in claim 9, a vector described in claim 10 or claim 11, a host cell described in claim 12 or claim 13, or a pharmaceutical composition described in claim 14.

23. A method for monitoring therapeutic efficacy or disease status in a subject diagnosed with a CD1a-expressing malignancy, i. providing a biological sample obtained from said subject; ii. Determining the binding level of one or more antibodies or antigen-binding fragments according to any one of claims 1 to 8 to CD1a-expressing cells in the sample obtained from the subject before treatment, between treatments, or during periods without treatment. iii. A method comprising: if tumor volume or the binding level of one or more antibodies or antigen-binding fragments of the present invention to CD1a-expressing cells decreases after treatment, between treatments, or during periods without treatment, optionally determining that the treatment is effective or the disease state is improving if the decrease in tumor volume or the binding level of one or more antibodies or antigen-binding fragments described in any one of claims 1 to 6 to CD1a-expressing cells is 25% or more.

24. A method for diagnosing a subject having inflammatory skin and mucous membrane diseases or disorders, related systemic diseases or disorders, systemic inflammatory drug reactions, or CD1a-expressing malignant tumors, i. providing a biological sample obtained from said subject; ii. Determining the expression level of CD1a in the sample obtained from the subject using one or more antibodies or antigen-binding fragments thereof as described in any one of claims 1 to 8. iii. The expression level of CD1a in the sample obtained from the subject is compared with the expression level of CD1a in a positive or negative reference sample. iv. If the CD1a expression level in the sample obtained from the subject is higher than the CD1a expression level in the negative reference sample, or equal to or higher than the CD1a expression level in the positive reference sample, then the subject is determined to have an inflammatory skin and mucous membrane disease or disorder, or a related systemic disease or disorder, or a systemic inflammatory drug reaction, or a CD1a-expressing malignant tumor, or A method comprising determining that the subject does not have inflammatory skin and mucous membrane diseases or disorders, related systemic diseases or disorders, systemic inflammatory drug reactions, or CD1a-expressing malignancies, if the expression level of the CD1a in the sample obtained from the subject is equal to or lower than the expression level of the CD1a in the negative reference sample, or lower than the expression level of the CD1a in the positive reference sample.

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