Anti-CD1a antibodies

Abstract

The present invention relates to an antibody or an antigen-binding fragment thereof capable of binding CD1a. The antibodies, or antigen-binding fragments thereof, may be chimeric or humanized, and may be used to treat one or more inflammatory dermatological or mucosal disorders or diseases, or one or more related systemic diseases or disorders, or one or more inflammatory drug responses of systemic manifestations, or CD1a-expressing malignancies.

Description

TECHNICAL FIELD

[0001] The present application relates to antibodies, and their use in the treatment, prevention, diagnosis or monitoring of inflammatory skin and mucosal diseases or conditions, or associated systemic diseases or conditions, or inflammatory drug reactions, or CD1a expressing malignancies. BACKGROUND

[0002] Antigen presentation is one of the fundamental pillars of host immunity, with the immune system detecting threats including infections, tissue damage and disease through antigen presentation and orchestrating tailored defenses. Antigen presentation includes internalization, processing and display on the surface of specialized antigen presenting cells (APCs) by presentation molecules. Presentation of tissue antigens to achieve optimal activation of immune responses targeted at the antigen source and elimination of threats. Antigens include a broad range of molecules including peptides, lipids and metabolites among others. MHC I and MHC II are proteins expressed on the surface of APCs that bind peptide antigens and are abundantly present on CD8+ T cells and CD4+ T cells, respectively. These T cell subsets are induced to exert their effector functions upon cell surface T cell receptor (TCR) recognition of MHC-bound peptide antigens, enabling immunity to pathogens and cancer. However, dysregulated presentation of innocuous antigens such as allergens in allergic diseases or self-proteins in autoimmunity elicit host damage, inflammation and disease. Therefore, targeting the antigen presentation pathway is a powerful means to modulate the ensuing immune response.

[0003] CD1 molecules constitute a family of antigen-presenting molecules structurally similar to MHC I. In contrast, CD1 molecules are relatively non-polymorphic, and the CD1 antigen-binding groove is rich in hydrophobic amino acids capable of presenting lipid species. Lipids are important antigens that form a critical component of host and pathogen cell membranes and are less subject to mutation than protein-derived peptide antigens. The CD1 family consists of cell surface group 1 molecules CD1a / b / c and group 2 CD1d and group 3 CD1e. Most of the understanding of CD1 lipid presentation and T cell responses comes from studies of invariant natural killer T cell recognition of glycolipid-bound CD1d, in part because CD1d is the only CD1 normally expressed in mice. CD1d and MHC I molecules are widely expressed, whereas MHC II and group 1 CD1 expression is relatively restricted to APC. However, the unique CD1a among these molecules is highly specific to the skin and mucosa. CD1a is constitutively expressed by Langerhans cells (LCs) in the epidermis of the skin and mucosa (1) and is commonly used as an identifying marker for LCs in addition to langerin. Additionally, CD1a is expressed at lower levels on a subset of dermal dendritic cells (2-4) and can be expressed and upregulated on skin invariant lymphocytes (ILCs), particularly ILC2 (5). Importantly, CD1a was first described on the surface of immature thymocytes, but expression is typically lost upon T cell maturation (6). The high level of constitutive expression of CD1a in the skin indicates an important physiological role for CD1a-dependent surveillance and T cell activation in healthy and diseased human skin. Furthermore, the increase in CD1a expression in atopic dermatitis skin can underlie the increased activation of CD1a-reactive T cell populations in inflammatory skin disease.

[0004] T cell responses directed by CDla, CDlb, or CDlc molecules presenting mycobacterial lipid-based antigens are relevant to the human immune response to Mycobacterium tuberculosis and Mycobacterium leprae infection. The recognition of other more common pathogenic or commensal bacterial lipids by CDla-restricted T cells is the subject of ongoing research, some of which is provided herein. While TCR recognition of peptide antigens by MHC-restricted T cells is generally highly specific for the peptide antigen, the CD1 pattern of TCR recognition is more diverse, with highly lipid-specific responses (7) and cross-reactivity or even apparent lipid-independent signaling mediated by direct TCR-CD1 interactions (8-10), as is the case for CDla-autoreactive T cells. In some cases, CDla-autoreactive T cells are activated in recognizing CDla bearing small, hydrophobic host-derived lipids that are nestled within the antigen-binding groove without protruding, allowing the TCR to interact with the CDla protein itself rather than with the lipid. In this case, binding of a lipid with a large or charged head group would prevent the interaction between the autoreactive TCR and CDla, preventing T cell activation (11, 12).

[0005] CD1a is relatively non-polymorphic, and thus has broad population potential in the prevention and / or treatment of inflammatory skin and mucosal diseases and conditions, such as atopic dermatitis, psoriasis, lupus erythematosus or related systemic diseases or conditions, or systemically manifested inflammatory drug reactions, in which the frequency of CD1a-expressing dendritic cell subsets is altered, and the migration pattern of LCs or responding T cells is altered (13-15). In addition, CD1a is implicated in other systemic conditions, including inflammatory bowel disease, multiple sclerosis, Guillain-Barre syndrome, thyroiditis, and neurodegeneration (Al-amodi Inflammatory Bowel Diseases 2018 24:1225-1236; 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). In addition, CD1a can be expressed by certain malignancies, including Langerhans cell histiocytosis, Langerhans cell sarcoma, subsets of T cell lymphomas, subsets of thymomas, and rare descriptions of other malignancies, such as subsets of mastocytosis.

[0006] It is an object of the present application to provide anti-CD1a antibodies. Such antibodies are particularly useful for the treatment or prevention of inflammatory diseases or conditions of the skin or mucosa, such as psoriasis, dermatitis, lupus erythematosus, or drug reactions manifested as inflammatory skin or mucosal diseases or conditions. Such antibodies can also be beneficial for the treatment or prevention of related systemic diseases or conditions, or systemically manifested inflammatory drug reactions, or for the treatment of malignancies expressing CD1a. SUMMARY

[0007] The present application relates to an antibody or antigen-binding fragment thereof capable of binding CD1a. The antibody or antigen-binding fragment thereof can specifically bind CD1a. The antibody or antigen-binding fragment thereof can preferentially bind CD1a. In some embodiments, the antibody or antigen-binding fragment thereof induces cell death of CD1a-expressing cells. In some embodiments, the antibody or antigen-binding fragment thereof blocks the binding of a ligand to CD1a.

[0008] In one aspect, the antibody or antigen-binding fragment thereof can be a chimeric antibody comprising or consisting of:

[0009] a) a heavy chain variable region comprising:

[0010] a CDR1 of SEQ ID NO: 33,

[0011] a CDR2 of SEQ ID NO: 34, and

[0012] a CDR3 of SEQ ID NO: 35,

[0013] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto, and / or

[0014] a light chain variable region comprising:

[0015] a CDR1 of SEQ ID NO: 36,

[0016] a CDR2 of SEQ ID NO: 37, and

[0017] a CDR3 of SEQ ID NO: 38,

[0018] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0019] b) a heavy chain variable region comprising:

[0020] a CDR1 of SEQ ID NO: 1,

[0021] a CDR2 of SEQ ID NO: 2, and

[0022] a CDR3 of SEQ ID NO: 3,

[0023] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto, and / or

[0024] a light chain variable region comprising:

[0025] a CDR1 of SEQ ID NO: 4,

[0026] a CDR2 of SEQ ID NO: 5, and

[0027] a CDR3 of SEQ ID NO: 6,

[0028] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0029] c) a heavy chain variable region comprising:

[0030] CDR1 of SEQ ID NO: 9,

[0031] CDR2 of SEQ ID NO: 10, and

[0032] CDR3 of SEQ ID NO: 11,

[0033] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto, and / or

[0034] a heavy chain variable region comprising:

[0035] CDR1 of SEQ ID NO: 12,

[0036] CDR2 of SEQ ID NO: 13, and

[0037] CDR3 of SEQ ID NO: 14,

[0038] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0039] d) a heavy chain variable region comprising:

[0040] CDR1 of SEQ ID NO: 17,

[0041] CDR2 of SEQ ID NO: 18, and

[0042] CDR3 of SEQ ID NO: 19,

[0043] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto, and / or

[0044] a heavy chain variable region comprising:

[0045] CDR1 of SEQ ID NO: 20,

[0046] CDR2 of SEQ ID NO: 21, and

[0047] CDR3 of SEQ ID NO: 22,

[0048] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0049] e) a heavy chain variable region comprising:

[0050] CDR1 of SEQ ID NO: 25,

[0051] CDR3 of SEQ ID NO: 27,

[0052] CDR3 of SEQ ID NO: 27,

[0053] or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or

[0054] a heavy chain variable region comprising:

[0055] CDR1 of SEQ ID NO: 28,

[0056] CDR2 of SEQ ID NO: 29, and

[0057] CDR3 of SEQ ID NO: 30,

[0058] or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; or

[0059] f) a heavy chain variable region comprising:

[0060] CDR1 of SEQ ID NO: 91,

[0061] CDR2 of SEQ ID NO: 92, and

[0062] CDR3 of SEQ ID NO: 93,

[0063] or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or

[0064] a heavy chain variable region comprising:

[0065] CDR1 of SEQ ID NO: 94,

[0066] CDR2 of SEQ ID NO: 95, and

[0067] CDR3 of SEQ ID NO: 96,

[0068] or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0069] In any of the above, as an antibody or antigen-binding fragment thereof that is a chimeric antibody, any combination of CDRs can be employed. Alternatively, as an antibody or antigen-binding fragment thereof that is a chimeric antibody, only the CDR3 of the above heavy chain variable region and light chain variable region can be included.

[0070] In another aspect, the antibody or antigen-binding fragment thereof can be a chimeric antibody comprising or consisting of:

[0071] (a) a heavy chain comprising or consisting of SEQ ID NO: 21 1, SEQ ID NO: 212, or SEQ ID NO: 213, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0072] a light chain comprising or consisting of SEQ ID NO: 210,

[0073] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0074] (b) a heavy chain comprising or consisting of SEQ ID NO: 215, SEQ ID NO: 216, or SEQ ID NO: 217, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0075] a light chain comprising or consisting of SEQ ID NO: 214,

[0076] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0077] (c) a heavy chain comprising or consisting of SEQ ID NO: 219, SEQ ID NO: 220, or SEQ ID NO: 221, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0078] a light chain comprising or consisting of SEQ ID NO: 218,

[0079] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; or

[0080] (d) a heavy chain comprising or consisting of SEQ ID NO: 254, SEQ ID NO: 255, or SEQ ID NO: 256, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0081] a light chain comprising or consisting of SEQ ID NO: 253,

[0082] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0083] The antibody or antigen-binding fragment thereof can be a chimeric antibody comprising or consisting of:

[0084] a) a heavy chain comprising or consisting of: SEQ ID NO: 211, SEQ ID NO: 212, or SEQ ID NO: 213; and

[0085] b) a light chain comprising or consisting of: SEQ ID NO: 210.

[0086] The antibody or antigen-binding fragment thereof can be a chimeric antibody comprising or consisting of:

[0087] a) a heavy chain comprising or consisting of: SEQ ID NO: 215, SEQ ID NO: 216, or SEQ ID NO: 217; and

[0088] b) a light chain comprising or consisting of: SEQ ID NO: 214.

[0089] The antibody or antigen-binding fragment thereof can be a chimeric antibody comprising or consisting of:

[0090] a) a heavy chain comprising or consisting of: SEQ ID NO: 219, SEQ ID NO: 220, or SEQ ID NO: 221; and

[0091] b) a light chain comprising or consisting of: SEQ ID NO: 218.

[0092] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0093] a) a heavy chain variable region comprising:

[0094] CDR1 of SEQ ID NO: 9,

[0095] CDR2 of SEQ ID NO: 10, and

[0096] CDR3 of SEQ ID NO: 11,

[0097] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0098] b) a light chain variable region comprising:

[0099] CDR1 of SEQ ID NO: 12,

[0100] CDR2 of SEQ ID NO: 13, and

[0101] CDR3 of SEQ ID NO: 14, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109,

[0102] or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto.

[0103]

[0104] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0105] a) a heavy chain variable region comprising:

[0106] CDR1 of SEQ ID NO: 17,

[0107] CDR2 of SEQ ID NO: 18, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134, and

[0108] CDR3 of SEQ ID NO: 19, or SEQ ID NO: 110,

[0109] or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100% identity thereto; and / or

[0110] b) a light chain variable region comprising:

[0111] CDR1 of SEQ ID NO: 20,

[0112] CDR2 of SEQ ID NO: 21, and

[0113] 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, or a sequence having at least 80%, 90%, 95%, 98%, 99%, or 100%

[0114] identity thereto.

[0115] ​The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0116] a) a heavy chain variable region comprising:

[0117] a CDR1 of SEQ ID NO: 25,

[0118] a CDR2 of SEQ ID NO: 26, and

[0119] a CDR3 of SEQ ID NO: 27,

[0120] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0121] b) a light chain variable region comprising:

[0122] a CDR1 of SEQ ID NO: 28,

[0123] a CDR2 of SEQ ID NO: 29, and

[0124] a CDR3 of SEQ ID NO: 30, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122,

[0125] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100%

[0126] identical thereto.

[0127] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0128] a) a heavy chain variable region comprising:

[0129] a CDR1 of SEQ ID NO: 33,

[0130] a CDR2 of SEQ ID NO: 34, and

[0131] a CDR3 of SEQ ID NO: 35,

[0132] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0133] b) a light chain variable region comprising:

[0134] a 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,

[0135] a CDR2 of SEQ ID NO: 37, and

[0136] a CDR3 of SEQ ID NO: 38, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131,

[0137] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0138] The antibody or antigen binding fragment thereof can be a humanized antibody comprising or consisting of:

[0139] a) a heavy chain variable region comprising:

[0140] a CDR1 of SEQ ID NO: 1,

[0141] 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 IDNO: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 CDR2 of 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

[0142] CDR3 of SEQ ID NO: 3,

[0143] or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or

[0144] b) a light chain variable region comprising:

[0145] CDR1 of SEQ ID NO:4,

[0146] CDR2 of SEQ ID NO:5, SEQ ID NO:140 or SEQ ID NO:141, and

[0147] CDR3 of SEQ ID NO: 6,

[0148] or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto.

[0149] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0150] a) a heavy chain variable region comprising:

[0151] a CDR1 of SEQ ID NO: 91,

[0152] a CDR2 of SEQ ID NO: 92, SEQ ID NO: 257, SEQ ID NO: 258, or SEQ ID NO: 259, and

[0153] a CDR3 of SEQ ID NO: 93,

[0154] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0155] b) a light chain variable region comprising:

[0156] a CDR1 of SEQ ID NO: 94,

[0157] a CDR2 of SEQ ID NO: 95, and

[0158] a CDR3 of SEQ ID NO: 96,

[0159] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0160] The CDRs of any of the antibodies or antigen-binding fragments disclosed herein can be associated with any framework region. Preferably, the framework region is human-derived.

[0161] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0162] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0163] b) a light chain variable region comprising or consisting of SEQ ID NO:8, SEQ ID NO: 195, SEQ ID NO: 196, or SEQ ID NO: 197,

[0164] or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0165] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0166] a) 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or

[0167] b) a light chain variable region comprising or consisting of SEQ ID NO: 98 or SEQ ID NO: 260, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0168] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0169] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 157; and

[0170] b) a light chain variable region comprising or consisting of SEQ ID NO: 16, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, or SEQ ID NO: 156.

[0171] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0172] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 23, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, or SEQ ID NO: 173, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0173] b) 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0174] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0175] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 31 or SEQ ID NO: 178, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0176] b) a light chain variable region comprising or consisting of SEQ ID NO: 32, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, or SEQ ID NO: 177, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0177] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0178] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 39 or SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0179] b) 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0180] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0181] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0182] b) a light chain variable region comprising or consisting of SEQ ID NO: 8, SEQ ID NO: 195, SEQ ID NO: 196, or SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0183] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0184] a) 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0185] b) a light chain variable region comprising or consisting of SEQ ID NO: 98 or SEQ ID NO: 260, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0186] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0187] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0188] b) a light chain variable region comprising or consisting of SEQ ID NO: 179, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0189] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0190] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0191] b) a light chain variable region comprising or consisting of SEQ ID NO: 180, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0192] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0193] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0194] b) a light chain variable region comprising or consisting of SEQ ID NO: 181, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0195] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0196] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0197] b) a light chain variable region comprising or consisting of SEQ ID NO: 182, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0198] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0199] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0200] b) a light chain variable region comprising or consisting of SEQ ID NO: 183, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0201] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0202] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0203] b) a light chain variable region comprising or consisting of SEQ ID NO: 184, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0204] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0205] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0206] b) a light chain variable region comprising or consisting of SEQ ID NO: 185, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0207] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0208] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0209] b) a light chain variable region comprising or consisting of SEQ ID NO: 186, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0210] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0211] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0212] b) a light chain variable region comprising or consisting of SEQ ID NO: 187, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0213] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0214] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0215] b) a light chain variable region comprising or consisting of SEQ ID NO: 188, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0216] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0217] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0218] b) a light chain variable region comprising or consisting of SEQ ID NO: 189, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0219] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0220] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0221] b) a light chain variable region comprising or consisting of SEQ ID NO: 190, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0222] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0223] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0224] b) a light chain variable region comprising or consisting of SEQ ID NO: 191, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0225] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0226] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0227] b) a light chain variable region comprising or consisting of SEQ ID NO: 192, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0228] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0229] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 194, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0230] b) a light chain variable region comprising or consisting of SEQ ID NO: 193, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0231] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0232] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 198, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0233] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0234] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0235] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 198, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0236] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0237] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0238] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 198, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0239] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0240] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0241] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 199, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0242] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0243] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0244] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 199, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0245] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0246] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0247] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 199, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0248] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0249] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0250] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 200, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0251] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0252] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0253] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 200, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0254] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0255] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0256] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 200, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0257] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0258] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0259] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 201, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0260] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0261] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0262] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 201, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0263] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0264] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0265] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 201, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0266] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0267] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0268] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 202, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0269] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0270] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0271] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 202, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0272] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0273] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0274] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 202, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0275] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0276] The antibody, or antigen-binding fragment thereof, can be a humanized antibody comprising or consisting of:

[0277] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 203, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0278] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0279] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0280] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 203, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0281] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0282] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0283] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 203, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0284] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0285] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0286] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 204, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0287] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0288] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0289] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 204, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0290] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0291] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0292] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 204, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0293] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0294] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0295] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 205, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0296] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0297] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0298] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 205, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0299] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0300] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0301] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 205, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0302] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0303] The antibody or antigen binding fragment thereof can be a humanized antibody comprising or consisting of:

[0304] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 206, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0305] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0306] The antibody or antigen binding fragment thereof can be a humanized antibody comprising or consisting of:

[0307] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 206, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0308] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0309] The antibody or antigen binding fragment thereof can be a humanized antibody comprising or consisting of:

[0310] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 206, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0311] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0312] The antibody or antigen binding fragment thereof can be a humanized antibody comprising or consisting of:

[0313] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 207, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0314] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0315] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0316] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 207, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0317] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0318] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0319] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 207, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0320] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0321] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0322] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 208, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0323] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0324] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0325] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 208, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0326] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0327] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0328] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 208, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0329] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0330] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0331] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 209, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0332] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0333] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0334] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 209, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0335] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0336] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0337] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 209, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0338] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0339] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0340] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 210, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0341] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0342] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0343] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 210, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0344] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0345] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0346] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 210, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0347] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0348] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0349] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 211, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0350] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0351] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0352] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 211, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0353] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0354] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0355] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 211, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0356] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0357] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0358] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 212, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0359] b) a light chain variable region comprising or consisting of SEQ ID NO: 195, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0360] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0361] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 212, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0362] b) a light chain variable region comprising or consisting of SEQ ID NO: 196, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0363] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0364] a) a heavy chain variable region comprising or consisting of SEQ ID NO: 212, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0365] b) a light chain variable region comprising or consisting of SEQ ID NO: 197, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0366] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0367] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0368] b) a Fc region.

[0369] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0370] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0371] b) a Fc region.

[0372] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0373] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0374] b) an Fc region.

[0375] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0376] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0377] b) an Fc region.

[0378] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0379] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0380] b) an Fc region.

[0381] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0382] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0383] b) an Fc region.

[0384] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0385] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0386] b) an Fc region.

[0387] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0388] 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: 273, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0389] b) a Fc region.

[0390] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0391] 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: 274, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0392] b) a Fc region.

[0393] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0394] 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: 275, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0395] b) a Fc region.

[0396] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0397] 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: 276, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0398] b) a Fc region.

[0399] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0400] 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: 277, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0401] b) an Fc region.

[0402] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0403] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0404] b) an Fc region.

[0405] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0406] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0407] b) an Fc region.

[0408] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0409] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0410] b) an Fc region.

[0411] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0412] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0413] b) an Fc region.

[0414] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0415] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0416] b) an Fc region.

[0417] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0418] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0419] b) an Fc region.

[0420] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0421] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0422] b) an Fc region.

[0423] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0424] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0425] b) an Fc region.

[0426] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0427] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0428] b) an Fc region.

[0429] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0430] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0431] b) a Fc region.

[0432] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0433] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0434] b) a Fc region.

[0435] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0436] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0437] b) a Fc region.

[0438] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0439] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0440] b) a Fc region.

[0441] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0442] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0443] b) an Fc region.

[0444] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0445] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0446] b) an Fc region.

[0447] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0448] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0449] b) an Fc region.

[0450] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0451] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0452] b) an Fc region.

[0453] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0454] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0455] b) an Fc region.

[0456] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0457] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0458] b) an Fc region.

[0459] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0460] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0461] b) an Fc region.

[0462] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0463] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0464] b) an Fc region.

[0465] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0466] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0467] b) an Fc region.

[0468] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0469] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0470] b) an Fc region.

[0471] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0472] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0473] b) a Fc region.

[0474] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0475] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0476] b) a Fc region.

[0477] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0478] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0479] b) a Fc region.

[0480] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0481] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0482] b) a Fc region.

[0483] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0484] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0485] b) an Fc region.

[0486] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0487] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0488] b) an Fc region.

[0489] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0490] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0491] b) an Fc region.

[0492] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0493] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0494] b) an Fc region.

[0495] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0496] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0497] b) an Fc region.

[0498] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0499] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0500] b) an Fc region.

[0501] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0502] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0503] b) an Fc region.

[0504] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0505] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0506] b) an Fc region.

[0507] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0508] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0509] b) an Fc region.

[0510] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0511] a) an 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0512] b) an Fc region.

[0513] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0514] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0515] b) a Fc region.

[0516] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0517] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0518] b) a Fc region.

[0519] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0520] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0521] b) a Fc region.

[0522] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0523] 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0524] b) a Fc region.

[0525] The antibody can comprise a human Fc region. More specifically, the antibody or antigen-binding fragment thereof can be a full-length antibody. More specifically, the antibody can be of IgG isotype. More specifically, the antibody can be IgG1 or IgG4. Most preferably, the Fc region comprises or consists of SEQ ID NO: 297.

[0526] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0527] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0528] b) a light chain comprising or consisting of SEQ ID NO: 266, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0529] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0530] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0531] b) a light chain comprising or consisting of SEQ ID NO: 267, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0532] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0533] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0534] b) a light chain comprising or consisting of SEQ ID NO: 268, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0535] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0536] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0537] b) a light chain comprising or consisting of SEQ ID NO: 269, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0538] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0539] a) a heavy chain comprising or consisting of SEQ ID NO:300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and270

[0540] b) a light chain comprising or consisting of SEQ ID NO:271, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0541] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0542] a) a heavy chain comprising or consisting of SEQ ID NO:300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0543] b) a light chain comprising or consisting of SEQ ID NO:271, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0544] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0545] a) a heavy chain comprising or consisting of SEQ ID NO:300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0546] b) a light chain comprising or consisting of SEQ ID NO:272, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0547] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0548] a) a heavy chain comprising or consisting of SEQ ID NO:300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0549] b) a light chain comprising or consisting of SEQ ID NO:273, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0550] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0551] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0552] b) a light chain comprising or consisting of SEQ ID NO: 274, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0553] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0554] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0555] b) a light chain comprising or consisting of SEQ ID NO: 275, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0556] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0557] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0558] b) a light chain comprising or consisting of SEQ ID NO: 276, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0559] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0560] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0561] b) a light chain comprising or consisting of SEQ ID NO: 277, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0562] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0563] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0564] b) a light chain comprising or consisting of SEQ ID NO: 278, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0565] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0566] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0567] b) a light chain comprising or consisting of SEQ ID NO: 279, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0568] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0569] a) a heavy chain comprising or consisting of SEQ ID NO: 300, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0570] b) a light chain comprising or consisting of SEQ ID NO: 280, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0571] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0572] a) a heavy chain comprising or consisting of SEQ ID NO: 301, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0573] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0574] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0575] a) a heavy chain comprising or consisting of SEQ ID NO: 301, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0576] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0577] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0578] a) a heavy chain comprising or consisting of SEQ ID NO: 301, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0579] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0580] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0581] a) a heavy chain comprising or consisting of SEQ ID NO: 302, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0582] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0583] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0584] a) a heavy chain comprising or consisting of SEQ ID NO: 302, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0585] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0586] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0587] a) a heavy chain comprising or consisting of SEQ ID NO: 302, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0588] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0589] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0590] a) a heavy chain comprising or consisting of SEQ ID NO: 303, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0591] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0592] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0593] a) a heavy chain comprising or consisting of SEQ ID NO: 303, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0594] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0595] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0596] a) a heavy chain comprising or consisting of SEQ ID NO: 303, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0597] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0598] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0599] a) a heavy chain comprising or consisting of SEQ ID NO: 304, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0600] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0601] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0602] a) a heavy chain comprising or consisting of SEQ ID NO: 304, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0603] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0604] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0605] a) a heavy chain comprising or consisting of SEQ ID NO: 304, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0606] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0607] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0608] a) a heavy chain comprising or consisting of SEQ ID NO: 305, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0609] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0610] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0611] a) a heavy chain comprising or consisting of SEQ ID NO: 305, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0612] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0613] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0614] a) a heavy chain comprising or consisting of SEQ ID NO: 305, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0615] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0616] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0617] a) a heavy chain comprising or consisting of SEQ ID NO: 306, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0618] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0619] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0620] a) a heavy chain comprising or consisting of SEQ ID NO: 306, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0621] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0622] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0623] a) a heavy chain comprising or consisting of SEQ ID NO: 306, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0624] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0625] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0626] a) a heavy chain comprising or consisting of SEQ ID NO: 307, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0627] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0628] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0629] a) a heavy chain comprising or consisting of SEQ ID NO: 307, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0630] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0631] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0632] a) a heavy chain comprising or consisting of SEQ ID NO: 307, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0633] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0634] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0635] a) a heavy chain comprising or consisting of SEQ ID NO:308, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0636] b) a light chain comprising or consisting of SEQ ID NO:282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0637] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0638] a) a heavy chain comprising or consisting of SEQ ID NO:308, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0639] b) a light chain comprising or consisting of SEQ ID NO:283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0640] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0641] a) a heavy chain comprising or consisting of SEQ ID NO:308, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0642] b) a light chain comprising or consisting of SEQ ID NO:284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0643] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0644] a) a heavy chain comprising or consisting of SEQ ID NO:309, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0645] b) a light chain comprising or consisting of SEQ ID NO:282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0646] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0647] a) a heavy chain comprising or consisting of SEQ ID NO: 309, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0648] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0649] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0650] a) a heavy chain comprising or consisting of SEQ ID NO: 309, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0651] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0652] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0653] a) a heavy chain comprising or consisting of SEQ ID NO: 310, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0654] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0655] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0656] a) a heavy chain comprising or consisting of SEQ ID NO: 310, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0657] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0658] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0659] a) a heavy chain comprising or consisting of SEQ ID NO:310, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0660] b) a light chain comprising or consisting of SEQ ID NO:284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0661] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0662] a) a heavy chain comprising or consisting of SEQ ID NO:311, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0663] b) a light chain comprising or consisting of SEQ ID NO:282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0664] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0665] a) a heavy chain comprising or consisting of SEQ ID NO:311, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0666] b) a light chain comprising or consisting of SEQ ID NO:283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0667] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0668] a) a heavy chain comprising or consisting of SEQ ID NO:311, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0669] b) a light chain comprising or consisting of SEQ ID NO:284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0670] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0671] a) a heavy chain comprising or consisting of SEQ ID NO: 312, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0672] b) a light chain comprising or consisting of SEQ ID NO: 282, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0673] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0674] a) a heavy chain comprising or consisting of SEQ ID NO: 312, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0675] b) a light chain comprising or consisting of SEQ ID NO: 283, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0676] The antibody or antigen-binding fragment thereof can be a humanized antibody comprising or consisting of:

[0677] a) a heavy chain comprising or consisting of SEQ ID NO: 312, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and

[0678] b) a light chain comprising or consisting of SEQ ID NO: 284, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0679] The constant region domains of the antibody, if present, can be selected according to the intended function of the antibody molecule, in particular the effector functions that can be desired. For example, the constant region domains can be human IgA, IgD, IgE, IgG, or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are desired, human IgG constant region domains, especially IgGl and IgG3 isotypes, can be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not desired, IgG2 and IgG4 isotypes can be used. It will be appreciated that sequence variants of these constant region domains can also be used. The skilled person will also be aware that antibodies can undergo a variety of post-translational modifications. The type and extent of these modifications will generally depend on the host cell line used to express the antibody and the cell culture conditions. Such modifications can include glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and changes in asparagine deamidation.

[0680] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0681] The antibody or antigen-binding fragment thereof can comprise or consist of:

[0682] An ScFv comprising or consisting of: SEQ ID NO: 101, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0683] The antibody or antigen-binding fragment thereof can comprise or consist of:

[0684] An ScFv comprising or consisting of: SEQ ID NO: 102, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0685] The antibody or antigen-binding fragment thereof can comprise or consist of:

[0686] An ScFv comprising or consisting of: SEQ ID NO: 103, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0687] The antibody or antigen-binding fragment thereof can comprise or consist of:

[0688] an ScFv comprising or consisting of SEQ ID NO: 104, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0689] an antibody or antigen binding fragment thereof can comprise or consist of:

[0690] an ScFv comprising or consisting of SEQ ID NO: 105, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0691] an antibody or antigen binding fragment thereof can comprise or consist of:

[0692] an ScFv comprising or consisting of SEQ ID NO: 106, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0693] In another aspect, there is provided an antibody or antigen binding fragment thereof that binds an epitope on CD1 a, which epitope comprises or consists of residues Arg 83, Tyr 84, His 86, Glu 87, Gln 89, Phe 90, Glu 91, Asn 139, Met 140, Lys 142, His 143, Lys 146, Val 147, and Gln 150 of CD1 a, and wherein the residue numbering is according to SEQ ID NO: 252.

[0694] In another aspect, there is provided an antibody or antigen binding fragment thereof that binds an epitope on CD1 a, which epitope comprises or consists of residues Glu 62, Glu 65, Leu 66, Thr 68, Leu 69, Ile 72, Asn 151, His 153, Glu 154, Ile 157, Asn 160, Asp 164, Thr 165, and Arg 168 of CD1 a, and wherein the residue numbering is according to SEQ ID NO: 252.

[0695] In another aspect, there is provided an antibody or antigen binding fragment thereof that binds an epitope on CD1 a, which epitope comprises or consists of residues Glu 79, Arg 82, Arg 83,

[0696] His 86, Glu 87, Gln 89, Phe 90, Glu 91, Tyr 92, Val 147, and Asn 150 of CD1 a, and wherein the residue numbering is according to SEQ ID NO: 252.

[0697] More specifically, the antibody or antigen-binding fragment thereof binds one or more of the residues disclosed above, more specifically binds to 5 or more residues.

[0698] Epitopes can be identified in combination with any one of the antibodies provided herein by any suitable epitope mapping method known in the art. Examples of such methods include screening peptides of varying lengths derived from the full-length target protein for binding to an antibody or fragment thereof of the disclosure, and identifying the minimal fragment that can specifically bind the antibody, which contains the sequence of the epitope recognized by the antibody. Target peptides can be produced synthetically. Peptides that bind the antibody can be identified by, e.g., mass spectrometric analysis. In another example, NMR spectroscopy or X-ray crystallography can be used to identify the epitope bound by an antibody of the application. Generally, when epitope determination is performed by X-ray crystallography, amino acid residues of the antigen that are within 3.5 A of the CDRs of the antibody are considered part of the amino acid residues of the epitope. Once identified, the epitope can be used to make fragments that bind the antibody of the application, and if desired, used as an immunogen to obtain additional antibodies that bind the same epitope.

[0699] Epitopes can be determined using a variety of techniques available and known to one of skill in the art, such as X-ray crystallography.

[0700] By using routine methods known in the art, it is easy to determine whether an antibody binds to the same epitope as a reference antibody or competes for binding with a reference antibody. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to a protein or peptide under saturating conditions. Next, the ability of the test antibody to bind to the protein or peptide is assessed. If the test antibody is able to bind to the protein or peptide after saturating binding by the reference antibody, then it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to the protein or peptide after saturating binding by the reference antibody, then the test antibody can bind to the same epitope as the reference antibody bound.

[0701] In one embodiment, an antibody or antigen-binding fragment thereof is provided that competes for binding to CD1a with an antibody or antigen-binding fragment thereof.

[0702] The term "antibody or antigen-binding fragment thereof that competes with a reference antibody or antigen-binding fragment thereof" means an antibody or antigen-binding fragment thereof that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay.

[0703] ​To determine if an antibody competes for binding with a reference antibody, the above binding methods are performed in both directions. In the first direction, the reference antibody is allowed to bind to the protein / peptide under saturating conditions, and then the binding of the test antibody to the protein / peptide molecule is assessed. In the second direction, the test antibody is allowed to bind to the protein / peptide under saturating conditions, and then the binding of the reference antibody to the protein / peptide is assessed. If, in both directions, only the first (saturating) antibody is able to bind to the protein / peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be appreciated by those in the art, an antibody that competes for binding with a reference antibody can not necessarily bind to the same epitope as the reference antibody, but can sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0704] Two antibodies bind to the same or overlapping epitopes 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 essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0705] Additional routine experimentation (e.g., peptide mutagenesis 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 the cause of the observed lack of binding. Such 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.

[0706] The antibodies of the application, or antigen-binding fragments thereof, can be isolated.

[0707] In any aspect, “an antibody or antigen-binding fragment thereof” can refer to one or more, such as two, of the described antibodies or antigen-binding fragments thereof. For example, in any aspect, two antibodies or antigen-binding fragments thereof are contemplated, e.g., that bind CD1a at different binding sites.

[0708] For example, in any of the therapeutic applications disclosed herein and / or in any of the monitoring methods disclosed herein, any combination of antibodies or antigen-binding fragments can be utilized. For example, Ab 116 and 16 can be used in combination. Alternatively, Ab 16 and 110 can be used in combination.

[0709] In another embodiment, Ab 116 can be used in any of the therapeutic applications disclosed herein and Ab 16 can be used to monitor the same subject. Alternatively, Ab 16 can be used in any of the therapeutic applications disclosed herein and Ab 116 can be used to monitor the same subject.

[0710] Any reference to an antibody or antigen-binding fragment thereof (e.g., Ab 116 or Ab 16) by its internal name also includes any chimeric or humanized form disclosed herein.

[0711] The term "antibody" as referred to herein refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (V H ) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (V L ) and a light chain constant region. The variable regions of the heavy and light chains contain the binding sites for the antigen. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). The constant regions of the antibodies can 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.

[0712] The term "antigen-binding fragment thereof" of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. The antigen-binding fragment thereof can be, but is not limited to, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, a Fv, a single domain antibody (e.g., VH or VL or VHH), a scFv, a bivalent, trivalent, or tetravalent antibody, a di-scFv, a diabody, a triabody, a tetrabody, and an epitope-binding fragment of any of the above (Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217). Methods for generating and manufacturing these antigen-binding fragments are well known in the art (see, e.g., Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0713] The term "chimeric" antibody refers to an antibody in which the variable domains of the heavy and / or light chains (or at least a portion thereof) are derived from a particular source or species, while the remainder of the heavy and / or light chains (i.e., constant domains) are derived from a different source or species. (Morrison; PNAS 81, 6851 (1984)). A chimeric antibody may, for example, comprise a non-human variable domain and a human constant domain. Chimeric antibodies are typically produced using recombinant DNA methods. A subcategory of "chimeric antibodies" is "humanized antibodies."

[0714] The term "humanized" antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment comprising amino acid residues from non-human HVR and amino acid residues from human FR. Typically, the heavy chain and / or light chain contain one or more CDRs (if necessary, 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 transplanted into the heavy chain and / or light chain variable region framework of a receptor antibody (human antibody) (see, for example, Vaughan et al., Nature Biotechnology, 16, 535-539, 1998). The advantage of such humanized antibodies is that they reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Rather than transferring the entire CDR, one or more specificity-determining residues from any one of the above-mentioned CDRs are transferred to a human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from human FR. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

[0715] The term "framework" or "FR" refers to the variable domain residues other than the hypervariable region residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0716] The antibody or its antigen-binding fragment can be a monoclonal antibody, a full-length antibody, a bispecific antibody, a multispecific antibody, ScFv or other single chain or modified form, Fab, (Fab')2, Fv, dAb, Fd, nanobody, camel antibody or diabody. Preferably, the antibody or its antigen-binding fragment is a monoclonal antibody.

[0717] A bispecific antibody can comprise a CD1 a targeting moiety comprising an antibody or antigen binding fragment thereof of the present application and a T cell engaging moiety. The T cell engaging moiety can be a CD3 targeting moiety, such as antibody UCHT1 (SEQ ID NO: 298). The CD1 a targeting moiety can comprise a light chain variable region of SEQ ID NO: 314 and a heavy chain variable region of SEQ ID NO: 314, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto. The CD1 a targeting moiety can comprise a light chain of SEQ ID NO: 210 and a heavy chain of SEQ ID NO: 299, or a sequence at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto.

[0718] The term“full-length antibody” is used herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that contains 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) which is composed of three constant domains, CH1, CH2 and CH3, or four constant domains, CH1, CH2, CH3 and CH4, depending on the isotype. The constant regions of antibodies can 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.

[0719] The present inventors have targeted CD1a and its potential role in inflammatory skin and mucosal diseases and conditions, or related systemic diseases or conditions, or inflammatory drug responses with systemic manifestations by generating potent monoclonal antibodies. Because CD1a is highly expressed in the skin and mucosa, the use of such antibodies provides an opportunity to selectively treat inflammatory skin and mucosal diseases and conditions while minimizing off-target effects. CD1a is not expressed by mice, but is expressed by other mammals. Human CD1a (UniProtKB / Swiss-Prot: P06126-CD1A_HUMAN) is expressed worldwide by a dominant allele, with variants present in some Chinese ethnic groups (18). Targeting CD1a antigen presentation also intercepts inflammatory pathways upstream of other cytokine-directed antibody therapies (such as anti-IL17 therapy) or other immunotherapies, thereby providing a powerful means to modulate proinflammatory conditions early in the immune cascade. Furthermore, exploiting the specificity of CD1a for the skin may provide a means of directing additional therapies to the skin, for example by using bispecific or multispecific or conjugated antibody technology to specifically target small molecules, drugs, nucleic acids, peptides, antibodies or cell conjugate therapies. Furthermore, because CD1a is relatively non-polymorphic, the present invention offers general potential in the prevention and / or treatment of inflammatory skin and mucosal diseases such as atopic dermatitis and psoriasis, in which the frequency of CD1a-expressing dendritic cell subsets is increased and the migration pattern of LCs is altered (13-15), or in malignancies that express CD1a.

[0720] By modifying the number and function of cells expressing CD1a, the antibodies will have effects beyond lipid reactivity and affect all effects of cells expressing CD1a, including antigen presentation to peptide-specific T cells and innate pathways (e.g., neutrophils). The antibodies of the present invention are able to reduce Langerhans cells, despite their mouse IgG1 properties. This reduction provides a means of controlling a wide range of inflammatory pathways in the absence of complement / ADCC-related inflammation, which can provide therapeutic benefits. This is shown in the imiquimod model described herein, where the antibodies according to the present invention, for example, reduce inflammation to levels significantly lower than those in wild-type mice, demonstrating a profound anti-inflammatory effect on pathways other than cells expressing CD1a (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-γ and IL-22) associated with a wide range of clinical diseases.

[0721] In another aspect, the present application provides nucleic acids encoding the antibodies of the present application or antigen binding fragments thereof. Such nucleic acids can be provided by any one of SEQ ID Nos: 51-90. The skilled person will appreciate that due to codon redundancy, many DNA sequences can be used to encode the antibodies of the present application or antigen binding fragments thereof. Alternatively, codon optimization of the nucleotide sequence can be used to improve the efficiency of translation in the expression system used to produce the antibodies of the present application or antigen binding fragments thereof.

[0722] In another aspect, the present application provides vectors comprising the nucleic acids of the present application. Suitable vectors can be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. The vector can be, for example, a plasmid or a virus. Further details see, for example, (Sambrook, J., E.F. Fritsch and T. Maniatis. (1989), Molecular cloning: a laboratory manual, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Many known techniques and protocols for the manipulation of nucleic acids, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in (Ausubel et al., Current protocols in molecular biology. New York: Greene Publishing Association; Wiley-Interscience, 1992). The vector can be an expression vector. The vector or expression vector can be a plasmid.

[0723] The nucleic acid molecules or vectors of the present application can be expressed using any suitable expression system, for example, in a suitable host cell or in a cell-free system.

[0724] In another aspect, the present application provides host cells comprising the antibodies of the present application or antigen binding fragments thereof, nucleic acids and / or vectors. The host cells can 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 cells of the present application are capable of producing the antibodies of the present application or antigen binding fragments thereof. The produced antibodies or antigen binding fragments thereof can be enriched by selection and / or isolation.

[0725] The antibodies of the present application or antigen binding fragments thereof can also be produced by chemical synthesis. The obtained antibodies or antigen binding fragments thereof can be enriched by selection and / or isolation.

[0726] According to another aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a nucleic acid, a vector and / or a host cell of the present application, optionally together with one or more pharmaceutically acceptable excipients or diluents.

[0727] The antibody or antigen-binding fragment thereof, nucleic acid, vector or host cell of the present application can be formulated into pharmaceutical compositions using established methods of preparation (Gennaro, A. L. and Gennaro, A. R. (2000) Remington: The Science and Practice of Pharmacy, 20thEdition, Lippincott Williams & Wilkins, Philadelphia, PA). For preparing pharmaceutical compositions, pharmaceutically inert, inorganic or organic excipients can be used. Lactose, talc, stearic acid, its salts, fats, waxes, solid or liquid polyols, natural and hardened oils are examples of pharmaceutically acceptable excipients that can be used for preparing, for example, pills, powders, gelatin capsules or suppositories. Suitable excipients for a solution, suspension, emulsion, aerosol mixture or a powder for reconstitution into a solution or aerosol mixture before use include water, alcohols, glycerol, polyols and suitable mixtures thereof and vegetable oils.

[0728] The pharmaceutical compositions of the present application can be administered by any parenteral or non-parenteral (enteral) route that is therapeutically effective. Parenteral administration methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, for example, in the form of injection solutions, infusion solutions or mixtures, as well as aerosol installations and inhalation, for example, in the form of aerosol mixtures, sprays or powders. The pharmaceutical compositions of the present application can be administered systemically or locally in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and the required vehicles. In the case of compounds having a relatively short or long serum half-life or requiring a rapid onset of action, a combination of intravenous and subcutaneous infusion and / or injection can be the most convenient. Preferably, the pharmaceutical composition is administered subcutaneously or intravenously. The pharmaceutical composition can be an aqueous solution, an oil-in-water emulsion or a water-in-oil emulsion.

[0729] For intravenous injection, or injection at the site of disease or other administration site, the active ingredient will be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free, and has suitable pH, isotonicity and stability. A person skilled in the relevant art is able to prepare a suitable solution using, for example, isotonic vehicles such as chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as required.

[0730] The composition is preferably administered to the individual in a "therapeutically effective amount", which is sufficient to show a benefit to the individual. The optimal dosage will depend on the biodistribution of the antibody or antigen-binding fragment thereof, the mode of administration, the severity of the disease / condition being treated, and the medical condition of the patient. If desired, the antibody or antigen-binding fragment thereof can be administered in the form of a sustained release formulation, such as a liposome dispersion or a hydrogel-based polymeric microsphere, such as PolyActive™ or OctoDEXTM (see Bos et al., Business Briefing: Pharmatech 2003: 1-6). Other useful sustained release formulations are, for example, PLGA-based polymers (PR drugs), PLA-PEG-based hydrogels (Medincell), and PEA-based polymers (Medivas). Prescription of treatment (e.g. determination of dosage, etc.) is within the responsibility of the medical practitioner, and typically takes into account the condition being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to medical practitioners.

[0731] The pharmaceutical composition can also contain additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and solvents or solubilizers or agents for achieving a depot effect. The latter is that the fusion protein can be incorporated into slow or sustained release or targeted delivery systems, such as liposomes and microcapsules.

[0732] In another aspect, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or pharmaceutical composition of the application can be used in the treatment or prevention of one or more diseases or conditions in a subject.

[0733] In one aspect, there is provided a method of treating or preventing one or more diseases or conditions in a subject, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or composition of the application.

[0734] In one aspect, there is provided the use of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or pharmaceutical composition of the application in the manufacture of a medicament for the treatment or prevention of one or more diseases or conditions in a subject.

[0735] In any aspect, the subject can be a mammal. The mammal can express a CD1 a ortholog. Preferably, the subject is a human.

[0736] The one or more diseases or conditions can be one or more inflammatory skin or mucosal conditions or diseases, or one or more related systemic diseases or conditions, or a systemic manifestation of one or more inflammatory drug reactions, or a malignant tumor expressing CD1 a.

[0737] The inflammatory skin or mucosal disease or condition can be selected from:

[0738] a) predominantly neutrophilic skin diseases such as acne, generalized pustular psoriasis, plaque psoriasis, guttate psoriasis, palmoplantar pustulosis, SAPHO syndrome, acute febrile neutrophilic dermatosis (Sweets syndrome), histiocytoid neutrophilic dermatitis, dorsal hand neutrophilic dermatosis, pyoderma gangrenosum, neutrophilic eccrine hidradenitis, pyogenic hidradenitis, persistent papulosis, Behcet disease, enterohemorrhagic dermatitis-arthritis syndrome, other infection-associated inflammation, neutrophilic urticarial dermatosis, fencepost neutrophilic granulomatous dermatitis, creeping eruption, neutrophilic annular erythema, acute generalized exanthematous pustulosis (AGEP), vasculitis, and the like;

[0739] 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, panniculitis, lichen planus, erythema multiforma, lichen sclerosus, other lichenoid and erythema multiforme-like diseases, vesiculosis psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, Guillain-Barre syndrome, thyroiditis, transverse myelitis, neurodegeneration, and the like;

[0740] c) mast cell disorders and eosinophilic disorders such as Muckle Wells syndrome, hypereosinophilic syndrome and systemic symptoms syndrome, urticaria, angioedema, keratoconjunctivitis, food allergies, other allergic or atopic diseases including atopic dermatitis, rhinitis, conjunctivitis, asthma, eosinophilic esophagitis and other eosinophilic mucosal diseases, contact dermatitis, chronic obstructive airway disease, and the like;

[0741] d) adverse drug reactions manifesting as inflammatory skin or mucosal diseases or disorders such as Stevens Johnsons syndrome, toxic epidermal necrolysis, drug reactions with eosinophilia and systemic symptoms syndrome (DRESS) and acute generalized exanthematous pustulosis (AGEP), erythema multiforma, fixed drug eruptions, checkpoint inhibitor-associated skin and other inflammation, and the like;

[0742] e) graft versus host disease;

[0743] f) pruritus and pruritic conditions including nodular prurigo.

[0744] A malignant tumor expressing CD1a as referred to herein can be any malignant tumor in which CD1a expression can be detected. Such malignant tumors can include Langerhans cell histiocytosis, Langerhans cell sarcoma, a subset of T-cell lymphomas, a subset of thymomas or other rare cases of malignant tumors such as a subset of mastocytosis. Preferably, the malignant tumor expressing CD1a is a subset of T-cell lymphomas.

[0745] Preferably, the one or more diseases or conditions comprise or consist of psoriasis, dermatitis, lupus erythematosus, neutrophilic dermatosis, a related systemic disease or condition and / or a systemically manifested inflammatory drug reaction or a malignant tumor expressing CD1a.

[0746] A related systemic disease or condition as used herein can refer to any non-skin site involvement that can be associated with an inflammatory skin or mucosal disease or condition as defined herein. This can include non-cutaneous lupus erythematosus.

[0747] A systemically manifested inflammatory drug reaction can be at a non-skin site, such as the spleen. The related systemic disease or condition or systemically manifested inflammatory drug reaction can be a result of an inflammatory response. The inflammatory response can be, for example, an inflammatory response to a drug such as Aldara (5% imiquimod cream). The inflammatory response can 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-1 b and / or MCP-1, and / or a decrease in IL-10 and / or IL-27.

[0748] Furthermore, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition of the application can be administered alone or in combination with one or more other therapeutic agents, either simultaneously, sequentially or separately, depending on the condition to be treated. The one or more other therapeutic agents can be selected from the group comprising a cytotoxic agent, an immune-activating agent (such as a checkpoint inhibitor or a TLR agonist), an anti-inflammatory agent (such as a steroid), a CAR-T cell (such as a regulatory or cytolytic CAR-T cell) or other cell expressing or presenting one or more antibodies or antigen-binding fragments of the application.

[0749] In another aspect, there is provided a method of monitoring the efficacy of treatment or the disease status of a subject diagnosed with a malignant tumor expressing CD1a, comprising:

[0750] i. providing a biological sample obtained from the subject;

[0751] ii. determining the level of binding of one or more antibodies or antigen-binding fragments of the application to cells expressing CD1a in a sample obtained from the subject prior to treatment, or at intervals between treatments, or at intervals of time in the absence of treatment;

[0752] iii. if the tumor volume is reduced or the level of binding of one or more antibodies or antigen-binding fragments of the application to CD1a-expressing cells is reduced after treatment, or between treatment intervals, or in the absence of treatment intervals, the treatment is determined to be effective or the disease state is determined to be improving.

[0753] A biological sample as referred to herein can be a blood or serum sample, a tissue biopsy, cerebrospinal fluid, a saliva or urine sample. Preferably, the biological sample can be a blood or serum sample.

[0754] The level of binding of one or more antibodies or antigen-binding fragments of the application to CD1a-expressing cells in a sample can be determined using any method known to the skilled person. One such method is for example using flow cytometry or any other technique that makes use of detectable labels to be able to determine the number of CD1a-expressing cells in a sample.

[0755] The tumor volume can be determined by any suitable technique known to the skilled person.

[0756] The reduction in tumor volume or reduction in the level of binding of one or more antibodies or antigen-binding fragments of the application to CD1a-expressing cells can be by 10% or more, such as 25% or more, 50% or more, 75% or more or 90% or more.

[0757] The treatment interval or the absence of treatment intervals can be two weeks or more, such as four weeks or more, 8 weeks or more, 12 weeks or more, six months or more or 12 months or more.

[0758] In another aspect, there is provided a method of diagnosing a subject having an inflammatory skin and mucosal disease or disorder, or an associated systemic disease or disorder, or a systemic manifestation of an inflammatory drug reaction, or a malignant tumor expressing CD1a, comprising:

[0759] i. providing a biological sample obtained from the subject;

[0760] ii. determining the level of expression of CD1a in the sample obtained from the subject using one or more antibodies or antigen-binding fragments thereof of the application;

[0761] iii. comparing the level of expression of CD1a in the sample obtained from the subject to the level of expression of CD1a in a positive or negative reference sample;

[0762] iv. If the expression level of CD1 a in the sample obtained from the subject is higher than the expression level of CD1 a in the negative reference sample, or equal to or higher than the expression level of CD1 a in the positive reference sample, the subject is determined to have an inflammatory skin and mucosal disease or disorder, or an associated systemic disease or condition, or a systemically manifested inflammatory drug reaction, or a CD1 a-expressing malignancy.

[0763] Alternatively, in step iv, if the expression level of CD1 a in the sample obtained from the subject is equal to or lower than the expression level of CD1 a in the negative reference sample, or lower than the expression level of CD1 a 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 condition, or a systemically manifested inflammatory drug reaction, or a CD1 a-expressing malignancy.

[0764] The negative reference sample can refer to a biological sample taken from a healthy subject known not to have an inflammatory skin and mucosal disease or disorder, or an associated systemic disease or condition, or a systemically manifested inflammatory drug reaction, or a CD1 a-expressing malignancy. The positive reference sample can refer to a biological sample taken from a subject who has been diagnosed with an inflammatory skin and mucosal disease or disorder, or an associated systemic disease or condition, or a systemically manifested inflammatory drug reaction, or a CD1 a-expressing malignancy.

[0765] The expression level of CD1 a in the diagnostic method can refer to the level of CD1 a molecules expressed on a given cell in a population, or the percentage of cells in a population or sample determined to express CD1 a.

[0766] 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, lgG2, etc.). 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, which alone are capable of binding an antigen. The antibody or antigen-binding fragment thereof can be chimeric, nanobody, single-chain, and / or humanized. The antibody or antigen-binding fragment thereof can be of human IgG1 isotype or human IgG4 isotype or other natural or modified isotype. The antibody can be monoclonal (mAb) or polyclonal.

[0767] The antibody or antigen-binding fragment thereof can be modified to alter in vivo stability and / or half-life. The modification can be, for example, PEGylation.

[0768] The antibody or antigen-binding fragment thereof can be an antibody-like molecule, which includes the use of CDRs, alone or in combination, in synthetic molecules such as SMIPs and small antibody mimetics.

[0769] 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 in the first sequence for optimal alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is that of the two sequences which results in the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides within the sequences (i.e., % identity = number of identical positions / total number of positions x 100).

[0770] Determination of the percent identity between two sequences can be accomplished using mathematical algorithms known to persons of skill in the art. An example of a mathematical algorithm that can be used is the algorithm of Karlin and Altschul, 1990, PNAS, 87(6): 2264-8, as modified 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, have been adapted to use this algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to nucleic acid molecules of the application. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to protein molecules of the application. 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 which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, 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 that can be used for sequence comparison is the algorithm of Myers and Miller. The ALIGN program (version 2.0) of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms known to persons of skill in the art for sequence analysis 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.

[0771] An antibody or antigen-binding fragment thereof of the application can comprise one or more mutated amino acid residues. The terms "mutated", "mutant" and "mutation" with respect to a nucleic acid or antibody or antigen-binding fragment thereof of the application refer to a substitution, deletion or insertion of one or more nucleotides or amino acids as compared to a "native" existing nucleic acid or polypeptide, i.e., as compared to a reference sequence that can be used to define the wild type.

[0772] The amino acid variations in the CDR sequences can be conservative amino acid substitutions.

[0773] The mutation can be a substitution, wherein the substitution is a conservative substitution. A conservative substitution is typically one of the following substitutions listed according to the amino acid to be mutated, each followed by one or more replacements that can be considered conservative: Ala→Gly, Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gin→Asn; Glu→Asp; Gly→Ala; His→Arg, Asn, Gin; He→Leu, Val; Leu→Ile, Val; Lys→Arg, Gin, 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 allowed and can be determined empirically or according to other known conservative or non-conservative substitutions.

[0774] One, two or three conservative substitutions can be made in the CDRs of the antibody or antigen binding fragment thereof of the application.

[0775] Methods of making antibodies or antigen binding fragments thereof are well known in the art. The skilled person can use, for example, hybridoma technology or can clone the respective antibody sequence into a vector, such as an expression vector, using recombinant DNA technology. Methods of making bispecific antibody molecules are known in the art, for example recombinant DNA technology, chemical conjugation of two different monoclonal antibodies or chemical conjugation of, for example, two antibody fragments, for example, two Fab fragments. Alternatively, bispecific antibody molecules are made by cell hybridoma technology, which produces hybridomas of the parent antibodies by fusion. Due to the random assortment of H and L chains, a potential mixture of ten different antibody structures is created, of which only one has the desired binding specificity. The bispecific antibody molecule of the application can act as a monoclonal antibody (mAb) against each target. The antibody or antigen binding fragment thereof can be chimeric, humanized or fully human. The antibody or antigen binding fragment thereof can be of human IgGl isotype or human IgG4 isotype or other natural or modified isotype. The bispecific antibody molecule or multispecific antibody can be, for example, a bispecific tandem single-chain Fv, a bispecific Fab2 or a bispecific diabody.

[0776] Reference to“OX16”,“OX116”,“OX110”,“OX111”,“OX77a” or“OX25” refers to antibody 16, 116, 110, 111, 77a or 25, respectively (as defined in Table 11).

[0777] All features disclosed in this specification can be combined in any combination, including with any aspect or any embodiment. BRIEF DESCRIPTION OF DRAWINGS

[0778] Figure 1 - Display of inhibition of polyclonal T cell responses by a panel of anti-CD1a antibodies. A. Dose titration curves of polyclonal T cell IFNy responses with increasing concentrations of anti-CD1a antibodies (0.01-10 pg / ml) (n=6 donors). B. IC50 values calculated for the panel of newly generated anti-CD1a antibodies and commercial antibodies (OKT6, HI149 and SK9) (n=6 donors).

[0779] Figure 2 - Display of 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 K562 transfected with empty vector (EV) or CD1a presenting endogenous ligands. Inhibition of IFNy (A.) or IL-22 (B.) by the panel of newly generated anti-CD1a antibodies was assessed by flow cytometry. C. IFNy secretion responses of CD1a-restricted enriched T cell lines induced by CD1a coated beads presenting endogenous ligands. Inhibition by the panel of newly generated anti-CD1a antibodies was assessed by flow cytometry. (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, where * indicates significance compared to “CD1a”).

[0780] Figure 3- Characterization of CD1a transgenic mice. A. Representative flow cytometry plots, and B. graphical summary of CD1a protein expression on cells of wild-type (WT) and CD1a transgenic (CD1a) mice. CD1a protein expression was evaluated on (left-to-right) total live ear skin cells, CD45+ skin cells, dermal dendritic cells (dDC, CD45+ / CD11c+ / langerin-), and Langerhans cells (LC, CD45+ / CD11c+ / langerin+). C. CD1a protein expression within ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice as observed by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red), scale bars from left to right are 50 pm, 50 pm, and 10 pm. D. Exemplary PCR genotyping of CD1a transgenic mouse littermates using CD1a forward and reverse primers and tail genomic DNA (lanes A-F). The expected CD1a band is at 655 bp. Lane G: positive control genomic DNA from founder mouse. Lane H: negative control lacking DNA template. E. Representative flow cytometry plots of thymus CD1a protein expression of wild-type (WT) and CD1a transgenic (CD1a) mice.

[0781] Figure 4 - Characterization of anti-CD1a antibodies in vivo. A. Schematic of imiquimod-induced skin inflammation and prophylactic administration of anti-CD1a. B. Daily measurements of CD1a transgenic mouse (CD1a) ear swelling by imiquimod treatment i.p. injected with mouse IgG1 isotype control and CD1a transgenic injected with the panel of modified anti-CD1a antibodies as shown in schematic A. (N=6, two-way ANOVA with Dunnett’s test, **, P<0.01; ****, P<0.0001 indicates significance at day 6 or as indicated compared to “CD1a”).

[0782] Figure 5 - Demonstration of the effect of anti-CD1a on imiquimod-induced skin immune responses. A-C. Flow cytometry analysis of ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice treated with mouse IgG1 isotype and CD1a transgenic injected with the panel of modified anti-CD1a antibodies following prophylactic model of administration. Skin T cells (A.) were counted and cell surface CD69 expression (B.) was evaluated, and skin neutrophil (C.) and eosinophil (D.) frequencies were determined. (N=4, one-way ANOVA with Dunnett’s test, *, P<0.05; **, P<0.01; ***, P<0.001).

[0783] Figure 6- Display the effect of anti-CD1 a on imiquimod-induced cellular Langerhans cell skin and lymph node responses. Flow cytometry analysis of ear skin (A-B.) and draining cervical lymph nodes (C-D.) of wild type (WT) and CD1 a transgenic (CD1 a) mice treated with mouse IgG1 isotype and CD1 a transgenic mice injected with improved anti-CD1 a antibody groups after prophylactic model administration. Skin LCs (A.) were counted and cell surface CD1 a expression (B.) was evaluated. Lymph node LCs (C.) were counted and cell surface CD1 a expression (D.) was evaluated. (N=4, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0784] Figure 7- Displayed antibody-dependent depletion (phenotypic change). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell reduction (such as death). Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 pg / ml) was incubated with EV or CD1a-K562 for 48 hours as indicated and the percentage of antibody-induced reduction was calculated relative to the reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell reduction with increasing concentration of anti-CD1a antibody (0.625 pg / ml - 5 pg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 pg / ml) was incubated with MoDC (upper panel) and MoLC (lower panel) for 5 days with addition of antibody and cytokines at day 0 or day 2 and using Incucyte live cell imaging, the percentage of antibody-induced reduction was calculated relative to isotype control as measured by percentage of confluence (N=4, two-way ANOVA with Tukey test.)(C.) and representative images of MoDC (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 hours and stained with Annexin V and analyzed by flow cytometry. (N=3-4, one-way ANOVA with Tukey test.) F. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum for 3 hours at 37°C in the presence of 5 pg / ml isotype control antibody or indicated antibodies. The percentage of cytotoxicity was calculated relative to the reference population of untreated K562 and normalized to isotype control treated cells. (N=6, one-way ANOVA with Tukey test.) G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMC at 1 :50 ratio for 5 hours at 37°C in the presence of 5 pg / ml isotype control antibody or indicated antibodies. The percentage of cytotoxicity was calculated relative to the reference population of untreated K562 and normalized to isotype control treated cells. (N=4-6, one-way ANOVA with Tukey test.) H. NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 pg isotype control antibody or indicated antibodies at day 6, day 10 and day 14. Tumor volume was measured over time. (N=6-15, two-way ANOVA with Tukey test, asterisks indicate significance compared to “CD1a-iso” at day 18). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0785] Figure 8 (A) - is a heatmap from CD1a epitope analysis. Matrix heatmap representation of CD1a antibody binding by flow cytometry as measured by CD1a-AF647 mean fluorescence intensity (MFI). Relevant purified antibodies were incubated with cells to assess interference of AF647-conjugated antibodies to CD1a binding prior to staining CD1a-K652 with anti-CD1a antibody conjugated to fluorophore AF647. Gray scale shows the degree of interference, where shades in top row (-) indicate no interference. (B) - demonstrates results of in vivo CD1a antibody epitope competition assay. A. Flow cytometry plots of CD1a expression as measured by staining with anti-CD1a antibody SK9 (left panel) or HI149 (right panel). Anti-CD1a antibody 116 (100 pg i.p.) was administered on days 0, 2, and 4, and ear skin tissue was collected, processed, and stained for CD1a on day 5.

[0786] Figure 9 - demonstrates effectiveness of anti-CD1a antibodies in treating imiquimod-induced inflammation. A. Schematic of imiquimod-induced inflammation model with therapeutic anti-CD1a administration. B. Daily measurements of ear swelling, and C. Representative images of CD1a transgenic induced inflammation (day 8) by imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a), followed by i.p. treatment with mouse IgG1 isotype control or injection of improved anti-CD1a antibody groups as shown in schematic A (arrows at day 3) (N=2-10, two-way ANOVA with Dunnett’s test, **, P<0.01; ****, P<0.0001 indicates significance at day 8 or as shown compared to “CD1a”). D. Ear and epidermis thickness and CD1a protein expression within ear skin of wild type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) as observed by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red), scale bar 10 pm upper panel and 100 pm lower panel. E-G. Flow cytometry analysis of ear skin of mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a) and CD1a transgenic injected with improved anti-CD1a antibody groups following administered therapeutic model. Skin T cells were counted and cell surface CD11a expression was assessed (E.), and neutrophil (F.) and eosinophil (G.) frequencies were determined. (N=7-9, one-way ANOVA with Dunnett’s test, *, P<0.05; **, P<0.01; ***, P<0.001).

[0787] Figure 10- Demonstrating the CD1a dependence of the systemic effects exerted by imiquimod. A. Wild-type (WT) and CD1a transgenic mice (CD1a) were treated with imiquimod on day 8, followed by ip treatment with a mouse IgG1 isotype control or as shown in the schematic ( Figure 9 (A) shows the CD1a transgenic mice injected with the modified anti-CD1a antibody group, with spleen weight (mg) measurements and representative images. B-E. Flow cytometric analysis of spleens of wild-type (WT) and CD1a transgenic mice (CD1a) treated with the IgG1 isotype, as well as those injected with the CD1a transgenic mice with the modified anti-CD1a antibody group after administration of the therapeutic model. Splenic CD4 (B.) and CD8 (C.) T cell CD69 expression was assessed, and neutrophils (D.) and eosinophils (E.) were counted. (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 blood of wild-type (WT) and CD1a transgenic (CD1a) mice treated with IgG1 isotype, and CD1a transgenic injected with anti-CD1a antibody after administration of the therapeutic model (N=7-9, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0788] Figure 11 - Demonstrates CD1a dependence of the systemic effects exerted by imiquimod. AE. Blood cell analysis of wild-type (WT) and CD1a transgenic (CD1a) mice treated with IgG1 isotype, as well as CD1a transgenic mice injected with an improved anti-CD1a antibody panel following administration of a therapeutic model. Circulating T cells (A), CD4+ (B), and CD8+ (C), neutrophils (D), and eosinophils (E) were counted. (N = 5-7, one-way ANOVA with Dunnett's test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0789] Figure 12 - Shows that imiquimod does not constitute a CD1a ligand. Isoelectric point-dependent migration of mock and imiquimod "loaded" CD1a protein on isoelectric focusing (IEF) gels pH 3-7. Mock: Vehicle control TBS 2% CHAPS 7% DMSO.

[0790] Figure 13- Effectiveness of the use of anti-CD1 a antibodies in the sustained control of imiquimod-induced inflammation. A. Schematic of the model of imiquimod re-challenge without subsequent anti-CD1 a administration. B. Daily measurement of CD1 a transgenic induced ear swelling injected with the improved anti-CD1 a antibody panel and IL-17A. dx = days of the model to reach significance compared to CD1 a transgenic ear thickness. Figure 13 Daily measurement of CD1 a transgenic induced ear swelling injected with the improved anti-CD1 a antibody panel (two-way ANOVA with Dunnett's test, *, P < 0.05; **, P < 0.01 indicates significance compared to "CD1 a" isotype on day 7 imiquimod re-application).

[0791] Figure 14 - Effectiveness of the use of anti-CD1 a antibodies in the treatment of imiquimod-induced inflammation compared to the standard of care. Wild type (WT) and CD1 a transgenic mice (CD1 a) were treated with imiquimod followed by i.p. treatment with mouse IgG1 isotype control (CD1 a) or as Figure 9 Daily measurement of CD1 a transgenic induced ear swelling injected with the improved anti-CD1 a antibody panel and IL-17A. dx = days of the model to reach significance compared to CD1 a transgenic ear thickness.

[0792] Figure 15- Comparative analysis of the effectiveness of anti-CD1 a antibodies in the treatment of imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with therapeutic anti-CD1 a administration. B. Daily measurements of CD1 a transgenic induced ear swelling by imiquimod treatment of wild type (WT) and CD1 a transgenic mice (CD1 a), followed by i.p. treatment with mouse IgG1 isotype control or injection of CD1 a transgenic with the panel of improved anti-CD1 a antibodies or CR2113 as shown in schematic A. N=2-7, two-way ANOVA with Dunnett’s test, *, P<0.05; **, P<0.01; ****, P<0.0001 indicates significance compared to “CD1 a” on day 8 or OX116 on day 8 compared to CR2113. C. Data presented in (B) and comparison, corrected for WT. D. Schematic of MC903-induced inflammation and prophylactic MC903-induced inflammation. E. Daily measurements of CD1 a transgenic induced ear swelling by MC903 treatment of wild type (WT) and CD1 a transgenic mice (CD1 a), followed by i.p. treatment with mouse IgG1 isotype control or injection of CD1 a transgenic with 16, 110 or 116 anti-CD1 a antibodies or CR2113 as shown in schematic D. N=3-4, two-way ANOVA with Dunnett’s test, *, P<0.05, indicates significance compared to “CD1 a” on day 7. F. Skin T cell percentages 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.

[0793] Figure 16 - Comparative analysis of the role of anti-CD1 a antibodies in skin and systemic immune responses with imiquimod-induced inflammation. Analysis of ear skin, draining cervical lymph nodes and plasma samples from CD1 a transgenic injected with the panel of improved anti-CD1 a antibodies following the therapeutic model of administration as shown in schematic Figure 15 A. Analysis of skin T cell IL-17A expression (left panel) using intracellular cytokine expression detected directly ex vivo by flow cytometry and counting of cervical lymph node eosinophils (right panel) from CD1 a transgenic injected with the panel of improved anti-CD1 a antibodies following the therapeutic model of administration as shown in schematic A. B-C. Plasma (B) and skin digest (C) cytokine levels 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).

[0794] Figure 17 - Crystal structure analysis of OX16, OX110, OX116

[0795] Summary of the crystal structure of human CD1a in complex with three different antibody fragments. Left panel: the heavy chain of CD1a is shown in gray, the β2-microglobulin in blue, and the respective scFv molecule is depicted in green for OX16, in yellow for OX110, and in pink for OX116. The segments of each scFv fragment corresponding to the V H domains are shown in darker colors, and the V L segments in lighter shades. Right panel: surface representation of the CD1a region (gray) recognized by each antibody fragment. The CD1a residues that contact each scFv molecule are shown in the depicted color. Contact residues are considered to be within a distance of 3.5 A from the interacting chain.

[0796] Figure 18 - Role of lipids in the binding of OX16 and OX116 to CD1a

[0797] The interaction between OX16 or OX116 scFv and CD1a carrying 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) over flow cells containing OX16-scFv (top) or OX116-scFv (bottom). The Bmax values are in relative response units, and the K D values are provided in nM.

[0798] Figure 19 - Role of anti-CD1a antibodies in blocking polyclonal and clonal T cell function

[0799] ​A-B. Assay of the ability of anti-CD1a antibody variants to inhibit CD1a-dependent activation polyclonal T cell IFNy (A.) and IL-22 (B.) production. T cells were isolated from donor PBMC by CD3 microbead separation. T cells were co-cultured with CD1a-K562 or EV-K562 overnight and IFNy or IL-22 production was detected by ELISpot in the presence of 10 pg / ml of anti-CD1a antibodies. Antibody formats were compared to mouse IgGl isotype control (top row statistics) or to the corresponding isotype (bottom row statistics). The % of blockade was calculated when comparing antibody treatment and isotype control after subtraction of the EV background level of 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 response of CD1a-restricted T cell clones induced by K562 presenting endogenous ligands, either transfected with empty vector (EV) or CD1a. IFNy inhibition by anti-CD1a antibodies was assessed by flow cytometry and normalized to 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).

[0800] Figure 20 Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of anti-CD1a antibodies

[0801] A. Flow cytometry analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum for 3h at 37°C in the presence of 5 pg / ml of isotype control antibody or the indicated antibodies. The percentage of cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells.

[0802] B. Flow cytometry analysis of antibody-dependent cellular cytotoxicity (ADCC). K562-CD1a cells were incubated with PBMC at an effector / target ratio of 50:1 for 5h at 37°C in the presence of 5 pg / ml of isotype control or the indicated antibodies and 0.5% FCS and 100 U / ml IL-2. The percentage of cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control-treated cells.

[0803] (N=4-6, one-way ANOVA with Tukey’s test.)*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0804] Figure 21 Inhibition of TCR binding to CD1a by OX116

[0805] A. Schematic of 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 gammadelta TCR - red; BK6 alpha beta TCR - blue CO22 gammadelta TCR - green) with CD1a bound to OX116 Ab fragments.

[0806] Figure 22 Cloning of OX25 CD1a binding characteristics

[0807] A. Investigation of CD1a-transfected cells and recombinant proteins (and controls) binding to antibody OX25 by flow cytometry (geometric mean of fluorescence intensity) and ELISA (optical density). Target cells and proteins include major and minor variants of CD1a as well as cynomolgus CD1a, and cells naturally expressing CD1a (MOLT4). B. ELISA (optical density) of full length CD1a compared to chimeric CD1a with human alpha 1 and alpha 2 domains fused to murine alpha 3 domain of CD1d using size exclusion library B. Surface plasmon resonance of OX25 interaction with CD1a. D. Heavy and light chain CDR3 regions of OX25.

[0808] Figure 23 Humanized antibodies can deplete CD1a expressing transfectants. Modified human IgGl anti-CD1a antibodies or isotype control (5 pg / ml) were incubated with K562-CD1a for 48 hours as indicated. The percentage of reduction induced by the antibodies was calculated relative to the isotype control using Incucyte live cell imaging, as measured by the percentage of confluence. (N=3, two-way ANOVA with Dunnett’s test, **, P<0.01; ****, P<0.0001, where * indicates significance compared to “CD1a”).

[0809] Figure 24 Humanized antibodies can inhibit CD1a autoreactive T cells. Cytokine secretion response of CD1a-restricted enriched T cell lines induced by K562 presenting endogenous ligands transfected with empty vector (EV) or CD1a. IFNy inhibition of modified human IgGl anti-CD1a antibody panel and isotype control (5 pg / ml) was assessed by flow cytometry. (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”).

[0810] Figure 25 - OX25 binds the a-3 domain of CD1a. Characterization of anti-CD1a antibody OX25. A. ELISpot analysis of OX25 and commercially available comparators (SK9 - non-blocking antibody, OKT6 and HI148 - blocking antibodies). IFNy response of polyclonal T cells to overnight co-culture with CD1a-transfected (CD1a) or empty vector (EV) K562 model antigen presenting cells. Effect of anti-CD1a antibodies (10 pg / ml) on T cell activation was measured by IFNy (IFNy spot) ELISpot (n = 8 T cell donors). B. Matrix heat map representation of CD1a antibody binding by flow cytometry as measured by CD1a-AF647 mean fluorescence intensity (MFI). Relevant purified antibodies were incubated with K562 cells prior to staining CD1a-K652 or empty vector (EV) K562 with anti-CD1a antibodies conjugated to fluorophore AF647 to assess interference of AF647-conjugated antibodies with CD1a binding. Grey scale shows degree of interference, with shades in top row (-) indicating no interference, 100% binding.

[0811] Figure 26 - Demonstration of OX116 effectiveness 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 measurement of ear swelling induced by imiquimod treatment i.p. injected with IgG1 mouse isotype control, anti-PD1 (J43) or anti-CD1a OX116 in wild-type (WT) and CD1a transgenic mice (CD1a) as indicated in schematic A. (N = 4, two-way ANOVA with Dunnett’s test, *, P < 0.05, **, P < 0.01; indicates significance as shown on day 6). C. Intracellular flow cytometry analysis of ear skin of wild-type (WT) and CD1a transgenic (CD1a) treated with or without imiquimod and i.p. injected with mouse IgG1 isotype control, anti-PD1 (J43) or anti-CD1a (OX116). Skin T cells were identified by CD3+ surface staining and IL-17 production was analyzed ex vivo by intracellular staining with anti-IL17 antibody.

[0812] Figure 27A. Pruritus and itch cytokines were significantly reduced after administration of anti-CD1 a antibodies. hCD1 a and WT mice were topically treated with 1 nmol MC903 / EtOH on days 0, 2 and 5. OX116, OX16 and isotype control were administered intraperitoneally at 100 ug / 100 ul on days -2, 0, 2 and 4. (A) Pruritus frequency was assessed at endpoint. Each data plot represents a single ear. Graphs 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 were reduced in skin tissue after administration of anti-CD1 a antibodies. hCD1 a and WT mice were topically treated with 1 nmol MC903 / EtOH on days 0, 2 and 5. OX116, OX16 and isotype control were administered intraperitoneally at 100 ug / 100 ul on days -2, 0, 2 and 4. Levels of TSLP (left panel) and IL-33 (right panel) (pg / ml per ear) were assessed at endpoint. Each data plot represents a single ear. Graphs 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. TM Levels of TSLP (left panel) and IL-33 (right panel) (pg / ml per ear) were assessed at endpoint. Each data plot represents a single ear. Graphs 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.

[0813] Figure 28- shows that the 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 expressing an activation reporter (NFAT-GFP) (50,000 each at effectors: targets 1 : 1). WIMM-3 bispecific antibody was added and incubated for 18 hours at 37°C, 5% C02. Cells were stained with a 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 each) and co-cultured with 125,000 human CD8+ T cells that had been isolated from PBMC and stimulated with anti-CD3 and anti-CD28 beads for 12 days. Co-cultures were incubated for 48 hours. B. Anesthetized NSG mice (n=7 per group) were injected subcutaneously in the flank with 500,000 K562 cells expressing CD1a. Four days later, mice were injected intravenously with human CD8+ T cells that had been isolated from PBMC and stimulated with anti-CD3 / anti-CD28 beads 14 days earlier. Mice were also injected with 100 μl of bispecific antibody WIMM-3 (0.5 mg / Kg) or PBS. Fourteen days after the first injection, mice were treated a second time with human CD8+ T cells and WIMM-3 or PBS. The left panel shows the mean tumor size, the right panel shows the data for individual mice, and the lower panel shows survival. Student's t-test was used at each time point. P value <0.0001 ****, <0.001 ***, <0.005 **, <0.05 *.

[0814] Materials and Methods

[0815] Mice

[0816] All mice were housed in specific pathogen-free facilities. In individual experiments, the age, sex, and background strain of mice were matched with wild-type littermates used as matched controls. All experiments performed in this study were performed under approval of the UK Home Office.

[0817] Generation of CD1a transgenic mice

[0818] Mice were generated at the Wellcome Trust Centre for Human Genetics, Oxford. A 5.7 kb genomic fragment encompassing the entire CD1A, including 0.8 kb of upstream sequence and 0.8 kb of downstream sequence, was amplified by PCR from human genomic DNA using primers 5'-ATGGTACCAAGAGGAATGTAAATGTGTCCGGC-3' and 5'-AAGCGGCCGCGATCATGTTAACCAAGGTCAGGAA-3', and subcloned into the Litmus 28 vector (NEB) by Kpnl and Notl sites incorporated into these PCR primers. After sequence verification of the coding exon sequences, the fragment transgene was excised from the vector backbone, purified and resuspended at 2 ng / ul in microinjection buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA) and microinjected into the pronuclei of fertilised oocytes prepared from C57BL / 6J mice. After overnight incubation, the resulting 2-cell embryos were surgically implanted into the oviducts of pseudopregnant CD1 outbred dams and allowed to go to term. Transgenic offspring were identified by PCR using transgene-specific primers and bred as separate lines with wild-type C57BL / 6J mice.

[0819] CD1a genotyping

[0820] Rough genomic DNA preparation was performed on ear notch samples from CD1a transgenic mice. 100 μl of DirectPCR ear lysis buffer (Viagen) supplemented with 0.4 mg / ml proteinase K (Sigma) was added to the ear notch and incubated overnight at 55°C. The enzyme was then heat inactivated at 85°C for 1 hour. The samples were centrifuged to pellet debris and the lysate was transferred to a clean tube. 1 μl of lysate was used as template for genotyping. The following PCR reaction was used for genotyping. The PCR products were loaded with Syber Safe onto a 1% TAE agarose gel, run electrophoretically and the gel was imaged under UV. If the expected band at 655 bp was detected, the mouse was considered positive for the CD1a transgene.

[0821]

[0822] 1. Initial denaturation 95°C, 2 minutes Cycling: Steps 2-4 X34 2. Denaturation 95°C, 20 seconds 3. Annealing 60°C, 15 seconds 4. Extension 72°C, 20 seconds 5. Final extension 72°C, 2 minutes 6. Hold 4℃

[0823] Cell lines

[0824] K562 cells trans fected with empty vector (EV-K562) and CD1a (CD1a-K562) cells (kindly gifted by B. Moody, Brigham and Women's Hospital, Harvard Medical School, Boston, MA) were maintained in RPMI 1640 medium supplemented with 10% FCS, 100 IU / ml penicillin, 100 pg / ml streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 1 x non-essential amino acids (NEAA) (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 500 mM 2-mercaptoethanol (Gibco), and 200 pg / ml G418 antibiotic (Thermo Fisher Scientific).

[0825] ELISpot analysis

[0826] ELISpot assays (IFNy ELISpot kit, Mabtech, AB) were used to detect activation-induced cytokine secretion from polyclonal T cells following co-culture with antigen presenting cells expressing model CD1a. PBMC from healthy donor blood were isolated by density gradient (Lymphoprep) and T cells purified using anti-CD3 beads according to the manufacturer's protocol (MACS, Miltenyi). All study participants gave full informed consent [National Health Service (NHS) National Research Ethics Service (NRES) Research Ethics Committee 14 / SC / 0106]. T cells were then cultured with IL-2 (200 U / ml) for 3 days to expand numbers before being co-cultured overnight with either un-pulsed / endogenous lipid loaded CD1a transfected K562 (CD1a-K562) or control empty vector transfected K562 (EV-K562). To assess functionality of anti-CD1a antibodies, K562 were incubated with 10 pg / ml anti-CD1a antibodies for 1 hour prior to and during co-culture with polyclonal T cells in anti-IFNy or anti-IL-22 capture antibody coated ELISpot plates (Millipore Corp., MA). IFNy and IL-22 secretion was detected with biotinylated anti-cytokine detection antibodies and visualised with streptavidin-alkaline phosphatase colour development. Resulting spots indicate cytokine producing T cells and were counted using an automated ELISpot reader (Autimmun Diagnostika gmbh ELISpot Reader Classic) and % blockade calculated when comparing antibody treated and untreated groups after subtracting the EV background level of cytokine producing spots. EV-K562 contribution was subtracted from CD1a IFNy / IL-22 spot counts. Adjusted CD1a-K562 antibody treated group spot counts were then divided by the no antibody group's CD1a and used to calculate % blockade.

[0827] CD1a-reactive T cell generation and activation analysis :

[0828] CD1a restricted T cells were isolated by fluorescence activated cell sorting. T cells were co-cultured with EV-K562 of CD1a-K562, and cytokine-producing responder T cells were detected using the Miltenyi MACS cytokine secretion assay according to the manufacturer's instructions. Briefly, after 6 hours of culture with CD1a-K562, T cells were coated with anti-cytokine (IL-22 or IFNγ) antibodies to detect CD1a-dependent autocrine cytokine production. The live responder cells were then sorted into culture plates. CD1a restricted T cells were amplified using mixed lymphocyte reaction, and the purity and CD1a responsiveness were assessed using the above-mentioned FACS-based cytokine secretion assay using analytical flow cytometry. The activation of CD1a restricted T cells was analyzed as follows. 2×10 5 K562 cells and 1-5 × 10 5 CD1a autoreactive T cell clones were co-cultured for 4 hours. Accessory cytokines were added to the co-cultures to support CD1a-dependent cytokine production. IFNγ-producing T cell cultures were supplied 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 cytokine production by T cells using a cytokine secretion assay (Miltenyi Biotec) according to the manufacturer's instructions.

[0829] Murine imiquimod administration

[0830] Mice were lightly anesthetized with isoflurane and were treated with 40 mg / kg dapoxetine on days 0, 1, 2, 3, 4, and 5 ( Figure 4 A) or in the treatment model at day 0, day 1, day 2 and day 4, day 5, day 6, day 7 ( Figure 9 A) 15 mg of Aldara cream containing 5% imiquimod was applied to the dorsal and ventral sides of the auricle. In the prevention model, the patients were treated with 15 mg of Aldara cream containing 5% imiquimod on days -5, -3, -1, 1, 3, 5, and 10 days. Figure 4 A) or in the treatment model at day 3, day 5, day 7 ( Figure 9 A) 100 μg of anti-CD1a antibody or mouse IgG1 isotype control was administered intraperitoneally. Figure 4 A) or days 0-8 in the treatment model ( Figure 9 Ear thickness measurements were performed daily using a micrometer (Mitutoyo) throughout the duration of A). Mice were sacrificed 24 hours after challenge and tissues were removed.

[0831] Murine MC903 administration

[0832] Mice were lightly anesthetized with isoflurane and 2 nmol / dose of MC903 applied to the ear ventral and dorsal sides (10 μΐ per side of ear) daily for 7 days. As Figure 15 100 μg of anti-CD1 a antibody or mouse IgG1 isotype control was administered intraperitoneally as indicated in D. Caliper measurements (Mitutoyo) were performed daily.

[0833] Tissue processing

[0834] Mice were sacrificed 24 hours after the last imiquimod challenge and tissues were taken. Ears, cervical lymph nodes (cLN) and spleen were collected for immunophenotyping analysis or imaging. Cell suspensions of spleen and cLN were obtained by passing the tissues through a 70 μιη mesh and washing with RPMI containing 10% FCS. Red blood cells were removed from the spleen cell suspension by incubation with RBC lysis solution (eBioscience).

[0835] Ears skin tissue was washed in HBSS to remove excess imiquimod, split ventrally, cut into <0.5 mm pieces and digested with 1 mg / mL collagenase P (Roche) and 0.1 mg / mL DNase I (Sigma-Aldrich) DMEM for 3 x 30 minutes with agitation, with dispersinase 5 mg / mL added to the final 30 minute digestion step. Single cell suspensions were obtained by washing through a 70 μιη filter with DMEM containing 10% FCS prior to analysis by flow cytometry.

[0836] Flow cytometry

[0837] For FACS surface staining, cells were labeled with the following anti-mouse antibodies (Biolegend origin, 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, PECy7: 153108), CD69 (H1.2F3, BV650: 104541), Langerin (4C7, PE: 144204), Ly6C (RB6-8C5, 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).

[0838] Flow cytometry: Epitope competition assay

[0839] CD1a-K562 cells were incubated with purified, newly generated and commercially available primary anti-CD1a antibodies on ice for 30 min (25 pg / ml), then unbound antibodies were washed away, then Alexa-Fluor-647 conjugated versions of the different antibodies were incubated with the cells in a matrix arrangement on ice for 30 min (10 pg / ml). Mean fluorescence intensity (MFI) was used to assess the degree of binding of fluorophore-conjugated antibodies.

[0840] Confocal imaging

[0841] Mouse ear skin was frozen in embedding compound at optimal cutting temperature and stored at -80 °C. 10 pm frozen sections were cut using a Leica cryostat and collected onto Superfrost Plus glass slides to air dry for 30 minutes before storage at -80 °C. Slides were rehydrated in PBS for 10 minutes prior to staining. Endogenous peroxidase activity of the samples was quenched by the addition of 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 non-specific binding of antibodies. Anti-CD1 a antibody was used for confocal microscopy (1 :100, OKT6; in-house produced and conjugated to biotin). The Alexa Fluor 594 Tyramide SuperBoost kit (streptavidin; Thermo Fisher Scientific) was used to enhance the signal, following the manufacturer’s instructions. Briefly, slides were incubated with primary antibody overnight at 4 °C. Following washing, HRP-conjugated streptavidin was added to the sections and incubated overnight at 4 °C. Excess streptavidin-HRP was washed away, the tissue was incubated with tyramide working solution for 8 minutes at room temperature and the reaction was stopped with the reaction stop reagent. Following staining, slides were mounted with an anti-fade mounting medium with DAPI (Vector Laboratories Ltd), a coverslip was applied and the slides were refrigerated in the dark until analysis by confocal microscopy (Zeiss LSM 780 confocal microscope-inverted microscope; 25x / 0.8 Imm Korr DIC M27; room temperature; Axiocam camera; Zen software) and Fiji was used for image processing.

[0842] Cell phenotype and cytotoxicity assay :

[0843] Anti-CD1a antibodies (5 μg / ml) and / or commercially available comparator NA1 / 34 (5 μg / ml) were incubated with CD1a-expressing K562 or EV control K562 for 48 hours and the cells were reduced by flow cytometry. In order to measure direct antibody-induced cell reduction, K562 was fluorescently labeled with CellTraceViolet before incubation with anti-CD1a antibodies for 48 hours. Before the reduction was assessed by flow cytometry, the reference population of untreated CFSE-labeled K562 was added to the antibody-treated K562 at a 1:1 ratio. The percentage of induced reduction was then calculated using the following equation by comparing the frequencies of viable cells of the different populations analyzed, antibody-treated and untreated reference CD1a+ and EVK562. % 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 cells expressing CD1a, K562-CD1a or K562-EV were incubated with an isotype control or anti-CD1a antibody (5 μg / ml) and stained for Annexin-V (Biolegend) 24 hours after incubation.

[0844] Complement-mediated lysis (CDC) and antibody-dependent cellular cytotoxicity ADCC assay :

[0845] For CDC assay, K562-CD1a cells (5 × 10 4 Cells / well) were pretreated with 5 μg / ml isotype control or indicated antibodies for 30 minutes and incubated with 10% normal human serum at 37°C in 5% CO2 for 3 hours. For ADCC assay, PBMCs were used. K562-CD1a cells (5×10 3 cells / well) and PBMC (2.5×10 5 Cells were cultured with IL-2 (100 U / ml) and 5 μg / ml isotype control antibody or the indicated antibody combination for 5 hours at 37°C in 5% CO2 (effector / target ratio of 50:1). Cytotoxicity was determined by calculating the percentage of surviving target K562-CD1a using the following equation: % cytotoxicity = 100 - ((% live cells of CD1a-K562 treated with CD1a antibody / % live reference K562) / (% live cells of CD1a-K562 treated with isotype antibody / % live reference K562) × 100).

[0846] In vivo CD1a+ cell depletion :

[0847] “NSG” (NOD-scid IL2Rγ nullMice were injected subcutaneously in the flank with ECM gel (Merck) suspension containing 250,000 CD1a-K562 cells (volume = 100 μΐ) and tumors were allowed to develop for 18 days. At day 6, day 10 and day 14, mice were treated intraperitoneally with 100 μg of isotype control antibody or the indicated antibodies and tumor size was measured.

[0848] Isoelectric focusing assay (IEF) :

[0849] Lipid loading was assessed by incubating 10 μg of CD1a with a 100X molar excess of imiquimod (Invivogen) dissolved in Tris-buffered saline and 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 protein were loaded onto IEF pH 3-7 gels (Novex) and then run at 100V for 1 hour, 200V for 1 hour and finally 500V for 30 minutes. Gels were then fixed with 12% TCA and stained with SimplyBlue SafeStain for 7 minutes and destained overnight in DI water.

[0850] Statistical analysis :

[0851] One-way and two-way ANOVA tests were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software). Error bars represent the standard deviation shown.

[0852] Generation and selection of therapeutic anti-CD1a antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834)

[0853] Many animals of 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, animals were sacrificed and PBMCs, spleen, bone marrow and lymph nodes were harvested. Serum binding to HEK-293 cells expressing human CD1a and human B2M was monitored by flow cytometry.

[0854] Memory B cell cultures were established (associated with 77A (VR11851), 110 (VR12112), 111 (VR12113) and 116 (VR12117)) and supernatants were first screened on the TTP Labtech Mirrorball system in a bead-based assay for their ability to bind to HEK-293 cells transiently transfected with human CD1a. This is a multiplex assay using HEK-293 cells expressing human CD1a and human B2M, which are stained with a cell dye and screened against reverse-stained HEK-293 cells expressing CD1b, CD1c or CD1d using a goat anti-species Fc-FITC conjugate as a display agent.

[0855] 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 further characterised by:

[0856] • ELISA to confirm binding to human CD1a protein (details below)

[0857] • ELISA to confirm binding to the CD1a lipid binding domain on a chimeric CD1a protein (human lipid binding domain of CD1a, mouse Ig domain of CD1d) (details below)

[0858] • Flow cytometry to confirm binding to human CD1a expressed on HEK-293 cells (co-expressed with human b2M) (details below)

[0859] V region recovery was performed on wells that demonstrated binding in the above assays using the fluorescent panning method.

[0860] The ability of plasma cells from bone marrow to bind human CD1a was also directly screened using fluorescent panning (associated with 16 (VR11834)). Here, B cells secreting CD1a specific antibodies were selected on biotinylated human CD1a immobilised on streptavidin beads using a goat anti-species Fc-FITC conjugate display reagent. Approximately 300 direct panning foci were selected.

[0861] Following reverse transcription (RT) and PCR on the selected cells, ‘transcriptionally active PCR’ (TAP) products encoding the antibody V regions were generated and used to transiently transfect HEK-293 cells. The resultant TAP supernatants containing recombinant antibodies were further characterised by:

[0862] • 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)

[0863] • Flow cytometry to confirm binding to human CD1a expressed on HEK-293 cells (co-expressed with human b2M) and to counter screen for cross-binding to related similar proteins CD1b, CD1c or CD1d expressed on HEK-293 cells (co-expressed with human b2M). (Details below)

[0864] Heavy and light chain variable region genes from TAP products of interest were then cloned as rabbit or mouse full length antibodies and re-expressed in the HEK-293 transient expression system. 119 V regions were co-cloned and banked. Recombinant cloned antibodies were then further characterized by:

[0865] • Repeat flow cytometry and ELISA assays described above.

[0866] • Flow cytometry to assess binding to CD1a expressed on a variety of cell lines. This gives an initial indication that binding is lipid independent. Supernatants were screened for binding to:

[0867] - Stable transduced C1R cells expressing CD1a or empty vector (co-expressed with human b2M). These were related to 110 (VR12112), 111 (VR12113) and 116 (VR12117). (Details below)

[0868] - MOLT4 cells endogenously expressing CD1a, CD1b, CD1c, CD1d and b2M. These were related to 110 (VR12112), 111 (VR12113) and 116 (VR12117). (Details below)

[0869] - MOLT4 cells endogenously expressing CD1a, CD1b, CD1c, CD1d and b2M. These were related to 110 (VR12112), 111 (VR12113) and 116 (VR12117). (Details below)

[0870] - MOLT4 cells endogenously expressing CD1a, CD1b, CD1c, CD1d and b2M. These were related to 110 (VR12112), 111 (VR12113) and 116 (VR12117). (Details below)

[0871] • Biacore was used to perform kinetic analysis to estimate off-rate and affinity (Details below)

[0872] Antibodies that showed binding and <100 nM affinity in the above assays were selected for purification. Protein A affinity purification was used to purify cell culture supernatants. Purified samples were buffer exchanged into 10 mM PBS pH 7.4 and analyzed for recovery and purity using UV spectroscopy, analytical size exclusion chromatography, SDS Page electrophoresis and LAL endotoxin assay respectively. Where required, samples were subjected to a second round of purification to increase monomer levels. Final samples were sterile filtered and stored in 10 mM PBS pH 7.4

[0873] Following purification, all 5 antibodies were then characterized by:

[0874] • Repeat the above flow cytometry, ELISA, and BIAcore assays

[0875] • ELISA to assess binding to cynomolgus CD1a protein and Chinese common human CD1a protein variant (18) (details below)

[0876] • Flow cytometry to assess binding to HEK-293 cells transiently transfected with:

[0877] (details below)

[0878] - cynomolgus CD1a co-transfected with cynomolgus beta2M

[0879] - Chinese common human CD1a variant co-transfected with human beta2M

[0880] 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 at various stages of antibody discovery (Tables 1-9). The only exception was 116 (VR12117), which was shown not to bind to recombinant or cell-expressed cynomolgus CD1a (Table 4 and Table 9). The inclusion of antibody 116 in subsequent in vitro and in vivo analyses was not considered to be significant, but was still a deliberate step in order to focus on the epitope binding region where the lipid binding domain is different between human and cynomolgus with potentially different functional effects. None of the antibodies demonstrated binding to CD1b, CD1c, or CD1d expressed on HEK-293 cells (Table 5), indicating that these antibodies are CD1a specific. Confirmation of CD1a, CD1b, CD1c, and CD1d expression in HEK-293 cells with a commercially available antibody supported this conclusion (data not shown). Binding to CD1a expressed on multiple cell types (HEK, C1R, and MOLT4) gave an initial indication that antibody binding can be lipid independent, as CD1a can be loaded with different lipid pools from each cell line.

[0881] Following antibody discovery, antibodies were evaluated for in vitro function in T cell assays as follows.

[0882] DNA encoding the heavy and light chain V regions of 77A (VR11851), 110 (VR12112), 111 (VR12113), and 116 (VR12117) on a mouse IgG1 backbone were synthesized at ATUM and expressed in-house in a HEK-293 transient expression system. Antibodies were then purified and endotoxin-removed and tested in in vivo assays as described below.

[0883] 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) bind to human CD1aAffinity of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) to human CD1a

[0884] The affinity of purified antibodies to human CD1a was assessed using a Biacore T200 instrument (GE Healthcare) by capturing antibodies against immobilized anti-species IgG F(ab')2, followed by titration of human CD1a. Affinipure goat anti-species IgG-F(ab')2 fragment specific (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip (GE Healthcare) by 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 running buffer at a flow rate of 10 μL / min. A 10 μL injection of 0.5 μg / mL of test antibody was used for capture by the immobilized goat anti-species Fab. Human CD1a was titrated over the captured 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.

[0885] The surface was regenerated between cycles by injection of 2 x 10 μL of 40 mM HC1, in between 10 μL of 5 mM NaOH was injected at a flow rate of 10 μL / min. Background subtracted binding curves were analyzed using Biacore T200 evaluation software following standard procedures. Kinetic parameters were determined by fitting algorithms. This assay was performed at the clone supernatant and purified antibody stage. Kinetic parameters of antibody binding to human CD1a are shown in Table 10.

[0886] Binding of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) assessed by ELISA

[0887] CD1a specific antibodies were identified by ELISA. ELISA plates were coated overnight at 4°C with 2 pg / mL of the protein of interest (human CD1a library B, chimeric CD1a library B [human lipid binding domain and mouse CD1d Ig domain], Chinese common variant CD1a, or cynomolgus CD1a] (20 pL / well) and then washed with wash buffer (PBS with 0.2% (v / v) Tween-20, pH 7.4). Plates were then blocked with 80 pL / well blocking buffer (1% (w / v) bovine serum albumin) for 1 hour at room temperature and then washed in wash buffer. Twenty pL of antibody sample (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) dilutions were transferred to the ELISA plates and incubated for 1 hour at room temperature and then washed with wash buffer. Twenty pL / well of peroxidase-conjugated goat anti-species IgG Fc-specific F(ab’)2 fragment (Jackson ImmunoResearch) diluted 1 :5000 in blocking buffer was added and incubated for 1 hour at room temperature and then washed with wash buffer. TMB substrate (EMD Millipore) (20 pL / well) was added to visualize binding and the reaction was incubated for 5 minutes at room temperature and then the optical density at 630 nM was measured using a microplate reader. The assay was performed at the B cell supernatant stage (human CD1a library B), TAP supernatant stage (human CD1a library B, chimeric CD1a library B), clone supernatant stage (human CD1a library B, chimeric CD1a library B), and purified antibody stage (human CD1a library B, chimeric CD1a library B, Chinese common variant CD1a, cynomolgus CD1a). Data for purified antibodies are shown in Tables 1-4.

[0888] Binding of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) to human CD1a by flow cytometry Table 1. Antibody binding to human CD1a pool B protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind human CD1a protein in ELISA. Antibodies were titrated by dilution series and compared to control rabbit IgG antibody. All 5 antibodies bound to human CD1a pool B protein. Data for purified antibodies are shown

[0889] CD1a specific antibodies were identified by flow cytometry. Binding to proteins expressed on HEK, C1R, and MOLT4 cell lines was evaluated. HEK-293 cells were transfected with the protein of interest (CD1a, CD1b, CD1c, CD1d, Chinese common variant CD1a, or cynomolgus CD1a) and species specific b2M (as described above). Transfection was performed using Expifectamine 293 kit (Gibco) and incubated overnight. On the day of need, C1R-CD1a, C1R- empty vector, and MOLT4 cell lines were washed in 1x PBS. All cell lines were counted and resuspended in 1x PBS and then stained with DiI or DiO cell stain (Invitrogen) for 30 minutes at 37°C. Cells were washed with flow cytometry buffer (PBS with 1% bovine serum albumin, 2mM EDTA, and 0.1% sodium azide) and then the 2 DiI stained and DiO stained populations were mixed together. Cells (20 mΐ / well) were then added to dilutions of antibody samples (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) (20 mΐ / well) and incubated in flow cytometry assay plates for 1 hour at 4°C, then washed with flow cytometry buffer. 10 mΐ / 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, then washed with wash buffer. Fluorescence intensity was then measured on an iQue screener PLUS. This assay was performed at the B cell supernatant stage (HEK-293 cells expressing human CD1a), TAP supernatant stage (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d), clone supernatant stage (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d; C1R cells expressing human CD1a or empty vector; MOLT4 cell line), and purified antibody stage (HEK-293 cells expressing human CD1a, CD1b, CD1c, CD1d, Chinese common variant CD1a, or cynomolgus CD1a; C1R cells expressing human CD1a or empty vector; MOLT4 cells). Data for purified antibodies are shown in Tables 5-9.

[0890] Table 2. Antibody binding to chimeric CD1a pool B protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind chimeric CD1a [human CD1a lipid binding domain, mouse CD1d Ig domain] protein in ELISA. Antibodies were titrated by dilution series and compared to control rabbit IgG antibody. All 5 antibodies bound to chimeric CD1a pool B protein. Data for purified antibodies are shown Table 3. Antibody binding to Chinese common human CD1a protein variant. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind Chinese common variant CD1a protein in ELISA. Antibodies were titrated by dilution series and compared to control rabbit IgG antibody. All 5 antibodies bound to Chinese common variant CD1a protein. Data for purified antibodies are shown ​ .

[0891]

[0892] ​ ​​ ​

[0893] ​ 。

[0894]

[0895]

[0896] ​ ​ ​ ​ 。

[0897]

[0898] Table 4. Antibody binding to cynomolgus CD1a protein. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were tested for their ability to bind cynomolgus CD1a protein in ELISA. Antibodies were titrated through dilution series and compared to control rabbit IgG antibody. All 5 antibodies bound cynomolgus CD1a protein except 116 (VR12117). Data shown for purified antibodies Table 5. Antibody binding 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 co-transfected with human β2Μ. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through dilution series and tested for binding to transfected proteins. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. All 5 antibodies bound human CD1a expressed on HEK-293 cells. No binding to CD1b, CD1c, or CD1d expressed on HEK-293 cells was observed. Table 6. Antibody binding 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 dilution series and tested for binding to C1R cells stably transduced with human CD1a or empty vector and human β2Μ. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. All 5 antibodies bound human CD1a expressed on C1R cells. No binding to C1R cells expressing empty vector was observed. Table 7. Antibody binding to MOLT4 cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through dilution series and tested for binding to MOLT4 cells which endogenously express CD1a, CD1b, CD1c, CD1d, and β2Μ. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. All 5 antibodies bound to MOLT4 cell surface protein, most likely CD1a. Data shown for purified antibodies 。

[0899]

[0900]

[0901] Table 8. Antibody binding to Chinese common variant CD1a expressed on HEK-293 cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) were titrated through dilution series and tested for binding to Chinese common variant CD1a expressed on HEK-293 cells. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. ​ ​ ​ ​ ​

[0902] ​ 。

[0903]

[0904]

[0905] ​ ​ ​ ​ ​ 。

[0906]

[0907] ​ ​ ​ ​ ​ 。

[0908]

[0909] ​ Titration of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) and testing of binding to HEK-293 cells transiently transfected with Chinese common variant CD1a (18) and human β2M. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. All 5 antibodies bound to Chinese common variant CD1a expressed on HEK-293 cells. Data shown for purified antibodies Table 9. Antibody binding to cynomolgus CD1a expressed on HEK-293 cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) were titrated by dilution series and tested for binding to HEK-293 cells transiently transfected with cynomolgus CD1a and cynomolgus β2M. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. All 5 antibodies bound to cynomolgus CD1a expressed on HEK-293 cells. Data shown for purified antibodies Table 9. Antibody binding to cynomolgus CD1a expressed on HEK-293 cells. 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) were titrated by dilution series and tested for binding to HEK-293 cells transiently transfected with cynomolgus CD1a and cynomolgus β2M. Binding was quantified as fold change in geometric mean fluorescence intensity relative to background as assessed by flow cytometry. All 5 antibodies bound to cynomolgus CD1a expressed on HEK-293 cells. Data shown for purified antibodies Table 10. Antibody affinity for human CD1a. Biacore was used to assess the affinity of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) for human CD1a. A 1:1 binding model was used to fit the data in all cases except for 16 (VR11834) which required a heterogeneous ligand binding model. Affinity required to be <100 nM to be considered for progression. Data shown for purified antibodies 。

[0910]

[0911]

[0912] Production of antibodies Crystallization Surface plasmon resonance Antibody binding detected by ELISA Antibody binding detected by flow cytometry .

[0913]

[0914] Method of antibody humanization of antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) Antibody 77A http: / / www.imgt.org / http: / / www.imgt.org / .

[0915]

[0916]

[0917] Antibody 110

[0918] For surface plasmon resonance and crystallization studies, single chain variable regions (ScFv) of OX16, OX110 and OX116 were generated using a flexible glycine-serine linker between the heavy and light chains. For in vitro functional assays, a number of constructs were generated as previously described, including existing mouse or rabbit variable regions on a human IgGl Fc region: wild type; Leu234Ala, Leu235Ala and Gly237Ala“LALAGA”; afucosylated; and Fab format. These were established for OX16, OX110, OX116 as well as comparative antibodies CR2113 and mAB571 (US10844118 and WO / 2022 / 077021). All antibodies were expressed in-house in the HEK-293 transient expression system, after which the antibodies underwent purification and endotoxin removal.

[0919] http: / / www.imgt.org /

[0920] CD1a / β2m heterodimers carrying fos-jun levers were expressed in HEK293S cells and purified through a nickel affinity and size exclusion chromatography step. CD1a was deglycosylated using EndoH (NEB) and fos-jun levers and the BirA tag and His tag were cleaved using thrombin overnight at room temperature. In the case of OX scFv, the BirA and His tags were cleaved using 3C protease overnight at 4°C. CD1a proteins used in crystallisation trials 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 through nickel and size exclusion chromatography. CD1a and antibody fragments were mixed at a 1 : 1 molar ratio and incubated overnight at 4°C. Sitting drop crystallisation trials were performed at the Monash Macromolecular Crystallisation Facility and the sample concentrations used in each case were in the range 5-10 mg / ml. Initial hits were further optimised by hanging drop method in manual trays. Crystals of OX16-CD1a appeared in 0.2 M sodium malonate, 20% PEG 3350 and diffracted up to 2.2 A resolution. Crystals of OX110-CD1a were obtained in 0.1 M MES pH 6, 20% PEG 8000, 0.2 M sodium acetate and diffracted up to 2.2 A resolution. Crystals of OX116-CD1a grew in 1.5 M ammonium sulphate, 0.1 M Bis-Tris pH 6 and diffracted up to 2.2 A resolution. In each case, structures were solved by molecular replacement using the CD1a binary structure (PDB: 6NUX) and Alphafold generated models of the respective antibody fragments. Structures were then refined through cycles of manual improvement in Coot followed by automatic improvement in Phenix.

[0921] http: / / www.imgt.org /

[0922] SPR experiments were performed on a Biacore 3000 using streptavidin-coated chips (Cytiva). Antibody fragments expressing a biotinylation tag at their C-terminus were biotinylated overnight using BirA ligase. According to the experiment, biotinylated scFv molecules were coupled onto the chip surface until a total of 150 or 1000 response units were reached per flow cell. To assess the impact of lipid antigen head groups on binding of OX16 and OX116 antibodies, increasing concentrations of deglycosylated CD1 a-endo or CD1 a loaded with specific lipids were injected onto each flow cell. Lipid loading of CD1 a 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), phosphatidylcholine (Avanti 850375). Each lipid was dissolved to 5-10 mM in 20 mM Tris pH8, 150 mM NaCl and 0.5% CHAPS. CD1 a-endo was incubated with a 15-40X molar excess of lipid overnight at room temperature. The mixtures were subsequently purified by anion exchange chromatography using a MonoQ column (GE Healthcare). Fractions corresponding to lipid-loaded CD1 a were pooled together and concentrated to 50 mM. Serial dilutions of CD1 a (up to 10 mM) were injected for 60 seconds in 20 mM Tris pH8, 150 mM NaCl buffer at 25°C. Dissociation times between injections were 5 minutes to 1 hour. To allow TCR binding to CD1 a-Ab complexes, 1000 response units of biotinylated OX116 were coupled to the SA chip. Each injection cycle consisted of a 60 seconds injection of 1 mM CD1 a only, followed immediately by increasing concentrations of TCR (0 mM to 50 mM) supplemented with 100 nM CD1 a to prevent further dissociation of CD1 a from the coupled Ab fragments. Runs were performed in 20 mM Tris pH8, 150 mM NaCl, 0.5% BSA buffer at 25°C. In all SPR experiments, the relative binding response was calculated by subtracting the non-specific response on a reference cell where no relevant protein was coupled. Binding curves were obtained by fitting the measured responses to a 1 : 1 specific binding model in GraphPad.

[0923] Antibody 111

[0924] ELISA plates were coated overnight at 4°C with 2 pg / mL of the protein of interest (human CD1a library B, chimeric CD1a library B [human lipid binding a1 / 2 domains and mouse CD1d a3 Ig domains], minor variant CD1a, or cynomolgus CD1a) (20 pL / well), then washed with wash buffer (PBS with 0.2% (v / v) Tween-20, pH 7.4). Plates were then blocked with 80 pL / well of blocking buffer (1% (w / v) bovine serum albumin) for 1 hour at room temperature, then washed in wash buffer. Twenty pL of antibody sample (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) dilutions were transferred to the ELISA plates and incubated for 1 hour at room temperature, then washed with wash buffer. Twenty pL / well of peroxidase-conjugated goat anti-species IgG Fc-specific F(ab’)2 fragment (Jackson ImmunoResearch) diluted 1 :5000 in blocking buffer was added and incubated for 1 hour at room temperature, then washed with wash buffer. TMB substrate (EMD Millipore) (20 pL / well) was added to visualize binding, and the reaction was incubated for 5 minutes at room temperature, then the optical density at 630 nM was measured using a microplate reader.

[0925] http: / / www.imgt.org /

[0926] 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 common variant CD1a, or cynomolgus CD1a) and species specific b2M (as described above). Transfection was performed using Expifectamine 293 kit (Gibco) and incubated overnight. On the day of need, C1R-CD1a, C1R- empty vector, and MOLT4 cell lines were washed in 1x PBS. All cell lines were counted and resuspended in 1x PBS and then stained with DiI or DiO cell stain (Invitrogen) for 30 minutes at 37°C. Cells were washed with flow cytometry buffer (PBS with 1% bovine serum albumin, 2mM EDTA, and 0.1% sodium azide) and then the 2 DiI stained and DiO stained populations were mixed together. Cells (20ul / well) were then added to dilutions of antibody samples (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) (20ul / well) and incubated in flow cytometry assay plates for 1 hour at 4°C, then washed with flow cytometry buffer. 10ul / 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, then washed with wash buffer. Fluorescence intensity was then measured on an iQue screener PLUS. This assay was performed at the B cell supernatant stage (HEK-293 cells expressing human CD1a), TAP supernatant stage (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d), clone supernatant stage (HEK-293 cells expressing human CD1a, CD1b, CD1c, or CD1d; C1R cells expressing human CD1a or empty vector; MOLT4 cell line), and purified antibody stage (HEK-293 cells expressing human CD1a, CD1b, CD1c, CD1d, Chinese common variant CD1a, or cynomolgus CD1a; C1R cells expressing human CD1a or empty vector; MOLT4 cells).

[0927] http: / / www.imgt.org / Antibody 116

[0928] Humanized antibodies are humanized by transplanting the cdr from rabbit and mouse antibody v district onto the human germline antibody v district framework. In order to restore the activity of the antibody, many framework residues from rabbit and mouse V district are also retained in the humanized sequence. These residues are selected using the scheme outlined in Adair et al. (1991) (Humanised antibodies.WO91 / 09967). Except for CDRH1, the CDR transplanted from the donor to the acceptor sequence is as defined by Kabat (Kabat et al., 1987), wherein the Chothia / Kabat definition (see Adair et al., 1991Humanised antibodies.WO91 / 09967) of the combination is used. Typically, the VH gene of rabbit antibodies is shorter than the selected human VH receptor gene. When compared with the human receptor sequence, the framework 1 in the rabbit antibody VH district generally lacks the N-terminal residue retained in the humanized antibody. Framework 3 of the rabbit antibody VH region also typically lacks one or two residues (75, or 75 and 76) in the loop between β-sheet strands D and E: in the humanized antibody, the gaps are filled by the corresponding residues from the chosen human acceptor sequence.

[0929] http: / / www.imgt.org /

[0930] Selected human V regions IGKV1-5 plus IGKJ4 J region (IMGT, http: / / www.imgt.org / ) as an acceptor for the light chain CDRs of antibody 11851. 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), respectively: alanine (A1), valine (V2), glutamic acid (E3), and tyrosine (Y71). In some cases, CDRL3 can be mutated to remove the unpaired cysteine ​​residue at position 90 (Kabat numbering) (C90, CDRL3 variant, SEQ ID NO: XY).

[0931] Select human V region IGHV3-23 plus IGHJ5 J region (IMGT, Antibody 16 ) as

[0932] Acceptor for the heavy chain CDRs of antibody 11851. 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), respectively: valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), valine (V78), and arginine (R94).

[0933] http: / / www.imgt.org /

[0934] Select human V region IGKV1-D13 plus IGKJ4 J region (IMGT, http: / / www.imgt.org / ) as an 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), respectively: glutamine (Q2), valine (V3), and glutamine (Q70). In some cases, CDRL3 can be mutated to remove the disulfide bond between the cysteine ​​residues at positions 94 and 95d (Kabat numbering) (C94 and C95d, CDRL3 variant, SEQ ID NO: XY).

[0935] Select human V region IGHV3-48 plus IGHJ2 J region (IMGT, Example 1 - Anti-CD1a panel improvement: functional evaluation of anti-CD1a antibodies ) as

[0936] Acceptor for the heavy chain CDRs of antibody 12112. In addition to the CDRs, 0, 1, 2, 3, 4, 5, or 6 of the following framework residues (donor residues) from the 12112 VH gene can be retained at positions 24, 48, 49, 71, 73, and 78 (Kabat numbering), respectively: valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), and valine (V78). In some cases, CDRH2 can be mutated to remove potential N-linked glycosylation sites (CDRH2 variants, SEQ ID NO: XY). In some cases, CDRH3 can be mutated to modify potential aspartic acid-proline hydrolysis sites (CDRH3 variants, SEQ ID NO: XY).

[0937] Figure 1

[0938] Selected human V regions IGKV1-5 plus IGKJ4 J region (IMGT, Figure 1 ) as an acceptor for the antibody 12113 light chain CDRs. In addition to the CDRs, 0, 1, 2, 3, or 4 of the following framework residues (donor residues) from the 12113 VK gene can be retained at positions 1, 2, 3, and 71 (Kabat numbering), respectively: alanine (A1), valine (V2), glutamic acid (E3), and tyrosine (Y71). In some cases, CDRL3 can be mutated to remove the unpaired cysteine ​​residue at position 90 (Kabat numbering) (C90, CDRL3 variant, SEQ ID NO: XY).

[0939] the human V region IGKV1-D13 plus IGKJ4 J region (IMGT, Example 2 - Anti-CD1a panel improvement: inhibition of CD1a-restricted enriched T cell line responses ) as acceptor for the heavy chain CDRs of antibody 12113. In addition to the CDRs, zero, one, two, three, four, five, or six 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), respectively: isoleucine (I48), glycine (G49), lysine (K71), serine (S73), valine (V78), and arginine (R94).

[0940]

[0941] Figure 2

[0942] the human V region IGKV1-D13 plus IGKJ4 J region (IMGT, Figure 2 ) as acceptor for the light chain CDRs of antibody 12117. In addition to the CDRs, zero, one, two, or three of the following framework residues (donor residues) from the 12117 VK gene can be retained at positions 2, 3, and 70 (Kabat numbering), respectively: glutamine (Q2), valine (V3), and glutamine (Q70). In some cases, CDRL1 can be mutated to modify a potential deamidation site (CDRL1 variant, SEQ ID NO: X-Y). In some cases, CDRL3 can be mutated to remove the disulfide bond between the cysteine residues at positions 94 and 95d (Kabat numbering) (C94 and C95d, CDRL3 variant, SEQ ID NO: X-Y).

[0943] the human V region IGKV1-D13 plus IGKJ4 J region (IMGT, ​ ) as acceptor for the heavy chain CDRs of antibody 12117. In addition to the CDRs, zero, one, two, three, four, five, or six 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), respectively: valine (V24), isoleucine (I48), glycine (G49), lysine (K71), serine (S73), and valine (V78).

[0944]

[0945] ​ the human V region IGKV1-D13 plus IGKJ4 J region (IMGT,

[0946] ​ ​​) as the acceptor for the light chain CDRs of antibody 11834. 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, respectively (Kabat numbering): valine (V48), glutamine (Q70), tyrosine (Y71), and arginine (R85). In some cases, CDRL2 can be mutated to remove a potential aspartate isomerization site (CDRL 2 variant, SEQ ID NO: X).

[0947] Select human V region IGHV3-23 plus IGHJ4 J region (IMGT, ​ ) as

[0948] Acceptor for the heavy chain CDRs of antibody 11834. 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, respectively (Kabat numbering): arginine (R44), alanine (A49), and arginine (R94). In some cases, CDRH2 can be mutated to modify two potential asparagine deamidation sites (CDRH2 variants, SEQ ID NO: XY). Example

[0949] ​

[0950] Following CD1a binding assessment, a large panel of anti-CD1a antibodies was screened for inhibitory function. T cell cytokine production was measured in an in vitro antigen presentation model using EliSpot. These data are summarized in ​ It was determined that many of the newly generated antibodies were more effective than the commercial anti-CD1a antibodies OKT6, HI149, and SK9 in inhibiting CD1a T cell responses. Notably, antibodies 16, 22, 39, 46, 77, 87, 110, and 116 all had an IC50 that was at least one log lower than that of OKT6. ​ B), which is 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.

[0951] ​

[0952] To aid in the shortlisting of antibody candidates for in vivo analysis, a different approach was taken to assess CD1a T cell responses; CD1a-restricted enriched T cell lines were isolated and expanded to analyze isolated CD1a responses rather than in a mixed polyclonal T cell background, where low signal-to-noise ratios can partially mask the potential of inhibitory antibodies.

[0953] In these assays, antibodies 116 and 16 stand out as effective inhibitory antibodies, with 16 uniquely inhibiting IL-22 autoreactivity / endogenous production ​ A and Figure 2 B). This improvement shows the possibility of using the antibodies in conditions where IL-22 plays a pathogenic role and in conditions where there is an effect on IFNy. Surprisingly, different effects on different cytokines were observed. In addition, an antibody-dependent inhibition of CD1a-restricted T cell activation was assessed using an APC-free system. CD1a-coated beads were used as a substitute for APCs, and the resulting T cell IFNy production was measured by flow cytometry. This assay revealed significant inhibition of CD1a-dependent cytokine responses by all antibodies, but particularly 77a, 87, 110, 111, and 116 Figure 2 C).

[0954] Example 3 - In vivo evaluation of inhibitory antibodies in skin inflammation

[0955] The aim of this study was to generate antibodies that would have clinical use in the treatment of human diseases and conditions, and thus it was necessary to determine efficacy in a complex immune system analogous to human disease. From the analysis of the data described above, a highly accurate best set of newly generated antibodies was selected (antibodies 16, 77a, 110, 111, and 116), and an attempt was made to determine their potential in in vivo models of psoriasis, dermatitis, lupus, and as a model of drug reactions manifesting as inflammatory skin or mucosal diseases or conditions, or related systemic diseases or conditions, or systemic manifestations of one or more inflammatory drug reactions. Experimental psoriasis and dermatitis have been shown to worsen in CD1a transgenic mice compared to WT, and CD1a-dependent inflammation can be improved by administration of anti-CD1a antibodies (Kim et al., 2016). It should also be noted that some individuals develop skin / mucosal inflammatory drug reactions to imiquimod, which is used topically for many skin conditions; such drug reactions include psoriatic reactions, dermatitis reactions, bullous diseases, alopecia, vesiculation, lichenoid reactions, neutrophilic diseases, lupus erythematosus, erythema multiforme, oral erosions, and severe drug reactions such as DRESS, AGEP, Stevens-Johnson syndrome, and toxic epidermal necrolysis (19-29).

[0956] Generation of CD1a transgenic mice

[0957] To evaluate the possible role of CD1a in skin and related systemic inflammation, the inventors generated CD1a transgenic mice. CD1a does not exist in the mouse genome, and therefore the human CD1a locus with 0.8kb 5' and 0.8kb 3' flanking regions (including promoter elements) was cloned and the transgene was inserted by microinjection, similar to the published CD1a transgenic model, but requiring additional transgenic fragment suture (Illing et al., Nature 486, 554-558 (2012)). Breed genotype-positive founder mice and screen for lines expressing CD1a transgenes. The inventors continued to phenotypic analysis of the mice and determine whether CD1a protein expression follows the expected pattern and whether it represents human CD1a cell expression. The ear skin of wild-type and CD1a transgenic (CD1aTg) mice was collected and enzymatically treated to allow analysis of the skin cell environment by flow cytometry ( Figure 3 A). CD1a expression was detected in the skin, accounting for 4.2% (+ / - 1.79) of total skin cells and 23.6% (+ / - 6.68) of CD45+ cells. To assess cellular regulation of expression, CD1a protein was assessed in dermal DCs (dDCs) and Langerhans cells (LCs). It has been reported that dermal DC subsets express CD1a, and Langerhans cells are characteristically constitutively CD1a. high CD1a was found to be expressed by 41.5% (+ / - 20.38) of dDCs and 88% (+ / - 4.606) of LCs ( Figure 3 AB). Further characterization of CD1a protein expression in the skin by immunofluorescence revealed characteristic epidermal locations and cells with dendrites typical of LC ( Figure 3 C). CD1a genotype was confirmed ( Figure 3 D), and CD1a expression in the thymus was mainly observed by the proportion of CD4+CD8+ double-positive thymocytes ( Figure 3 E) CD1aTg mice do not display abnormal skin inflammation at steady state. In summary, the present inventors generated CD1a transgenic mice that display CD1a expression in a manner phenotypically similar to human tissue expression.

[0958] This model is used to test anti-CD1a antibodies for the prevention of inflammatory skin diseases and conditions ( Figure 4A). Application of Aldara cream containing 5% imiquimod, a TLR7 / 8 agonist, is an established model that induces psoriasis-like, dermatitis-like, lupus-like skin inflammation represented by skin thickening, scaling and redness (30, 31). Ear inflammation in CD1a transgenic mice was found to be significantly higher than the WT counterpart responding to Aldara. Moreover, all anti-CD1a antibodies applied prior to imiquimod reduced subsequent ear thickening, whereas antibodies 116 and 16 ablated CD1a-dependent inflammation to at least WT levels Figure 4 B). At the end of the experiment, CD1a transgenic (-Tg) mice treated with antibodies 16 and 110 showed reduced inflammation to WT levels of ear thickening. Surprisingly and unexpectedly, antibody 116 treatment reduced the level of CD1a-Tg ear skin inflammation to levels significantly below WT skin Figure 4 B).

[0959] Example 4 - In vivo effect of inhibitory antibodies on skin immune responses Figure 5 .

[0960] It was found that skin T cell infiltration is elevated in CD1a transgenic mice and the frequency of this population is reduced by anti-CD1a antibodies, in particular antibodies 116, 16 and 110 in the prophylactic model Figure 5 A). Notably, 16 and 116 were able to reduce skin T cell infiltration below wild type levels, indicating an improvement and profound impact on inflammation in vivo. Moreover, the activation marker CD69 was increased on the surface of skin T cells in CD1a transgenic mice and suppressed by some anti-CD1a antibodies, in particular 116 and 16 in the prophylactic model Figure 5 B). Neutrophils are known to be important cells in many inflammatory conditions, including the psoriasis response and the murine imiquimod model. Here, an elevated frequency of neutrophils was found in the skin upon imiquimod treatment and further increased in CD1a transgenic mice, which was reduced to WT levels or below using anti-CD1a antibodies 116 and 16 in the prophylactic model Figure 5 C). A reduction of skin eosinophils in response to the antibodies was also noted, which is of interest given the known role of eosinophils in many forms of drug reactivity Figure 6 D). This unexpected finding represents an improvement, as no effect on eosinophils has been observed previously.

[0961] Langerhans cells (defined here as CD11c+Langerin+) were also increased in the skin after imiquimod challenge of CD1a transgenic mice compared to WT, as observed in human skin inflammatory conditions. Skin LC counts were reduced in the prophylactic model with the application of antibodies 16, 116, 111 and non-significant 110 (Figure 6 A). Notably, antibody 116 reduced the number of skin LCs below the number of LCs in wild-type skin, showing an improved and surprising level of effect. As the main CD1a expressing population, the effect of the antibodies on LC CD1a expression was evaluated. Notably, antibodies 110 and 116 had reduced staining, but this was due to interference of the 110 / 116 antibodies with the binding of the HI149 detection antibody Figure 6 B). This shows sustained binding of the antibodies in vivo, which is a surprising effect and correlates with a therapeutic advantage. These findings also raise the possibility of using the antibodies for diagnostic or prognostic purposes or to monitor CD1a expressing cells before and during treatment. This observation was not observed with the non-competitive SK9 detection antibody, as shown below. The observed reduction in LCs can be due to antibody-dependent LC death or migration or phenotypic change. Therefore, the presence of CD11c+Langerin+LCs in cervical lymph nodes was analyzed. It was found that the number of LCs in the lymph nodes of CD1a transgenic mice was increased compared to WT, however migration to the LN did not seem to explain the reduction of skin LCs in mice treated with antibodies 110 and 116 Figure 6 C). Notably, the immunological improvement brought by antibody 116 was close to those in wild-type skin, showing an improved and surprising level of effect. Interestingly, the expression level of CD1a on lymph node-derived LCs followed a similar pattern as in the skin, as LCs had reduced staining due to interference of the 110 / 116 antibodies with the binding of the HI149 detection antibody Example 5 - Cytotoxicity observed with anti-CD1a antibodies expressed in terms of effect on CD1a expressing cell phenotypes D), as discussed further below. This was not observed in the non-competitive SK9 detection antibody. Notably, lymph node-derived LCs expressed less CD1a per cell than skin-derived LCs, which can be a control mechanism to prevent systemic inflammation. Therefore, the antibodies maintained their effect on LCs in vivo in the skin, even after migration to the lymph nodes. This is an important enhancement, as the clinical effect will be more durable.

[0962] Figure 7

[0963] Given that the enhanced migration did not fully explain the reduction in skin LCs, the possibility of antibody-induced phenotypic change of CD1a+cells was investigated, despite the murine IgG1 nature.

[0964] It was demonstrated that all anti-CD1a antibodies, but particularly 110 and 116, were able to reduce the number of CD1a+K562 cells lacking MHC class I and II in vitro and thus allowed comparison of the responses Figure 7 A). Antibodies 110 and 116 were tested in more detail, which showed a dose-dependent reduction Figure 7B), which is an improvement and surprising for the murine IgGl isotype. This is in clear contrast to the published CR2113 antibody (16, 18) (US10844118B2 and CA 2924882 Al), which is said to require complement and / or antibody dependent cellular cytotoxicity. Specifically, it states that "CR2113 does not directly induce apoptosis" (17), and notes that NA1 / 34 does not induce direct killing. However, since the different Fc regions affect effector functions, the comparative effect of CR2113 on the murine IgGl background will be set out directly below. The inventors continued to assess the ability of the antibodies to induce direct reduction of primary human CD1a expressing cells. Using the cytokines IL-4 / GM-CSF and IL-4 / GM-CSF / TGF-β, respectively, DC-like and LC-like cells were generated by 5-day in vitro differentiation of monocytes with the addition of anti-CD1a antibodies on day 0 or day 2 of culture. It was observed that antibodies 110 and 116 reduced LC and to a lesser extent DC in vitro (respectively Figure 7 C top and bottom panels). In exploring potential mechanisms for this reduction, the inventors found that this reduction was associated with a significant cell cluster culture phenotype morphology Figure 7 D). The reduction in number can be partially explained by this clustering, but in addition, it was tested whether the antibodies could induce apoptosis of CD1a expressing target cells and compared to CR2113 (on a murine IgGl background). Figure 7 E shows that 110 and 116 (but not 16) and CR2113 (on a murine IgGl background) induced annexin V expression of K562 expressing CD1a, even in the absence of complement or ADCC. This indicates that the 110, 116 and CR2113 antibodies can mediate K562 cell death to some extent. To investigate the role of complement mediated lysis (CDC) and antibody dependent cellular cytotoxicity (ADCC), K562-CD1a were incubated with complement Figure 7 F) and / or with human PBMCs Figure 7 G). Despite the murine IgGl nature of the antibodies, there is evidence of complement mediated lysis and ADCC. The effect of the antibodies on the human IgGl Fc region was investigated below. To further investigate the in vivo mechanisms, a new model was established using K562-CD1a subcutaneous tumors in an immunodeficient NSG model, where there is a wide defect in lymphocyte response and other effects. The data show that all three antibodies reduced the size of the lymphocyte tumors by day 10, with the effect persisting to day 15-20 for 16 and 116 (25% or more reduction in volume of CD1a expressing tumor cells), but not for CR2113 Figure 2H). Differences in the responses in vitro and in vivo can be explained by other cofactors present in vivo, such as complement, the many innate cell subsets that carry FcRs specific for different Fc, differential target cell density, reduced antibody half-life in vivo, and altered tissue pathways. This direct alteration in the phenotype of the target cells expressing CD1a can contribute to a less inflammatory response of CD1a-expressing cells. Thus, the reduction of LCs in the skin of CD1a-Tg mice treated with 110 and 116 can be partially explained by a direct antibody-dependent change in the CD1a+ LC phenotype and contributes to the clinical effect, e.g., in 116, reducing inflammation below that of wild-type. This data also raises the possibility that the antibodies can be useful in treating malignancies expressing CD1a, including Langerhans cell histiocytosis and some forms of T-cell lymphoma and some forms of thymoma. However, the change in phenotype of the target cells cannot explain the reduction in T-cell functional responses shown in Figures 1 1 A-C, because the CD1a-bead assay (Figure 1 1 D) is not affected by any depleting effect. Figure 2 C) would not be affected by any depleting effect. Example 6 - Epitope binding analysis of CD1a antibodies

[0965] Figure 8 The data presented herein demonstrate that the five newly generated anti-CD1a antibodies have a range of functionalities, and an attempt was made to determine if the antibodies had overlapping binding sites using a flow cytometry cross-blocking assay. In addition, the epitope overlap was assessed with the commercially available antibodies OKT6, HI149, SK9, and NA1 / 34, which are known to have overlapping binding sites with CR2113, as described above.

[0966] CD1a-K562 cells were incubated with purified primary anti-CD1a antibodies (Y-axis

[0967] A, 25 μg / ml), then unbound antibody was washed away, then Alexa-Fluor-647 conjugated versions of the different antibodies were incubated with the cells in a matrix arrangement of A (X-axis, 10 μg / ml). Mean fluorescence intensity (MFI) was used to assess the degree of fluorophore-conjugated antibody binding, so any steric interference caused by the binding of the primary purified antibodies would be represented by a reduction in MFI. The results indicate that antibodies HI149, OKT6, 110, and 116 can have overlapping or closely related epitopes, and a second group containing antibodies NA1 / 34, 77a, 111, and 16 can have closely related binding sites. This suggests that the reduction in CD1a expression observed in vivo (Figures 1 1 B and D) is due to interference of the 110 / 116 antibodies with the binding of the HI / 149 detection antibodies. Indeed, this effect was not observed with the non-competitive SK9 detection antibody (Figure 1 1 E). Figure 8 B and D) is due to interference of the 110 / 116 antibodies with the binding of the HI / 149 detection antibodies. Indeed, this effect was not observed with the non-competitive SK9 detection antibody (Figure 1 1 E). Figure 6 Figure 8 B and D) is due to interference of the 110 / 116 antibodies with the binding of the HI / 149 detection antibodies. Indeed, this effect was not observed with the non-competitive SK9 detection antibody (Figure 1 1 E). Figure 8 ​B). Importantly and unexpectedly, the antibodies thus remain present on LCs in the skin in vivo even after migration to lymph nodes and after enzymatic digestion of skin tissue. This would likely correlate with longer and more pronounced clinical benefits. Since the antibodies fall into two major groups that do not compete, Example 7 - Demonstration of effectiveness of antibodies of the application for the treatment of imiquimod-induced inflammation and systemic related inflammation (A and B) show that combinations of antibody members selected from each group can be used together, e.g., as therapeutic / monitoring or combination therapeutics. One such combination would be 116 and 16.

[0968] Therapeutic potential of anti-CD1a antibodies Figure 9 .

[0969] Given the skin dominant expression of CD1a, most studies have focused on skin specific functional effects, although the presence of circulating CD1a reactive T cells has been demonstrated (11). The role of CD1a in inflammation of tissues other than the skin has not been extensively studied. Furthermore, CD1a is known to amplify the imiquimod skin response (16), but the associated systemic sequelae have not been studied. The present inventors generated new CD1a transgenic mice and CD1a reactive T cells and used human and mouse assays, respectively, to characterize the functionality of anti-CD1a antibodies in vitro and in vivo. These findings demonstrate that CD1a dependent effects extend to systemic effects, implicating treatment of systemic correlates of skin diseases including adverse inflammatory drug reactions.

[0970] Figure 9

[0971] To further assess the therapeutic potential of the newly generated anti-CD1a antibodies, the present inventors 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 the establishment of imiquimod-induced inflammation Figure 9 A). Despite ongoing imiquimod application, all three antibodies rapidly improved the clinical response after initiation Figure 9 B-C). For 116, the response was most pronounced, reducing ear thickness Figure 9 B). The entire skin (top panel) and epidermal (bottom panel) thickening was observed by confocal microscopy Figure 8 D), which confirmed the micrometer assessment Figure 9 B). CD1a protein expression was assessed in CD1a transgenic epidermis (anti-CD1a OKT6 AF-594, red) and noted to be reduced by cell death and epitope competition in 110 and 116 treated skin Figure 9 A and CD1a involvement in systemic immune response to imiquimod D). Upon analysis of the skin cellular immune response after the imiquimod treatment model, a reduction in skin T cell counts and activation, skin LCs and skin neutrophils were observed upon introduction of the antibodies (Figure 10 E-G).

[0972] Figure 10

[0973] Imiquimod treatment human effects can extend beyond the skin and have been shown to induce splenomegaly in murine models. The contribution of CD1a to this pathway was assessed. Strikingly, spleen weight was increased in CD1a Tg mice treated with imiquimod compared to wild type and antibodies reduced spleen size and weight, consistent with systemic effects beyond the skin Figure 10 A). Furthermore, antibodies reduced CD4 and CD8 T cell activation as determined by CD69 expression (116 and 110, Figure 10 B-C), splenic neutrophil (non-significant trend) and eosinophil frequency (16, 110, 116) (respectively Figure 11 D and 10E). Plasma cytokine levels were assessed on day 8. Significant increases in IL-23, IL-12p70, IL-1b, IL-1a6 and MCP-1 were observed in CD1a transgenic mice treated with imiquimod and reduced in some or all of the 16, 110 and 116 treated groups Figure 12 F). Plasma immunoregulatory cytokines IL-10 and IL-27 were increased in the presence of antibodies 16 and 116 respectively (and same trend with others). The impact on circulating immune cells was then determined. Similar to the spleen, blood CD4 and CD8 T cell counts, neutrophilia and eosinophilia were increased in CD1a transgenic groups treated with imiquimod. This increase was significantly blocked following treatment with 16, 110 or 116 Figure 13 A-E). Finally, the inventors investigated whether imiquimod itself can be a CD1a ligand and showed that this is not the case, suggesting a more widespread autoimmune and autoinflammatory role of the CD1a pathway Figure 13 ). Thus, it can be shown that a widespread systemic inflammatory immune response is initiated or influenced by CD1a in the skin.

[0974] To investigate whether anti-CD1a antibodies can produce a sustained resetting of skin inflammation following imiquimod application, the schematic Figure 9 A depicted model was performed where imiquimod was re-challenged in the absence of re-administration of anti-CD1a antibodies Figure 14B) Surprisingly, 16, 110, and 116 all produced sustained improvements in ear thickness in the absence of repeated antibody administration, consistent with a sustained immune effect. The immune response also persisted, with significant decreases 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), lymph node eosinophils (116), and 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).

[0975] To compare the performance of the antibody with the current standard of care in the management of moderate-to-severe psoriasis, a repeated imiquimod treatment model ( Figure 15 A), anti-IL-17A (IgG1 isotype) was administered simultaneously at the same time and dose (100 μg) as anti-CD1a antibody ( Figure 15 All anti-CD1a antibodies again showed significant improvement in ear thickness results, with all antibodies producing significant improvement earlier than anti-IL-17A. It was noted that anti-IL-17A did not significantly reduce the frequencies of skin T cells, skin Langerhans cells, skin eosinophils, lymph node T cells, lymph node neutrophils, lymph node eosinophils, plasma IL-23, MCP-1, or IL-6 compared to the different anti-CD1a antibodies.

[0976] To directly compare the skin and systemic inflammatory outcomes between the antibodies described herein and CR2113, an imiquimod skin treatment model ( Figure 15 A). All anti-CD1a antibodies had a beneficial effect on ear thickness, but antibody 116 showed a significant improvement relative to CR2113 ( Figure 15 To extend the study of the improvement of anti-CD1a antibodies 16, 110 and 116 relative to CR2113, an additional model of skin inflammation (i.e., MC903-induced inflammation) was compared ( Figure 15 D), and significant benefits were observed for antibodies 16, 110, and 116, but not for CR2113, thus showing improvement ( Figure 16 E). Note that 16 and 116 showed significant reductions in skin T cell percentage and skin eosinophil counts, whereas CR2113 did not show a significant reduction (Figure 16 F) Skin extract cytokines were significantly reduced, with CR2113 showing no significant reduction in IL-5 (16, 110, 116), IL-6 (16, 110, 116), IL-9 (16), IL-23 (116), IL-17F (16, 110, 116).

[0977] It was further observed that 116 showed a consistent improvement over CR2113 in reducing the cutaneous, lymph node and plasma inflammatory responses to imiquimod Example 8 - Crystal structure of anti-CD1a antibodies For some results, 16 also significantly improved over CR2113 Figure 17 Specifically, antibody 116 improved over CR2113 in reducing IL-17A expression by cutaneous T cells and in the frequency of eosinophils in draining lymph nodes. 116 also improved over CR2113 in reducing plasma IFNy, IL-1a, IL-1b, IL-5, IL-9, IL-17A, IL-17F, IL-22 and skin digest IL-1a, IL-22 and TNFa. 16 improved over CR2113 in a strong trend in reducing lymph node eosinophils, plasma IL-1b, IL-22, IL-9 and IL-5 and skin digest IL-1a and IL-17A. In summary, the data demonstrate that the antibodies described herein are able to inhibit the cutaneous and systemic inflammatory responses to imiquimod and MC903.

[0978] Example 9 - Role of lipids in OX16 and OX116 binding to CD1a

[0979] The crystal structure of CD1a bound to the single chain variable construct of OX16, OX110 and OX116 antibodies was solved at resolutions of and respectively Figure 18 The electron density maps of CD1a, b2m and scFv chains were of good quality and allowed a detailed molecular analysis of the interactions. The molecular details of the complexes are as follows:

[0980] OX16-CD1a: OX16 binds directly on top of CD1a, spanning the entire A' top, docking on the a1 and a2 helices. The total buried surface area (BSA) of the interface is ( for OX16 And for CD1a 747). The CD1a residues that contribute to the interaction are: Glu 62, Glu 65, Leu 66, Thr 68, Leu 69, Ile 72 on the a1 helix and Asn 151, His 153, Glu 154, Ile 157, Asn 160, Asp 164, Thr 165 and Arg 168 on the a2 helix. The heavy chain provides 70% of the interaction and 30% corresponds to the light chain of the antibody. The variable loops of the antibody that participate in the interaction are heavy chain: H1 (Tyr 34), H3 (Arg 100 to Trp 106; Arg 100, Tyr 103, Tyr 104, Tyr 106), light chain: L1 (Tyr 169), L2 (Tyr 186), L3 (Tyr 229, Trp 233). The CDR3 loop of the heavy chain is central to the interaction as it contains 60% of all buried surface area.

[0981] The blocking ability of OX16 appears to be apparent as its epitope has a large overlap with the epitope of one of the self-reactive TCRs BK6, the only aP TCR with known crystal structure that binds to CD1a. Most of the CD1a residues recognized by BK6 overlap with the residues central to the OX16-CD1a interaction (Glu 62, Glu 65, Ile 157, Asn 160, Asp 164, Thr 165, Arg 168 (Birkinshaw et al., Nature Immunology 2015)) thus making the binding of OX16 incompatible with BK6. Even if the Ab does not block the F' portal, the L1 loop is almost directly above it, leaving limited space for the protruding head group, which prompted the SPR experiments to investigate whether the size and ‘volume’ of the head group could have an impact on the recognition of CD1a by OX16.

[0982] OX110-CD1a. The OX110 antibody binds CD1a on the edge of the a1 domain, on only one side of the F' pocket, and is reminiscent of the recently published binding by the gammadelta T cell receptor CO3 (Wegrecki et al., Nat comm 2022). In this crystal structure, four CD1a-antibody complexes are observed in the asymmetric unit, and there are surprisingly minor differences between them in terms of the interacting side chains, yet the overall docking pattern remains almost identical. For example, the loop Tyr19-Trp23 of CD1a can adopt variable conformations and interact with the antibody in two copies of the complex, but not with the antibody in the other two copies. This suggests that the recognition of CD1a by OX110 has a degree of flexibility, thus demonstrating that the interaction is very robust. From a functional perspective, this can be important because even minor conformational changes in CD1a when it binds a specific lipid ligand on the cell surface are unlikely to affect the recognition of CD1a by OX110. For clarity, the analysis is focused on the complex with the best electron density maps within the asymmetric unit. The total buried area at complex formation is wherein corresponding to and corresponding to CD1a. The residues from the a1 helix of CD1a that contact the antibody are: Glu 79, Arg 82, Arg 83, His 86, Glu 87, Gln 89, Phe 90, Glu 91, Tyr 92, and from the a2 domain, Val 147, Asn 150. His 86 appears to be central to this interaction as it establishes H bonds and salt bridges with 3 residues from the heavy chain of OX110, which explains why the point mutant CD1a[H86A] completely destroys the binding of OX110 to CD1a, as seen in epitope mapping experiments. The antibody contributions from the heavy and light chains are 68% and 32%, respectively. The variable loops that interact with CD1a are: H1 (Ser 31, Ser 32), H2 (Asn 53, Ser 54, Ser 55), H3 (Asp 97, Tyr 99, Tyr 101, Tyr 103, Gly 104, Trp 105), L1 (Phe 165, Asn 166), and L3 (Glu 228, Phe 229, Ser 230, Cys 231). Again, the majority of the antibody contribution comes from the H3 loop, which provides 30% of the total buried area. Interestingly, L3 contains an intra-loop disulfide bond between Cys 231-Cys 236, which is common in single chain antibodies, where it stabilizes the long CDR3 loop. Here, it does not play an obvious role as L3 has only a small contribution to the interface of the interaction.

[0983] OX116-CD1a. The OX116 antibody also binds to the side of CD1a that is transverse to the F' pocket. This epitope overlaps with one of OX110, however OX116 spans the a1 domain and a2 domain of CD1a. The buried area of assembly is (CD1a provides and OX116 provides ). The CD1a residues that interact with the antibody include: Arg 83, Tyr 84, His 86, Glu 87, Gln 89, Phe 90, Glu 91 on the a1 domain and Asn 139, Met 140, Lys 142, His 143, Lys 146, Val 147, Gln 150 on the a2 domain. In this case, even though His 86 is in contact with the antibody, these do not include H bonds and consist only of weak an der Waals contact, which explains why the CD1a[H86A] mutant does not affect the interaction in the epitope binding experiment. The OX116 residues that participate in complex formation belong to H1 (Ser 31, Asn 32, Ala 34), H2 (Tyr 53, Thr 54, Thr 55, Gly 56, Phe 57, Tyr 59), H3 (Ala 99, Thr 100, Tyr 101, Val 102, Pro 104), L1 (Tyr 166, Asn 167) and L3 (Glu 229, Phe 230, Ser 231, Cys 232). As with the other two complexes, here the VH domain comprises 75% of the assembly interface and the VL provides the remaining 25%. However, the H3 loop, which dominates the interaction in OX16-CD1a and OX110-CD1a, here provides only 25% of the total interaction area. Surprisingly, 35% of the BSA comes from the germline-encoded H2 loop, which has minimal contribution (16% of the BSA) in OX110-CD1a and none in OX16-CD1a. Here again there is an intra-loop disulfide bond within L3. In fact, the sequence of loop L3 is almost identical between OX110 (GEFSCSSTDCVTF) and OX116 (GEFSCSSVDCATF) and in each case the same residues from L3 contact the same segment of CD1a (Gln 89), however the angle of the interaction is different and the heavy chain docks on a different epitope.

[0984] Even though both OX110 and OX116 bind the F' pocket side of CD1a, their binding modes are different. OX110 binding around His86 induces a conformational change in this portion of the a1 helix that adopts a different conformation than any other structure of CD1a. In addition, it affects the amino-terminal portion of the a1 helix in the A' roof region, and slightly alters the way the a1 and a2 helices interact to form the A' roof. No similar effects are observed when OX116 binds the a1-a2 interface on the F' side of the cleft. Overall, the shape of the binding cleft of CD1a appears to be unaffected by association with OX16 or OX116, whereas binding of OX110 affects the a1 helix and its association with the a2 helix.

[0985] Figure 18

[0986] CD1a was loaded with different lipids, known to be permissive (endogenous "endo", lysophosphatidylcholine 18:1 (LPC)) or non-permissive (sphingomyelin 24:1 (SM24:1)) or a large headgroup lipid control (ganglioside GD3). OX16 (A) and OX116 (B) antibody binding to CD1a loaded with lipids was then tested using surface plasmon resonance. Example 10 - Effect of anti-CD1a antibodies on blocking polyclonal and clonal T cell function A) and OX116 (B) antibody binding to CD1a loaded with lipids was then tested using surface plasmon resonance. Figure 19 A) and OX116 (B) antibody binding to CD1a loaded with lipids was then tested using surface plasmon resonance. As described in Example 8, this can be explained by the OX16 protruding end of the F' portal, which can limit binding to non-permissive lipids with large protruding headgroup antigens. Thus, OX16 binding to CD1a follows the molecular pattern described previously for self-reactive aP T cell receptors exactly. OX116 shows binding to all tested lipids, including endogenous lipids ("endo"), SM24:1, LPC, GD3, sulfatide and phosphatidylcholine, without preferentially binding to known permissive or non-permissive classes of ligands. This is unexpected given the proximity of OX116 binding to the F' portal. In addition, it demonstrates that the recognition of CD1a by OX116 is robust and less likely to be affected by the identity of the antigenic lipids present in the cleft of CD1a at the cellular level. Overall, these data show that OX16 and OX116 can bind to CD1a loaded with multiple lipids.

[0987] Figure 19

[0988] K562 cells expressing CD1a or empty vector control (EV) were incubated overnight with different anti-CD1a antibodies and polyclonal T cells isolated from a healthy adult donor. The number of cells expressing IFNg or IL-22 was measured using ELISpot and the percentage of inhibition was compared to isotype control Example 11 - CDC and ADCC effect of anti-CD1a antibodies A-B). Wild-type human IgGl Fc showed a significant decrease in IL-22 production for all antibodies, but a significant decrease in IFNg production for only antibodies OX16, OX110, OX116. Afucosylated IgGl showed a significant decrease in IL-22 for OX16, OX110, OX116, CR2113 and mAb571. Afucosylated IgGl showed an increase in IFNg production for OX16, OX110, CR2113 and mAb571, which is consistent with the known enhanced Fc effector function of afucosylated IgGl. Notably, despite the enhanced effector function of afucosylated IgGl, antibody OX116 did not show a significant increase in IFNg induction. The potential mechanisms are explored below. Overall, these data show that the Fab format of the antibodies can inhibit polyclonal CD1a-dependent T cell reactivity, with improved evidence for OX16, OX110 and OX116 relative to CR2113 and mAb571 highlighted by comparison using different IgGl Fc region comparators. The ability of the antibodies to modulate IFNg production by CD1a-responsive T cell clones was next investigated Figure 7 C). All antibodies showed the ability to inhibit IFNg production by CD1a-responsive T cell clones, both on wild-type human IgGl Fc region and as Fab variants.

[0989] Figure 20

[0990] In view of Figure 20 The ability of the antibodies to induce complement-mediated cytotoxicity on different human Fc backgrounds compared to CR2113 and mAb571 was investigated using a murine Fc region in the assay Figure 19A). When placed on all changes in the human IgGl background, OX16 and OX110 do not induce CDC, but OX116 induces significant killing in the presence of complement. However, when using the Fab form of OX116, no CDC was observed, suggesting an Fc-dependent effect. When placed on different IgGl Fc regions, antibody OX116 showed improvement over the published antibodies CR2113 and mAb571, which is relevant to the use of the antibody for specific indications, for example, where cytotoxicity of cells expressing CD1a can be beneficial to the patient, such as in the case of CD1a-expressing malignancies. Next, the ability of anti-CD1a antibodies on different human Fc backgrounds to induce antibody-dependent cellular cytotoxicity (ADCC) was tested. All anti-CD1a antibodies, human IgGl and a-fucosylated IgGl, showed evidence of ADCC of CD1a-expressing target cells Figure 19 B). No significant ADCC was observed for the Fab form of the antibodies. Notably, ADCC cannot alone explain the findings in Example 12 - Inhibition of TCR binding to CD1a by OX116 , as in the latter, the effector population consists of T cells and does not include NK cells, and a different effector:target ratio was used. Moreover, inhibitory responses were observed with the Fab form of the antibodies in Figure 21 . Thus, the anti-CD1a antibodies show T cell blocking function as well as some Fc forms of the antibodies that show ADCC. Moreover, as co-illustrated above, OX116 can also induce direct killing of CD1a-expressing cells.

[0991] Figure 21

[0992] Next, it was investigated whether OX116 can inhibit the binding of known CD1a-reactive TCRs. Biotinylated ScFv of OX116 was captured on a streptavidin chip Figure 21 A), followed by injection of CD1a, and then injection of 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)). BK6 αβ TCR binds to the A' top of CD1a, and CO3 γδ TCR recognizes the α1 domain of CD1a within the region overlapping with the epitope of OX110 and OX116 antibodies. The CO22 binding site was not established, but is independent of the A' top and requires the α3 domain of CD1a. As expected, CO22 TCR bound to the CD1a-OX116 complex Figure 21B green curve). To explore whether binding of OX116 to the side of CD1a could have an indirect distal effect on the shape of the A' cusp and its recognition by self-reactive TCRs, we used the BK6 TCR. However, in this case, binding was still detectable Example 13 - Generation of OX25 antibodies binding to the α3 domain of CD1a B blue curve), indicating that OX116 does not interfere with the recognition of the A' cusp. We used scFv fragments in the SPR experiments, so it is possible that binding of full-length antibodies would result in more pronounced steric hindrance, with an inhibitory effect on self-reactive TCRs. As expected, the interaction of the CO3 TCR, which is known to bind close to the F' portal, was completely abolished by OX116 Figure 22 B, red curve), confirming that the binding of TCRs and antibodies is mutually exclusive due to the proximity of the adjacent epitopes on the surface of CD1a. Overall, these data show that OX116 can inhibit the engagement of TCRs with CD1a.

[0993] Example 14 - Humanized antibodies can deplete transfectants expressing CD1a and inhibit CD1a autoreactive T cells

[0994] Having established the footprints of OX16, OX110 and OX116 anti-CD1a antibodies against the al and a2 domains, we sought to discover antibodies against the a3 domain. Ab 25 was generated and selected according to the "Generation and selection of therapeutic anti-CD1a antibodies" Materials and Methods section Figure 23 ).

[0995] Figure 24

[0996] Humanized variants of antibodies (Ab 1-51) were generated and tested for binding to K562-CD1a transfectants. Ab 1-15 were derived from OX116 and Ab 16-51 from OX16. All 51 variant antibodies showed evidence of binding to CD1a expressed by the transfectants. However, it was noted that there was a range of mean fluorescence intensity (MFI) and that the top antibodies were progressed towards functional analysis. Example 15 - Activity of antibody 25 K562-CD1a transfectants were shown to be depleted in the presence of anti-CD1a antibodies. Humanized antibodies derived from OX116 (12345) all retained the ability to deplete and those derived from OX16 (161721, 222831, 34363839, 414246474851) did not have intrinsic depleting activity, except variants 28 and 51 which showed significant depleting ability. Variants were next tested for their ability to block CD1a reactive T cell clones Figure 22 ), and most showed significant ability to block IFNg production, with antibody 51 performing the strongest. Humanized antibodies can have direct diagnostic / monitoring / therapeutic utility, or can be included as part of other approaches including bi-specific or multi-specific molecules or as part of cell therapies.

[0997] Figure 25

[0998] The antibody OX25, which binds the a3 domain of CD1a, has been established ( Example 16 - CD1a antibodies can treat skin inflammation ) and next tested for its ability to block CD1a reactive T cells ( Figure 26 ). This demonstrated that polyclonal CD1a self-reactive T cell production of IFNg was not inhibited by OX25 as expected. Furthermore, OX25 was found to compete with CD1a for binding to SK9, but not with antibodies that bind the a1 and a2 domains of CD1a. These data demonstrate the juxtramembrane binding site of OX25, which can provide diagnostic, monitoring and / or therapeutic utility where non-competitive juxtramembrane domains are advantageous, as observed with certain checkpoint agonists.

[0999] Figure 26

[1000] Checkpoint inhibitors are increasingly used to manage malignancies, but can be limited by side effects, including various inflammatory skin reactions, such as psoriasiform inflammation. This can sometimes necessitate cessation of checkpoint inhibitors, resulting in potential malignancy management, or use of broad immunosuppressants, which also have potential impact on immune control of potential malignancies. Methods to treat tissue inflammation to allow continued use of checkpoint inhibitors would have therapeutic utility. It was investigated whether CD1a and Langerhans cell pathways can be involved by using OX116 and anti-PD-1 antibodies, alone or in combination, in a model of imiquimod-induced skin inflammation. Figure 26 A shows a schematic of the method, which determined a significant reduction in ear thickness of PD-1 associated inflammation in the presence of OX116 ( Example 17 - CD1a antibodies can treat itch B) and reduction in percentage of skin IL-17A+ T cells ( Figure 27 C). The degree of reduction in ear thickness was surprising and significant, and revealed that these pathways are relevant.

[1001] Figure 27

[1002] A major impact of inflammatory skin diseases and other conditions on quality of life is itch. CD1a has not previously been associated with itch, and so establishing relevance of the pathways would represent a new area of biology. Next the role of CD1a and Langerhans cells in itch was evaluated using a model of MC903-induced skin inflammation. Example 18 - CD1a antibodies can be coupled to other antibodies to increase effectiveness A shows that both OX116 and OX16 significantly reduce itch, which is accompanied by reduction in alarmin cytokines known to play a role in itch ( Figure 28B). These data are surprising, novel and inventive as CD1a pathway has not been implicated in pruritus previously. These findings will support the use of anti-CD1a antibodies in the prevention and treatment of pruritus and related diseases.

[1003] Figure 28

[1004] Anti-CD1a antibodies can act as a method of targeting bispecific modalities, including 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 co-cultured with CD1a expressing transfectants. Table B shows the sequences of WIMM-3 used. Figure 28 A (left panel) shows that WIMM-3 T cell engager successfully activates reporter T cells in vitro. Discussion A (right panel) shows that WIMM3 promotes potent CD8+T cell mediated cytotoxicity of CD1a expressing target cells. Summary B shows that this is also reflected in an immunodeficient xenograft model, where CD1a expressing target cells are administered with human CD8+T cells. WIMM-3 CD1a-T cell engager potently inhibits tumor growth, which is associated with a significant improvement in survival. These data show that anti-CD1a antibodies can potently 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 modalities to CD1a expressing cells.

[1005] References

[1006] Skin inflammation (such as dermatitis, psoriasis, and lupus) is a common condition with significant associated physical and psychological morbidity. Cutaneous adverse reactions to drugs are also common, with 1.8-7 per 1000 inpatients. Severe cutaneous adverse reactions with broad and systemic effects (such as SJS, TEN, AGEP, and DRESS) are less common; for example, SJS / TEN has an incidence of about 1-6 per million individuals per year (M. Mockenhaupt, Allergol Select 1, 96-108 (2017)). Gell and Coombs defined a classification of hypersensitivity reactions in the 1960s, in which the delayed type IV hypersensitivity reaction requires the action 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, 2nd edition, 1968)). Although it is increasingly recognized that this classification does not explain all aspects of drug hypersensitivity reactions, there remains a major focus on the altered recognition of covalent hapten or non-covalently modified peptide / MHC molecules. However, the current model does not explain the preponderance of skin and mucosal involvement in drug hypersensitivity reactions (M. Mockenhaupt, Allergol Select 1, 96-108 (2017)).

[1007] By generating CD1a transgenic mice and autoreactive human CD1a-restricted enriched T cell lines, and characterizing functional anti-CD1a antibodies, the data provided herein show that CD1a presentation of endogenous lipid ligands is induced. This leads to autoreactive T cell-mediated skin and systemic inflammation. Anti-CD1a antibodies have clinical and immunological effects, whether they are blocking or blocking / modulating, indicating that CD1a lipid presentation to T cells is important. TLR7 can recognize single-stranded RNA, and it is of interest that reactivity to viral infection can mimic the clinical phenotype of different severe forms of skin inflammation, including psoriasis, dermatitis, lupus, and adverse inflammatory reactions to drugs, including SJS and TEN. Such a common final common clinical presentation can indicate that many precipitants can facilitate CD1a autoreactivity and autoinflammation. The model can also help explain the increased risk of autoimmunity associated with certain drug reactions, including lupus erythematosus and DRESS syndrome. Furthermore, the finding will imply that CD1a autoreactivity disrupts broader T cell tolerance.

[1008] In addition to the effect on the T cell response to the imiquimod containing drug Aldara, an increase in neutrophil and eosinophil responses in the skin, draining lymph nodes and spleen was observed in CD1a transgenic mice. These effects were inhibited by administration of the antibodies of the application, in particular 16, 110 and 116. This implicates a CD1a dependent immune cascade which reaches more broadly than initially anticipated. Reduction of neutrophils has been shown to ameliorate 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)).

[1009] Aldara / imiquimod application recapitulates key aspects of different forms of skin inflammation and associated systemic diseases and conditions, including psoriasis, dermatitis, lupus and severe skin hypersensitivity reactions, including T cell and neutrophil infiltration as described above. The data demonstrated herein show that imiquimod dependent eosinophil infiltration of the skin, lymph nodes and spleen is enhanced in CD1a transgenic mice and reduced by administration of the antibodies of the application, in particular 16, 110 and 116.

[1010] Furthermore, it was reported that the number of LCs in lesional skin is increased compared to non-lesional skin of patients with different forms of inflammatory skin diseases or conditions, including psoriasis, dermatitis, lupus and maculopapular drug eruptions, and decreases to non-lesional levels as the skin rash resolves (D.I. Dascalu, Y. Kletter, M. Baratz, S. Brenner, Acta Derm Venereol 72, 175-177 (1992)). Interestingly, psoriasis is associated with altered LC migration, suggesting that despite the well-studied and effective murine models of psoriasis and lupus and dermatitis, it is also applicable to include adverse drug inflammatory drug reactions. Here, the inventors show that CD1a antibody-dependent modulation of LCs is associated with reduced skin inflammation following administration of the antibodies of the invention, particularly 110 and 116, which can have therapeutic importance for the treatment of psoriasis, dermatitis, lupus, inflammatory drug reactions and other conditions. Epitope analysis highlights the potential therapeutic importance of the epitope binding sites; anti-CD1a antibodies are divided into two groups based on the binding sites and resulting effector functions. Epitope sites can promote clustering and changes in phenotypic effects seen with antibodies 77a, 111 and 16, which are not major blocking antibodies, but rather antibodies 110 and 116. Clustering can indeed lead to cross-linking / agglutination-like cell morphology, which can also explain the reduction of CD1a-transfected K562 and monocyte-derived LCs, as both cell types express high levels of CD1a, higher than monocyte-derived DCs. The different antibody binding sites of the two groups do not compete, so a combination selected from each of the two groups has utility, for example, in treatment / monitoring or combination therapy.

[1011] The role of CD1a in the pathogenesis of skin inflammation and related systemic diseases implicates its role in many diseases, including psoriasis, dermatitis and lupus erythematosus and drug hypersensitivity reactions. Furthermore, the characterization of CD1a blocking and modulating antibodies provides new potential avenues for the development of prophylaxis and treatment of skin inflammation and CD1a-expressing malignancies.

[1012] The data shown herein define the CD1a contact points for anti-CD1a antibodies OX16, OX110 and OX116. The binding site for OX110 and OX116 is proximal to the F’ portal and is distinct from other published structures of anti-CD1a antibodies (US10844118 and WO / 2022 / 077021) that bind at the A’ top. OX16 and OX116 are able to bind CD1a loaded with different lipids, including permissive and non-permissive ligands. Given the proximity of OX116 to the F’ portal of CD1a, this is surprising but suggests that OX116 can have broad utility in CD1a binding and / or CD1a blockade. The binding of OX16 is negatively impacted by lipid antigens with bulky protruding head groups that mimic the behavior of self-reactive T cells and create the possibility of using OX16 as a blocker of CD1a that carries only self-reactive permissive / small lipids but not lipids with properties in which co-recognition of the head group is expected to trigger a desired immune response (e.g. response to Mtb lipids). These findings are consistent with the ability of OX16, OX110 and OX116 antibodies to suppress polyclonal CD1a-dependent reactivity (including responses to IFNy and IL-22) with broad relevance. Comparison of functional suppression of polyclonal T cell responses with published antibodies CR2113 and mAb571 (US10844118 and WO / 2022 / 077021). Antibodies show the ability to broadly and significantly reduce CD1a-dependent self-reactivity with significant improvement of OX16, OX110 and OX116 relative to published antibody comparators.

[1013] The functional effects of OX16, OX110 and OX116 on CD1a expressing target cells were altered using different human IgGl Fc variants. While all three antibodies show clear improvement over published CR2113 and mAb571 antibodies, it is noted that OX116 produces the greatest CDC and confluence loss of CD1a expressing target cells, which suggests potential utility of OX116 in environments where depletion of CD1a expressing cells can be advantageous (e.g. in CD1a expressing malignancies). The use of antibodies in Fab form does not induce CDC or confluence loss, suggesting that the Fc region and / or dimerization is required for such effector functions.

[1014] Thus, in general, a family of antibodies with binding sites across CD1a is described. As shown in Examples 2-7 above, having a range of binding sites can have utility in detecting CD1a or modulating CD1a function in different ways, alone or in combination.

[1015] OX25. Antibodies were examined for their effect on IFNg and IL-22 production. These cytokines are broadly implicated in inflammatory skin diseases and related systemic diseases. For example, IFNg is known to promote T cell and neutrophil responses and Ig class switching, as well as MHC class I and II induction, thereby amplifying innate and adaptive immune responses. IL-22 is known to have broad effects on epithelial and stromal cells, promoting cell proliferation, antimicrobial peptide expression, and skin and systemic inflammation. IL-22 is implicated in many inflammatory diseases, including systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, and psoriasis (Dudakov JA et al., Ann Review Immunol 33:747-85 (2015)).

[1016] Antibodies OX16, OX110, OX116, and OX25 have different binding footprints, with different associated functions. The ability to bind the a1, a2, or a3 domains of CD1a provides opportunities to identify CD1a and modulate CD1a function, alone or in combination. This can be through the use of linked formats or separate anti-CD1a antibodies, or as part of other bispecific constructs (or other binding agents) or cell-based therapeutic approaches. Different binding sites also provide the potential to exploit combinations in diagnosis or monitoring of treatment.

[1017] Table 11 - Sequence ID

[1018] In summary, the inventors have generated an improved set 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 showed efficacy in exemplary inflammatory skin disease prevention and treatment models, which have features of psoriasis, dermatitis, lupus erythematosus, and drug reactions manifesting as inflammatory skin or mucosal diseases or disorders, and those drug reactions that are systemic (non-skin), and in xenograft tumor models. The success of the antibody discovery process in identifying improved antibodies can be attributed to the combination of: a) screening a large number of hits (3500); b) use of a novel chimeric immunogen, whereby the human CD1a lipid binding domain was fused to the host organism CD1d Ig domain, thereby targeting antibody production to the lipid binding domain for which functional inhibitory potential is likely to exist; c) examination of multiple polyclonal and enriched T cell assays for different functional outcomes.

[1019] In vitro human functional assays showed that the antibodies were more potent than commercially available antibodies as measured by IC50 evaluation of primary polyclonal T cell response inhibition. Furthermore, using a highly sensitive human CD1a restricted T cell clone assay, it was determined that anti-CD1a antibodies 16 and 116 were able to block IL-22 production, which is a key regulator of inflammatory skin and mucosal diseases. This activity is an improvement and surprising because it was not shown in prior publications or patents for anti-CD1a CR2113 ((16, 17), US 10844118B2 and CA 2924882 A1) where IL-17 or IFNy production was induced and inhibited in murine systems. IL-22 inhibition is an important advantage of the antibodies because IL-22 is a key regulator of skin and mucosal diseases.

[1020] Parallel analysis of human and in vivo murine models provides a robust means to assess the therapeutic benefits of newly generated antibodies. In vivo, imiquimod is used to induce psoriasiform, dermatitis-like, lupus-like, drug reaction-like phenotypes and provides a model skin inflammation series, and can be more broadly applicable to many inflammatory diseases and conditions and related systemic diseases or conditions and systemic manifestations of inflammatory drug reactions. Here it is shown that antibodies 110, 116 and 16 significantly reduce CD1a-dependent inflammation induced by imiquimod with improvements over the standard of care (anti-IL-17A) and comparator anti-CD1a antibody (CR2113) in the same murine IgG1 background. Importantly, and unexpectedly, antibody 116 reduces skin inflammation to below that of WT imiquimod treated mice and normalizes many skin and systemic immune markers to WT skin and systemic immune markers, suggesting that anti-CD1a 116 has mechanisms of action beyond inhibition of CD1a-TCR signaling. Immunophenotyping of the skin revealed a reduction in T cell numbers and activation, and neutrophil infiltration to WT levels upon 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 CR2113, highlighting the potential of antibodies 110, 116 and 16.

[1021] Importantly, when analyzing LC populations in the skin, a significant reduction of CD11c+Langerin+LC was observed after administration of antibodies 110 and 116. This reduction cannot be explained by an enhanced migration to draining lymph nodes. However, it is possible that antibodies 110 and, to a greater extent, 116 are able to directly reduce CD1a+cells in vivo, which explains the reduction of skin LC in vivo and is demonstrated by a significant reduction of human CD1a+cells in vitro. This is a surprising result considering the mouse IgGl isotype of the antibodies, where a mouse IgG2a isotype is more likely to cause cytotoxicity through complement-mediated lysis or antibody-dependent cellular cytotoxicity, and further the proprietary and published anti-CD1a CR2113 has been reported to not directly deplete (17), although here apoptosis of CD1a expressing cells can also be induced by CR2113 in the mouse IgGl context. The modulating ability of these antibodies can help explain the reduction of imiquimod-induced inflammation below that of WT isotype treated mice. Antibody 116 not only blocks the interaction of CD1a with the TCR, but also modifies LC, reduces / reset the inflammatory potential of the skin, and normalizes many skin and systemic immune markers to those of the WT. This can explain the improved effect beyond the CD1a-dependent response to improve beyond the wild type, whereas anti-CD1a CR2113 does not.

[1022] Furthermore, the data suggest that the 16, 110 and / or 116 antibodies presented herein have utility in treating malignant tumors expressing CD1a, such as Langerhans cell histiocytosis or some forms of T cell lymphomas and thymomas. This can be through a direct action, or where the anti-CD1a antibody is coupled or associated with one or more other therapeutic agents selected from the group comprising: cytotoxic agents, anti-inflammatory agents such as steroids, and CAR-T cells such as regulatory or cytolytic CAR-T cells or other cells expressing or presenting antibodies or antigen binding fragments.

[1023] This study demonstrates that antibody 16 is a highly potent blocking antibody that eliminates CD1a-dependent inflammation in vivo without inducing direct cell apoptosis, 110 is a highly potent blocking and modifying antibody that significantly reduces CD1a-dependent inflammation in vivo, 116 is a highly potent blocking and modifying antibody that reduces inflammation below WT levels and normalizes many skin and systemic immune markers to WT markers. This grouping of antibodies is consistent with the basic epitope analysis where the direct modifying antibodies 110 and 116 cluster and the blocking antibodies 77a, 111 and 16 cluster. The epitope analysis also reveals that the group 77a, 111 and 16 overlap with the epitope recognized by the non-depleting NA1 / 34; this is important because NA1 / 34 has been shown to cross-block the binding of the anti-CD1a CR2113. Antibodies 110 and 116 do not cross-block NA1 / 34 and therefore likely represent different epitope regions. The antibodies remain on the LC in the skin in vivo and even after migration to the lymph nodes. This is an important enhancement because the clinical effect will be more durable.

[1024] With these data, the inventors demonstrate the potential of this improved improved set of anti-CD1a antibodies in the prevention and treatment of inflammatory skin and mucosal disorders including but not limited to psoriasis, dermatitis, lupus, and for the treatment and / or prevention of one or more associated systemic diseases or disorders or one or more systemic manifestations of an inflammatory drug reaction. The effects on the broad cascade of inflammation including LCs, T cells and neutrophils, particularly of antibodies 110, 116 and 16, will have broad effects in inflammatory skin and mucosal disorders including psoriasis, dermatitis, lupus and drug reactions manifested as inflammatory skin or mucosal disease or disorders or malignancies expressing CD1a.

[1025] Here, the structural basis of antibodies that bind to CD1a is defined and the lipid dependence of the binding is examined, as well as the in vitro functional effects of different human IgGl variants on function. It is shown that OX16 and OX116 bind at different sites and that OX116 binding is completely lipid antigen independent, despite proximity to the F' portal, which explains the broad effects on CD1a blocking. However, the interaction of CD1a with OX16 is modestly affected by the head group of the prominent lipid, which suggests a possible mechanism of selective self-reactive recognition of the CD1a molecule that only carries certain classes of small permissive self-lipids but not those with larger head groups that can be immunologically problematic. An anti-CD1a antibody OX25 is generated that has a binding site on the a3 domain of CD1a; the data collectively present a series of antibodies with different associated functions that bind to CD1a. An improvement of the antibodies over the other published antibodies CR2113 and mAb571 is also shown, which is consistent with the role of the antibodies in the diagnosis, monitoring, prevention and treatment of CD1a-dependent diseases.

[1026] In summary, the inventors demonstrate that the improved anti-CD1 a antibodies 16, 77a, 110, 111 and 116 are useful as a method for preventing and treating inflammatory skin and mucosal diseases or conditions, or associated systemic diseases or conditions, or systemic manifestations of inflammatory drug reactions, or CD1 a-expressing malignancies, by blocking CD1 a and / or modifying the phenotype / function of CD1 a+ cells.

[1027] Figure 2

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[1058] 31. Stockenhuber K, Hegazy AN, West NR, Ilott NE, Stockenhuber A, Bullers SJ, Thornton EE, Arnold IC, Tucci A, Waldmann H, Ogg GS, Powrie F. Foxp3+ T reg cells control psoriasiform inflammation by restraining an IFN-I-driven CD8+ T cell response. J Exp Med. 215, 1987-1998 (2018).

[1059] All references cited herein, including patents, patent applications, papers, textbooks, and references cited therein, are hereby incorporated by reference in their entirety to the extent not previously incorporated by reference.

[1060] Figure 2 Example 3 - In vivo evaluation of inhibitory antibodies in skin inflammation Generation of CD1a transgenic mice Figure 3 Figure 3 Figure 3 Figure 3 Figure 3 Figure 4 Figure 4 Figure 4 Example 4 - In vivo effect of inhibitory antibodies on skin immune responses Figure 5 Figure 5 Figure 5 Figure 5 Figure 6 Figure 6 Figure 6 Figure 6 Example 5 - Cytotoxicity observed with anti-CD1a antibodies expressed in terms of effect on CD1a expressing cell phenotypes Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 2 Figure 2 Example 6 - Epitope binding analysis of CD1a antibodies Figure 8 Figure 8 Figure 6 Figure 8 Figure 8 Example 7 - Demonstration of effectiveness of antibodies of the application for the treatment of imiquimod-induced inflammation and systemic related inflammation Therapeutic potential of anti-CD1a antibodies Figure 9 Figure 9 Figure 9 Figure 9 Figure 9 Figure 8 Figure 9 Figure 9 CD1a involvement in systemic immune response to imiquimod Figure 10 Figure 10 Figure 10 Figure 10 Figure 11 Figure 12 Figure 13 Figure 13 Figure 9 Figure 14 Figure 15 Figure 15 Figure 15 Figure 15 Figure 15 Figure 16 Figure 16 Example 8 - Crystal structure of anti-CD1a antibodies Figure 17 Example 9 - Role of lipids in OX16 and OX116 binding to CD1a Figure 18 Figure 18 Example 10 - Effect of anti-CD1a antibodies on blocking polyclonal and clonal T cell function Figure 19 Figure 19 Example 11 - CDC and ADCC effect of anti-CD1a antibodies Figure 7 Figure 20 Figure 20 Figure 19 Figure 19 Example 12 - Inhibition of TCR binding to CD1a by OX116 Figure 21 Figure 21 Figure 21 Figure 21 Example 13 - Generation of OX25 antibodies binding to the α3 domain of CD1a Figure 22 Example 14 - Humanized antibodies can deplete transfectants expressing CD1a and inhibit CD1a autoreactive T cells Figure 23 Figure 24 Example 15 - Activity of antibody 25 Figure 22 Figure 25 Example 16 - CD1a antibodies can treat skin inflammation Figure 26 Figure 26 Figure 26 Example 17 - CD1a antibodies can treat itch Figure 27 Figure 27 Example 18 - CD1a antibodies can be coupled to other antibodies to increase effectiveness Figure 28 Figure 28 Figure 28 Discussion Summary References Table 11 - Sequence ID

[1061]

[1062]

[1063]

[1064]

[1065]

[1066]

[1067]

[1068]

[1069]

[1070]

[1071]

[1072]

[1073]

[1074]

[1075]

[1076]

[1077]

[1078]

[1079]

[1080]

[1081]

[1082]

[1083]

[1084]

[1085]

[1086]

[1087]

[1088]

[1089]

[1090]

[1091]

[1092] The underlined portions of any of the above DNA sequences represent signal sequences.

Claims

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

2. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof comprising or consisting of: (a) a heavy chain comprising or consisting of: SEQ ID NO:219, SEQ ID NO:220 or SEQ ID NO:221, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain comprising or consisting of SEQ ID NO: 218, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical 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 at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain comprising or consisting of SEQ ID NO: 214, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical 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 at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain comprising or consisting of SEQ ID NO: 210, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical 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 at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain comprising or consisting of SEQ ID NO: 253, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto.

3. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof comprising or consisting of: (a) a heavy chain variable region comprising: CDR1 of SEQ ID NO:33, CDR2 of SEQ ID NO:34, and CDR3 of SEQ ID NO:35, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain variable region comprising: 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 The CDR3 of SEQ ID NO:38, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130 or SEQ ID NO:131, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (b) a heavy chain variable region comprising: 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 SEQ ID NO: 19 or SEQ ID NO: 110, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain variable region comprising: CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:21, and The 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, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (c) a heavy chain variable region comprising: CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO: 27, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain variable region comprising: CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO:30, SEQ ID NO:120, SEQ ID NO:121 or SEQ ID NO:122, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (d) a heavy chain variable region comprising: CDR1 of SEQ ID NO:9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain variable region comprising: 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 sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (e) a heavy chain variable region comprising: CDR1 of SEQ ID NO: 1, 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 CDR2 of 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 sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain variable region comprising: 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 sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (f) a heavy chain variable region comprising: 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 sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or c) a light chain variable region comprising: CDR1 of SEQ ID NO:94, CDR2 of SEQ ID NO:95, and CDR3 of SEQ ID NO:96, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto.

4. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof comprising or consisting of: (a) a heavy chain variable region comprising or consisting of: SEQ ID NO:39 or SEQ ID NO:194, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or 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 at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (b) a heavy chain variable region comprising or consisting of SEQ ID NO:23, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172 or SEQ ID NO:173, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or 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 at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (c) a heavy chain variable region comprising or consisting of SEQ ID NO: 31 or SEQ ID NO: 178, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or a light chain variable region comprising or consisting of SEQ ID NO: 32, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176 or SEQ ID NO: 177, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (d) a heavy chain variable region comprising or consisting of SEQ ID NO: 15 or SEQ ID NO: 157, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or a light chain variable region comprising or consisting of SEQ ID NO: 16, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155 or SEQ ID NO: 156, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; or (e) 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 at least 80%, 90%, 95%, 98%, 99%, or 100% identical thereto; and / or a light chain variable region comprising or consisting of SEQ ID NO:8, SEQ ID NO:195, SEQ ID NO:196, or SEQ ID NO:197, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical 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 at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto; and / or A light chain variable region comprising or consisting of SEQ ID NO: 98 or SEQ ID NO: 260, or a sequence at least 80%, 90%, 95%, 98%, 99% or 100% identical thereto.

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

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

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

8. An antibody or antigen-binding fragment thereof, which binds to an epitope on CD1a, or which competes with an antibody or antigen-binding fragment thereof for binding to CD1a, wherein the epitope of CD1a comprises or consists of: (a) CD1a residues Arg 83, Tyr 84, His 86, Glu 87, Gln 89, Phe 90, Glu 91, Asn 139, Met 140, Lys 142, His 143, Lys 146, Val 147 and Gln 150, and wherein the residue numbering is according to SEQ ID NO: 252; or (b) residues Glu 62, Glu 65, Leu 66, Thr 68, Leu 69, Ile 72, Asn 151, His 153, Glu 154, Ile 157, Asn 160, Asp 164, Thr 165 and Arg 168 of CD1a, and wherein the residue numbering is according to SEQ ID NO: 252; or (c) residues Glu 79, Arg 82, Arg 83, His 86, Glu 87, Gln 89, Phe 90, Glu 91, Tyr 92, Val 147 and Asn 150 of CD1 , and wherein the residue numbering is according to SEQ ID NO:

252.

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

10. A vector comprising the nucleic acid according to claim 9. 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 claim 1 , 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. 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. 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, for use in treating or preventing one or more inflammatory skin or mucosal diseases or conditions, or one or more related systemic diseases or conditions, or one or more systemically manifested inflammatory drug reactions, or a CD1a-expressing malignancy.

17. One or more antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells or pharmaceutical compositions for use according to claim 16, wherein: (a) the one or more inflammatory skin or mucosal diseases or conditions are one or more of the following: (i) mainly neutrophilic skin diseases, such as acne, generalized pustular psoriasis, plaque psoriasis, guttate psoriasis, palmoplantar pustulosis, SAPHO syndrome, acute febrile neutrophilic dermatosis (Sweet syndrome), histiocytic neutrophilic dermatitis, neutrophilic dermatosis of the dorsal hand, pyoderma gangrenosum, neutrophilic hidradenitis, hidradenitis suppurativa, erythema protuberans persistent, Behçet's disease, enterodermatitis-arthritis syndrome, other infection-related inflammation, neutrophilic urticarial dermatosis, palisade neutrophilic granulomatous dermatitis, serpiginous erythema gyri, neutrophilic annular erythema, acute generalized exanthematous pustulosis (AGEP), vasculitis, etc.; (ii) autoimmune disorders, such as connective tissue diseases (e.g., lupus, dermatomyositis, scleroderma / systemic sclerosis, Tschag-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, generalized hair loss, alopecia totalis, panniculitis, lichen planus, erythema multiforme, lichen sclerosus, other lichenoid and erythema multiforme-like diseases, vesicular 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 Mueller-Weiss syndrome, hypereosinophilia and systemic symptom syndrome, urticaria, angioedema, keratoconjunctivitis, food allergies, other allergic or atopic diseases, 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 conditions, such as Stevens-Johnson syndrome, toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms syndrome (DRESS) and acute generalized keratopustulosis (AGEP), erythema multiforme, bullae, fixed drug eruptions, checkpoint inhibitor-associated skin and other inflammatory diseases; (v) graft-versus-host disease; (vi) pruritus and pruritic conditions, including prurigo nodularis, (b) one or more related systemic diseases or conditions, or one or more systemic manifestations of an inflammatory drug reaction, or an inflammatory reaction to Aldara (imiquimod); or (c) The CD1a-expressing malignancy is one or more of the following: Langerhans cell histiocytosis, Langerhans cell sarcoma, T-cell lymphoma subsets, thymoma subsets, or rare cases of other malignancies, such as mastocytosis subsets.

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 conditions are one or more of psoriasis, dermatitis, lupus erythematosus or a drug reaction manifesting as an inflammatory skin or mucosal disease or condition.

19. 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 for administration alone or in combination with one or more other therapeutic agents.

20. One or more antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells or pharmaceutical compositions for use according to claim 19, wherein the one or more other therapeutic agents are selected from the group consisting of: cytotoxic agents; anti-inflammatory agents, such as steroids; and CAR-T cells, such as regulatory or cytolytic CAR-T cells, or other cells expressing or presenting one or more antibodies or antigen-binding fragments 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 treating or preventing one or more inflammatory skin or mucosal diseases or conditions, or one or more related systemic diseases or conditions, or one or more systemically manifested inflammatory drug reactions, or one or more CD1a-expressing malignancies.

22. A method of treating one or more inflammatory skin or mucosal diseases or conditions, or one or more related systemic diseases or conditions, or one or more systemically manifested inflammatory drug reactions, 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 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.

23. A method of monitoring the efficacy of treatment or the disease state of a subject diagnosed with a CD1a-expressing malignancy, the method comprising: i. providing a biological sample obtained from the subject; ii. determining the level of binding of one or more antibodies or antigen-binding fragments according to any one of claims 1 to 8 to cells expressing CD1a in the sample obtained from the subject before treatment, or at an interval between treatments, or at a time interval in the absence of treatment; iii. If after treatment, or between treatment intervals, or in time intervals in the absence of treatment, the tumor volume is reduced or the level of binding of one or more antibodies or antigen-binding fragments of the invention to cells expressing CD1a is reduced, then the treatment is determined to be effective or the disease state is improving, optionally wherein the reduction in tumor volume or the reduction in the level of binding of one or more antibodies or antigen-binding fragments according to any one of claims 1 to 6 to cells expressing CD1a is 25% or more.

24. A method of diagnosing a subject having an inflammatory skin and mucosal disease or condition, or a related systemic disease or condition, or a systemically manifested inflammatory drug reaction, or a CD1a-expressing malignancy, the method comprising: i. providing a biological sample obtained from the 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 according to any one of claims 1 to 8; iii. comparing the expression level of CD1a in the sample obtained from the subject with the expression level of CD1a in a positive or negative reference sample; iv. 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, then determining that the subject has an inflammatory skin and mucosal disease or disorder, or a related systemic disease or disorder, or an inflammatory drug reaction with systemic manifestations, or a malignant tumor expressing CD1a; or 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 is determined not to have inflammatory skin and mucosal diseases or disorders, or related systemic diseases or disorders, or systemic inflammatory drug reactions, or malignancies expressing CD1a.

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