Antibody

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof capable of binding to CD1a, which is particularly suitable for the treatment or prevention of one or more inflammatory skin or mucosal disorders, or one or more associated systemic diseases or disorders, or one or more systemically manifested inflammatory drug reactions, or CD1a-expressing malignancies.

Description

[Technical field]

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

[0002] Antigen presentation is one of the fundamental pillars of host immunity, where the immune system detects threats, including infection, tissue damage and disease, and orchestrates a targeted defense. Antigen presentation involves the internalization, processing and presentation of antigens by presentation molecules on the surface of specialized antigen-presenting cells (APCs). Antigen presentation is orchestrated to achieve optimal activation of immune responses targeting the antigen source and eliminating the threat. Antigens encompass a wide range of molecules, including peptides, lipids and metabolites. MHC I and MHC II are proteins expressed on the surface of APCs, which bind and present peptide antigens, mostly to CD8+ and CD4+ T cells, respectively. These T cell subsets are induced to exert their effector functions upon recognition of MHC-bound peptide antigens by cell surface T cell receptors (TCRs), enabling immunity against pathogens and cancer. However, dysregulation of presentation of non-invasive antigens, e.g., allergens in allergic diseases, or self-proteins in autoimmunity, leads to host damage, inflammation and disease. Targeting the antigen presentation pathway is therefore a powerful means of modulating the resulting immune response.

[0003] CD1 molecules constitute a family of antigen-presenting molecules structurally similar to MHCI. In contrast, CD1 molecules are relatively non-polymorphic, and the CD1 antigen-binding groove is enriched in hydrophobic amino acids that allow the presentation of lipid species. Lipids are important antigens that form an integral component of host and pathogen cell membranes and are less susceptible to mutations than protein-derived peptide antigens. The CD1 family comprises the 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 has come from studies of inmutated natural killer T cell recognition of glycolipid-bound CD1d, in part because CD1d is the only CD1 normally expressed in mice. CD1d and MHCI molecules are widely expressed, whereas MHCII and group 1 CD1 expression is relatively restricted to APCs. However, unique among such molecules, CD1a is highly specific for skin and mucous membranes. CD1a is constitutively expressed by epidermal and mucosal Langerhans cells (LCs) of the skin (1) and is commonly used as an identifying marker for LCs in addition to langerin. In addition, CD1a is expressed at low levels on a subset of dermal dendritic cells (2-4) and can be expressed and upregulated on cutaneous innate lymphoid cells (ILCs), particularly ILC2s (5). Importantly, CD1a was first described as present on the surface of immature thymocytes, but expression is typically lost upon T cell maturation (6). High levels of constitutive expression of CD1a in the skin indicate an important physiological role for CD1a-dependent surveillance and T cell activation in healthy and diseased human skin. Moreover, elevated CD1a expression in atopic dermatitis skin may underlie enhanced activation of CD1a-reactive T cell populations in inflammatory skin diseases.

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

[0005] CD1a is relatively non-polymorphic, and as a result there is a potential for population-wide prevention and / or treatment of inflammatory skin and mucosal diseases and disorders, such as atopic dermatitis, psoriasis, lupus erythematosus, or related systemic diseases or disorders, or systemically manifested inflammatory drug reactions, where the frequency of CD1a-expressing dendritic cell subsets is altered and the migration patterns of LCs or responding T cells are modified (13-15). Moreover, CD1a has been associated with other systemic disorders 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 may be expressed by certain malignancies including Langerhans cell histiocytosis, a subset of T cell lymphomas, a subset of thymomas and rare forms of other malignancies, e.g., a subset of mastocytosis. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide an anti-CD1a antibody. Such an antibody is particularly useful in the treatment or prevention of inflammatory diseases or disorders of the skin or mucosa, such as psoriasis, dermatitis, lupus erythematosus, or drug reactions that manifest as inflammatory diseases or disorders of the skin or mucosa. Such an antibody may also be beneficial in the treatment or prevention of related systemic diseases or disorders, or inflammatory drug reactions that manifest systemically, or in the treatment of CD1a-expressing malignancies. [Means for solving the problem]

[0007] In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof capable of binding to CD1a. The antibody or antigen-binding fragment thereof may specifically bind to CD1a. The antibody or antigen-binding fragment thereof may preferentially bind to CD1a. The antibody or antigen-binding fragment thereof may induce cell death of cells expressing CD1a. The antibody or antigen-binding fragment thereof may block binding of a ligand to CD1a.

[0008] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising complementarity determining region (CDR) 3 (CDR3) of SEQ ID NO:3 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; and / or The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a CDR3 of SEQ ID NO:6 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto.

[0009] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a CDR3 of SEQ ID NO: 11 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; and / or The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a CDR3 of SEQ ID NO: 14 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto.

[0010] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a CDR3 of SEQ ID NO: 19 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; and / or The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a CDR3 of SEQ ID NO:22 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto.

[0011] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a CDR3 of SEQ ID NO:27 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; and / or The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a CDR3 of SEQ ID NO: 30 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto.

[0012] The antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a CDR3 of SEQ ID NO: 35 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; and / or The antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising a CDR3 of SEQ ID NO: 38, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto.

[0013] The antibody or antigen-binding fragment thereof may be a) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO:2, and CDR3 of SEQ ID NO:3, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. and / or b) CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO:5, and CDR3 of SEQ ID NO:6, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. A light chain variable region comprising It may comprise or consist of:

[0014] The antibody or antigen-binding fragment thereof may be a) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. and / or b) CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 14, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. A light chain variable region comprising It may comprise or consist of:

[0015] The antibody or antigen-binding fragment thereof may be a) CDR1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. and / or b) CDR1 of SEQ ID NO: 20, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO:22 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. A light chain variable region comprising It may comprise or consist of:

[0016] The antibody or antigen-binding fragment thereof may be a) CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. and / or b) CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto A light chain variable region comprising It may comprise or consist of:

[0017] The antibody or antigen-binding fragment thereof may be a) CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto and / or b) CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38, or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto A light chain variable region comprising It may comprise or consist of:

[0018] The CDRs may be combined with any framework region. Preferably, the framework regions are of human origin.

[0019] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO: 7 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain variable region comprising or consisting of SEQ ID NO: 8 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; It may comprise or consist of:

[0020] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO: 15 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain variable region comprising or consisting of SEQ ID NO: 16 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; It may comprise or consist of:

[0021] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO: 23 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain variable region comprising or consisting of SEQ ID NO: 24 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; It may comprise or consist of:

[0022] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO: 31 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain variable region comprising or consisting of SEQ ID NO: 32 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; It may comprise or consist of:

[0023] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO: 39 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain variable region comprising or consisting of SEQ ID NO: 40 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto; It may comprise or consist of:

[0024] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO:7, and b) a light chain variable region comprising or consisting of SEQ ID NO:8 It may consist of:

[0025] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO: 15, and b) a light chain variable region comprising or consisting of SEQ ID NO: 16 It may consist of:

[0026] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO:23, and b) a light chain variable region comprising or consisting of SEQ ID NO: 24 It may consist of:

[0027] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO:31, and b) a light chain variable region comprising or consisting of SEQ ID NO: 32 It may consist of:

[0028] The antibody or antigen-binding fragment thereof may be a) a heavy chain variable region comprising or consisting of SEQ ID NO:39, and b) a light chain variable region comprising or consisting of SEQ ID NO: 40 It may consist of:

[0029] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 41 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain comprising or consisting of SEQ ID NO: 42 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. It may comprise or consist of:

[0030] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 43 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain comprising or consisting of SEQ ID NO: 44 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. It may comprise or consist of:

[0031] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 45 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain comprising or consisting of SEQ ID NO: 46 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. It may comprise or consist of:

[0032] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 47 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain comprising or consisting of SEQ ID NO: 48 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. It may comprise or consist of:

[0033] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 49 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto, and / or b) a light chain comprising or consisting of SEQ ID NO: 50 or a sequence having at least 80%, 90%, 95%, 98%, 99% or 100% identity thereto. It may comprise or consist of:

[0034] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 41, and b) a light chain comprising or consisting of SEQ ID NO: 42 It may consist of:

[0035] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 43, and b) a light chain comprising or consisting of SEQ ID NO: 44 It may consist of:

[0036] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 45, and b) a light chain comprising or consisting of SEQ ID NO: 46 It may consist of:

[0037] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 47, and b) a light chain comprising or consisting of SEQ ID NO: 48 It may consist of:

[0038] The antibody or antigen-binding fragment thereof may be a) a heavy chain comprising or consisting of SEQ ID NO: 49, and b) a light chain comprising or consisting of SEQ ID NO: 50 It may consist of:

[0039] The antibody or antigen-binding fragment thereof of the present invention may be isolated.

[0040] In any embodiment, an "antibody or antigen-binding fragment thereof" can refer to one or more, e.g., two, of the listed antibodies or antigen-binding fragments thereof. For example, in any embodiment, two antibodies or antigen-binding fragments thereof may be envisaged, each 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 having at least 80% identity thereto; 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 having at least 80% identity thereto; A first antibody or antigen-binding fragment thereof, 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 having at least 80% identity thereto; 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 having at least 80% identity thereto; or a second antibody or antigen-binding fragment thereof, b) a first antibody or antigen-binding fragment thereof, having a heavy chain variable region comprising or consisting of SEQ ID NO: 39, and a light chain variable region comprising or consisting of SEQ ID NO: 40, or a sequence having at least 80% identity thereto; and A second antibody or antigen-binding fragment thereof having a heavy chain variable region comprising or consisting of SEQ ID NO:7 and a light chain variable region comprising or consisting of SEQ ID NO:8, or a sequence having at least 80% identity thereto; or c) a first antibody or antigen-binding fragment thereof, having a heavy chain comprising or consisting of SEQ ID NO: 49 and a light chain comprising or consisting of SEQ ID NO: 50, or a sequence having at least 80% identity thereto; and A second antibody or antigen-binding fragment thereof having a heavy chain comprising or consisting of SEQ ID NO: 41 and a light chain comprising or consisting of SEQ ID NO: 42, or a sequence having at least 80% identity thereto. It comprises or consists of:

[0041] For example, any combination of antibodies or antigen-binding fragments may be used in any of the therapeutic applications disclosed herein and / or any of the monitoring methods disclosed herein. Preferably, Ab116 and 16 are used in combination.

[0042] In another embodiment, Ab116 may be used in any of the therapeutic applications disclosed herein and Ab16 may be used in monitoring the same subject. Alternatively, Ab16 may be used in any of the therapeutic applications disclosed herein and Ab116 may be used in monitoring the same subject.

[0043] The term "antibody" as referred to herein refers to a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains that are mutually bound by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. The variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The constant regions of the antibody 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.

[0044] The term "antigen-binding fragment thereof" of an antibody refers to one or more antibody fragments that retain the ability to selectively bind to an antigen. The antigen-binding fragment may be, but is not limited to, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibody (e.g., VH or VL or VHH), scFv, bivalent, trivalent or tetravalent antibody, bis-scFv, diabody, triabody, tetrabody and any of the above epitope-binding fragments (Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136;Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for generating and producing such antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0045] The antibody or antigen-binding fragment thereof may be a monoclonal antibody, a bispecific antibody, a multispecific antibody, an ScFv or other single chain or modified format, a Fab, (Fab')2, an Fv, a dAb, an Fd, a nanobody, a camelid antibody or a diabody. Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0046] The inventors have targeted CD1a and its potential role in inflammatory skin and mucosal diseases and disorders, or related systemic diseases or disorders, or inflammatory drug responses manifested systemically, by generating an effective monoclonal antibody. Because CD1a is highly expressed in skin and mucosa, the use of such antibodies provides an opportunity to selectively treat inflammatory skin and mucosal diseases and disorders while minimizing off-target effects. CD1a is not expressed in mice, but is expressed in other mammals. Human CD1a (UniProtKB / Swiss-Prot: P06126-CD1A_HUMAN) is expressed worldwide with a dominant allele, with variants present in some ethnic groups in China (18). Targeting CD1a antigen presentation also disrupts inflammatory pathways upstream of other cytokine-specific antibody therapies, such as anti-IL17 therapy or other immunotherapies, thus providing a powerful means to modulate pro-inflammatory disorders early in the immune cascade. Moreover, exploiting the specificity of CD1a for the skin may provide a means to direct additional therapies to the skin, for example by using bispecific or multispecific or conjugated antibody technology to specifically target small molecule, drug, nucleic acid, peptide, antibody or cell conjugate therapies.Furthermore, because CD1a is relatively non-polymorphic, the present invention offers universal potential in the prevention and / or treatment of inflammatory skin and mucosal diseases, such as atopic dermatitis and psoriasis (13-15), or CD1a-expressing malignancies, in which the frequency of CD1a-expressing dendritic cell subsets is increased and the migration pattern of LCs is altered.

[0047] By modifying the number and function of CD1a expressing cells, the antibodies have effects that go beyond lipid responsiveness and affect all roles of CD1a expressing cells, including antigen presentation to peptide-specific T cells and innate pathways (e.g., neutrophils). The antibodies of the invention are capable of reducing Langerhans cells despite their murine IgG1 nature. Such reduction provides a means of controlling a wide range of inflammatory pathways without complement / ADCC-associated inflammation, which may provide therapeutic benefit. This is shown in the imiquimod model described herein, where the antibodies of the invention reduce inflammation to levels significantly below wild-type mice, for example, and demonstrate a profound anti-inflammatory effect on innate pathways, including CD1a expressing cells, including neutrophils and eosinophils. The antibodies of the invention also inhibit the production of a variety of cytokines, including IFN-gamma and IL-22, that are associated with a wide range of clinical diseases.

[0048] In another aspect, the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention. Such a nucleic acid may be provided by any of SEQ ID NOs: 51-90. Those skilled in the art will appreciate that due to codon redundancy, many DNA sequences can be used to encode the antibody or antigen-binding fragment thereof of the present invention. Alternatively, codon optimization of nucleotide sequences can be used to improve the efficiency of translation in an expression system for the production of the antibody or antigen-binding fragment thereof of the present invention.

[0049] In another aspect, the present invention provides a vector comprising the nucleic acid of the present invention. A suitable vector can be selected or constructed, including appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as necessary. The vector can be, for example, a plasmid or a virus. For further details, see, for example, (Sambrook, J., EF Fritsch, and T. Maniatis. (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). For example, many known techniques and protocols for preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and manipulating nucleic acids in gene expression, and analyzing proteins are described in detail in (Ausubel et al., Current protocols in molecular biology. New York: Greene Publishing Association; Wiley-Interscience, 1992). The vector can be an expression vector. The vector or expression vector can be a plasmid.

[0050] The nucleic acid molecules or vectors of the invention may be expressed using any suitable expression system, for example in a suitable host cell or a cell-free system.

[0051] In another aspect, the present invention provides a host cell comprising the antibody or antigen-binding fragment thereof, nucleic acid and / or vector of the present invention. The host cell may be selected from a bacterial host cell (prokaryotic system), such as E. Coli, or a eukaryotic cell, such as a yeast, fungal cell, insect cell or mammalian cell. Preferably, the host cell of the present invention is capable of producing the antibody or antigen-binding fragment thereof of the present invention. The produced antibody or antigen-binding fragment thereof may be enriched by means of selection and / or isolation.

[0052] Alternatively, the antibodies or antigen-binding fragments thereof of the present invention may be produced by chemical synthesis. The resulting antibodies or antigen-binding fragments thereof may be enriched by selection and / or isolation means.

[0053] According to a further aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a nucleic acid, a vector and / or a host cell of the invention, optionally together with one or more pharma- ceutically acceptable excipients or diluents.

[0054] The antibody or antigen-binding fragment thereof, nucleic acid, vector or host cell of the present invention can be formulated into a pharmaceutical composition using established preparation methods (Gennaro, AL and Gennaro, AR (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). Pharmaceutically inert inorganic or organic excipients can be used to prepare pharmaceutical compositions. For example, for the preparation of pills, powders, gelatin capsules or suppositories, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils are examples of pharma- ceutically acceptable excipients that can be used. Suitable excipients for the production of solutions, suspensions, emulsions, aerosol mixtures, or powders for reconstitution of the solution or aerosol mixture before use include water, alcohol, glycerol, polyols and suitable mixtures thereof, as well as vegetable oils.

[0055] The pharmaceutical composition of the present invention may be administered via any therapeutically effective parenteral or oral (enteral) route. Parenteral application methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal or intravenous injection and infusion techniques, for example, in the form of injection solutions, infusions or mixtures, and aerosol introduction and inhalation, for example, in the form of aerosol mixtures, sprays or powders. The pharmaceutical composition of the present invention may be administered systemically or locally by a formulation containing conventional non-toxic pharma- ceutical acceptable excipients or carriers, additives and vehicles as required. A combination of intravenous and subcutaneous infusion and / or injection may be most convenient when the compound has a relatively short or long serum half-life or requires a rapid onset of action. Preferably, the pharmaceutical composition is administered subcutaneously or intravenously. The pharmaceutical composition may be an aqueous solution, an oil-in-water emulsion or a water-in-oil emulsion.

[0056] For intravenous injection or injection at affected area or other administration site, active ingredient is in the form of parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art are familiar with the preparation of suitable solution, for example, using isotonic medium, for example, sodium chloride injection, Ringer's injection, lactate Ringer's injection.Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as necessary.

[0057] The composition is preferably administered to an individual in a "therapeutically effective amount" sufficient to provide benefit to the individual. The optimal dosage depends on the biodistribution of the antibody or antigen-binding fragment thereof, the method of administration, the severity of the disease / disorder being treated, and the medical condition of the patient. If desired, the antibody or antigen-binding fragment thereof may be administered in a sustained release formulation, such as a liposomal dispersion or hydrogel-based polymer microspheres, such as PolyActive™ or OctoDEX™ (see Bos et al., Business Briefing: Pharmatech 2003: 1-6). Other sustained release formulations available are, for example, PLGA-based polymers (PRpharmaceuticals), PLA-PEG-based hydrogels (Medincell), and PEA-based polymers (Medivas). Prescription of therapeutic agents, such as determining dosage, is within the responsibility of the physician and typically takes into account the disorder being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0058] The pharmaceutical composition may also contain additives such as fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, additional solvents or solubilizers or agents for achieving a depot effect, etc. The latter may incorporate the fusion protein into slow or sustained release or targeted delivery systems such as liposomes and microcapsules.

[0059] In another aspect, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition of the invention may be for use in the treatment or prevention of one or more diseases or disorders in a subject.

[0060] In one aspect, there is provided a method of treating or preventing one or more diseases or disorders in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or composition of the invention.

[0061] In one aspect, there is provided the use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of one or more diseases or disorders in a subject.

[0062] In any embodiment, the subject may be a mammal. The mammal may express a CD1a ortholog. Preferably, the subject is a human.

[0063] The one or more diseases or disorders may be one or more inflammatory skin or mucosal disorders or diseases, or one or more associated systemic diseases or disorders, or one or more systemically manifested inflammatory drug reactions, or CD1a-expressing malignancies.

[0064] The inflammatory skin or mucosal disease or disorder may be selected from: a) Neutrophil-dominant skin diseases, such as acne, generalized pustular psoriasis, psoriasis vulgaris, guttate psoriasis, palmoplantar pustulosis, SAPHO syndrome, acute febrile neutrophilic dermatosis (Sweet's syndrome), histiocytic neutrophilic dermatitis, dorsal neutrophilic dermatosis, pyoderma gangrenosum, neutrophilic eccrine hidradenitis, hidradenitis suppurativa, erythema elevatum, Behçet's disease, enteroarthritis syndrome, inflammation associated with other infections, neutrophilic urticarial dermatosis, palisadian neutrophilic granulomatous dermatitis, erythema gyratum repens, neutrophilic annular erythema, acute generalized exanthematous pustulosis (AGEP), vasculitis, etc.; b) autoimmune disorders, e.g. connective tissue diseases (e.g. lupus, dermatomyositis, scleroderma / systemic sclerosis, Churg-Strauss syndrome), panniculitis, vasculitis, autoimmune blistering conditions (e.g. bullous pemphigoid, pemphigus, linear IgA disease), dermatitis herpetiformis, celiac disease, some autoinflammatory diseases, vitiligo, alopecia areata, alopecia universalis, alopecia totalis, panniculitis, lichen planus, erythema multiforme, lichen sclerosus, other lichenoid and erythema multiforme-like diseases, vesicular psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, Guillain-Barré syndrome, thyroiditis, transverse myelitis, neurodegeneration, etc.; c) Mast cell disorders and eosinophilic disorders, e.g. Muckle-Wells syndrome, eosinophilia and systemic symptom syndrome, urticaria, angioedema, keratoconjunctivitis, food allergies, other allergies or atopy including atopic dermatitis, rhinitis, conjunctivitis, asthma, eosinophilic oesophagitis and other eosinophilic mucosal diseases, contact dermatitis etc; d) Adverse drug reactions manifesting as inflammatory skin or mucosal diseases or disorders, e.g. Stevens-Johnson syndrome, toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms syndrome (DRESS) as well as acute generalized exanthematous pustulosis (AGEP), erythema multiforme, bullous diseases, fixed drug eruptions, etc.; e) Graft-versus-host disease.

[0065] CD1a expressing malignant disease as referred to herein can be any malignant disease that can detect CD1a expression.Such malignant disease can include Langerhans cell histiocytosis, a subset of T-cell lymphoma, a subset of thymoma, or other rare cases of malignant disease, such as a subset of mastocytosis.Preferably, CD1a expressing malignant disease is a subset of T-cell lymphoma.

[0066] Preferably, the one or more diseases or disorders comprise or consist of psoriasis, dermatitis, lupus erythematosus, a neutrophilic skin disease, a related systemic disease or disorder, and / or a systemically manifested inflammatory drug reaction, or a CD1a-expressing malignancy.

[0067] As used herein, an associated systemic disease or disorder may refer to any non-skin site complication that may be associated with an inflammatory skin or mucosal disease or disorder as defined herein, which may include non-cutaneous lupus erythematosus.

[0068] The inflammatory drug reaction that is systemically manifested may be a reaction at a non-skin site, for example, the spleen.The associated systemic disease or disorder or the inflammatory drug reaction that is systemically manifested may be the result of an inflammatory response.The inflammatory response may be, for example, to a drug, for example, Aldara (Imiquimod 5% cream).The inflammatory response may result in an increase in the number or activity of CD4 T cells, CD8 T cells, neutrophils or eosinophils, and / or an increase in the level of IL-23, IL-12, IL-1β and / or MCP-1, and / or a decrease in IL-10 and / or IL-27.

[0069] Moreover, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell or pharmaceutical composition of the present invention may 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 may be selected from the group including cytotoxic agents, immune activators, such as checkpoint inhibitors or TLR agonists, anti-inflammatory agents, such as steroids, 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 of the present invention.

[0070] In another aspect, i. providing a biological sample from a subject; ii. determining the level of binding of one or more antibodies or antigen-binding fragments of the invention to CD1a-expressing cells in a sample obtained from the subject before treatment or during an interval or period of no treatment; iii. determining that the treatment is effective or that the disease condition is improved if there is a decrease in the tumor burden or the binding level of one or more antibodies or antigen-binding fragments of the present invention to CD1a-expressing cells after the treatment or during a period between or without treatment; The present invention provides a method for monitoring therapeutic efficacy or disease status in a subject diagnosed with a CD1a-expressing malignancy, comprising:

[0071] The biological sample may be a blood or serum sample, a tissue biopsy, cerebrospinal fluid, saliva or a urine sample. Preferably, the biological sample may be a blood or serum sample.

[0072] The level of binding of one or more antibodies or antigen-binding fragments of the present invention to CD1a-expressing cells in a sample can be determined using any method known to those skilled in the art. One such method, for example, allows for the determination of the number of CD1a-expressing cells in a sample using flow cytometry or any other technique that utilizes a detectable label.

[0073] Tumor burden may be determined by any suitable technique known to those of skill in the art.

[0074] The reduction in tumor burden or binding level of one or more antibodies or antigen-binding fragments of the invention to CD1a-expressing cells can be a reduction of 10% or more, for example, 25% or more, 50% or more, 75% or more, or 90% or more.

[0075] The period between treatments or without treatment may be 2 weeks or more, such as 4 weeks or more, 8 weeks or more, 12 weeks or more, 6 months or more, or 12 months or more.

[0076] Techniques for the production of antibodies and antigen-binding fragments thereof are well known in the art. The term "antibody" also includes immunoglobulins (Ig) of various classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (e.g., IgG1, IgG2, 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 a VL domain capable of binding to an antigen alone. The antibody or antigen-binding fragment thereof may be a chimeric, nanobody, single chain and / or humanized antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be a human IgG1 or human IgG4 isotype or other natural or modified isotype. The antibody can be a monoclonal (mAb) or a polyclonal antibody.

[0077] The antibody or antigen-binding fragment thereof may be modified to alter its stability and / or half-life in vivo. The modification may be, for example, PEGylation.

[0078] An antibody or antigen-binding fragment thereof may be an antibody-like molecule, which includes the use of CDRs separately or in combination with synthetic molecules, such as SMIPs and small antibody mimetics.

[0079] The percent identity of two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the first sequence for best alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of two sequences that results in the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides in the sequences (i.e., % identity=number of identical positions / total number of positions×100).

[0080] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, PNAS, 87(6):2264-8, modified by Karlin and Altschul, 1993, PNAS, 90(12):5873-5877, etc. The NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol., 215:403-10 incorporate such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). Alternatively, PSI-Blast can be used to perform iterative searches, thereby detecting distant relationships between molecules (ibid.). When using BLAST, GappedBLAST and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm used for sequence comparison is the Myers and Miller algorithm. The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, incorporates such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994), and FASTA as described in Pearson and Lipman (1988).In FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0081] The antibodies or antigen-binding fragments thereof of the invention may contain one or more mutated amino acid residues. The terms "mutated", "mutant" and "mutation" with respect to the nucleic acids or antibodies or antigen-binding fragments thereof of the invention refer to a substitution, deletion or insertion of one or more nucleotides or amino acids, respectively, compared to a "naturally" occurring nucleic acid or polypeptide, i.e., a reference sequence available for defining the wild type.

[0082] The amino acid mutations in the CDR sequences may be conservative amino acid substitutions.

[0083] Mutation can be a substitution, in which case the substitution is a conservative substitution.Conservative substitutions are generally the following substitutions, listed according to the amino acid to be mutated, each followed by one or more substitutions (multiple substitutions) that can be considered as conservative: Ala→Gly, Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp; Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln, Glu; Met→Leu, Tyr, He; Phe→Met, Leu, Tyr; Ser→Thr; Thr→Ser; Trp→Tyr; Tyr→Trp, Phe; Val→He, Leu.Other substitutions are also acceptable and can be determined empirically or in accordance with other known conservative or non-conservative substitutions.

[0084] One, two or three conservative substitutions may be made in the CDRs of an antibody or antigen-binding fragment thereof of the present invention.

[0085] Methods for generating antibodies or antigen-binding fragments thereof are well known in the art. A person skilled in the art can use, for example, hybridoma technology or can use recombinant DNA technology to clone each antibody sequence into a vector, for example, an expression vector. Methods for generating bispecific antibody molecules are known in the art, including, for example, recombinant DNA technology, chemical conjugation of two different monoclonal antibodies, or even chemical conjugation of, for example, two antibody fragments, for example, two Fab fragments. Alternatively, bispecific antibody molecules are generated by quadroma technology, which is by fusion of hybridomas that produce the parent antibodies. Random combination of H and L chains generates a potential mixture of 10 different antibody structures, only one of which has the desired binding specificity. The bispecific antibody molecules of the present invention can act as monoclonal antibodies (mAbs) for each target. The antibody or antigen-binding fragment thereof can be a chimeric, humanized or fully human antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can be a human IgG1 or human IgG4 isotype, or other natural or modified isotype. A bispecific antibody molecule or a multispecific antibody can be, for example, a bispecific tandem single chain Fv, a bispecific Fab2 or a bispecific diabody.

[0086] All features disclosed in this specification may be combined in any combination, including in any aspect or combination with any embodiment. [Brief description of the drawings]

[0087] [Figure 1] Figure 1 shows inhibition of polyclonal T cell responses by a panel of anti-CD1a antibodies. A. Dose titration curves of polyclonal T cell IFNγ responses with increasing concentrations of anti-CD1a antibodies (0.01-10 μg / ml) (n=6 donors). B. Calculated IC50 values ​​for a panel of newly generated anti-CD1a antibodies and commercially available antibodies (OKT6, HI149 and SK9, n=6 donors). [Figure 2-1]Figure 2 demonstrates inhibition of CD1a-restricted enriched T cell line responses by a panel of anti-CD1a antibodies. A-B. Cytokine secretion responses of CD1a-restricted enriched T cell lines induced by empty vector (EV) or CD1a-transfected K562 presenting endogenous ligands. Inhibition of IFNγ (A.) or IL-22 (B.) was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. C. IFNγ secretion responses of CD1a-restricted enriched T cell lines induced by CD1a-coated beads presenting endogenous ligands. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies (N = 4 19 enriched T cell lines, two-way ANOVA with Tukey's test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, where * indicates significance for comparison against "CD1a"). [Figure 2-2] Figure 2 demonstrates inhibition of CD1a-restricted enriched T cell line responses by a panel of anti-CD1a antibodies. A-B. Cytokine secretion responses of CD1a-restricted enriched T cell lines induced by empty vector (EV) or CD1a-transfected K562 presenting endogenous ligands. Inhibition of IFNγ (A.) or IL-22 (B.) was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. C. IFNγ secretion responses of CD1a-restricted enriched T cell lines induced by CD1a-coated beads presenting endogenous ligands. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies. Inhibition was assessed by flow cytometry for a panel of newly generated anti-CD1a antibodies (N = 4 19 enriched T cell lines, two-way ANOVA with Tukey's test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, where * indicates significance for comparison against "CD1a"). [Figure 3-1]FIG. 3 demonstrates the characterization of CD1a transgenic mice. A. Respective flow cytometry plots and B. Summary of CD1a protein expression by cells of wild-type (WT) and CD1a transgenic (CD1a) mice. CD1a protein expression was elevated in (from left to right) total live ear skin cells, CD45+ skin cells, dermal dendritic cells (dDCs, CD45+ / CD11c+ / Langerin-) and Langerhans cells (LCs, CD45+ / CD11c+ / Langerin+). C. CD1a protein expression in ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, from left to right, 50 μm, 50 μm and 10 μm. D. Example PCR genotyping of CD1a transgenic mouse strain littermates (lanes A-F) using CD1a forward and reverse primers and tail genomic DNA. Expected CD1a band 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 thymic CD1a protein expression by wild type (WT) and CD1a transgenic (CD1a) mice. [Figure 3-2]FIG. 3 demonstrates the characterization of CD1a transgenic mice. A. Respective flow cytometry plots and B. Summary of CD1a protein expression by cells of wild-type (WT) and CD1a transgenic (CD1a) mice. CD1a protein expression was elevated in (from left to right) total live ear skin cells, CD45+ skin cells, dermal dendritic cells (dDCs, CD45+ / CD11c+ / Langerin-) and Langerhans cells (LCs, CD45+ / CD11c+ / Langerin+). C. CD1a protein expression in ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, from left to right, 50 μm, 50 μm and 10 μm. D. Example PCR genotyping of CD1a transgenic mouse strain littermates (lanes A-F) using CD1a forward and reverse primers and tail genomic DNA. Expected CD1a band 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 thymic CD1a protein expression by wild type (WT) and CD1a transgenic (CD1a) mice. [Figure 4] Figure 4. Characterization of anti-CD1a antibodies in vivo. A. Schematic of imiquimod-induced skin inflammation and anti-CD1a prophylaxis. B. Daily measurements of ear swelling induced by imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) injected ip with mouse IgG1 isotype control and CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies as in schematic panel A. (N=6, 2-way ANOVA with Dunnett's test, **, P<0.01; ****, P<0.0001, significance indicated for comparison against "CD1a" at day 6 or the days indicated). [Diagram 5]Figure 5 demonstrates the effect of anti-CD1a on imiquimod-induced cutaneous immune responses. A-C. Flow cytometric analysis of ear skin from mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies according to the prophylactic dosing model. Cutaneous T cells were enumerated (A.) and assessed for cell surface CD69 expression (B.), and cutaneous 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). [Figure 6] Figure 6 demonstrates the effect of anti-CD1a on imiquimod-induced cellular Langerhans cell skin and lymph node responses. Flow cytometric analysis of ear skin (A-B.) and draining cervical lymph nodes (C-D.) of mouse IgG1 isotype-treated wild type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies according to the prophylactic dosing model. Skin LCs were enumerated (A.) and assessed for cell surface CD1a expression (B.). Lymph node LCs were enumerated (C.) and assessed for cell surface CD1a expression (D.) (N=4, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 7-1]Figure 7 demonstrates antibody-dependent depletion (phenotypic changes). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibodies or mouse IgG1 isotype control (iso, 5 μg / ml) were incubated with EVs or CD1a-K562 for 48 h as indicated, and the percentage of antibody-induced depletion was calculated relative to a reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion by increasing concentrations of anti-CD1a antibodies (0.625-5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days as indicated, with antibody and cytokines added on day 0 or 2, and the percentage of antibody-induced reduction was calculated relative to isotype control measured by percentage confluence using Incucyte live cell imaging (N=4, two-way ANOVA with Tukey's test) (C.) and representative images of MoLCs (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 h, stained for Annexin V, and analyzed by flow cytometry (N=3-4, one-way ANOVA with Tukey's test). F. Flow cytometric analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of either isotype control or indicated antibodies at 5 μg / ml for 3 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMCs at a ratio of 1:50 in the presence of either isotype control or indicated antibodies at 5 μg / ml for 5 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=4-6, one-way ANOVA with Tukey's test).H.NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies on days 6, 10, and 14. Tumor burden was measured over time (N=6-15, two-way ANOVA with Tukey's test, asterisks indicate significance for comparison to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-2]Figure 7 demonstrates antibody-dependent depletion (phenotypic changes). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibodies or mouse IgG1 isotype control (iso, 5 μg / ml) were incubated with EVs or CD1a-K562 for 48 h as indicated, and the percentage of antibody-induced depletion was calculated relative to a reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion by increasing concentrations of anti-CD1a antibodies (0.625-5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days as indicated, with antibody and cytokines added on day 0 or 2, and the percentage of antibody-induced reduction was calculated relative to isotype control measured by percentage confluence using Incucyte live cell imaging (N=4, two-way ANOVA with Tukey's test) (C.) and representative images of MoLCs (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 h, stained for Annexin V, and analyzed by flow cytometry (N=3-4, one-way ANOVA with Tukey's test). F. Flow cytometric analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of either isotype control or indicated antibodies at 5 μg / ml for 3 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMCs at a ratio of 1:50 in the presence of either isotype control or indicated antibodies at 5 μg / ml for 5 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=4-6, one-way ANOVA with Tukey's test).H.NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies on days 6, 10, and 14. Tumor burden was measured over time (N=6-15, two-way ANOVA with Tukey's test, asterisks indicate significance for comparison to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-3]Figure 7 demonstrates antibody-dependent depletion (phenotypic changes). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibodies or mouse IgG1 isotype control (iso, 5 μg / ml) were incubated with EVs or CD1a-K562 for 48 h as indicated, and the percentage of antibody-induced depletion was calculated relative to a reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion by increasing concentrations of anti-CD1a antibodies (0.625-5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days as indicated, with antibody and cytokines added on day 0 or 2, and the percentage of antibody-induced reduction was calculated relative to isotype control measured by percentage confluence using Incucyte live cell imaging (N=4, two-way ANOVA with Tukey's test) (C.) and representative images of MoLCs (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 h, stained for Annexin V, and analyzed by flow cytometry (N=3-4, one-way ANOVA with Tukey's test). F. Flow cytometric analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of either isotype control or indicated antibodies at 5 μg / ml for 3 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMCs at a ratio of 1:50 in the presence of either isotype control or indicated antibodies at 5 μg / ml for 5 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=4-6, one-way ANOVA with Tukey's test).H.NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies on days 6, 10, and 14. Tumor burden was measured over time (N=6-15, two-way ANOVA with Tukey's test, asterisks indicate significance for comparison to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-4]Figure 7 demonstrates antibody-dependent depletion (phenotypic changes). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibodies or mouse IgG1 isotype control (iso, 5 μg / ml) were incubated with EVs or CD1a-K562 for 48 h as indicated, and the percentage of antibody-induced depletion was calculated relative to a reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion by increasing concentrations of anti-CD1a antibodies (0.625-5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days as indicated, with antibody and cytokines added on day 0 or 2, and the percentage of antibody-induced reduction was calculated relative to isotype control measured by percentage confluence using Incucyte live cell imaging (N=4, two-way ANOVA with Tukey's test) (C.) and representative images of MoLCs (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 h, stained for Annexin V, and analyzed by flow cytometry (N=3-4, one-way ANOVA with Tukey's test). F. Flow cytometric analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of either isotype control or indicated antibodies at 5 μg / ml for 3 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMCs at a ratio of 1:50 in the presence of either isotype control or indicated antibodies at 5 μg / ml for 5 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=4-6, one-way ANOVA with Tukey's test).H.NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies on days 6, 10, and 14. Tumor burden was measured over time (N=6-15, two-way ANOVA with Tukey's test, asterisks indicate significance for comparison to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-5]Figure 7 demonstrates antibody-dependent depletion (phenotypic changes). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibodies or mouse IgG1 isotype control (iso, 5 μg / ml) were incubated with EVs or CD1a-K562 for 48 h as indicated, and the percentage of antibody-induced depletion was calculated relative to a reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion by increasing concentrations of anti-CD1a antibodies (0.625-5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days as indicated, with antibody and cytokines added on day 0 or 2, and the percentage of antibody-induced reduction was calculated relative to isotype control measured by percentage confluence using Incucyte live cell imaging (N=4, two-way ANOVA with Tukey's test) (C.) and representative images of MoLCs (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 h, stained for Annexin V, and analyzed by flow cytometry (N=3-4, one-way ANOVA with Tukey's test). F. Flow cytometric analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of either isotype control or indicated antibodies at 5 μg / ml for 3 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMCs at a ratio of 1:50 in the presence of either isotype control or indicated antibodies at 5 μg / ml for 5 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=4-6, one-way ANOVA with Tukey's test).H.NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies on days 6, 10, and 14. Tumor burden was measured over time (N=6-15, two-way ANOVA with Tukey's test, asterisks indicate significance for comparison to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 7-6]Figure 7 demonstrates antibody-dependent depletion (phenotypic changes). A. Flow cytometry analysis of antibody-induced CD1a-dependent cell depletion (e.g., death). Anti-CD1a antibodies or mouse IgG1 isotype control (iso, 5 μg / ml) were incubated with EVs or CD1a-K562 for 48 h as indicated, and the percentage of antibody-induced depletion was calculated relative to a reference population of untreated K562 and normalized to EV control cells. B. Dose titration curve of antibody-induced CD1a-K562 cell depletion by increasing concentrations of anti-CD1a antibodies (0.625-5 μg / ml). C-D. Anti-CD1a antibody or mouse IgG1 isotype control (iso, 5 μg / ml) was incubated with MoDCs (upper panel) and MoLCs (lower panel) for 5 days as indicated, with antibody and cytokines added on day 0 or 2, and the percentage of antibody-induced reduction was calculated relative to isotype control measured by percentage confluence using Incucyte live cell imaging (N=4, two-way ANOVA with Tukey's test) (C.) and representative images of MoLCs (D.). E. K562-CD1a or K562-EV (empty vector) were incubated with anti-CD1a antibody for 24 h, stained for Annexin V, and analyzed by flow cytometry (N=3-4, one-way ANOVA with Tukey's test). F. Flow cytometric analysis of complement-dependent cytotoxicity (CDC). K562-CD1a cells were incubated with 10% normal human serum in the presence of either isotype control or indicated antibodies at 5 μg / ml for 3 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=6, one-way ANOVA with Tukey's test). G. Flow cytometry analysis of antibody-dependent cell-mediated cytotoxicity (ADCC). K562-CD1a cells were co-cultured with PBMCs at a ratio of 1:50 in the presence of either isotype control or indicated antibodies at 5 μg / ml for 5 h at 37°C. Percentage cytotoxicity was calculated relative to a reference population of untreated K562 and normalized to isotype control treated cells (N=4-6, one-way ANOVA with Tukey's test).H.NSG mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells and allowed to develop tumors for 18 days. Mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies on days 6, 10, and 14. Tumor burden was measured over time (N=6-15, two-way ANOVA with Tukey's test, asterisks indicate significance for comparison to "CD1a-iso" on day 18). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. [Figure 8-1] FIG. 8(A) is a heat map from CD1a epitope analysis. Matrix heat map representation of CD1a antibody binding by flow cytometry measured by CD1a-AF647 mean fluorescence intensity (MFI). Prior to staining of CD1a-K652 with anti-CD1a antibody conjugated to fluorophore AF647, relevant purified antibodies were incubated with cells to assess interference of AF647-conjugated antibodies in CD1a binding. Grey scale indicates degree of interference, top row (-) shade indicates no interference. (B) demonstrates in vivo CD1a antibody epitope competition assay results. A. Flow cytometry plots of CD1a expression measured by staining with anti-CD1a antibody SK9 (left panel) or HI149 (right panel). Anti-CD1a antibody 116 (100 μg ip) was administered on days 0, 2 and 4, and ear skin tissue was harvested, processed and stained for CD1a on day 5. [Figure 8-2]FIG. 8(A) is a heat map from CD1a epitope analysis. Matrix heat map representation of CD1a antibody binding by flow cytometry measured by CD1a-AF647 mean fluorescence intensity (MFI). Prior to staining of CD1a-K652 with anti-CD1a antibody conjugated to fluorophore AF647, relevant purified antibodies were incubated with cells to assess interference of AF647-conjugated antibodies in CD1a binding. Grey scale indicates degree of interference, with top row (-) shade indicating no interference. (B) demonstrates in vivo CD1a antibody epitope competition assay results. A. Flow cytometry plots of CD1a expression measured by staining with anti-CD1a antibody SK9 (left panel) or HI149 (right panel). Anti-CD1a antibody 116 (100 μg ip) was administered on days 0, 2 and 4, and ear skin tissue was harvested, processed and stained for CD1a on day 5. [Figure 9-1]FIG. 9 demonstrates the efficacy of application of anti-CD1a antibodies in the treatment of imiquimod-induced inflammation. A. Schematic of imiquimod-induced inflammation model with anti-CD1a formulation administration. B. Daily measurements of ear swelling and C. Representative images of inflammation induced by ip treatment with mouse IgG1 isotype control (day 8) after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) or injection with improved panel of anti-CD1a antibodies as in schematic panel A (arrow at day 3) of CD1a transgenic mice (N=2-10, two-way ANOVA with Dunnett's test, **, P<0.01; ****, P<0.0001, indicating significance for comparison against "CD1a" at day 8 or the days indicated). D. CD1a protein expression in ears and epidermal thickness and ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, 10 μm for upper panels and 100 μm for lower panels. E-G. Flow cytometric analysis of ear skin of mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with a modified panel of anti-CD1a antibodies according to the therapeutic dosing model. Cutaneous T cells were enumerated and assessed for cell surface CD11a expression (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). [Figure 9-2]FIG. 9 demonstrates the efficacy of application of anti-CD1a antibodies in the treatment of imiquimod-induced inflammation. A. Schematic of imiquimod-induced inflammation model with anti-CD1a formulation administration. B. Daily measurements of ear swelling and C. Representative images of inflammation induced by ip treatment with mouse IgG1 isotype control (day 8) after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) or injection with improved panel of anti-CD1a antibodies as in schematic panel A (arrow at day 3) of CD1a transgenic mice (N=2-10, two-way ANOVA with Dunnett's test, **, P<0.01; ****, P<0.0001, indicating significance for comparison against "CD1a" at day 8 or the days indicated). D. CD1a protein expression in ears and epidermal thickness and ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, 10 μm for upper panels and 100 μm for lower panels. E-G. Flow cytometric analysis of ear skin of mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with a modified panel of anti-CD1a antibodies according to the therapeutic dosing model. Cutaneous T cells were enumerated and assessed for cell surface CD11a expression (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). [Figure 9-3]FIG. 9 demonstrates the efficacy of application of anti-CD1a antibodies in the treatment of imiquimod-induced inflammation. A. Schematic of imiquimod-induced inflammation model with anti-CD1a formulation administration. B. Daily measurements of ear swelling and C. Representative images of inflammation induced by ip treatment with mouse IgG1 isotype control (day 8) after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) or injection with improved panel of anti-CD1a antibodies as in schematic panel A (arrow at day 3) of CD1a transgenic mice (N=2-10, two-way ANOVA with Dunnett's test, **, P<0.01; ****, P<0.0001, indicating significance for comparison against "CD1a" at day 8 or the days indicated). D. CD1a protein expression in ears and epidermal thickness and ear skin of wild-type (WT) and CD1a transgenic (CD1a) mice treated with imiquimod (Imiq) or untreated (U) visualized by immunofluorescence. Frozen sections were stained with DAPI (blue) and anti-CD1a AF-594 (OKT6, red). Scale bars, 10 μm for upper panels and 100 μm for lower panels. E-G. Flow cytometric analysis of ear skin of mouse IgG1 isotype-treated wild-type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with a modified panel of anti-CD1a antibodies according to the therapeutic dosing model. Cutaneous T cells were enumerated and assessed for cell surface CD11a expression (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). [Figure 10-1]Figure 10 demonstrates the CD1a dependency of the systemic effects of imiquimod application. A. Spleen weight (mg) measurements and representative images on day 8 after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) following ip treatment with mouse IgG1 isotype control or injection with an improved panel of anti-CD1a antibodies as in schematic illustration of CD1a transgenic mice (Figure 9A). B-E. Flow cytometric analysis of spleens of mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a), as well as CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies according to the therapeutic dosing model. Splenic CD4 (B.) and CD8 (C.) T cell CD69 expression was assessed, and neutrophils (D.) and eosinophils (E.) were enumerated (N=7-9, one-way ANOVA with Dunnett's test, *,P<0.05; **,P<0.01; ***,P<0.001; ****,P<0.0001). F. Plasma cytokine levels in blood of mouse IgG1 isotype-treated wild type (WT) and CD1a transgenic (CD1a), as well as CD1a transgenic mice injected with anti-CD1a antibodies according to the therapeutic dosing model (N=7-9, one-way ANOVA with Dunnett's test, *,P<0.05; **,P<0.01; ***,P<0.001; ****,P<0.0001). [Figure 10-2]Figure 10 demonstrates the CD1a dependency of the systemic effects of imiquimod application. A. Spleen weight (mg) measurements and representative images on day 8 after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) following ip treatment with mouse IgG1 isotype control or injection with an improved panel of anti-CD1a antibodies as in schematic illustration of CD1a transgenic mice (Figure 9A). B-E. Flow cytometric analysis of spleens of mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a), as well as CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies according to the therapeutic dosing model. Splenic CD4 (B.) and CD8 (C.) T cell CD69 expression was assessed, and neutrophils (D.) and eosinophils (E.) were enumerated (N=7-9, one-way ANOVA with Dunnett's test, *,P<0.05; **,P<0.01; ***,P<0.001; ****,P<0.0001). F. Plasma cytokine levels in blood of mouse IgG1 isotype-treated wild type (WT) and CD1a transgenic (CD1a), as well as CD1a transgenic mice injected with anti-CD1a antibodies according to the therapeutic dosing model (N=7-9, one-way ANOVA with Dunnett's test, *,P<0.05; **,P<0.01; ***,P<0.001; ****,P<0.0001). [Figure 11] Figure 11 demonstrates the CD1a dependency of the systemic effect of imiquimod application. A-E. Blood cell analysis of blood of mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a), as well as CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies according to the therapeutic dosing model. Circulating T cells (A.), CD4+ (B.) and CD8 (C.), neutrophils (D.) and eosinophils (E.) were enumerated (N=5-7, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 12] Figure 12 shows that imiquimod does not constitute a CD1a ligand. Isoelectric point dependent migration of mock and imiquimod "spiked" CD1a protein on isoelectric focusing (IEF) gels pH 3-7. Sham: vehicle control TBS 2% CHAPS 7% DMSO. [Figure 13-1] Figure 13: Efficacy of anti-CD1a antibody application in sustained control of imiquimod-induced inflammation. A. Schematic of imiquimod re-exposure model without subsequent anti-CD1a administration. B. Daily measurements of ear swelling induced by imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) injected ip with mouse IgG1 isotype control and CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies as in schematic panel 13A (2-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01, indicating significance for comparison against "CD1a" isotype on day 7 after imiquimod re-application). [Figure 13-2] Figure 13: Efficacy of anti-CD1a antibody application in sustained control of imiquimod-induced inflammation. A. Schematic of imiquimod re-exposure model without subsequent anti-CD1a administration. B. Daily measurements of ear swelling induced by imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a) injected ip with mouse IgG1 isotype control and CD1a transgenic mice injected with an improved panel of anti-CD1a antibodies as in schematic panel 13A (2-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01, indicating significance for comparison against "CD1a" isotype on day 7 after imiquimod re-application). [Figure 14] Figure 14: Efficacy of anti-CD1a antibody application in treating imiquimod-induced inflammation compared to standard treatment. Daily measurements of ear swelling induced by ip treatment with mouse IgG1 isotype control (CD1a) after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a), or injection of CD1a transgenic mice with an improved panel of anti-CD1a antibodies as in schematic panel Figure 9A and anti-IL-17A. dx = number of days in the model when significance was achieved compared to ear thickness in CD1a transgenic mice. [Figure 15-1]FIG. 15 is a comparative analysis of the efficacy of anti-CD1a antibody application in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with anti-CD1a formulation administration. B. Daily measurements of ear swelling induced by ip treatment with mouse IgG1 isotype control after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a), or injection of CD1a transgenic mice with an improved panel of anti-CD1a antibodies as in schematic panel A or CR2113. N=2-7, two-way ANOVA with Dunnett's test, *, P<0.05, **, P<0.01; ****, P<0.0001, indicating significance for "CD1a" on day 8 or comparison to OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B), corrected for WT. D. Schematic of MC903-induced inflammation with MC903-induced inflammation preventive drugs. E. Daily measurements of ear swelling induced by MC903 treatment after ip treatment with mouse IgG1 isotype control in wild type (WT) and CD1a transgenic mice (CD1a), or injection with 16, 110 or 116 anti-CD1a antibodies or CR2113 as in schematic panel D in CD1a transgenic mice. Corrected for WT. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05, indicates significance for comparison to "CD1a" on day 7. F. Cutaneous T cell percentages and eosinophil counts measured by flow cytometry. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 15-2]FIG. 15 is a comparative analysis of the efficacy of anti-CD1a antibody application in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with anti-CD1a formulation administration. B. Daily measurements of ear swelling induced by ip treatment with mouse IgG1 isotype control after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a), or injection of CD1a transgenic mice with an improved panel of anti-CD1a antibodies as in schematic panel A or CR2113. N=2-7, two-way ANOVA with Dunnett's test, *, P<0.05, **, P<0.01; ****, P<0.0001, indicating significance for "CD1a" on day 8 or comparison to OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B), corrected for WT. D. Schematic of MC903-induced inflammation with MC903-induced inflammation preventive drugs. E. Daily measurements of ear swelling induced by MC903 treatment after ip treatment with mouse IgG1 isotype control in wild type (WT) and CD1a transgenic mice (CD1a), or injection with 16, 110 or 116 anti-CD1a antibodies or CR2113 as in schematic panel D in CD1a transgenic mice. Corrected for WT. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05, indicates significance for comparison to "CD1a" on day 7. F. Cutaneous T cell percentages and eosinophil counts measured by flow cytometry. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 15-3]FIG. 15 is a comparative analysis of the efficacy of anti-CD1a antibody application in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with anti-CD1a formulation administration. B. Daily measurements of ear swelling induced by ip treatment with mouse IgG1 isotype control after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a), or injection of CD1a transgenic mice with an improved panel of anti-CD1a antibodies as in schematic panel A or CR2113. N=2-7, two-way ANOVA with Dunnett's test, *, P<0.05, **, P<0.01; ****, P<0.0001, indicating significance for "CD1a" on day 8 or comparison to OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B), corrected for WT. D. Schematic of MC903-induced inflammation with MC903-induced inflammation preventive drugs. E. Daily measurements of ear swelling induced by MC903 treatment after ip treatment with mouse IgG1 isotype control in wild type (WT) and CD1a transgenic mice (CD1a), or injection with 16, 110 or 116 anti-CD1a antibodies or CR2113 as in schematic panel D in CD1a transgenic mice. Corrected for WT. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05, indicates significance for comparison to "CD1a" on day 7. F. Cutaneous T cell percentages and eosinophil counts measured by flow cytometry. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 15-4]FIG. 15 is a comparative analysis of the efficacy of anti-CD1a antibody application in treating imiquimod / MC903-induced inflammation. A. Schematic of imiquimod-induced inflammation with anti-CD1a formulation administration. B. Daily measurements of ear swelling induced by ip treatment with mouse IgG1 isotype control after imiquimod treatment of wild type (WT) and CD1a transgenic mice (CD1a), or injection of CD1a transgenic mice with an improved panel of anti-CD1a antibodies as in schematic panel A or CR2113. N=2-7, two-way ANOVA with Dunnett's test, *, P<0.05, **, P<0.01; ****, P<0.0001, indicating significance for "CD1a" on day 8 or comparison to OX116 vs. CR2113 on day 8. C. Data and comparisons shown in (B), corrected for WT. D. Schematic of MC903-induced inflammation with MC903-induced inflammation preventive drugs. E. Daily measurements of ear swelling induced by MC903 treatment after ip treatment with mouse IgG1 isotype control in wild type (WT) and CD1a transgenic mice (CD1a), or injection with 16, 110 or 116 anti-CD1a antibodies or CR2113 as in schematic panel D in CD1a transgenic mice. Corrected for WT. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05, indicates significance for comparison to "CD1a" on day 7. F. Cutaneous T cell percentages and eosinophil counts measured by flow cytometry. N=3-4, 2-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001. [Figure 16-1]Figure 16: Comparative analysis of the effects of anti-CD1a antibodies on cutaneous and systemic immune responses due to imiquimod-induced inflammation. Ear skin, draining cervical lymph node and plasma samples from mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with a modified panel of anti-CD1a antibodies according to the therapeutic dosing model shown in Schematic Figure 15A were analyzed. A. Cutaneous T cell IL-17A expression was analyzed directly ex vivo using intracellular cytokine expression detected by flow cytometry (left panel) and cervical lymph node eosinophils were enumerated (right panel). B-C. Plasma (B) and skin lysate (C) cytokine levels were measured by ELISA (N=2-7, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 16-2] Figure 16: Comparative analysis of the effects of anti-CD1a antibodies on cutaneous and systemic immune responses due to imiquimod-induced inflammation. Ear skin, draining cervical lymph node and plasma samples from mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with a modified panel of anti-CD1a antibodies according to the therapeutic dosing model shown in Schematic Figure 15A were analyzed. A. Cutaneous T cell IL-17A expression was analyzed directly ex vivo using intracellular cytokine expression detected by flow cytometry (left panel) and cervical lymph node eosinophils were enumerated (right panel). B-C. Plasma (B) and skin lysate (C) cytokine levels were measured by ELISA (N=2-7, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). [Figure 16-3]Figure 16: Comparative analysis of the effects of anti-CD1a antibodies on cutaneous and systemic immune responses due to imiquimod-induced inflammation. Ear skin, draining cervical lymph node and plasma samples from mouse IgG1 isotype treated wild type (WT) and CD1a transgenic (CD1a) and CD1a transgenic mice injected with a modified panel of anti-CD1a antibodies according to the therapeutic dosing model shown in Schematic Figure 15A were analyzed. A. Cutaneous T cell IL-17A expression was analyzed directly ex vivo using intracellular cytokine expression detected by flow cytometry (left panel) and cervical lymph node eosinophils were enumerated (right panel). B-C. Plasma (B) and skin lysate (C) cytokine levels were measured by ELISA (N=2-7, one-way ANOVA with Dunnett's test, *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] material and method mouse All mice were housed in a specific pathogen-free facility. In each experiment, mice were age-, sex- and background-matched with wild-type littermates used as matched controls. All experiments performed in this study were approved by the UK Home Office.

[0089] Generation of CD1a transgenic mice Mice were generated by the Wellcome Trust Centre for Human Genetics, Oxford. A 5.7 kb genomic fragment encompassing the entire CD1A gene, including 0.8 kb of upstream and 0.8 kb of downstream sequences, was amplified by PCR from human genomic DNA using primers 5'-ATGGTACCAAGAGGAATGTAAATGTGTCCGGC-3' and 5'-AAGCGGCCGCGATCATGTTAACCAAGGTCAGGAA-3' and subcloned into Litmus28 vector (NEB) via KpnI and NotI sites engineered into the PCR primers. After sequence verification of the coding exons, the transgene fragment was excised from the vector backbone, purified, resuspended at 2 ng / μl in microinjection buffer (10 mM Tris-HCl, pH 7.4, 0.25 mM EDTA) and microinjected into pronuclei of fertilized eggs prepared from C57BL / 6J mice. After overnight culture, the resulting 2-cell embryos were surgically transferred into the oviducts of pseudopregnant CD1 foster mothers and allowed to grow to term. Transgenic pups were identified by PCR using transgene-specific primers and housed as individual strains with wild-type C57BL / 6J mice.

[0090] CD1a genotyping Crude genomic DNA preparation was performed on chopped ear 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 chopped ear and incubated overnight at 55°C. The enzyme was then heat inactivated at 85°C for 1 hour. Samples were centrifuged to pellet the pieces and the lysate was transferred to a clean tube. 1 μl of the lysate was used as template for genotyping. The following PCR reaction was used for genotyping: PCR products were loaded onto a 1% TAE agarose gel with SyberSafe, electrophoresis was performed and the gel was imaged under UV. Mice were considered positive for the CD1a transgene if the expected band was detected at 655 bp.

[0091] [Table 1]

[0092] [Table 2]

[0093] cell line Empty vector-transfected K562 (EV-K562) and CD1a-transfected K562 (CD1a-K562) cells (a gift from 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 μg / ml streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Gibco), 1× non-essential amino acids (NEAA) (Gibco), 1 mM sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 500 μM 2-mercaptoethanol (Gibco), and 200 μg / ml G418 antibiotic (Thermo Fisher Scientific).

[0094] ELISpot analysis ELISpot assays were used to detect activation-induced cytokine secretion by polyclonal T cells upon co-culture with model CD1a-expressing antigen-presenting cells. PBMCs from healthy donor blood were isolated by density gradient (Lymphoprep) and T cells were purified (MACS, Miltenyi) using anti-CD3 magnetic bead sorting according to the manufacturer's protocol. Full informed written consent was obtained from all study participants [National Health Service (NHS) National Research Ethics Service (NRES) Research Ethics Committee 14 / SC / 0106]. T cells were then cultured with IL-2 (200U / ml) for 3 days to expand their numbers, before co-cultured overnight with non-pulsed / endogenous lipid-linked CD1a-transfected K562 (CD1a-K562) or control empty vector-transfected K562 (EV-K562) at a ratio of 25,000 K562 cells to 50,000 polyclonal T cells. To assess the functionality of anti-CD1a antibodies, K562 were incubated with 10 μg / ml of anti-CD1a antibodies 1 h before and during co-culture with polyclonal T cells in anti-IFNγ capture antibody-coated ELISpot plates. IFNγ secretion was detected by biotinylated anti-IFNγ detection antibody and visualized by streptavidin-alkaline phosphatase development. The resulting spots represented cytokine-producing T cells. They were enumerated using an automated ELISpot reader (ELISpot Reader Classic from Autimmun Diagnostika gmbh) and % blocking was calculated when comparing antibody-treated and untreated groups after subtraction of EV background levels of cytokine-producing spots. The contribution of EV-K562 (with and without antibody) was subtracted from the CD1a IFNγ spot count (with and without antibody, respectively). The corrected spot count of the CD1a-K562 antibody-treated group was then divided by the CD1a antibody-free group and used to calculate % blocking.

[0095] CD1a-reactive T cell generation and activation analysis: CD1a-restricted T cells were isolated by fluorescence-activated cell sorting. T cells were co-cultured with CD1a-K562 EV-K562 and cytokine-producing responding T cells were detected using Miltenyi's MACS cytokine secretion assay according to the manufacturer's instructions. Briefly, T cells were coated with anti-cytokine (IL-22 or IFNγ) antibodies after 6 h of culture with CD1a-K562 to detect CD1a-dependent autocrine cytokine production. Live responding cells were then sorted into culture plates. Composite lymphocyte reactions of CD1a-restricted T cells were expanded to assess purity and CD1a responsiveness using an analytical flow cytometer by the FACS-based cytokine secretion assay method described above. The activity of CD1a-restricted T cells was analyzed as follows: 2 × 105 K562 cells were co-cultured with 1–5 × 105 CD1a autoreactive T cells for 4 h. Helper cytokines were added to the co-culture to support CD1a-dependent cytokine production. IFNγ-producing T cell cultures were supplemented with IL-12 (1 ng / mL, BioLegend), IL-18 (1 ng / mL, BioLegend) and IL-2 (25 U / mL, BioLegend), and IL-22-producing T cell cultures were supplemented with IL-6 (5 ng / mL, BioLegend), TNFγ (5 ng / mL, BioLegend) and IL-2 (25 U / mL, BioLegend). T cell activation was assessed by T cell cytokine production using the above secretion assay (Miltenyi Biotec) according to the manufacturer's instructions.

[0096] Administration of imiquimod to mice Mice were lightly anesthetized with isoflurane and 15 mg of Aldara cream containing 5% imiquimod was applied to the dorsal and ventral sides of the ear pinna on days 0, 1, 2, 3, 4, and 5 in the preventive model (Figure 4A) or days 0, 1, 2 and 4, 5, 6, and 7 in the treatment model (Figure 9A). 100 μg of anti-CD1a antibody or mouse IgG1 isotype control was administered intraperitoneally on days -5, -3, -1, 1, 3, and 5 in the preventive model (Figure 4A) or days 3, 5, and 7 in the treatment model (Figure 9A). Ear thickness measurements were taken daily using a micrometer (Mitutoyo) during the period of Aldara application from days 0 to 6 in the preventive model (Figure 4A) or days 0 to 8 in the treatment model (Figure 9A). Mice were sacrificed 24 hours after exposure and tissues were collected.

[0097] Administration of MC903 to mice Mice were lightly anesthetized with isoflurane and 2 nmol per dose of MC903 was applied to the ventral and dorsal ears (10 microliters per ear) daily for 7 days. 100 μg of anti-CD1a antibody or mouse IgG1 isotype control was administered intraperitoneally as shown in FIG. 15D. Ear thickness measurements were taken daily using a micrometer (Mitutoyo).

[0098] Tissue processing Mice were sacrificed 24 hours after the final imiquimod exposure and tissues were harvested. Ears, cervical lymph nodes (cLNs) and spleens were harvested for immunophenotyping or imaging. Spleen and cLN cell suspensions were obtained by passing the tissue through a 70 μm strainer and washing with RPMI containing 10% FCS. Red blood cells in splenic cell suspensions were removed by incubation with RBC lysis solution (eBioscience).

[0099] Ear skin tissue was washed with HBSS to remove excess imiquimod, split ventrally, cut into sub-0.5 mm cubes, and digested with DMEM containing 1 mg / mL collagenase P (Roche) and 0.1 mg / mL DNase I (Sigma-Aldrich) for 3×30 min with stirring, with 5 mg / mL dispase added for a final digestion step of 30 min, washed through a 70 μm strainer with DMEM containing 10% FCS to obtain single cell suspensions, and then analyzed by flow cytometry.

[0100] Flow cytometry For FACS surface staining, cells were labeled with the following anti-mouse antibodies (supplied by Biolegend unless otherwise stated): CD3 (500A2, BUV495: 741064 BD Pharmingen), CD11b (M1 / 70, BUV395: 563553 BD Pharmingen), CD11c (N418, BV711: 117349), CD8 (53-6.7, BUV805: 612898 BD Pharmingen), and CD11c (N418, BV711: 117349). Pharmingen), CD4 (GK1.5, AF700:100430), CD45 (2D1, FITC:368507), CD11a (I21 / 7, P ECy7:153108), 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).

[0101] Flow cytometry: epitope competition assay CD1a-K562 cells were incubated with freshly produced purified anti-CD1a primary antibody and commercial anti-CD1a antibody (25 μg / ml) for 30 min on ice, then unbound antibody was washed away, and Alexa-Fluor-647 conjugated forms of the various antibodies were then incubated with the cells by matrix deposition for 30 min on ice (10 μg / ml). Mean fluorescence intensity (MFI) was used to assess the extent of binding of the fluorophore-conjugated antibodies.

[0102] Confocal imaging Mouse ear skin was frozen in optimal cutting temperature embedding compound and stored at -80°C. 10 μm frozen sections were cut using a Leica cryostat and collected onto Superfrost Plus slides, air-dried for 30 min, and then stored at -80°C. Slides were rehydrated in PBS for 10 min before staining. Endogenous peroxidase activity in the samples was quenched by adding 0.15% hydrogen peroxide solution for 5 min at room temperature. Endogenous biotin was blocked with an avidin / biotin blocking kit (Vector Laboratories Ltd), and 10% goat serum was used to reduce nonspecific binding of antibodies. Anti-CD1a antibody was used for confocal microscopy (1:100, OKT6; produced in-house and conjugated to biotin). Signal was enhanced using Alexa Fluor594 Tyramide SuperBoost Kit (Streptavidin; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, slides were incubated with primary antibodies overnight at 4°C. After washing, HRP-conjugated streptavidin was added to the sections and incubated overnight at 4°C. Excess streptavidin-HRP was washed away, tissues were incubated with working tyramide solution at room temperature for 8 min, and the reaction was stopped with stop reagent. After staining, slides were mounted using anti-fade mounting medium with DAPI (Vector Laboratories Ltd), coverslips were applied, and slides were refrigerated in the dark until analysis by confocal microscopy (Zeiss LSM780 confocal microscope-inverted microscope; 25× / 0.8 Imm Korr DIC M27; room temperature; Axiocam camera; Zen software) and image processing was performed using Fiji.

[0103] Cell phenotype and cytotoxicity assays: Anti-CD1a antibody (5 μg / ml) and commercial control NA1 / 34 (5 μg / ml) were incubated with CD1a-expressing K562 or EV control K562 for 48 h, and cell depletion was evaluated by flow cytometry. To measure direct antibody-induced cell depletion, K562 were fluorescently labeled with CellTraceViolet and then incubated with anti-CD1a antibody for 48 h. A reference population of untreated CFSE-labeled K562 was added to antibody-treated K562 in a 1:1 ratio before evaluation of depletion by flow cytometry. The percentage of induced depletion was then calculated by comparing the frequency of viable cells in the different populations analyzed, antibody-treated and untreated reference CD1a+ and EVK562, using the following formula: % reduction = 100 - ((% antibody-treated CD1a-K562 live cells / % control CFSE-labeled K562 live cells) / (% untreated CD1a-K562 live cells / % control CFSE-labeled K562 live cells) x 100). To examine the effect of anti-CD1a antibodies on apoptosis of CD1a-expressing cells, K562-CD1a or K562-EVs were incubated with either isotype control or anti-CD1a antibodies (5 μg / ml) and stained for Annexin V (Biolegend) 24 h after incubation.

[0104] Complement-Mediated Lysis (CDC) and Antibody-Dependent Cellular Cytotoxicity (ADCC) Assays: For CDC assays, K562-CD1a cells (5x104 cells per well) were pretreated with either isotype control or indicated antibodies at 5μg / ml for 30 min and incubated with 10% normal human serum at 37°C with 5% CO2 for 3 h. For ADCC assays, fresh PBMCs were used. K562-CD1a cells (5x103 cells per well) were combined with either isotype control or indicated antibodies at 5μg / ml and co-cultured with PBMCs (2.5x105 cells per well) in the presence of IL-2 (100U / ml) for 5 h at 37°C with 5% CO2 (effector / target ratio 50:1). Cytotoxicity was determined by calculating the percentage of viable K562-CD1a targets using the following formula: % cytotoxicity = ((% CD1a antibody-treated CD1a-K562 viable cells / % reference viable cells) / (% isotype antibody-treated CD1a-K562 viable cells / % reference viable cells) × 100).

[0105] In vivo CD1a+ cell depletion: "NSG" (NOD-scid IL2Rgammanull) mice were injected subcutaneously in the flank with 250,000 CD1a-K562 cells in ECM gel (Merck) suspension (vol=100 μl) and allowed to develop tumors for 18 days. On days 6, 10 and 14, mice were treated intraperitoneally with 100 μg of isotype control or indicated antibodies and tumor size was measured.

[0106] Isoelectric focusing assay (IEF): Lipid loading was assessed by incubating 10 μg of CD1a with 100× molar excess of imiquimod (Invivogen) or vehicle alone (mock) dissolved in Tris-buffered saline and 2% CHAPS, 7% DMSO for 2 h at 37 °C and overnight at room temperature. CD1a samples were separated by isoelectric focusing (IEF). Briefly, CD1a-imiquimod and CD1a-mock proteins were loaded onto an IEF pH 3-7 gel (Novex), which was then run at 100 V for 1 h, 200 V for 1 h and finally 500 V for 30 min. Gels were then fixed with 12% TCA, stained with SimplyBlue SafeStain for 7 min and destained overnight in DI water.

[0107] Statistical analysis: One-way and two-way ANOVA tests were performed using GraphPad Prism version 6.00 (GraphPad Software, Inc.) Error bars represent standard deviation as indicated.

[0108] Generation and selection of anti-CD1a antibody preparations 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117), and 16 (VR11834) A number of animals across a range of species were immunized, including mice and rabbits. 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, spleens, bone marrow and lymph nodes were harvested. Serum was monitored via flow cytometry for binding to HEK-293 cells expressing human CD1a and human B2M.

[0109] Memory B cell cultures were set up (related to 77A (VR11851), 110 (VR12112), 111 (VR12113) and 116 (VR12117)) and supernatants were first screened for their ability to bind to HEK-293 cells transiently transfected with human CD1a in a bead-based assay on the TTP Labtech Mirrorball system. This is a multiplex assay using HEK-293 cells expressing human CD1a and human B2M stained with a cell dye and counterscreened against counterstained HEK-293 cells expressing CD1b, CD1c or CD1d together with human B2M using goat anti-species Fc-FITC conjugate as revealer.

[0110] Approximately 3500 CD1a-specific positive hits were identified in the primary Mirrorball screen from a total of 10 x 200 plates of B culture experiments. Positive supernatants from this assay were then taken forward for further characterization by: ELISA, to confirm binding to human CD1a protein (details below) ELISA, to confirm binding to the CD1a lipid-binding domains (human lipid-binding domain of CD1a, mouse Ig domain of CD1d) on the chimeric CD1a protein (details below) Flow cytometry to confirm binding to human CD1a (co-expressed with human β2M) expressed on HEK-293 cells (details below)

[0111] Wells that demonstrated binding in the above assay were progressed for recovery of V regions using fluorescence focusing.

[0112] Bone marrow-derived plasma cells were also directly screened for their ability to bind human CD1a using fluorescent focusing (related to 16(VR11834)). Here, B cells secreting CD1a-specific antibodies were harvested onto biotinylated human CD1a immobilized on streptavidin beads using a goat anti-species Fc-FITC conjugate revealing reagent. Approximately 300 direct foci were harvested.

[0113] After reverse transcription (RT) and PCR of harvested cells, "transcriptionally active PCR" (TAP) products encoding the V-regions of the antibodies were generated and used to transiently transfect HEK-293 cells. The resulting TAP supernatants containing the recombinant antibodies were further characterized by: ELISA, to confirm binding to human CD1a protein and chimeric CD1a protein (human lipid-binding domain of CD1a, mouse Ig domain of CD1d) (details below) Flow cytometry to confirm binding to human CD1a (co-expressed with human β2M) expressed on HEK-293 cells and counter screen for cross-binding to related similar proteins: CD1b, CD1c or CD1d (co-expressed with human β2M) expressed on HEK-293 cells (details below)

[0114] The heavy and light chain variable region gene pairs from the interesting TAP products were then cloned as either rabbit or mouse full-length antibodies and re-expressed in the HEK-293 transient expression system. A total of 119 V regions were cloned and recorded. The recombinant clone antibodies were then further characterized by: Repeat the above flow cytometry and ELISA assays Flow cytometry to assess binding to CD1a expressed on multiple cell lines This was the first indication that the binding was lipid independent. Supernatants were screened for binding to: - stably transduced C1R cells expressing CD1a or empty vector (co-expressing human β2M), these relate to 110 (VR12112), 111 (VR12113) and 116 (VR12117) (details below). - MOLT4 cells that endogenously express CD1a, CD1b, CD1c, CD1d and β2M, which are related to 110 (VR12112), 111 (VR12113) and 116 (VR12117) (details below). BIAcore profiling to estimate off-rates and affinities (details below)

[0115] Antibodies that demonstrated binding in the above assays and affinities less than 100 nM were selected for purification. Cell culture supernatants were purified using Protein A affinity purification. Purified samples were buffer exchanged into PBS at 10 mM and pH 7.4 and analyzed for recovery and purity using UV spectroscopy, analytical size exclusion chromatography, SDS Page electrophoresis and LAL endotoxin assay, respectively. Now, required samples were subjected to a second round of purification to increase monomer levels. Final samples were sterile filtered and stored in PBS at 10 mM and pH 7.4.

[0116] After purification, all five antibodies were then further characterized by: Repeat the above flow cytometry, ELISA and BIAcore assays ELISA, assessing binding to cynomolgus monkey CD1a protein and a variant of human CD1a protein common in China (18) (details below) Flow cytometry, - Cynomolgus CD1a co-transfected with Cynomolgus β2M - A common variant of human CD1a in China co-transfected with human β2M Assess binding to transiently transfected HEK-293 cells (details below)

[0117] Antibodies 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) demonstrated the ability to bind to all tested forms of recombinant and cell-expressed CD1a protein during their respective antibody discovery stages (Tables 1-9). The only exception was 116 (VR12117), which showed no binding to recombinant or cell-expressed cynomolgus CD1a (Tables 4 and 9). Inclusion of antibody 116 in subsequent in vitro and in vivo analyses was not considered obvious, but was nevertheless a deliberate step to focus on epitope binding regions where the lipid binding domain differs between humans and cynomolgus monkeys and potentially has different functional effects. None of the antibodies demonstrated binding to CD1b, CD1c, or CD1d expressed on HEK-293 cells (Table 5), indicating that such antibodies were CD1a specific. CD1a, CD1b, CD1c, and CD1d expression on HEK-293 cells was confirmed with commercially available antibodies, supporting this conclusion (data not shown). Binding to CD1a expressed on multiple cell types (HEK, C1R, and MOLT4) was the first indication that antibody binding may be lipid-independent, as CD1a is likely added from a different lipid pool in each cell line.

[0118] Following antibody discovery, the antibodies were assessed for function in vitro in the following T cell assays.

[0119] DNA encoding the heavy and light chain V-regions of 77A (VR11851), 110 (VR12112), 111 (VR12113) and 116 (VR12117) on a mouse IgG1 backbone was synthesized in ATUM and expressed in an in-house HEK-293 transient expression system. The antibodies were then purified and endotoxin-free and tested in in vivo assays as follows.

[0120] Affinity of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) for human CD1a The affinity of purified antibodies to human CD1a was assessed by titrating human CD1a after capturing the antibodies to immobilized anti-species IgG F(ab')2 using a Biacore T200 instrument (GE Healthcare). Affinipure goat anti-species IgG-F(ab')2 specific fragments (Jackson ImmunoResearch) were immobilized onto a CM5 sensor chip (GE Healthcare) via amine coupling chemistry to a capture level of approximately 5000 response units (RU). HBS-EP+ buffer (10 mM and pH 7.4 HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, GE Healthcare) was used as the running buffer at a flow rate of 10 μL / min. A 10 μL injection of 0.5 μg / mL of test antibody was used for capture with immobilized goat anti-species Fab. Human CD1a was titrated against 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.

[0121] The surface was regenerated between cycles with 2 x 10 μL injections of 40 mM HCl and dispersed with 10 μL injections of 5 mM NaOH at a flow rate of 10 μL / min. Background subtracted binding curves were analyzed using Biacore T200 evaluation software according to standard procedures. Kinetic parameters were determined by a fitting algorithm. The assay was performed at the clone supernatant and purified antibody stages. Kinetic parameters for antibody binding to human CD1a are shown in Table 10.

[0122] Binding of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) assessed by ELISA CD1a-specific antibodies were identified by ELISA. ELISA plates were coated with 2 μg / mL of the protein of interest (human CD1a pool B, chimeric CD1a pool B [human lipid-binding domain and mouse CD1d Ig domain], Chinese mutant CD1a or cynomolgus CD1a) (20 μL / well) overnight at 4° C., then washed with wash buffer (0.2% (v / v) Tween-20 in PBS, pH 7.4). The plates were then blocked with 80 μl / well of blocking buffer (1% (w / v) bovine serum albumin) for 1 h at room temperature, then washed in wash buffer. 20 μL of antibody sample (B cell culture supernatant, TAP supernatant, clone supernatant, purified antibody solution) dilutions were transferred to the ELISA plate and incubated at room temperature for 1 h, followed by washing with wash buffer. 20 μl / 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 h at room temperature, followed by washing with wash buffer. Binding was visualized by adding TMB substrate (EMD Millipore) (20 μL / well) and the reaction was incubated for 5 min at room temperature before measuring the optical density at 630 nM using a microplate reader. The assay was performed at the B cell supernatant stage (human CD1a pool B), TAP supernatant stage (human CD1a pool B, chimeric CD1a pool B), clone supernatant stage (human CD1a pool B, chimeric CD1a pool B) and purified antibody stage (human CD1a pool B, chimeric CD1a pool B, Chinese mutant CD1a, cynomolgus CD1a). Data for purified antibodies are shown in Tables 1-4.

[0123] Binding of 77A (VR11851), 110 (VR12112), 111 (VR12113), 116 (VR12117) and 16 (VR11834) assessed by flow cytometry CD1a-specific antibodies were identified by flow cytometry. Binding to proteins expressed on HEK, C1R and MOLT4 cell lines was assessed. HEK-293 cells were transfected with the protein of interest (CD1a, CD1b, CD1c, CD1d, Chinese variant CD1a or cynomolgus CD1a) and species-specific β2M (as indicated above). Transfection was performed using the Expifectamine293 kit (Gibco) and incubated overnight. C1R-CD1a, C1R-empty vector and MOLT4 cell lines were washed in 1x PBS on the day required. All cell lines were counted and resuspended in 1x PBS, then stained with DiI or DiO cell stain (Invitrogen) for 30 min at 37°C. After washing the cells with flow cytometry buffer (1% bovine serum albumin, 2 mM EDTA and 0.1% sodium azide in PBS), the two DiI- and DiO-stained populations were mixed together. The cells (20 μl / well) were then added to dilutions of antibody samples (B cell culture supernatants, TAP supernatants, clone supernatants, purified antibody solutions) (20 μl / well) and incubated for 1 h at 4°C in the flow cytometry assay plate, followed by washing with flow cytometry buffer. 10 μl / well of Alexafluor647-conjugated goat anti-species IgG Fc-specific F(ab')2 fragments (JacksonImmunoResearch) were added at 1:2500 dilution in flow cytometry buffer, incubated for 30 min at 4°C, followed by washing with wash buffer. The fluorescence intensity was then measured on an iQue screener PLUS.The 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 mutant CD1a or cynomolgus CD1a; C1R cells expressing human CD1a or empty vector; MOLT4 cells). The data for purified antibodies are shown in Tables 5-9.

[0124] [Table 3]

[0125] [Table 4]

[0126] [Table 5]

[0127] [Table 6]

[0128] [Table 7-1] [Table 7-2]

[0129] [Table 8]

[0130] [Table 9]

[0131] [Table 10]

[0132] [Table 11]

[0133] [Table 12]

[0134] [Table 13] EXAMPLES

[0135] [Example 1]

[0136] Improved anti-CD1a panel: Functional evaluation of anti-CD1a antibodies After CD1a binding evaluation, a large panel of generated anti-CD1a antibodies was screened for inhibitory function. T cell cytokine production was measured by EliSpot in an in vitro antigen presentation model. A summary of such data is shown in Figure 1. Many of the newly generated antibodies were determined to be more potent than the commercially available anti-CD1a antibodies OKT6, HI149 and SK9 in inhibiting CD1a T cell responses. Of note, antibodies 16, 22, 39, 46, 77, 87, 110, 116 all had IC50s at least 1 log lower than OKT6 (Figure 1B), 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 immortal T cells. [Example 2]

[0137] Improved anti-CD1a panel: Inhibition of CD1a-restricted enriched T cell line responses To help shortlist antibody candidates for in vivo analysis, we took a different approach to assess CD1a T cell responses: CD1a-restricted enriched T cell lines were isolated and expanded to analyze CD1a responses in isolation rather than in a complex polyclonal T cell background where potential inhibitory antibodies may be partially masked by a low signal-to-noise ratio.

[0138] In such assays, antibodies 116 and 16 excelled as potent inhibitory antibodies, with 16 uniquely inhibiting autoreactive / endogenous production of IL-22 (Figures 2A and 2B). This improvement indicates the possibility of using the antibodies under conditions where IL-22 plays a pathogenic role in addition to those where IFNγ has a role to play. It was surprising to see differential effects on various cytokines. Furthermore, an APC-free system was used to evaluate antibody-dependent inhibition of CD1a-restricted T cell activation. CD1a-coated beads were used as a surrogate for APCs, and the resulting T cell IFNγ production was measured by flow cytometry. This assay revealed significant inhibition of CD1a-dependent cytokine responses by all antibodies, particularly 77a, 87, 110, 111 and 116 (Figure 2C). [Example 3]

[0139] In vivo evaluation of inhibitory antibodies in skin inflammation The aim of this study was to generate antibodies for clinical use in the treatment of human diseases and disorders, and thus it was essential to ascertain efficacy in a complex immune system resembling human disease. A highly refined panel of the best newly generated antibodies was selected from the analysis of the above data (antibodies 16, 77a, 110, 111 and 116) and we sought to determine their potential in in vivo models of psoriasis, dermatitis, lupus, and as models of drug responses manifested as inflammatory skin or mucosal diseases or disorders, or related systemic diseases or disorders, or one or more systemically manifested inflammatory drug responses. Experimental psoriasis and dermatitis were shown to be exacerbated in CD1a transgenic mice compared to WT, and CD1a-dependent inflammation could be reversed by administration of anti-CD1a antibodies (Kim et al., 2016). It is also noteworthy that some individuals develop cutaneous / mucosal inflammatory drug reactions to imiquimod used topically for a number of skin disorders, including psoriatic reactions, dermatitis reactions, bullous disease, alopecia, vesiculation, lichenoid reactions, neutrophilic disease, lupus erythematosus, erythema multiforme, oral erosions, and severe drug reactions such as DRESS, AGEP, Stevens-Johnson syndrome, and toxic epidermal necrolysis (19-29).

[0140] Generation of CD1a transgenic mice To evaluate the possible role of CD1a in skin and associated systemic inflammation, we generated CD1a transgenic mice. Since CD1a is not present in the mouse genome, we cloned the human CD1a locus with 0.8kb5' and 0.8kb3' flanking regions including the promoter element, and inserted this transgene by microinjection, similar to the published CD1a transgenic model but requiring additional transgene fragment stitching (Illing et al., Nature 486, 554-558 (2012)). Genotype-positive founder mice were bred and lines were screened for CD1a transgene expression. We further phenotyped the mice to determine whether CD1a protein expression followed the expected profile and was representative of human CD1a cell expression. Ear skin of wild-type and CD1a transgenic (CD1aTg) mice was harvested and enzymatically processed to allow analysis of skin cell rings by flow cytometry (Figure 3A). CD1a expression was detected in skin, accounting for 4.2% (+ / - 1.79) of total skin cells and 23.6% (+ / - 6.68) of CD45+ cells. To assess cellular expression regulation, dermal DCs (dDCs) and Langerhans cells (LCs) were assessed for CD1a protein. Dermal DC subsets have been reported to express CD1a, and Langerhans cells are characteristically constitutively CD1ahigh. CD1a was found to be expressed by 41.5% (+ / - 20.38) of dDCs and 88% (+ / - 4.606) of LCs (Figure 3A-B). CD1a protein expression in skin was further characterized by immunofluorescence, revealing a characteristic epidermal location and cells with dendrites typical of LCs (Figure 3C). The CD1a genotype was confirmed (Figure 3D), and CD1a expression in the thymus was observed mainly by a percentage of CD4+CD8+ double positive thymocytes (Figure 3E). CD1aTg mice did not show abnormal skin inflammation at steady state. In summary, we have generated CD1a transgenic mice that exhibit CD1a expression phenotypically similar to human tissue expression.

[0141] Using this model, anti-CD1a antibodies were tested for the prevention of inflammatory skin diseases and disorders (Figure 4A). Application of Aldara cream containing the TLR7 / 8 agonist imiquimod 5% is an established model that induces psoriasis-like, dermatitis-like, and lupus-like skin inflammation, typically manifested by skin thickening, scaling, and redness (30,31). Ear inflammation in CD1a transgenic mice was found to be significantly higher than that in WT counterparts in response to Aldara. Moreover, all anti-CD1a antibodies administered prior to imiquimod reduced subsequent ear thickening, while antibodies 116 and 16 reduced CD1a-dependent inflammation at least to WT levels (Figure 4B). By the end point of the experiment, CD1a transgenic (-Tg) mice treated with antibodies 16 and 110 showed reduced inflammation to WT levels of ear thickening. Remarkably and unexpectedly, antibody 116 treatment significantly reduced the levels of CD1a-Tg ear skin inflammation below that of WT skin (FIG. 4B). [Example 4]

[0142] In vivo effects of inhibitory antibodies on cutaneous immune responses. We therefore sought to analyze the contribution of skin immune populations to imiquimod-induced CD1a-dependent ear inflammation.

[0143] We found that skin T cell infiltration was elevated in CD1a transgenic mice and the frequency of this population was reduced by anti-CD1a antibodies, especially antibodies 116, 16 and 110 in the preventive model (Figure 5A). Of note, 16 and 116 were able to reduce skin T cell infiltration to levels below wild type, suggesting a significant amelioration of inflammation in vivo. Moreover, the activation marker CD69 was elevated on the surface of skin T cells in CD1a transgenic mice and was inhibited by some of the anti-CD1a antibodies, especially 116 and 16 in the preventive model (Figure 5B). Neutrophils have been found to be key cells in many inflammatory disorders, including psoriatic responses and the mouse imiquimod model. Here, we found an increase in neutrophil frequency in the skin upon imiquimod treatment and a further increase in CD1a transgenic mice, which was reduced to WT levels or below by anti-CD1a antibodies 116 and 16 in the preventive model (Figure 5C). There was also a reduction in skin eosinophils in response to the antibody, which is intriguing given the known role of eosinophils in many forms of drug reactivity (Figure 5D). This unexpected finding represents an improvement, as no effect on eosinophils has been observed previously.

[0144] As observed in human cutaneous inflammatory disorders, Langerhans cells, defined here as CD11c+Langerin+, were also increased in the skin of CD1a transgenic mice upon imiquimod exposure compared to WT. Administration of antibodies 16, 116, 111 and non-significantly 110 reduced the number of skin LCs in the prophylactic model (Figure 6A). Notably, antibody 116 reduced the number of skin LCs below the number in wild-type skin, showing a surprising level of improvement in efficacy. The effect of the antibodies on CD1a expression of LCs, as the main CD1a expressing population, was evaluated. It was notable that antibodies 110 and 116 reduced staining, but this was due to interference of the 110 / 116 antibodies with binding by the HI149 detection antibody (Figure 6B). This is a surprising effect and indicates persistent binding of the antibodies in vivo, which is associated with therapeutic benefit. This finding also raises the possibility of using the antibodies for diagnostic or prognostic purposes or for monitoring CD1a expressing cells before and during treatment. This observation was not seen with the non-competitive SK9 detection antibody, as shown below. The observed LC reduction could be due to antibody-dependent LC death or migration or phenotypic alteration. Therefore, cervical lymph nodes were analyzed for the presence of CD11c+Langerin+LC. However, although an increase in the number of LCs in lymph nodes of CD1a transgenic mice was found compared to WT, migration to LNs did not seem to explain the reduction in skin LCs in mice treated with antibodies 110 and 116 (Figure 6C). Of note, antibody 116 led to an immunological improvement close to that of wild-type skin, showing a surprising level of improvement in efficacy. Interestingly, the expression levels of CD1a on lymph node-derived LCs followed a similar pattern to those of the skin, in that there was reduced staining of LCs, which is due to interference of the 110 / 116 antibodies with binding by the HI149 detection antibody, as discussed further below (Figure 6D). This was not seen with the non-competitive SK9 detection antibody. It is noteworthy that lymph node-derived LC expressed less CD1a per cell than skin, which may be a regulatory mechanism to prevent systemic inflammation. Thus, antibodies maintain their effect on LC in vivo, both in the skin and even after migration to lymph nodes.This is an important enhancement since the clinical effect is more durable. [Example 5]

[0145] Anti-CD1a antibodies observed cytotoxicity expressed in response to CD1a-expressing cell phenotypes Given that enhanced migration did not fully explain the loss of dermal LCs, the possibility of antibody-induced alteration of the CD1a+ cell phenotype, regardless of the nature of the murine IgG1, was examined.

[0146] We demonstrated that all anti-CD1a antibodies, especially 110 and 116, were able to reduce the number of CD1a+K562 cells in vitro, allowing comparison of responses due to the lack of MHC class I and II (Figure 7A). Antibodies 110 and 116, when examined in more detail, showed a dose-dependent reduction (Figure 7B), which was an improvement and a surprise considering the IgG1 isotype. This appears to be different from the published CR2113 antibody (16, 18) (US Pat. No. 10,844,118 and Canadian Patent Publication No. 2,924,882), which was stated to require complement and / or antibody-dependent cellular cytotoxicity. In particular, it was stated that "CR2113 does not directly induce apoptosis" (17), and it was observed that NA1 / 34 does not induce direct killing. However, since effector functions are influenced by the different Fc regions, the comparable effects of CR2113 in a murine IgG1 background are addressed directly below. The inventors further evaluated the ability of the antibodies to induce a direct reduction of primary human CD1a-expressing cells. DC and LC-like cells were generated by 5-day in vitro differentiation of monocytes with cytokines IL-4 / GM-CSF and IL-4 / GM-CSF / TGF-β, respectively, with anti-CD1a antibodies added at day 0 or 2 of culture. We observed that antibodies 110 and 116 reduced LC and, to a lesser extent, DC in vitro (upper and lower panels, respectively, in Figure 7C). Investigating the mechanism underlying this reduction, we found a reduction associated with a striking phenotypic morphology of cell clustering cultures (Figure 7D). Although the reduction in numbers could be partially explained by this clustering, in addition, we tested whether the antibodies could induce apoptosis of CD1a-expressing target cells and compared them to CR2113 (in a mouse IgG1 background). Figure 7E shows that 110 and 116 (but not 16) and CR2113 (mouse IgG1 background) induce Annexin V expression by CD1a-expressing K562 even in the absence of complement or ADCC, suggesting that 110, 116 and CR2113 antibodies can mediate K562 cell death to some extent.To examine the role of complement-mediated lysis (CDC) and antibody-dependent cellular cytotoxicity (ADCC), K562-CD1a was incubated with complement (Figure 7F) and / or human PBMCs (Figure 7G). Despite the mouse IgG1 nature of the antibody, there was evidence of complement-mediated lysis and ADCC. To further examine the in vivo mechanism, a new model was established using K562-CD1a subcutaneous tumors in an immunodeficient NSG model that broadly lacks lymphocyte responses and other effects. Data showed that all three antibodies reduced lymphoid cell tumor size by day 10, with 16 and 116 maintaining their effect until days 15-20 (until a reduction in CD1a-expressing tumor cell burden of 25% or more), but CR2113 losing its effect (Figure 7H). The difference in response in vitro and in vivo may be explained by other cofactors present in vivo, such as complement, multiple innate cell subsets carrying FcR with different Fc specificity, differential target cell density, shortened antibody half-life in vivo, and altered tissue delivery. Such direct alteration of CD1a-expressing target cell phenotype may promote a low inflammatory response of CD1a-expressing cells. Thus, the reduction of LC in the skin of CD1a-Tg mice treated with 110 and 116 may be partially explained by direct antibody-dependent changes in the phenotype of CD1a+LC, which may contribute to clinical effects, such as the reduction of inflammation in 116 to below wild-type levels. The data also raise the possibility that the antibody may have utility in the treatment of CD1a-expressing malignancies, including Langerhans cell histiocytosis and some types of T-cell lymphoma and some types of thymoma. However, target cell phenotypic alteration cannot explain the reduced T cell functional responses shown in FIG. 2, since the CD1a bead assay (FIG. 2C) was unaffected by any depletion effect. [Example 6]

[0147] Epitope binding analysis of CD1a antibody The data presented herein demonstrate that the five newly generated anti-CD1a antibodies have different functionalities, and we sought to determine whether the antibodies have overlapping binding sites using flow cytometry cross-blocking assays. In addition, epitope overlap was assessed with the commercially available antibodies OKT6, HI149, SK9 and NA1 / 34 (which, as mentioned above, have known overlapping binding sites with CR2113).

[0148] CD1a-K562 cells were incubated with purified anti-CD1a primary antibody (Y-axis in Figure 8A, 25 μg / ml), then unbound antibody was washed away, and Alexa-Fluor-647 conjugated forms of various antibodies were then incubated with the cells in the matrix arrangement of Figure 8A (X-axis, 10 μg / ml). Mean fluorescence intensity (MFI) was used to assess the extent of binding of the fluorophore-conjugated antibodies, and any steric interference caused by binding of the purified primary antibody was represented by a decrease in MFI. The results showed that antibodies HI149, OKT6, 110 and 116 may have overlapping or closely binding epitopes, and a second group including antibodies NA1 / 34, 77a, 111 and 16 may have closely related binding sites. This suggests that the decrease in CD1a expression observed in vivo (Figures 6B and D) is due to interference of the 110 / 116 antibodies with binding by the HI / 149 detection antibody. Indeed, this effect was not seen with non-competitive SK9 detection antibodies (Figure 8B). Importantly and unexpectedly, this allows the antibody to maintain its presence in the skin and on LC in vivo, even after migration to lymph nodes and subsequent enzymatic degradation of skin tissue. This may be associated with longer-term and substantial clinical utility. As the antibodies are divided into two main groups that do not compete, Figure 8 (A and B) shows that combinations of antibody members selected from each group can be used together, for example, as treatment / monitoring or combination treatment. One such combination is 116 and 16. [Example 7]

[0149] Demonstration of the efficacy of the antibodies of the invention for the treatment of imiquimod-induced inflammation and also systemically related inflammation Although the presence of circulating CD1a-reactive T cells has been demonstrated (11), most studies have focused on skin-specific functional effects, given the skin-predominant expression of CD1a. The role of CD1a in inflammation of tissues beyond the skin has not been extensively examined. Moreover, although CD1a is known to amplify imiquimod skin responses (16), there are no studies of related systemic sequelae. We generate novel CD1a transgenic mice and CD1a-reactive T cells and characterize the functionality of anti-CD1a antibodies in vitro and in vivo using human and mouse assays, respectively. This finding confirms that CD1a-dependent effects extend to systemic effects and has implications for the treatment of systemically related skin diseases, including adverse inflammatory drug reactions.

[0150] Therapeutic potential of anti-CD1a antibodies To further evaluate the therapeutic potential of the newly generated anti-CD1a antibodies, we tested the three most clinically effective antibodies 16, 110 and 116 in an imiquimod-treated model, where anti-CD1a antibodies were introduced after the establishment of imiquimod-induced inflammation (Figure 9A). All three antibodies improved the clinical response quickly after initiation, despite ongoing imiquimod application (Figures 9B-C). The response was most prominent with 116, which reduced ear thickness (Figure 9B). Thickening of the whole skin (upper panel) and epidermis (lower panel) was visualized by confocal microscopy (Figure 9D), confirming the micrometer assessment (Figure 9B). CD1a protein expression was evaluated in CD1a transgenic epidermis (anti-CD1a OKT6 AF-594, red) and was found to be reduced in skin treated with 110 and 116 due to cell death and epitope competition (Figures 8A and 9D). When analyzing the skin cell immune response after the imiquimod treatment model, we observed a decrease in the number and activation of skin T cells after antibody introduction, a decrease in skin LCs, and a decrease in skin neutrophils (Figure 9E-G).

[0151] CD1a is involved in the systemic immune response to imiquimod The human effects of imiquimod treatment can extend beyond the skin, and in mouse models, it has been shown to induce splenomegaly. The contribution of CD1a to this pathway was evaluated. Strikingly, consistent with a systemic effect that does not remain confined to the skin, spleen weight was elevated in imiquimod-treated CD1a Tg mice compared to wild type, and the antibody reduced spleen size and weight (Figure 10A). Moreover, the antibody reduced CD4 and CD8 T cell activation as determined by CD69 expression (116 and 110, Figure 10B-C), splenic neutrophils (non-significant trend), and eosinophil frequency (16, 110, 116) (Figures 10D and 10E, respectively). Plasma cytokine levels were assessed on day 8. In imiquimod-treated CD1a transgenic mice, we observed significant increases in IL-23, IL-12p70, IL-1β, IL-1α and MCP-1, which were reduced in some or all of the 16, 110 and 116-treated groups (Figure 10F). In the presence of antibodies 16 and 116, respectively, plasma immunoregulatory cytokines IL-10 and IL-27 were elevated (similar trends elsewhere). We then examined the effects on circulating immune cells. As in the spleen, imiquimod-treated CD1a transgenic groups showed increases in blood CD4 and CD8 T cell counts, neutrophilia and eosinophilia. This increase was significantly blocked after treatment with 16, 110 or 116 (Figures 11A-E). Finally, we investigated whether imiquimod itself could be a CD1a ligand and showed that this was not the case, implicating a broader autoimmune and autoinflammatory effect of the CD1a pathway (Figure 12). It can therefore be suggested that a broad systemic inflammatory immune response is stimulated or influenced by CD1a in the skin.

[0152] To address whether anti-CD1a antibodies could result in a sustained reset of skin inflammation after imiquimod application, we attempted the model shown in Schematic Figure 13A, where re-exposure to imiquimod was performed without re-administration of anti-CD1a antibodies (Figure 13B). Surprisingly, consistent with a sustained immunological effect, 16, 110, and 116 all resulted in sustained improvements in ear thickness without repeated administration of the antibody. We also observed that the immunological response was sustained along with the following: 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 ), lymph node neutrophils ( 116 ), blood T cell frequency ( 110 , 116 ), blood T cell activation ( 116 ), blood eosinophils ( 110 , 116 ), plasma IL-1α ( 116 ), IFNγ ( 16 , 110 , 116 ), IL-1β ( 16 , 110 , 116 ), IL-6 ( 16 , 116 ), and IL-17A ( 16 , 110 , 116 ) were significantly decreased.

[0153] To compare the ability of the antibodies to the current standard of care in managing moderate-to-severe psoriasis, the imiquimod treatment model (Figure 9A) was repeated in parallel with administration of anti-IL-17A (IgG1 isotype) at the same time and in the same dose (100 μg) as the anti-CD1a antibodies (Figure 14). All anti-CD1a antibodies again showed significant improvement in ear thickness results, and all produced significant improvement earlier than anti-IL-17A. In contrast to the various anti-CD1a antibodies, anti-IL-17A was found to not significantly reduce the frequency of cutaneous T cells, cutaneous Langerhans cells, cutaneous eosinophils, lymph node T cells, lymph node neutrophils, lymph node eosinophils, plasma IL-23, MCP-1, or IL-6.

[0154] To directly compare the skin and systemic inflammation outcomes between the antibodies described herein and CR2113, an imiquimod skin treatment model was attempted (Figure 15A). All anti-CD1a antibodies had a beneficial effect on ear thickness, but antibody 116 was significantly elevated over CR2113 (Figures 15B-C). To expand the investigation of the improvement of anti-CD1a antibodies 16, 110 and 116 over CR2113, a comparison was made to an additional model of skin inflammation, namely MC903-induced inflammation (Figure 15D), where a significant benefit was observed for antibodies 16, 110 and 116, but not CR2113, thus showing improvement (Figure 15E). It was observed that 16 and 116, but not CR2113, showed a significant reduction in skin T cell percentage and skin eosinophil count (Figure 15F). Skin-extracted 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), or IL-17F (16, 110, 116).

[0155] It was further observed that 116 showed consistent improvement over CR2113 in reducing the skin, lymph node and plasma inflammatory response to imiquimod (Figure 16). In some results, 16 also improved significantly over CR2113 (Figure 16). In particular, antibody 116 improved over CR2113 in reducing IL-17A expression by skin T cells and frequency of draining lymph node eosinophils. 116 also improved over CR2113 in reducing plasma IFNγ, IL-1α, IL-1β, IL-5, IL-9, IL-17A, IL-17F, IL-22, and skin degradation products IL-1α, IL-22 and TNFα. 16 improved over CR2113 in reducing lymph node eosinophils, plasma IL-1β, IL-22, IL-9 and IL-5, and skin degradation products IL-1α and IL-17A with a strong trend. Overall, the data confirm that the antibodies described herein are capable of inhibiting cutaneous and systemic inflammatory responses to imiquimod and MC903.

[0156] Consideration Skin inflammation, e.g., dermatitis, psoriasis, and lupus, are common disorders that are significantly associated with physical and psychological morbidity. Cutaneous adverse reactions to drugs are also common, ranging from 1.8 to 7 per 1000 hospitalized patients. Severe cutaneous adverse reactions with widespread and systemic effects, e.g., SJS, TEN, AGEP, and DRESS, are less common, e.g., SJS / TEN, has an incidence of approximately 1 to 6 cases per million individuals per year (M. Mockenhaupt, Allergol Select 1, 96-108 (2017)). Gell and Coombs defined the classification of hypersensitivity in the 1960s, where delayed IV hypersensitivity required the role of effector T cells (RRA Coombs, Gell, PGH, Classification of allergic reactions responsible for drug hypersensitivity reactions. In Clinical Aspects of Immunology. (Davis, Philadelphia, ed. second, 1968)). There is increasing recognition that this classification does not explain all aspects of drug hypersensitivity, but still focuses largely on altered recognition of covalently bound haptens or non-covalently modified peptides / MHC molecules, however current models do not explain the predominance of cutaneous and mucosal complications of drug hypersensitivity (M. Mockenhaupt, Allergol Select 1, 96-108 (2017)).

[0157] By generating CD1a transgenic mice and autoreactive human CD1a-restricted enriched T cell lines, as well as characterizing functional anti-CD1a antibodies, the data presented here show the induction of CD1a presentation of endogenous lipid ligands. This leads to skin and systemic inflammation mediated by autoreactive T cells. Anti-CD1a antibodies, whether blocking or blocking / modulating, had clinical and immunological effects, suggesting the significance of CD1a lipid presentation to T cells. Since TLR7 can recognize single-stranded RNA, it is intriguing that reactivity to viral infections can mimic the clinical phenotypes of various severe forms of skin inflammation, including psoriasis, dermatitis, lupus, and adverse inflammatory responses to drugs, including SJS and TEN. This shared final general clinical observation may indicate that many precipitating agents can promote CD1a autoreactivity and autoinflammation. This model may also help explain the increased risk of autoimmunity associated with certain drug reactions, including lupus erythematosus and DRESS syndrome. Moreover, this finding implicates CD1a autoreactivity in the breakdown of broader T cell tolerance.

[0158] In addition to the effect on T cell response to imiquimod-containing drug Aldara, an increase in neutrophil and eosinophil responses in skin, draining lymph nodes, and spleen was observed in CD1a transgenic mice. Such effects were inhibited by administration of the antibodies of the present invention, particularly 16, 110, and 116. This implies that the CD1a-dependent immune cascade is more widespread than originally expected. It has been shown that depletion of neutrophils ameliorates 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)).

[0159] Application of Aldara / Imiquimod reproduces key aspects of various forms of skin inflammation and related systemic diseases and disorders, including psoriasis, dermatitis, lupus and severe cutaneous hypersensitivity reactions, including T cell and neutrophil infiltration as discussed above. Data demonstrated herein show that imiquimod-dependent eosinophil infiltration of skin, lymph nodes and spleen is enhanced in CD1a transgenic mice and reduced by administration of the antibodies of the invention, particularly 16, 110 and 116.

[0160] Moreover, it has been reported that the number of LCs in the lesional skin of patients with various forms of inflammatory skin diseases or disorders, including psoriasis, dermatitis, lupus, and maculopapular drug eruptions, is increased compared to non-lesional skin and decreases to non-lesional levels when the rash resolves (DI Dascalu, Y. Kletter, M. Baratz, S. Brenner, Acta Derm Venereol 72, 175-177 (1992)). Interestingly, psoriasis is associated with altered LC migration, suggesting that application of imiquimod and validated mouse models of psoriasis and lupus and dermatitis are well studied, but also has applicability to adverse drug inflammatory drug reactions. Here, we show that CD1a antibody-dependent modulation of LCs was associated with reduced skin inflammation upon administration of the antibodies of the invention, particularly 110 and 116, which may have therapeutic implications for the treatment of psoriasis, dermatitis, lupus, inflammatory drug reactions, and other conditions. Epitope analysis highlights the potential therapeutic significance of epitope binding sites and divides anti-CD1a antibodies into two groups based on the binding site and the resulting effector function. The epitope site may promote the clustering and altered phenotypic effects seen with 110 and 116, but not with antibodies 77a, 111 and 16, which were primarily blocking antibodies. Clustering may indeed cause a bridging / aggregation-like cell morphology, which may also explain the reduction of both cell types, as CD1a-transfected K562 and monocyte-derived LC express higher levels of CD1a than monocyte-derived DC. Since the various antibody binding sites of the two groups do not compete, there is utility, for example, in treatment / monitoring or combination therapy, for combinations selected from each of the two groups.

[0161] The role of CD1a in the pathogenesis of skin inflammation and related systemic diseases implicates this role in many diseases, including psoriasis, dermatitis and lupus erythematosus, as well as drug hypersensitivity. Moreover, the characterization of CD1a blocking and modulating antibodies provides new potential routes for the development of preventive and therapeutic approaches against skin inflammation and CD1a-expressing malignancies.

[0162] summary In summary, the inventors have generated an improved panel of anti-CD1a antibodies with therapeutic potential in the prevention and / or treatment of inflammatory skin and mucosal disorders. Five antibodies 16, 77a, 110, 111 and 116 have been shown to be potent inhibitors of human CD1a antigen presentation in vitro and have shown efficacy in exemplary inflammatory skin disease prevention and treatment models, as well as xenograft tumor models, with characteristics of drug reactions manifested as psoriasis, dermatitis, lupus erythematosus and inflammatory skin or mucosal diseases or disorders, as well as systemic (non-cutaneous) disorders. 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) using a novel chimeric immunogen, whereby the human CD1a lipid binding domain is fused to the host organism CD1d Ig domain, thus targeting antibody production against the lipid binding domain. In this case, functional inhibitory potential can be contingent on c) diverse polyclonal and enriched T cell analyses examining different functional outcomes.

[0163] In vitro human functional assays showed that this antibody was more potent than commercially available antibodies, as measured by IC50 evaluation of inhibition of primary polyclonal T cell responses. Moreover, using a highly sensitive human CD1a-restricted T cell clone assay, it was determined that anti-CD1a antibodies 16 and 116 can block IL-22 production, a key regulator of inflammatory skin and mucosal diseases. This was not shown in existing publications or patents for anti-CD1a CR2113 ((16, 17), U.S. Pat. No. 10,844,118 and Canadian Patent Publication No. 2,924,882), where such activity was improved and surprising, since IL-17 or IFNγ production was induced and inhibited in mouse systems. IL-22 inhibition is an important advantage of the antibody, since IL-22 is a key regulator of skin and mucosal diseases.

[0164] Parallel analysis of human and in vivo mouse models provides a powerful means of evaluating the therapeutic utility of newly generated antibodies. In vivo, imiquimod was utilized to induce psoriasis-like, dermatitis-like, lupus-like, and drug reaction-like phenotypes, resulting in a model skin inflammatory system. Imiquimod may also be more broadly applicable to many inflammatory diseases and disorders as well as related systemic diseases or disorders and inflammatory drug reactions that manifest systemically. Here, antibodies 110, 116, and 16 were shown to significantly reduce imiquimod-induced CD1a-dependent inflammation, improving over standard of care (anti-IL-17A) and a control, an anti-CD1a antibody (CR2113) in the same mouse IgG1 background. Importantly and unexpectedly, antibody 116 reduced skin inflammation below the levels of WT imiquimod-treated mice and normalized many of the skin and systemic immune markers to WT levels, suggesting a mechanism by which anti-CD1a 116 acts beyond inhibition of CD1a-TCR signaling. Skin immunophenotypes were determined and we observed a reduction in T cell numbers and activation, as well as neutrophil infiltration to WT levels with treatment with antibodies 110, 116 and 16. The observed reduction in neutrophils to WT levels is an unexpected improvement over the published anti-CD1a CR2113, thereby highlighting the potential of antibodies 110, 116 and 16.

[0165] Importantly, when analyzing LC populations in the skin, a significant reduction in CD11c+Langerin+LCs was observed after administration of antibodies 110 and 116. This reduction was not explained by enhanced migration to draining lymph nodes. However, antibodies 110 and to a greater extent 116 may be capable of directly depleting CD1a+ cells in vivo, explaining the reduction in skin LCs in vivo, as evidenced by the marked reduction in human CD1a+ cells in vitro. This is a surprising result considering the mouse IgG1 isotype of the antibody, where the mouse IgG2a isotype is more likely to cause cytotoxicity via complement-mediated lysis or antibody-dependent cellular cytotoxicity. It was also found here that apoptosis of CD1a-expressing cells could also be induced by CR2113 in a mouse IgG1 background, whereas a further patented and published anti-CD1a CR2113 reportedly does not allow direct depletion (17). The regulatory capacity of such antibodies may help explain the reduction in imiquimod-induced inflammation below the levels of WT isotype-treated mice. Antibody 116 not only blocks CD1a interaction with the TCR, but also modifies LC reducing / resetting the inflammatory potential of the skin and normalizing many of the skin and systemic immune markers to WT levels, which may explain the restorative effect beyond CD1a-dependent responses to improvement over wild type that is absent in anti-CD1a CR2113.

[0166] Moreover, data suggest that the 16, 110 and / or 116 antibodies presented herein have utility in treating CD1a-expressing malignancies, such as Langerhans cell histiocytosis or some types of T-cell lymphoma and thymoma. This may be by direct action or, in this case, the anti-CD1a antibody is conjugated or associated with one or more other therapeutic agents selected from the group including 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.

[0167] In this study, we demonstrate that antibody 16 is a highly effective blocking antibody that eliminates CD1a-dependent inflammation in vivo without directly inducing apoptosis, 110 modifies LC phenotype and function to significantly reduce CD1a-dependent inflammation in vivo, and 116 is a highly effective blocking and modifying antibody that reduces inflammation below WT levels and normalizes many of the skin and systemic immune markers to WT levels. This grouping of antibodies is consistent with basic epitope analysis, which directly modifies the 110 and 116 cluster and blocks the 77a, 111, and 16 cluster. This epitope analysis also revealed that groups 77a, 111, and 16 recognize epitopes that overlap with non-depleting NA1 / 34. This is important to note, since NA1 / 34 is known to cross-block anti-CD1a CR2113 binding. Antibodies 110 and 116 do not cross-block NA1 / 34 and thus have the potential to represent different epitope regions. The antibodies maintain their presence on LC in vivo, even in the skin and after migration to lymph nodes. This is an important enhancement since the clinical effect is more durable.

[0168] With such data, the inventors demonstrate the potential of this improved panel of improved anti-CD1a antibodies for use in the prevention and treatment of inflammatory skin and mucosal conditions, including but not limited to psoriasis, dermatitis, lupus, and in the treatment and / or prevention of one or more associated systemic diseases or disorders, or one or more systemically manifested inflammatory drug reactions. The effects on the broad linkage of inflammation involving LCs, T cells and neutrophils, particularly the effects of antibodies 110, 116 and 16, have broad-reaching effects on inflammatory skin and mucosal disorders, including psoriasis, dermatitis, lupus, and drug reactions manifesting as inflammatory skin or mucosal diseases or disorders, or CD1a-expressing malignancies.

[0169] In conclusion, the inventors demonstrate the improvement of anti-CD1a antibodies 16, 77a, 110, 111 and 116 as a method for preventing and treating inflammatory skin and mucosal diseases or disorders, or associated systemic diseases or disorders, or systemically manifested inflammatory drug reactions, or CD1a-expressing malignancies, by blocking CD1a and / or modifying the phenotype / function of CD1a+ cells.

[0170] References All references cited herein, including patents, patent applications, articles, texts, and the like, and the references cited therein, are hereby incorporated by reference in their entirety, to the extent not already incorporated.

[0171] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8] [Table 14-9] [Table 14-10]

Claims

1. a) a heavy chain variable region comprising a CDR3 of SEQ ID NO: 35 or a sequence having at least 80% identity thereto, and / or a light chain variable region comprising a CDR3 of SEQ ID NO: 38 or a sequence having at least 80% identity thereto, or b) a heavy chain variable region comprising a CDR3 of SEQ ID NO: 3 or a sequence having at least 80% identity thereto, and / or a light chain variable region comprising a CDR3 of SEQ ID NO: 6 or a sequence having at least 80% identity thereto, or c) a heavy chain variable region comprising a CDR3 of SEQ ID NO: 11 or a sequence having at least 80% identity thereto, and / or a light chain variable region comprising a CDR3 of SEQ ID NO: 14 or a sequence having at least 80% identity thereto, or d) a heavy chain variable region comprising a CDR3 of SEQ ID NO: 19 or a sequence having at least 80% identity thereto, and / or a light chain variable region comprising a CDR3 of SEQ ID NO: 22 or a sequence having at least 80% identity thereto, or e) a heavy chain variable region comprising a CDR3 of SEQ ID NO: 27 or a sequence having at least 80% identity thereto, and / or a light chain variable region comprising a CDR3 of SEQ ID NO: 30 or a sequence having at least 80% identity thereto An antibody or antigen-binding fragment thereof that binds to CD1a.

2. a) CDR1 of SEQ ID NO: 33, CDR2 of SEQ ID NO: 34, and CDR3 of SEQ ID NO: 35, or a sequence having at least 80% identity thereto comprising a heavy chain variable region, and / or CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38, or a sequence having at least 80% identity thereto comprising a light chain variable region, or b) CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3, or a sequence having at least 80% identity thereto comprising a heavy chain variable region, and / or CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6, or a sequence having at least 80% identity thereto comprising a light chain variable region, or c) CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 10, and CDR3 of SEQ ID NO: 11, or a sequence having at least 80% identity thereto A heavy chain variable region comprising, and / or CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO: 14, or a sequence having at least 80% identity thereto A light chain variable region comprising, or d) CDR1 of SEQ ID NO: 17, CDR2 of SEQ ID NO: 18, and CDR3 of SEQ ID NO: 19, or a sequence having at least 80% identity thereto A heavy chain variable region comprising, and / or CDR1 of SEQ ID NO: 20, CDR2 of SEQ ID NO: 21, and CDR3 of SEQ ID NO: 22, or a sequence having at least 80% identity thereto A light chain variable region comprising, or e) CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27, or a sequence having at least 80% identity thereto A heavy chain variable region comprising, and / or CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, or a sequence having at least 80% identity thereto A light chain variable region comprising An antibody or an antigen-binding fragment thereof that binds to CD1a, comprising. **Claim 3** a) A heavy chain variable region comprising or consisting of SEQ ID NO: 39, and / or a light chain variable region comprising or consisting of SEQ ID NO: 40 or a sequence having at least 80% identity thereto, or b) A heavy chain variable region comprising or consisting of SEQ ID NO: 7, and / or a light chain variable region comprising or consisting of SEQ ID NO: 8 or a sequence having at least 80% identity thereto, or c) A heavy chain variable region comprising or consisting of SEQ ID NO: 15, and / or a light chain variable region comprising or consisting of SEQ ID NO: 16 or a sequence having at least 80% identity thereto, or d) A heavy chain variable region comprising or consisting of SEQ ID NO: 23, and / or a light chain variable region comprising or consisting of SEQ ID NO: 24 or a sequence having at least 80% identity thereto, or e) A heavy chain variable region comprising or consisting of SEQ ID NO: 31, and / or a light chain variable region comprising or consisting of SEQ ID NO: 32 or a sequence having at least 80% identity thereto An antibody or an antigen-binding fragment thereof that binds to CD1a, comprising. **Claim 4** a) a heavy chain comprising or consisting of SEQ ID NO: 49 and / or a light chain comprising or consisting of SEQ ID NO: 50, or a sequence having at least 80% identity thereto, or b) a heavy chain comprising or consisting of SEQ ID NO: 41 and / or a light chain comprising or consisting of SEQ ID NO: 42, or a sequence having at least 80% identity thereto, or c) a heavy chain comprising or consisting of SEQ ID NO: 43 and / or a light chain comprising or consisting of SEQ ID NO: 44, or a sequence having at least 80% identity thereto, or d) a heavy chain comprising or consisting of SEQ ID NO: 45 and / or a light chain comprising or consisting of SEQ ID NO: 46, or a sequence having at least 80% identity thereto, or e) a heavy chain comprising or consisting of SEQ ID NO: 47 and / or a light chain comprising or consisting of SEQ ID NO: 48, or a sequence having at least 80% identity thereto An antibody or antigen-binding fragment thereof that binds to CD1a, comprising the same. **Claim 5** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a ScFv or other modified format. **Claim 6** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is modified to stabilize and / or extend the half-life, and the modification may be PEGylation. **Claim 7** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a chimeric antibody or humanized antibody or antigen-binding fragment thereof. **Claim 8** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a human IgG1 isotype or human IgG4 isotype or other natural or modified isotype. **Claim 9** The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a bispecific or multispecific antibody or antigen-binding fragment thereof. **Claim 10** A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. **Claim 11** A vector comprising the nucleic acid according to claim 10, which is, as appropriate, an expression vector, plasmid, or viral vector. **Claim 12** A host cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, a nucleic acid encoding the antibody or antigen-binding fragment thereof, and / or a vector comprising the nucleic acid, which is optionally a bacterial cell or a mammalian cell.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, a nucleic acid encoding the antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid, and / or a host cell comprising the antibody or antigen-binding fragment thereof, the nucleic acid or the vector.

14. Use in the treatment or prevention of one or more inflammatory skin or mucosal diseases or disorders, or one or more associated systemic diseases or disorders, or one or more systemically occurring inflammatory drug reactions, or CD1a-expressing malignancies, of the pharmaceutical composition according to claim 13.

15. The antibody or antigen-binding fragment thereof comprises or consists of two antibodies or antigen-binding fragments thereof, each of which 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 having at least 80% identity thereto and 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 having at least 80% identity thereto and a first antibody or antigen-binding fragment thereof having, and 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 having at least 80% identity thereto and 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 having at least 80% identity thereto and a second antibody or antigen-binding fragment thereof having, or b) a first antibody or antigen-binding fragment thereof having a heavy chain variable region comprising or consisting of SEQ ID NO: 39 and a light chain variable region comprising or consisting of SEQ ID NO: 40, or a sequence having at least 80% identity thereto, and A second antibody or an antigen-binding fragment thereof having a heavy chain variable region comprising or consisting of SEQ ID NO: 7 and a light chain variable region comprising or consisting of SEQ ID NO: 8, or a sequence having at least 80% identity thereto, or c) A first antibody or an antigen-binding fragment thereof having a heavy chain comprising or consisting of SEQ ID NO: 49 and a light chain comprising or consisting of SEQ ID NO: 50, or a sequence having at least 80% identity thereto, and A second antibody or an antigen-binding fragment thereof having a heavy chain comprising or consisting of SEQ ID NO: 41 and a light chain comprising or consisting of SEQ ID NO: 42, or a sequence having at least 80% identity thereto The pharmaceutical composition according to claim 14, comprising or consisting of.

16. a) One or more inflammatory skin or mucosal diseases or disorders are i) Neutrophil-predominant skin diseases including acne, generalized pustular psoriasis, psoriasis vulgaris, guttate psoriasis, palmoplantar pustulosis, SAPHO syndrome, acute febrile neutrophilic dermatosis (Sweet syndrome), histiocytoid neutrophilic dermatitis, dorsal hand neutrophilic dermatosis, pyoderma gangrenosum, neutrophilic eccrine hidradenitis, hidradenitis suppurativa, persistent nodular erythema, Behçet's disease, enterocutaneous arthritis syndrome, inflammation associated with other infections, neutrophilic urticarial-like dermatosis, palisaded neutrophilic granulomatous dermatitis, erythema gyratum repens, neutrophilic annular erythema, acute generalized exanthematous pustulosis (AGEP), or vasculitis; ii) Connective tissue diseases, lupus, dermatomyositis, scleroderma / systemic sclerosis, Churg-Strauss syndrome, panniculitis, vasculitis, autoimmune blistering conditions, bullous pemphigoid, pemphigus, linear IgA disease, herpes-like dermatitis, celiac disease, some autoinflammatory diseases, vitiligo, alopecia areata, alopecia universalis, alopecia totalis, panniculitis, lichen planus, erythema multiforme, lichen sclerosus, other lichenoid and erythema multiforme-like diseases, psoriatic arthritis, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, Guillain-Barré syndrome, transverse myelitis, thyroiditis, or autoimmune disorders including neurodegeneration; iii) Other allergies or atopies including Maculopapular Urticaria Syndrome, eosinophilia and systemic symptoms syndrome, urticaria, angioedema, keratoconjunctivitis, food allergy, atopic dermatitis, rhinitis, conjunctivitis, asthma, eosinophilic esophagitis and other eosinophilic mucosal diseases, or mast cell disorders and eosinophilic disorders including contact dermatitis iv) graft-versus-host disease; and v) other drug reactions that appear as inflammatory skin or mucosal diseases or disorders, including Stevens-Johnson syndrome, toxic epidermal necrolysis, eosinophilia and systemic symptoms syndrome due to drug reactions (DRESS) and acute generalized exanthematous pustulosis (AGEP), erythema multiforme, vesicular diseases, fixed drug eruptions, and other drug reactions that appear as inflammatory skin or mucosal diseases or disorders is one or more of, or b) one or more related systemic diseases or disorders, or one or more systemically appearing inflammatory drug reactions are inflammatory reactions to Aldara (imiquimod), or c) the CD1a-expressing malignant disease is one or more of Langerhans cell histiocytosis, T cell lymphoma or thymoma, The pharmaceutical composition according to claim 14.

17. The pharmaceutical composition according to claim 14, wherein one or more inflammatory skin or mucosal diseases or disorders are one or more of psoriasis, dermatitis, erythematosus, or drug reactions that appear as inflammatory skin or mucosal diseases or disorders.

18. The antibody or its antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to claim 14 is administered alone or in combination with one or more other therapeutic agents.

19. The pharmaceutical composition according to claim 18, wherein one or more other therapeutic agents are selected from the group comprising cytotoxic substances, anti-inflammatory substances, steroids, CAR-T cells, regulatory or cytolytic CAR-T cells, or other cells that express or present the antibody or antigen-binding fragment.

20. i. preparing a biological sample obtained from the subject; and ii. determining the binding level of the antibody or its antigen-binding fragment according to any one of claims 1 to 4 to CD1a-expressing cells in a sample obtained from the subject before treatment, or during the intervals between treatments, or during a period without treatment; and iii. after treatment or during the intervals between treatments or during a period without treatment, the tumor mass or the binding level of the antibody or its antigen-binding fragment to CD1a-expressing cells decreases, and if appropriate, when the decrease in the tumor mass or the binding level of the antibody or its antigen-binding fragment to CD1a-expressing cells is 25% or more, determining that the treatment is effective or that the condition has improved A method for monitoring the therapeutic efficacy or disease state in a subject diagnosed with a CD1a-expressing malignant disease, which comprises

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