Anti-CD123 chimeric antigen receptor T cell for treating autoimmune disease

Abstract

The present invention relates to an isolated chimeric antigen receptor (CAR) molecule comprising an antibody or an antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain and an intracellular signaling domain comprising at least a stimulating domain, and wherein the anti-CD123 binding domain comprises a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence SEQ ID NO: 1, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence SEQ ID NO: 2, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence SEQ ID NO: 3, and a light chain, the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity with the amino acid sequence SEQ ID NO: 4, a complementarity determining region 2 (CDR2) having at least 90% identity with the amino acid sequence serine-threonine-serine (STS), and a complementarity determining region 3 (CDR3) having at least 90% identity with the amino acid sequence SEQ ID NO: 5, the separated chimeric antigen receptor molecule is used for treating autoimmune diseases.

Description

[0001] The present invention relates to anti-CD123 CAR-T cells for use in the treatment of autoimmune diseases. More specifically, the present invention relates to said CAR-T cells in diseases mediated by plasmacytoid dendritic cells (pDC).

[0002] Autoimmune diseases are widespread among the global population. Basically, autoimmune diseases are known to occur in situations where the body's natural immune system fails to distinguish between healthy cells from the own body and unhealthy foreign pathogens and / or cells. As a result, the immune system erroneously attacks healthy body cells.

[0003] There are more than 80 types of autoimmune diseases. Some of them are known to be associated with plasmacytoid dendritic cells (pDC), which are cells from the innate immune system that are involved in the first line of defense against foreign pathogens and / or cells that enter the body.

[0004] Plasmacytoid dendritic cells (pDC) are known to specifically produce type I interferons (IFN-I) in many autoimmune and inflammatory diseases, such as lupus erythematosus, inflammatory myopathies (dermatomyositis, polymyositis), systemic sclerosis or psoriasis; see Ganguly, D. et al. Self-RNA- antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8. J. Exp. Med. (2009); Bell, E. Plasmacytoid dendritic cells in psoriasis, Nat. Rev. Immunol. (2007); and Albanesi, C., Scarponi, C, Bosisio, D., Sozzani, S. & Girolomoni, G. Immune functions and recruitment of plasmacytoid dendritic cells in psoriasis, Autoimmunity (2010).

[0005] More specifically, in diseases related to pDC, in recent studies it has been described that pDCs infiltrate strongly in affected skin lesions and organs and their secretion of IFN-I, see Huang X et al., Predominant role of plasmacytoid dendritic cells in stimulating systemic autoimmunity, Front. Immunol (2015); Tucci M et al, Glomerular accumulation of plasmacytoid dendritic cells in active lupus nephritis: role of interleukin- 18. Arthritis Rheum (2008); and Kafaja S et al., pDCs in lung and skin fibrosis in bleomycin-induced model and patients with systemic sclerosis, JCI Insight (2018).

[0006] It is now known that the CD123 molecule is strongly expressed in pDCs. Moreover, the CD123 molecule appears to be almost specific to plasmacytoid dendritic cells; see Oon, S. et al., A cytotoxic anti-IL-3Ra antibody targets key cells and cytokines implicated in systemic lupus erythematosus, JCI Insight, (2016); and - Richard, E. et al., CD28 / 4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm, Leukemia (2020). This makes CD123 an interesting biomarker against target.

[0007] However, targeting biomarkers such as CD123 is often difficult due to many technical obstacles such as specificity issues, transduction and proliferation efficiency and cytotoxicity.

[0008] Recently, in another technical field, namely cancer research, methods using specifically engineered T cells (or T lymphocytes) have been developed; Singh, A. K. & McGuirk, J. P. CAR T cells: continuation in a revolution of immunotherapy. Lancet Oncol, (2020). These T cells comprise so-called chimeric antigen receptors, which are commonly referred to as CARs (or CARs). CARs are able to fight cancer because they are directed against target antigens expressed by tumor cells. However, the field of cancer research and the field of autoimmune diseases are very different in nature and generally use radically different treatment methods.

[0009] The present invention improves this situation by using anti-CD123 CAR-T cells as described in EP3753954 from the applicant (application number EP19305816, also published as WO2020254682). The present inventors surprisingly found that CAR-T cells that have been specifically engineered to fight an aggressive cancer called blastic plasmacytoid dendritic cell neoplasm (BPDCN) are able to improve immunotherapy of autoimmune diseases.

[0010] Therefore, the present invention focuses on the use of specific CAR-T cells directed against CD123 in the treatment of autoimmune diseases.

[0011] For this purpose, it is an object of the present invention an isolated chimeric antigen receptor (CAR) molecule comprising an antibody or antibody fragment, the antibody or antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain and an intracellular signaling domain, the intracellular signaling domain comprising at least a stimulatory domain, and wherein the anti-CD123 binding domain comprises a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence SEQ ID NO: 1 (GYSITSDYT), a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence SEQ ID NO: 2 (ISFSGST) and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence SEQ ID NO: 3 (ARGLDY), and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence SEQ ID NO: 4 (SSISSSY), a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence Serine-Threonine-Serine (STS) and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence SEQ ID NO: 5 (HQLHRSPWT), for use in the treatment of an autoimmune disease.

[0012] According to an embodiment of the application, the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

[0013] According to another embodiment of the application, the anti-CD123 binding domain is selected from an antibody, an Fv, an scFv, a Fab or other antibody fragment, preferably an scFv.

[0014] According to another embodiment of the application, the intracellular signaling domain is CD3-zeta (CD3y), optionally comprising a costimulatory domain selected from CD28, 4.1BB, inducible T cell co-stimulator (ICOS), OX-40 or a combination thereof.

[0015] According to an embodiment, the isolated chimeric antigen receptor of the application has at least 90% identity, preferably 100% identity, with the nucleic acid sequence SEQ ID NO: 6.

[0016] According to another aspect, it is an object of the present application a expression vector comprising a nucleic acid molecule having SEQ ID NO: 6, wherein the vector is selected from a DNA, a RNA, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector, said expression vector for use in the treatment of an autoimmune disease. According to an embodiment, the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

[0017] According to another aspect, it is an object of the present application an engineered immune cell comprising a nucleic acid molecule having SEQ ID NO: 6 or the above-mentioned vector, said engineered immune cell for use in the treatment of an autoimmune disease. According to an embodiment, the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

[0018] According to another aspect, it is an object of the present application a pharmaceutical composition comprising the isolated chimeric antigen receptor (CAR) as described above, the expression vector as described above and / or the engineered immune cell as described above and a pharmaceutically acceptable excipient, said pharmaceutical composition for use in the treatment of an autoimmune disease. According to an embodiment, the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

[0019] Other characteristics and advantages of the present application will become apparent and / or will be clarified in the light of the following description and accompanying drawings, which contain specific embodiments given by way of illustration and not limitation, in which:

[0020] Figure 1The nucleotide and amino acid sequences of the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of the heavy chain and light chain of the antibodies of the present application are shown;

[0021] Figure 2 The nucleotide and amino acid sequences of the framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) of the heavy chain of the antibodies of the present application are shown;

[0022] Figure 3 The nucleotide and amino acid sequences of the framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) of the light chain of the antibodies of the present application are shown;

[0023] Figure 4 The consensus sequence of the light chain and the consensus sequence of the heavy chain of the antibodies of the present application are shown;

[0024] Figure 5 The nucleic acid sequence of the chimeric antigen receptor of the present application is shown; and

[0025] Figure 6 Results for the anti-CD123 CAR-T of the present application against different blood samples are shown.

[0026] The drawings and the specification herein contain, in general, illustrative elements of a nature that is determinative. Thus, the description and drawings are to be regarded as being illustrative in nature and, where appropriate, are helpful in defining the application.

[0027] In this specification, the term "and / or" is to be interpreted as including one or more of the items it connects. For example, the phrase "a protein or protein sequence can be prepared using standard recombinant and / or synthetic methods" means that a protein or protein sequence can be prepared using standard recombinant and synthetic methods, or that a protein or protein sequence can be prepared using standard recombinant methods, or that a protein or protein sequence can be prepared using synthetic methods. More generally, the term "and / or" as used in the phrases "A and / or B" is intended to encompass A and B, A or B, A, and B. Likewise, the term "and / or" as used in the phrase "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0028] The term "comprising" is to be interpreted as including all particular recited features and optional, additional, unrecited features. More generally, the terms "comprise", "comprises" and "comprising" are to be construed as specifying the presence of stated steps or elements or a group of steps or elements, but do not preclude the presence or addition of one or more other steps or elements or groups thereof. The term "consisting of should be interpreted as excluding features other than the recited features. Also, the indefinite articles "a" or "an" do not exclude a plurality.

[0029] When referring to "one embodiment", "an embodiment", "certain embodiments", "certain implementation", "further embodiments", "another embodiment" or "further embodiments" or "one aspect", "an aspect", "certain aspects", "certain implementation", "further aspects", "another aspect" or "further aspects" and combinations thereof throughout this specification, it is meant to refer to a particular described feature, structure, or characteristic associated with an embodiment. The articles "a", "an" and "the" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the articles.

[0030] The term "gene" refers to a DNA sequence (nucleic acid sequence or nucleotide sequence) that encodes a particular amino acid sequence. A gene comprises all or part of one or more proteins or enzymes and can include regulatory DNA sequences such as promoter sequences that at least partially determine the conditions for expression of the gene. Some genes that are not structural genes can be transcribed from DNA to RNA but are not translated into an amino acid sequence. Other genes can act as regulators of structural genes or as regulators of the transcription of DNA. In particular, the term gene can mean a genomic sequence that encodes a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

[0031] In the context of two or more nucleic acid sequences or amino acid sequences, the term "identical" or percent (%) "identity" means that two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured by using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain an alignment of amino acid or nucleotide sequences. One non-limiting example of such a sequence alignment algorithm is the algorithm set forth in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990, as modified in Karlin et al., Proc. Natl. Acad. Sci. 90:5873-5877, 1993, and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1991). Gapped BLAST can also be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453, 1970) (e.g., using the Blossum 62 matrix or PAM250 matrix, and a gap penalty of 16, 14, 12, 10, 8, 6, or 4, and a gap length penalty of 1, 2, 3, 4, 5). Alternatively, the percent identity between nucleotide or amino acid sequences can be determined using the algorithm of Myers and Miller (CABIOS, 4:11-17, 1989). For example, the percent identity between sequences can be determined using the ALIGN program (version 2.0) and using PAM120 and residue table, a gap length penalty of 12, and a gap penalty of 4. Those of skill in the art can determine appropriate parameters for maximum alignment using a particular alignment software. In general, the default parameters of the alignment software are used.

[0032] A sequence that is "at least 85% identical to" or "something that has at least 85% identity to" a reference sequence is a sequence that has 85% or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the full length of the reference sequence over its full length. In this context, "percent identity" in the present specification is calculated using pairwise alignment (i.e. comparing the full length of two sequences). Methods of comparing the identity of two or more sequences are well known in the art as described above. For example, pairwise sequence alignment of the present application can be generated using a bioinformatics tool or program such as EMBOSS Needle available on the website ebi.ac.uk. Typically, many bioinformatics tools and programs use the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimal alignment (including gaps) of two sequences when considering their full length. However, other algorithms can be implemented in various bioinformatics tools or programs.

[0033] More generally, for the purposes of the present application, "sequence identity" or "sequence homology" is calculated by comparing two aligned sequences in a comparison window. Sequence alignment enables the number of positions (nucleotides or amino acids) in the comparison window that are common to both sequences to be determined. The number of common positions is then divided by the total number of positions in the comparison window and multiplied by one hundred to obtain the percentage of homology. Sequence identity percentage can be determined manually or using well-known bioinformatics computer programs.

[0034] According to the present application, the percentage of identity between two polypeptides can be calculated by using the EMBOSS:needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5 and a Blosum62 matrix.

[0035] A protein having (or consisting of) an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to" a reference sequence or a portion thereof can comprise mutations, such as deletions, insertions and / or substitutions, compared to the reference sequence.

[0036] In the case of substitution, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can correspond to a homologous sequence (i.e.,“homology”) derived from another species than the reference sequence. The substitution of amino acids can be conservative or non-conservative. A conservative substitution is one in which an amino acid is replaced with another amino acid having similar side chain, i.e., similar structure and / or similar chemical properties. In this regard, conservative substitutions can occur between:

[0037] - amino acids with non-polar side chains: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), proline (Pro), phenylalanine (Phe), methionine (Met), tryptophan (Trp);

[0038] - amino acids with uncharged polar side chains: asparagine (Asn), glutamine (Gin), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys);

[0039] - amino acids with acidic side chains: aspartic acid (Asp), glutamic acid (Glu);

[0040] - amino acids with basic side chains: lysine (Lys), arginine (Arg), histidine (His);

[0041] - amino acids with beta-branched side chains: threonine (Thr), valine (Val), isoleucine (lie);

[0042] - amino acids with aromatic side chains: tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), histidine (His).

[0043] Non-conservative substitutions can occur randomly between the above. Depending on the position of the individual amino acid within the three-dimensional shape of the protein, other definitions of conservative or non-conservative substitutions can be given.

[0044] The techniques used herein are known to those skilled in the art of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, genetic and / or biotechnological cell engineering, immunology, and cell biology, unless otherwise stated. Such methods are described for illustrative purposes when needed. Most of these methods and other techniques are described in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley and Sons, 4 thEd. 1999; DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Techniques for the Analysis of Complex Genomes, Anand, Academic Press, 1992; Transcription and Translation, B. Names & S. Higgins, 1984; A Practical Guide to Molecular Cloning, Perbal, 1984; Antibodies, Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1998; Current Protocols in Immunology, Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, 1991; Annual Review of Immunology. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred embodiments of composition, methods and materials are described herein.

[0045] As understood by those skilled in the art and as described herein, an "antibody" (also referred to as an "immunoglobulin") comprises two heavy chains and two light chains. Each heavy chain is comprised of a variable region and a first, second, and third constant regions, while each light chain is comprised of a variable region and a constant region. The two heavy chains are connected to each other by disulfide bonds, with each heavy chain connected to a light chain by a disulfide bond.

[0046] In mammals, there are two types of light chains, lambda (l) and kappa (k). In addition, there are five major classes (or isotypes) of heavy chains, classified as a, d, e, y, and m, which determine the functional activity of the antibody molecule: IgA, IgD, IgE, IgG, and IgM.

[0047] Each chain comprises different sequence domains. The light chain comprises two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). More precisely, the intact antibody forms a “Y” shape. The base or stem of the Y is composed of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) that are held together by disulfide bonds, sometimes referred to as interchain disulfide bonds that form a hinge. The heavy chain y and d have constant regions composed of three tandem (in a line) Ig domains, and have a hinge region for increased flexibility; the heavy chain m and e have constant regions composed of four immunoglobulin domains. The second and third constant regions are referred to as the “CH2 domain” and the “CH3 domain,” respectively. Each arm or branch of the Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. More generally, the variable regions of both the light (VL) and heavy (VH) chains determine the binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain association, secretion, transplacental migration, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and is composed of the variable portions of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining site is composed of residues from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and, thus, the combining site. Complementarity determining regions or CDRs refer to the amino acid sequences within the naturally occurring Fv region of an antibody that contribute to the binding affinity and specificity of the antibody. The light and heavy chains of an immunoglobulin each have three CDRs, often referred to as CDR1, CDR2, and CDR3, or more precisely as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Other nomenclatures can be found in the literature. Thus, the CDRs located in the variable region of the heavy chain of an antibody can be referred to as CDRH1, CDRH2, and CDRH3, while the CDRs located in the variable region of the light chain of an antibody can be referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Thus, a conventional antibody antigen binding site includes six CDRs, including the CDR sets from each of the heavy and light V regions.

[0048] As mentioned above, the light and heavy chain variable regions contain so-called "framework" regions interrupted by hypervariable CDR regions. The CDRs can be defined or identified by methods, e.g. according to Kabat et al., Wu, TT and Kabat, E.A., J Exp Med, 132(2): 211-50, 1970; Borden, P. and Kabat E.A., PNAS, 84: 2440-2443, 1987; Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991 by sequence- or according to Chothia et al. - Choithia, C. and Lesk, A.M., JMol. Biol., 196(4): 901-917, 1987; Choithia, C. et al., Nature, 342: 877-883, 1989 by structure.

[0049] The sequences of the framework regions of different light or heavy chains are relatively conserved within a single species, e.g. humans. More generally, "framework regions" (FR) are relatively conserved among different immunoglobulins of a single species. The framework regions of an antibody, which are certain combinations of all framework regions constituting the light and heavy chains, serve to position and align the CDRs in three-dimensional space, i.e. when the antibody is folded and in the active state. The CDRs are primarily responsible for binding to the epitope of an antigen. As mentioned above, the CDRs of each chain are referred to as CDR1, CDR2, and CDR3. In fact, the CDRs are sequentially numbered starting from the N-terminus. The light and heavy chains of an immunoglobulin each have four FRs, referred to as FR1, FR2, FR3, and FR4, or more precisely for the light chain FR1-L, FR2-L, FR3-L, FR4-L, and for the heavy chain FR1-H, FR2-H, FR3-H, FR4-H, respectively. Thus, the light chain variable region can be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable region can be referred to as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Knowing the amino acid sequence of the CDRs, the skilled person can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L, and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0050] In the present specification, a "V HReferences to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein. References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein. L References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein. References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein.

[0051] As used herein, the term "antibody" or "immunoglobulin" primarily refers to conventional antibodies, in particular monoclonal antibodies and fragments thereof, and single domain antibodies and fragments thereof, in particular the variable heavy chain of a single domain antibody, and further to chimeric antibodies, humanized, bispecific or multispecific antibodies. The antibody is preferably a human, murine or humanized antibody.

[0052] A "monoclonal antibody" or "mAb" is an antibody produced by a single clone of B lymphocytes or cells transfected with the heavy and light chain genes of a single antibody. Monoclonal antibodies are produced by methods well known in the art, for example by preparing hybridoma cells from the fusion of myeloma cells with immunized spleen cells or by recombinant techniques, i.e. by protein engineering. Monoclonal antibodies include humanized monoclonal antibodies. More generally, a monoclonal antibody is a molecule of single amino acid composition directed against a specific antigen and should not be interpreted as requiring any particular method for producing the antibody other than those known in the art.

[0053] The term "chimeric antibody" refers in the broadest sense to an engineered antibody comprising one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, a chimeric antibody comprises a VH domain and a VL domain derived from an antibody of a non-human animal in association with a CH domain and a CL domain of another antibody, in particular a human antibody. As non-human animal, any animal can be used, for example a mouse, a rat, a hamster, a rabbit, etc. A chimeric antibody can also refer to a multispecific antibody having at least two different antigen specificities.

[0054] The term "antibody" or "immunoglobulin" also includes the meaning of "single domain antibody", which has been described and developed recently. A single domain antibody is an antibody whose complementarity determining regions (CDRs) are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally lacking a light chain, single domain antibodies derived from conventional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be derived from any species, in particular mouse, human or rabbit. Single domain antibodies can be naturally occurring single domain antibodies, known as heavy chain antibodies lacking a light chain. In particular, heavy chain antibodies lacking a light chain are produced by Camelidae species, such as camels, dromedaries, llamas, alpacas and guanacos.

[0055] The variable heavy chains of these single-domain antibodies, which lack light chains, are referred to in the art as "VHH" or "nanobody". Similar to a conventional VH domain, a VHH comprises four framework regions (FRs) and three complementarity determining regions (CDRs). One particular advantage of nanobodies over conventional antibodies is that they are about ten times smaller than IgG molecules. Thus, functional nanobodies that are correctly folded can be produced in high yield by in vitro expression. Furthermore, nanobodies are described in the art as being very stable and resistant to the action of proteases. The properties and production of nanobodies are reviewed, for example, in Harmsen and De Haard HJ, Appl. Microbiol. Biotechnol., 2007, Nov, 77(1): 13-22.

[0056] The term antibody also includes antigen-binding fragments thereof. Such "fragments" comprise a portion of an intact antibody, particularly the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab' fragments, F(ab)'2 fragments, Fv, single-chain Fv proteins ("scFv"), and portions of full-length antibodies that are responsible for antigen binding, bispecific antibodies, bispecific and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies can also be single domain antibodies, such as heavy chain antibodies or VHHs. The term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, in which about one-half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds in a fragment obtained by treating IgG with a protease, papain. The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, which is slightly larger than the Fab bound by disulfide bonds via the hinge region in a fragment obtained by treating IgG with a protease, pepsin. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and are in either order, i.e., VL-VH or VH-VL. Single chains can be cloned from the V region genes of a hybridoma specific for a desired target. Production of such hybridomas has become routine. Techniques useful for cloning variable region heavy (VH) and variable region light (VL) chains are described, for example, in Orlandi et al., PNAS, 1989, 86:3833-3837. Typically, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. More specifically, single-chain Fv ("scFv") polypeptides are covalently-linked VH::VL heterodimers that are typically expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. In some embodiments, human scFv fragments include CDRs, particularly maintained in the appropriate conformation by use of genetic recombination techniques. Bivalent and multivalent antibody fragments can form spontaneously by association of monovalent scFv, or can be generated by coupling monovalent scFv with a peptide linker, e.g., bivalent sc(Fv)2. "dsFv" is a VH::VL heterodimer stabilized by disulfide bonds. "(dsFv)2" denotes two dsFv coupled by a peptide linker. The term "bispecific antibody" or "BsAb" generally denotes an antibody that combines the antigen-binding sites of two antibodies within a single molecule. Thus, BsAbs are capable of simultaneously binding two different antigens. Genetic engineering has been used increasingly frequently to design, modify and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, e.g., as described in EP 2050764 A1. The term "multispecific antibody" denotes an antibody that combines the antigen-binding sites of two or more antibodies within a single molecule.The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0057] The present application provides immune effector cells genetically engineered with vectors designed to express chimeric antigen receptors (CARs) that redirect cytotoxicity to cells and / or molecules associated with autoimmune diseases. CARs are molecules that combine antibody-based specificity for a target antigen (e.g., an autoimmune disease-associated antigen) with intracellular domains of T cell receptor activation to create a chimeric protein that exhibits specific cellular immune activity. As used herein, the term "chimeric" describes something composed of parts from different proteins or DNA from different sources. The primary feature of CARs is their ability to redirect the specificity of an immune effector cell, triggering proliferation, cytokine production, phagocytosis, or the ability to mediate cell death of cells expressing the target antigen with molecules that can utilize monoclonal antibodies, soluble ligands, or cell-specific coreceptors in a major histocompatibility (MHC)-independent manner.

[0058] As used herein, the terms "binding domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and provide the ability of a CAR to specifically bind a target antigen of interest. A binding domain can comprise any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind a biomolecule, such as a cell surface receptor or an autoimmune disease-associated protein, lipid, polysaccharide, or other cell surface target molecule or component thereof. Binding domains include binding partners of any naturally occurring, synthetic, semi-synthetic, or recombinantly produced biological molecule. As used herein, the term "specific binding affinity" or "specifically binds" or "specifically bound" or "specifically binding" or "specifically targets" describes the binding affinity with which one molecule binds to another molecule above background. If a binding domain (or a CAR comprising a binding domain or a fusion protein containing a binding domain) binds to a target antigen with, for example, a binding affinity that is greater than or equal to about 10 5 M -1The affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction having a specific binding interaction of 1 / M unit) for a target molecule binds or associates with it, thus "specifically binding" the target molecule. The affinity of the binding domain peptides and CAR proteins according to this disclosure can be readily determined using conventional techniques such as competitive ELISA (enzyme-linked immunosorbent assay). Furthermore, the affinity-related binding mass, i.e., the so-called K, can be determined, quantified, and defined. D Value. K D Related to antibody affinity and sensitivity. K D It is the equilibrium dissociation constant between the antibody and its antigen, and is k. off / k on The ratio. K D It is inversely correlated with affinity. K D The value of K is related to the antibody concentration (the amount of antibody required for a specific experiment), therefore K D The lower the value (the lower the concentration), the higher the antibody affinity. K D It is the antibody dissociation rate (k) off ) and antibody association rate (k on The ratio of antibody to antigen is defined as follows: antibody dissociation rate is how quickly an antibody dissociates from its antigen, and antibody association rate is how quickly an antibody binds to its antigen. This is achieved by measuring the k-value of specific antibody / antigen interactions. on and k off Rate, and then calculate K using the ratio of these values. D The value is used to determine K in this specification. D More generally, antibody affinity is the strength of the binding between a single molecule and its ligand. It is typically expressed by the equilibrium dissociation constant (K0). D The equilibrium dissociation constant is used to assess and rank the strength of bimolecular interactions. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactants. At equilibrium, the rate of formation of the [antibody]|[antigen] complex is equal to the rate of dissociation into its components [antibody] + [antigen]. The measurement of the reaction rate constant can be used to define the equilibrium or affinity constant (1 / K). D In short, K D The smaller the value, the greater the affinity of the antibody for its target.

[0059] A CAR's binding domain is typically followed by one or more "hinge regions." The hinge region plays a role in positioning the antigen-binding domain away from the effector cell surface to achieve proper intercellular contact, antigen binding, and activation. A CAR may have one or more hinge regions between its binding domain and transmembrane domain. As described in the art, the hinge region can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0060] A "transmembrane domain" is a portion of a CAR that fuses the extracellular binding moiety and the intracellular signaling domain. The transmembrane domain anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane region can be derived from natural, synthetic, semi-synthetic, or recombinant sources, as described in the art.

[0061] In preferred embodiments, the CAR of the application comprises an intracellular signaling domain. An "intracellular signaling domain" refers to a portion of a CAR that is involved in the transduction of the information that the CAR has bound to the target antigen to the interior of the immune effector cell to elicit an effector cell function (e.g., activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to a target cell to which the CAR binds, or other cellular responses elicited by the binding of an antigen to the extracellular CAR domain). An "effector function" refers to a specialized function of a cell. The effector function of a T cell can be cytolytic activity or activity that includes cytokine secretion. Thus, in this specification, an "intracellular signaling domain" refers to a portion of a protein that transduces a signal for an "effector function" such that the cell is induced to perform a specialized function. As described in the art, either the entire intracellular signaling domain or a truncated portion of the intracellular signaling domain can be used. As will be appreciated by those in the art, a truncated portion must transduce the effector function signal in substantially the same way as the entire domain. Herein, the term "intracellular signaling domain" is meant to include any truncated portion of an intracellular signaling domain that is sufficient to transduce the effector function signal.

[0062] In preferred embodiments of the application, the CAR has a hinge region IgGl-CH2-CH3 (the first portion of IgGl is positioned in the membrane and the second portion of IgGl is positioned extracellularly) and CD28 as the transmembrane domain (the first portion of CD28 is positioned in the membrane and the second portion of CD28 is positioned in the cytoplasm intracellularly). CD8a can also be used as the transmembrane domain and hinge region. Sequences derived from IgG4 can also be used.

[0063] As is known in the art, a signal generated by the T cell receptor (TCR) alone is not sufficient to fully activate a T cell, and a secondary or costimulatory signal is also required. Thus, it can be said that T cell activation is mediated by two different classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation through the TCR (e.g., TCR / CD3 complex) and a costimulatory signaling domain that acts in an antigen-independent manner to provide a secondary or any costimulatory signal. In preferred embodiments, the CAR of the application comprises an intracellular signaling domain comprising one or more “costimulatory signaling domains.” Thus, in other preferred embodiments, the isolated nucleic acid molecule of the application encodes an intracellular signaling domain comprising at least one costimulatory domain, such that the resulting intracellular signaling domain therefore comprises at least one costimulatory domain.

[0064] As used herein, the term “costimulatory signaling domain” or “costimulatory domain” refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, when engaged by antigen, provide the second signal required for efficient T cell activation / function.

[0065] In this specification, the terms “polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are used interchangeably and have their conventional meaning, i.e., an amino acid sequence, unless otherwise indicated. Polypeptides are not limited to a particular length, and can comprise full-length protein sequences or fragments thereof. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. In addition, polypeptides can have post-translational modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a marker component, and, more generally, any naturally occurring and / or non-naturally occurring modification. In view of the fact that the polypeptides of the present application are based on antibodies, in some embodiments, the polypeptides can exist as single chains or associated chains. The polypeptides can be prepared using any technique known in the art, i.e., recombinant and / or synthetic techniques. More particularly, the polypeptides described herein include the CARs of the present disclosure, or sequences having a deletion, addition and / or substitution of one or more amino acids from the CARs disclosed herein.

[0066] As used herein, “isolated peptide” or “isolated polypeptide” and the like refer to a peptide or polypeptide molecule that is separated from the cellular environment and association with other cellular components in vitro. Similarly, “isolated cell” refers to a cell that has been obtained from a tissue or organ in vivo and is substantially free of extracellular matrix.

[0067] The term "vector" as used herein refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. More generally, a "vector" refers to a construct capable of delivering and expressing one or more genes or sequences in a host cell. The nucleic acid to be transported or transferred is linked to the vector nucleic acid molecule in any manner known in the art, for example by insertion. The vector can include sequences that direct autonomous replication in the cell, or can include sequences sufficient to permit integration into the host cell DNA. The present application also provides vectors comprising nucleic acid molecules encoding the CARs of the present application. The vector can be selected from a DNA, RNA, bacteriophage vector, plasmid, lentivirus vector, adenovirus vector, or retrovirus vector. The vector of the present application preferably comprises a promoter, for example an EF-1 alpha promoter. As used herein, the term "promoter" refers to the recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. As known by those skilled in the art, the RNA polymerase initiates transcription of the polynucleotide operably linked to the promoter. In a particular embodiment, it can be desirable to express the polynucleotide comprising the CAR from a promoter that provides stable and long-term CAR expression in T cells. CAR expression under the control of the promoter further ensures sufficient expression levels to direct the T cells against cells expressing the target antigen.

[0068] Retroviruses are a common tool for gene delivery. In some embodiments, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to a cell, preferably an immune cell. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus". The provirus serves as a template for RNA polymerase and directs the expression of RNA molecules encoding the structural proteins and enzymes required for production of new viral particles. Thus, T cells transduced with the vectors of the present application can produce a stable, long-term and persistent CAR-mediated T cell response. In particular embodiments, T cells are transduced with a lentiviral vector (i.e., a lentivirus) encoding a CAR according to the present application. The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, primarily derived from a lentivirus, including long terminal repeat sequences (LTRs). The term "lentivirus" refers to a group (or genus) of complex retroviruses. Well-known lentiviruses include human immunodeficiency virus (HIV, e.g., type 1 or type 2); visna-maedi virus (VMV); caprine arthritis- encephalitis virus (CAEV); equine infectious anaemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). The term "self-inactivating (SIN)" vector refers to a replication-defective vector, e.g., a retroviral or lentiviral vector, in which the 3' LTR enhancer-promoter region (termed the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.

[0069] The terms "purified" and "isolated" as used herein, when referring to a molecule (e.g., a polypeptide or antibody or nucleotide sequence of the application), means that the indicated molecule is present in the absence of other biological macromolecules of the same type. More specifically, as used herein, the term "purified" specifically means that at least 85%, 90%, 95%, or 98% by weight of the biological macromolecules of the same type are present. More specifically, an "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule can include some additional base components or moieties that do not materially affect the basic properties of the composition.

[0070] The terms "antigen," "target," or "target antigen" refer to a molecule or a portion of a molecule that is capable of being bound by an antibody or antibody-like binding protein. The term also refers to a molecule or a portion of a molecule that is capable of being used in an animal to produce an antibody that is capable of binding to an epitope of the antigen. A target antigen can have one or more epitopes. With respect to each target antigen recognized by an antibody or antibody-like binding protein, the antibody-like binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[0071] The terms "CD123," "IL-3Ra," or "IL-3R a," "interleukin-3 receptor alpha," or "interleukin-3 receptor a," are used interchangeably herein and refer to any native (human) IL-3Ra or CD123, unless otherwise indicated. The CD123 protein is the interleukin 3 specific subunit of the heterodimeric cytokine receptor, i.e., the IL-3 receptor or IL-3R. The IL-3R is composed of a ligand specific a subunit and a signal transducing common b subunit (also known as CD131), which is shared by the receptors for interleukin 3 (IL3), colony stimulating factor 2 (CSF2 / GM-CSF), and interleukin 5 (IL5). Binding of IL3 to CD123 / IL-3Ra is dependent on this b subunit. This b subunit is activated by ligand binding and is required for the biological activity of IL3. All of the aforementioned terms for CD123 can refer to the protein or nucleic acid sequence as indicated herein. The term "CD123 / IL-3Ra" includes "full-length," unprocessed CD123 / IL-3Ra as well as any form of CD123 / IL-3Ra that results from cellular processing. The term also includes naturally occurring variants of CD123 / IL-3Ra protein or nucleic acid, e.g., splice variants, allelic variants, and isoforms. The CD123 / IL-3Ra polypeptides and polynucleotides described herein can be isolated from a variety of sources, e.g., from a human tissue type or from another source, or prepared by recombinant or synthetic methods. Examples of CD123 / IL-3Ra sequences include, but are not limited to, NCBI Reference Nos. NP_002174 & NM_002183 (protein and nucleic acid sequences for human CD123 variant 1) and NP_001254642 & NM_001267713 (protein and nucleic acid sequences for human CD123 variant 2).

[0072] The terms "anti-CD 123 antibody," "anti-IL-3Ra antibody," or "anti-IL-3Ra antibody," "antibody that specifically binds CD 123," or "antibody that specifically binds IL-3Ra / IL-3Ra" refer to an antibody that is capable of binding CD 123 with sufficient affinity that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD 123. Unless otherwise indicated, the extent of the binding of an anti-CD 123 antibody to an unrelated, non-CD 123 protein is less than about 10% of the binding of the antibody to CD 123 as measured. More generally, the term "specifically binds" designates that an antibody binds to an epitope through its antigen binding domain, and that binding involves some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope with its antigen binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity of a certain antibody for a certain epitope. For example, antibody "A" can be said to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind an epitope "C" with a higher specificity than it does for a related epitope "D." Thus, an antibody or antigen binding fragment of the application specifically binds to a CD 123 antigen because it has a higher specificity for the CD 123 antigen (from any species) than for a non-CD 123 antigen. More specifically, an antibody or antigen binding fragment of the application specifically binds to a human CD 123 antigen because it has a higher specificity for the human CD 123 antigen than for a non-human CD 123 antigen (e.g., mouse or rat CD 123). Occasionally, the term "preferentially binds" designates that an antibody specifically binds an epitope more readily than it binds a related, similar, homologous, or analogous epitope. Thus, an antibody that preferentially binds a given epitope can be more likely to bind that given epitope than a related epitope, even though such an antibody can also cross-react with the related epitope. For example, an antibody or antigen binding fragment of the application can preferentially bind a human CD 123 antigen over a mouse CD 123.

[0073] From the above, it is readily deduced that the terms associated with an antibody, CAR, vector, or cell of the application. Indeed, the general context of the specification does not always require following the exact same phrasing as marked above. Some deviations can occur. For example, when referring to a CAR of a T cell, it is not always necessary to specify that the CAR comprises a moiety directed against a particular antigen, i.e., "anti-something." Thus, and more generally, a CAR of the application comprising a moiety that specifically binds CD 123 can be referred to not only as an anti-CD 123 CAR, but also as a CD 123 CAR. In the same way, a T cell transduced with a CAR that specifically binds CD 123 according to the application can be referred to as a CD 123 CAR T cell.

[0074] As used herein, "treatment" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and can include any reduction in one or more measurable markers of the disease or condition being treated, e.g., an autoimmune disease. Treatment can include a reduction in symptoms or any improvement in symptoms with respect to a disease or condition. Treatment can also delay progression of a disease or condition. Thus, the term "treatment" does not necessarily imply complete eradication or cure of the disease or condition, or associated symptoms thereof. With this in mind, the present disclosure provides for the treatment or prevention of BPDCN comprising administering to a subject in need thereof a therapeutically effective amount of the T cells of the present invention.

[0075] In this context, the T cells described herein can be administered alone or as a pharmaceutical composition (also known as a pharmaceutical preparation). The term "pharmaceutical composition" or "pharmaceutical preparation" refers to a preparation which is in a form which allows the biological activity of the active ingredients to be effective and which is free of additional components which would render the composition / preparation unacceptable for the subject to whom the composition / preparation is to be administered. Such preparation can be sterile. The pharmaceutical composition can comprise the T cells according to the application alone or in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates, such as glucose, mannose, sucrose or dextrans; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants and stabilizers. The use of the term "pharmaceutically acceptable" or "physiologically acceptable" in relation to a compound, material, composition or dosage form indicates that the reference material is compatible with the other materials that make up the composition or dosage form and that the reference material does not pose an unacceptable risk of toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The compositions of the application are preferably formulated for parenteral administration. "Parenteral administration" can refer to modes of administration other than enteral and topical administration. Generally, parenteral administration is by injection, for example, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinally and intrasternal injection and / or infusion. Preferably, the CAR modified T cells or compositions of the application are administered to a subject by direct injection into an inflammation site, lymph node, systemic circulation or site of infection. Generally, the application can be used to treat a subject diagnosed with a disease that overexpresses CD123, in particular a patient with BPDCN. The treatment comprises taking immune effector cells from a subject, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding a CAR as contemplated herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells are T cells. The amount and frequency of administration are determined by the physical condition of the patient (the patient's condition, the type and severity of the patient's disease). Appropriate dosages can also be determined with the aid of animal models followed by clinical trials. When referring to dosages, the term "effective amount" is an amount which is sufficient to achieve the particular stated purpose. The amount of active ingredient (e.g. genetically modified T cells) which constitutes a "therapeutically effective amount" can depend on such factors as the disease and its state, the age, sex and weight of the patient, and further on the ability of the cells to produce the desired response in the patient. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or the therapeutic cells are outweighed by the therapeutically beneficial effects. It can be stated in general that the pharmaceutical composition comprising the T cells described herein can be administered in a dose of 104 -10 9 cells / kg body weight, preferably 10 5 -10 6 cells / kg body weight, including all integer values within these ranges. More generally, the term "therapeutically effective amount" refers to the amount of an antibody, active ingredient, or other drug that is effective in treating a disease or condition in a subject.

[0076] In the case of autoimmune diseases, a therapeutically effective amount of a drug can reduce the number of cells associated with the pathophysiology of the disease and thus the number / concentration of inflammatory molecules; reduce the size of inflammation to suppress the disease (i.e., slow or stop the disease to some extent); alleviate to some extent one or more symptoms associated with the autoimmune disease and / or lead to an advantageous response, such as increasing progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), reduction in progressive disease (PD), reduction in time to progression (TTP), or any combination thereof. The term "prophylactically effective amount" refers to the amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, a prophylactically effective amount is less than a therapeutically effective amount since a prophylactic dose is used prior to or at an earlier stage of disease in a subject.

[0077] In particular embodiments, the immune effector cells described herein are manipulated or genetically modified in vitro prior to being obtained from a subject. In particular embodiments, the immune effector cells that express a CAR of the application on their membrane include T cells. T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any technique known to those of skill in the art, such as FICOLL TM separation). In another embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes (including T cells), monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be collected by washing to remove the plasma fraction and placing the cells in an appropriate buffer or medium for subsequent processing. In other embodiments, the red blood cells are lysed and the mononuclear cells are depleted (e.g., by PERCOLL TMT cells are isolated from peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque density gradient centrifugation. Specific subpopulations of T cells expressing one or several markers such as CD3, CD4 or CD8 can be further isolated by positive or negative selection techniques. For example, enrichment of the T cell population by negative selection can be achieved by a combination of antibodies against surface markers not expressed on the negatively selected cells.

[0078] In a more general context, the present specification highlights a cell therapy in which T cells are genetically engineered (or genetically modified) ex vivo to express a CAR, and the CAR T cells are infused to a patient, preferably a human, in need thereof.

[0079] The infused cells are able to kill cells in the patient by expressing a specific molecule, for example CD123. As mentioned above and unlike antibody therapy, T cells comprising a CAR are able to replicate in vivo, leading to long-term persistence, which can result in sustained control of the autoimmune disease. Moreover, CARs allow the redirection and activation of effector T cells to any cell surface molecule upon antibody-derivative receptor binding and are independent of MHC restriction (MHC restricted antigen recognition; MHC stands for Major Histocompatibility Complex). As mentioned above, the genetically engineered T cells of the present invention are constructed starting from T cells collected from the patient himself (autologous), but they can also be derived from other allogeneic donors to provide allogeneic genetically engineered T cells in the context of bone marrow or peripheral hematopoietic stem cell allogeneic transplantation (donor lymphocyte infusion). These T cells expressing a CAR molecule according to the present invention can be used to treat an autoimmune disease in a mammal, preferably a human.

[0080] The present invention is further described in detail by reference to experimental data and specific examples. The examples are provided for illustration purposes only, and are not intended to limit the application unless otherwise stated. While the following detailed description focuses on

[0081] The present invention can generally provide an improvement and / or a solution to autoimmune diseases in which CD123 overexpression occurs.

[0082] Thus, the main focus of the present invention is to provide a method of treatment of autoimmune diseases, in particular cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

[0083] However, the development of a treatment or a drug is accompanied by obstacles for the person skilled in the art. For example, a drug can be too toxic in result for the treatment of a non-fatal disease. Moreover, the development is often extremely expensive, which often drives the person skilled in the art away from diseases with only a small market.

[0084] Furthermore, treatments such as those involving CARs are difficult to develop. For example, constructing a CAR is an extremely difficult task. This is often due to the fact that CARs are constructed from a patient's cells, or at least by using specific cells (e.g. T lymphocytes) extracted from a patient. However, those cells are often abnormal in patients suffering from autoimmune diseases. This is a particular case of T lymphocytes in autoimmune diseases, as the cells themselves are auto-immune.

[0085] To consider CAR treatments, many other obstacles also need to be overcome. Another example is that, in some diseases, CARs need to diffuse into the skin, which can bring some additional obstacles.

[0086] According to embodiments of the application, CARs have been genetically engineered to be made of a single chain variable fragment (ScFv) of the heavy and light chains of a monoclonal antibody (MAb) linked to the intracellular signaling chain of a T cell receptor (TCR). Preferentially, the intracellular part of the CAR comprises CD3 zeta and co-stimulatory domains (e.g. CD28 and 4-1BB) which enable better activation and cell signaling. This CAR can be called a third generation CAR. For more details on CARs: see the review by Andrew D. Fesnak, Carl H. June and Bruce L. Levine, Engineered T cells: the promise and challenges of cancer immunotherapy, Nature, vol. 16, sept. 2016. ScFV allows the recognition of antigens without dependence on major histocompatibility complex (MHC) presentation.

[0087] An increasing number of clinical trials show that patients receiving CAR treatments develop toxicities. This is a major problem in the development of efficient CARs. The Applicant has developed a specific CAR which overcomes the problems of the prior art (see EP3753954). More specifically, the Applicant has engineered both third generation retroviral and lentiviral CARs. The CARs of the present application overcome the problems of the prior art and, in particular, those related to cytotoxicity. The CARs of the present application have improved functional activity on BPDCN cells (BPDCN cell lines, PDX cells and primary BPDCN cells) in several mouse models of BPDCN. The assessment of their potential cytotoxicity on CD123 low positive cells is surprisingly promising.

[0088] According to the present application, monoclonal antibodies (Mab) against human CD123 have been generated. The monoclonal antibodies used with the present application can be used in both allogeneic and autologous settings.

[0089] The method for producing the anti-CD123 monoclonal antibody involves immunizing mice with recombinant CD123 protein. More specifically, five mice are immunized with recombinant CD123, available from R&D Systems, Minnesota, USA, according to reference number #301-R3-025. Immunization is performed via footpad and / or intraperitoneal administration. Lymph node and / or spleen B cells are used to generate mab-secreting hybridomas. CD123 is then used. + and CD123 - Cell lines were selected for hybridomas based on the affinity and specificity of MAb.

[0090] For this invention, a specific antibody was selected. In this document, this antibody is named 18B4D5 (also referred to herein as AB1, or sometimes abbreviated as B4D5). Molecular characterization and DNA sequencing were performed according to the SANGER method.

[0091] The VDJ and VJ gene rearrangements were sequenced, and the shared nucleotide sequences were compared with those obtained using the VQUEST online tool. Identification was performed after database comparison; see Brochet X, Lefranc MP, Giudicelli V. IMGT / V-QUEST: the highly customized and integrated system for IG and TR-standardized VJ and VDJ sequence analysis, Nucleic acids research, 2008; 36 (Web Server issue): W503-8. The sequence (nucleotide and amino acid sequences) of antibody 18B4D5 is listed in... Figures 1 to 5 middle.

[0092] Figure 1 The nucleotide and amino acid sequences of complementarity-determining regions 1 (CDR1), 2 (CDR2), and 3 (CDR3) of both the heavy and light chains of antibody 18B4D5 used in this invention are shown.

[0093] Figure 2 The nucleotide and amino acid sequences of frame regions 1 (FR1), 2 (FR2), 3 (FR3), and 4 (FR4) of the heavy chain of antibody 18B4D5 used in this invention are shown.

[0094] Figure 3 The nucleotide and amino acid sequences of frame regions 1 (FR1), 2 (FR2), 3 (FR3), and 4 (FR4) of the light chain of antibody 18B4D5 used in this invention are shown.

[0095] Figure 4 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the antibody AB1 used with the present application designated 18B4D5 or B4D5 are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted in reading order from left to right, respectively.

[0096] The sequence homology of the heavy chain VH and JH and the light chain VK and JK of the antibodies used in the present application is shown in Table 1 using the VQUEST online tool as described above.

[0097] Table 1 : Sequence homology of the variable chains.

[0098]

[0099] By selecting this particular antibody, the Applicant designed a specific Chimeric Antigen Receptor T cell (CAR-T) that is very effective in the treatment of autoimmune diseases.

[0100] To do this, the present inventors initially based their research on the knowledge about CAR-T therapy. CAR-T therapy is one of the most promising cell-based therapies, with over 800 clinical studies being conducted worldwide to evaluate many targets and indications, see Singh, A.K. & McGuirk, J.P. CAR T cells: continuation in a revolution of immunotherapy - Lancet Oncol. 21, e168-e178 (2020). It was mainly developed in the field of oncology and in particular in the field of hematology. It provides promising results, enabling patients who relapse or are resistant to other treatments to have a high remission rate, see Maude S.L. et al., Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia - N. Engl. J. Med. 378, 439-448 (2018).

[0101] More recently, the use of CAR-T therapies has been extended to other fields, such as infectious diseases, cardiac fibrosis or autoimmune diseases (AID), see Aghajanian, H., Rurik, J. G. & Epstein, J. A. CAR-based therapies: opportunities for immuno-medicine beyond cancer - Nat. Metab. 4, 163-169 (2022). Moreover, a rapid and durable remission of a patient with severe lupus treated with CD19-targeted CAR-T has recently been reported, see Mougiakakos, D. et al., CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus - N. Engl. J. Med. 385, 567-569 (2021).

[0102] Therefore, the Applicant focused on the use of specific autologous CAR-T in autoimmune diseases. However, the main difficulty is related to the identification of an effective CAR-T which also shows cytotoxicity compatible with the treatment of the patient. Since there are many CAR-Ts available in the state of the art, this identification is particularly challenging among other difficulties.

[0103] Another study showed that other patients benefited from anti-CD19 CAR-T against their refractory lupus and obtained satisfactory clinical results, see Mackensen, A. et al., Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus - Nat. Med. 28, 2124-2132 (2022). Injected CAR-Ts seemed to have no major adverse effects on patients. Various preclinical studies are currently being conducted in the field of anti-CD19 CAR-Ts. Some CAR-Ts show preclinical efficacy in autoimmune diseases and are now in clinical trials: anti-CD19 (NCT03030976 in lupus), anti-CD19 / BCMA (NCT05030779 in lupus), anti-BCMA (NCT0414051 in general myasthenia gravis) and anti-DSG34.6 CAAR-T (NCT04422912 in pemphigus) Ellebrecht, C. T. et al., Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease - Science 353, 179-184 (2016).

[0104] The Applicant diverted its research towards CAR-Ts other than the above in order to identify a new generation of CAR-Ts actively against autoimmune diseases. By doing so, the Applicant managed to identify the general feasibility of CAR-T transfer and further identified a CAR-T not only tolerable to patients but also highly effective in systemic lupus erythematosus.

[0105] However, the development of CAR-T therapies is highly dependent on the selection of their antigen target. Therefore, it is challenging that the target must be as specific as possible against the cells to be eliminated in order to reduce the so-called “off-target on-tumor” adverse reactions.

[0106] The Applicant surprisingly found that an anti-CD123 CAR-T meets the above challenges and requirements.

[0107] The CD123 marker is the alpha subunit of the IL-3 heterodimeric receptor. It is strongly expressed in pDC and basophilic cells, while it is weakly expressed in other cell types such as monocytes, myeloid progenitor cells and endothelial cells. IL-3 signaling is essential for the survival of pDC, see -Richard, E. et al., CD28 / 4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm - Leukemia 34, 3228-3241 (2020) and Oon, S. et al., A cytotoxic anti-IL-3Ra antibody targets key cells and cytokines implicated in systemic lupus erythematosus - JCI Insight 1, (2016).

[0108] Applicants believe that pDCs represent 0.1-0.5% of circulating monocytes and further believe that pDCs are part of the innate immune system, see Bode, C. et al., Human plasmacytoid dentritic cells elicit a Type I Interferon response by sensing DNA via the cGAS-STING signaling pathway - Eur. J. Immunol. 46, 1615-1621 (2016). Furthermore, applicants believe that pDCs have a rather high capacity to secrete type I interferons (IFN-I). IFN-I is a major cytokine of the innate immune system, which is produced by stimulation of TLR7 and TLR9. TLR7 and TLR9 are cytosolic receptors of pDCs, which are mainly activated by RNA and DNA fragments, see Ah Kioon, M.D. et al., Plasmacytoid dendritic cells promote systemic sclerosis with a key role for TLR8 - Sci. Transl. Med. 10, eaam8458 (2018); Kafaja, S. et al., pDCs in lung and skin fibrosis in a bleomycin-induced model and patients with systemic sclerosis - JCI Insight 3, 98380 (2018); and Ganguly, D. et al., Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8. J. Exp. Med. 206, 1983-1994 (2009).

[0109] Some studies suggest that pDCs can be involved in the development of autoimmune and / or autoinflammatory diseases associated with an overproduction of IFN-I, see Li, S., Wu, J., Zhu, S., Liu, Y.-J. & Chen, J. Disease-Associated Plasmacytoid Dendritic Cells. Front. Immunol. 8, 1268 (2017).

[0110] Applicants also believe that pDCs have antigen-presenting functions and the function of secreting proinflammatory cytokines (IFN-I, IL-6, IL-12, CXCL8, CXCL10, CCl3 and CCL4). In addition, the research direction of the applicants is data showing that patients with specific autoimmune diseases have a lower rate of circulating pDCs than healthy people. However, in these patients, pDCs infiltrate damaged and inflamed tissues. Thus, pDCs appear to have a strong tissue infiltrating capacity, especially in the skin in systemic sclerosis (SSc) or in the kidney in lupus erythematosus, see Kokaji, A.I., Holland, S., Fairhurst, M.A., Thomas, T.E. & Guilbault, B.G. A simple one-step method for isolating highly purified plasmacytoid dendritic cells from human peripheral blood (78.33)- J. Immunol. 182, 78.33-78.33 (2009); Vermi, W. et al., Cutaneous distribution of plasmacytoid dendritic cells in lupus erythematosus. Selective tropism at the site of epithelial apoptotic damage - Immunobiology 214, 877-886 (2009); Albanesi, C., Scarponi, C., Bosisio, D., Sozzani, S. & Girolomoni, G. Immune functions and recruitment of plasmacytoid dendritic cells in psoriasis. Autoimmunity 43, 215-219 (2010); Nestle, F.O. et al., Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. J. Exp. Med. 202, 135-143 (2005); and Bell, E. Plasmacytoid dendritic cells in psoriasis. Nat. Rev. Immunol. 7, 839-839 (2007).

[0111] Other autoimmune diseases seem to be associated with pDC activity producing IFN-I, such as psoriasis and dermatomyositis, see Albanesi, C. et al., Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment - J. Exp. Med. 206, 249-258 (2009); and Tezak, Z. et al., Gene expression profiling in DQA1*0501+ children with untreated dermatomyositis: A novel model of pathogenesis - J. Immunol. 168, 4154-4163 (2002).

[0112] In SSc, pDCs infiltrate the lung, with secretion of IFN-a and CXCL4, which is considered a poor prognosis in humans and mice, associated with skin and lung fibrosis and more severe clinical features, see van Bon, L. et al., Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis - N. Engl. J. Med. 370, 433-443 (2014). In other words, high levels of cytokines / chimiokines (IFN-a and CXCL4) identified in patients are associated with skin and lung fibrosis and more severe clinical features. This suggests that pDCs secreting these cytokines play a detrimental role in this pathology - this is also observed in mouse models. Thus, pDC infiltration is a poor prognostic factor.

[0113] Figure 5 The nucleic acid sequence of the CAR according to the application is shown.

[0114] The CAR was constructed with the antibody AB1. The nucleic acids corresponding to the peptide signal, the HA tag, the heavy chain, the hinge 1, the light chain, the hinge 2, CD28, 4.1 BB and CD3z are highlighted with different shades in reading order from left to right, respectively. Further details on the construction of the CAR 123 of the application are disclosed in EP3753954 to which the reader is referred.

[0115] Therefore, one object of the present application is an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the nucleic acid molecule consists of a sequence having at least 85%, preferably 90%, more preferably 95% identity with SEQ ID NO: 6, for use in the treatment of autoimmune diseases.

[0116] Another object of the present application is an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the nucleic acid consists of SEQ ID NO: 6, for use in the treatment of autoimmune diseases.

[0117] Antibodies were tested for their affinity to CD123 on a streptavidin biosensor. In this context, seven different concentrations of CD123 were used on immobilized antibodies of the present application in order to determine the K D values. The results of the Octet system analysis available from FortéBio were analyzed.

[0118] The results of the antibody affinity are shown in Table 2.

[0119] Table 2: Antibody affinity.

[0120]

[0121] Applicants isolated mononuclear cells by Ficoll. T lymphocytes were isolated from mononuclear cells and then activated by magnetic sorting (anti-CD3 / CD28 magnetic beads, Gibco TM ). Activated T lymphocytes were cultured in RPMI 10% (supplemented with human serum) and IL-2 medium. pDC were isolated by negative sorting (EasySep TM Human pDCIsolation kit, StemCell) and phenotypic analysis was performed before and after sorting to determine the sorting purity before freezing at -80°C.

[0122] Two days after T lymphocyte activation, they were transduced with lentiviral vectors. Transduction efficiency (CD3+ / CD19+) was evaluated seven days after transduction. Non-transduced T lymphocytes (C0) were used as controls for all experiments.

[0123] CAR123 function with target cells (sorted pDC or CAL-1) after 6 hours of co-culture with T lymphocytes (C0 or CAR123) was investigated. The percentage of cytotoxicity was then evaluated by flow cytometry.

[0124] Figure 6Results of the analysis discussed above with respect to the CAR 123 of the application using different blood samples: healthy donors (HD), patients with systemic lupus (SLE) or cutaneous arthritic lupus (CLE), patients with dermatomyositis (DM), patients with psoriasis (PSO), and patients with systemic sclerosis (SSc) are shown.

[0125] Figure 6 A shows the T cell transduction efficiency, determined by CD3+ / CD19+ expression seven days after transduction. It corresponds to the percentage of T cells that have been transduced, i.e. that express the CAR of the application on their surface. No significant difference was observed between HD and patients.

[0126] Figure 6 B shows the T cell fold expansion determined after 9 days of culture in RPMI medium supplemented with human serum and IL-2. The results show that T cells from patients are able to efficiently activate and proliferate.

[0127] Figure 6 C shows that the percentage of pDCs determined by flow cytometry is the same when comparing HDs to patients. pDCs express CD123 and BDCA2.

[0128] Figure 6 D shows the ability to sort pDCs using EasyStem TM Human pDC Isolation Kit (StemCell).

[0129] Figure 6 E shows the percentage of frequency of killed target cells. The percentage of cytotoxicity was evaluated by the percentage of lysed target cells (pDCs or CAL-1) after 6 hours of co-culture with effectors (C0 or CAR-T) at an effector / target ratio of 5 / 1. CAL-1 was used as a positive control (CD123+ BPDCN cell line). We obtained strong cytotoxicity of CAR-T from patients against CAL-1 or pDCs, without difference with CAR-T from HD: these results show that we are able to obtain functional CAR-T from patients as well as from T lymphocytes from healthy patients.

[0130] Statistical tests: non-parametric t-test (Wilcoxon) was used for statistical analysis. Data are represented by mean and standard deviation. p-value < 0.05 was considered statistically significant, *** p < 0.001.

[0131] The results show that the autologous CAR123 of the application has a strong ability to eliminate circulating autologous pDC in vitro, which is prepared from a sample of a patient suffering from a pDC-mediated autoimmune disease. As known to the person skilled in the art, these results also apply to in vivo models.

[0132] Other cells express the CD123 molecule on their surface. This is the case, for example, for basophilic polymorphonuclear cells (high expression) and monocytes (low expression). These cells are therefore also targets of the CAR123 of the application. The Applicant has therefore investigated the CAR123 of the application on said autoimmune diseases.

[0133] More specifically, in lupus, the Applicant believes that monocytes and macrophages are an important infiltration in the inflammatory lesions of the kidney in patients with lupus nephritis. Moreover, the presence of monocytes (CD16+ subset) in the lesions increases the activation of autoreactive T and B lymphocytes, increases antigen presentation and participates in the poor clearance of apoptotic bodies and immune complexes, on the one hand. Moreover, they massively secrete proinflammatory cytokines; see Katsiari, C.G. et al., Aberrant expression of the costimulatory molecule CD40 ligand on monocytes from patients with systemic lupus erythematosus, Clin. Immunol. Orlando Fla 103, 54-62 (2002); Kwant, L.E. et al., Macrophages in Lupus Nephritis: Exploring a potential new therapeutic avenue, Autoimmun. Rev. 21, 103211 (2022); Miyagawa, F., Tagaya, Y., Ozato, K., Horie, K. & Asada, H. Inflammatory monocyte-derived dendritic cells mediate autoimmunity in murine model of systemic lupus erythematosus, J. Transl. Autoimmun. 3, 100060 (2020).

[0134] With respect to SSc, the Applicant believes that the involvement of monocytes / macrophages is of importance. Indeed, in SSc, the percentage of circulating monocytes is significantly higher compared to healthy donors and the subset of monocytes expressing CD16 is higher in diffuse SSc compared to limited SSc. This subset is also associated with greater severity of cutaneous fibrosis, lung fibrosis and lung dysfunction, suggesting a link between these monocytes and the pathogenesis of fibrosis in SSc; see Lescoat, A. et al., CD16-positive circulating monocytes and fibrotic manifestations of systemic sclerosis, Clin. Rheumatol. 36, 1649-1654 (2017); Laurent, P. et al., Innate Immunity in Systemic Sclerosis Fibrosis: Recent Advances, Front. Immunol. 9, (2018). Moreover, the Applicant believes that pro-fibrotic cells derived from circulating CD14+ monocytes in SSc are associated with interstitial lung disease and involved in the pathogenesis of SSc and the further fact that the presence of monocytes is associated with fibrotic manifestations accompanied by infiltration of CD14+ monocytes / macrophages in lung tissue; see Higashi-Kuwata, N. et al., Characterization of monocyte / macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis. Arthritis Res. Ther. 12, R128 (2010).

[0135] In lupus, the deleterious role of basophilic polymorphonuclear cells has been demonstrated. The presence of autoreactive IgE has been found in the serum of lupus patients and this presence is associated with a higher rate of clinical severity score in lupus nephritis patients. Basophilic polymorphonuclear cells have been shown to contribute to the maintenance of the pathogenic Th2 environment in lupus, leading to the maturation of autoreactive B lymphocytes and their production of autoreactive IgE. Another study suggests that basophilic polymorphonuclear cells and the Th2 environment are involved in the development of lupus nephritis; see Charles, N., Hardwick, D., Daugas, E., Illei, G. G. & Rivera, J., Basophils and the T helper 2 environment can promote the development of lupus nephritis, Nat. Med. 16, 701-707 (2010); Pellefigues, C. & Charles, N., The deleterious role of basophils in systemic lupus erythematosus, Curr. Opin. Immunol. 25, 10.1016 / j.coi.2013.10.003 (2013). In SSc, peripheral basophilic polymorphonuclear cells are able to stimulate LB and fibroblasts, participating in fibrosis, which suggests that basophilic polymorphonuclear cells play an important role in the pathophysiology of SSc; see Basophils Are Activated and Stimulate Both B Cells and Fibroblasts in Systemic Sclerosis. ACR Meeting Abstracts https: / / acrabstracts.org / abstract / basophils-are-activated-and-stimulate-both-b-cells-and-fibroblasts-in- systemic-sclerosis / .

[0136] The CAR 123 of the present application has an impact on different immune cells, which is an advantage in each of the diseases described above that involve different immune players in their pathogenesis.

Claims

1. An isolated chimeric antigen receptor (CAR) molecule comprising an antibody or antibody fragment, said antibody or antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signal transduction domain including at least a stimulatory domain, wherein said anti-CD123 binding domain comprises a heavy chain and a light chain, said heavy chain comprising a complementarity-determining region 1 (CDR1) having at least 90% identity with the amino acid sequence SEQ ID NO:1, a complementarity-determining region 2 (CDR2) having at least 90% identity with the amino acid sequence SEQ ID NO:2, and a complementarity-determining region 3 (CDR3) having at least 90% identity with the amino acid sequence SEQ ID NO:3, said light chain comprising a complementarity-determining region 1 (CDR1) having at least 90% identity with the amino acid sequence SEQ ID NO:4, a complementarity-determining region 2 (CDR2) having at least 90% identity with the amino acid sequence serine-threonine-serine (STS), and a complementarity-determining region 3 (CDR3) having at least 90% identity with the amino acid sequence SEQ ID NO:4, and a complementarity-determining region 4 (CDR3) having at least 90% identity with the amino acid sequence SEQ ID NO:4, ... NO:5 has a complementarity-determining region 3 (CDR3) with at least 90% identity, and the isolated chimeric antigen receptor molecule is used to treat autoimmune diseases.

2. The isolated chimeric antigen receptor according to claim 1, wherein the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

3. The isolated chimeric antigen receptor according to any one of the preceding claims, wherein the anti-CD123 binding domain is selected from antibodies, Fv, scFv, Fab or other antibody fragments, preferably scFv.

4. The isolated chimeric antigen receptor according to any one of the preceding claims, wherein the intracellular signal transduction domain is CD3-zeta (CD3ζ), optionally comprising a costimulatory domain selected from CD28, 4.1BB, inducible T cell costimulator (ICOS), OX-40, or combinations thereof.

5. The isolated chimeric antigen receptor of claim 1, which has at least 90% identity with the nucleic acid sequence SEQ ID NO:6, preferably 100% identity.

6. An expression vector comprising a nucleic acid molecule as defined in claim 5, wherein the vector is selected from DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector, the expression vector being used to treat an autoimmune disease.

7. The expression vector of claim 6, wherein the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis and systemic sclerosis.

8. An engineered immune cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6, said engineered immune cell being used to treat autoimmune diseases.

9. The engineered immune cells of claim 8, wherein the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis, and systemic sclerosis.

10. A pharmaceutical composition comprising an isolated chimeric antigen receptor (CAR) according to claims 1 to 5, an expression vector according to claims 6 and 7, and / or engineered immune cells according to claims 8 and 9, and a pharmaceutically acceptable excipient, said pharmaceutical composition for treating an autoimmune disease.

11. The pharmaceutical composition of claim 10, wherein the autoimmune disease is selected from cutaneous lupus erythematosus, dermatomyositis, psoriasis, and systemic sclerosis.

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