Anti-CD123 chimeric antigen receptor T cells for use in the treatment of autoimmune diseases

Abstract

The present invention relates to an isolated chimeric antigen receptor (CAR) molecule for use in treating an autoimmune disease, comprising an antibody or antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, 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 comprising a complementarity-determining region 1 (CDR1) having at least 90% identity to the amino acid sequence SEQ ID NO:4, 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.

Description

[Technical Field]

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

[0002] Autoimmune diseases are common in people all over the world. It is generally known that autoimmune diseases occur when the body's innate immune system is unable to distinguish between healthy body cells and unhealthy foreign bacteria and / or cells. As a result, the immune system mistakenly attacks healthy body cells.

[0003] There are over 80 different autoimmune diseases, some of which are known to be associated with plasmacytoid dendritic cells (pDCs), cells of the innate immune system that are involved in the first line of defense against foreign bacteria and / or cells that invade the body.

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

[0005] More specifically, for pDC-associated diseases, recent studies have demonstrated strong pDC infiltration and their IFN-I secretion in affected skin lesions and organs. See X Huang et al., "Predominant role of plasmacytoid dendritic cells in stimulating systemic autoimmunity" (Front.Immunol, 2015), M Tucci et al., "Glomerular accumulation of plasmacytoid dendritic cells in activating lupus nephritis: role of interleukin-18" (Arthritis Rheum, 2008), and S Kafaja et al., "pDCs in lung and skin fibrosis in a bleomycin-indicated model and patients with systemic sclerosis" (JCI Insight, 2018).

[0006] It is now known that CD123 is strongly expressed on pDCs. Furthermore, CD123 appears to be largely specific to plasmacytoid dendritic cells (see S. Oon et al., "A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus" (JCI Insight, 2016) and E. Bole-Richard et al., "CD28 / 4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasms" (Leukemia, 2020). Therefore, CD123 is an interesting biomarker to target. Summary of the Invention [Problem to be solved by the invention]

[0007] However, targeting biomarkers such as CD123 is generally difficult due to a number of technical obstacles, including specificity issues, transduction and expansion efficiency, and cytotoxicity.

[0008] Recently, in another technical field (i.e., cancer research), methods using specifically engineered T cells (or T lymphocytes) have been developed. See "CAR T cells: continuation in a revolution of immunotherapy" by A.K. Singh and J.P. McGuirk (Lancet Oncol, 2020). These T cells contain so-called chimeric antigen receptors, commonly referred to as CARs. CARs can attack cancer by specifically targeting target antigens expressed on tumor cells. However, the fields of cancer research and autoimmune disease research are very different in nature and generally use fundamentally different treatment approaches. [Means for solving the problem]

[0009] The present invention improves the situation by using anti-CD123 CAR-T cells, as described in the applicant's EP 3753954 (EP Application No. 19305816, also published as WO 2020254682). The inventors surprisingly discovered that CAR-T cells specifically engineered to attack an aggressive cancer called blastic plasmacytoid dendritic cell neoplasm (BPDCN) can improve immunotherapy in autoimmune diseases.

[0010] Therefore, the present invention focuses on the use of specific CAR-T cells targeting CD123 in therapy for autoimmune diseases.

[0011] Accordingly, an object of the present invention is an isolated chimeric antigen receptor (CAR) molecule for use in the treatment of autoimmune diseases, comprising an antibody or antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, wherein the anti-CD123 binding domain has a complementary determining region 1 (CDR1) having at least 90% identity to the amino acid sequence SEQ ID NO:1 (GYSITSDYT), a complementary determining region 2 (CDR2) having at least 90% identity to the amino acid sequence SEQ ID NO:2 (ISFSGST), and a complementary determining region 3 (CDR4) 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).

[0012] According to one embodiment of the present invention, the autoimmune disease is selected from the group consisting of cutaneous lupus erythematosus, dermatomyositis, psoriasis, and systemic sclerosis.

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

[0014] According to another embodiment of the invention, the intracellular signaling domain is CD3-zeta (CD3ζ), optionally comprising a costimulatory domain selected from the group consisting of CD28, 4.1BB, inducible T-cell costimulator (ICOS), OX-40, or a combination thereof.

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

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

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

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

[0019] Other characteristics and advantages of the invention will be apparent and / or become clear on reading the following description, including the specific examples given by way of example and non-limiting, and the drawings, in which: [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 shows the nucleotide and amino acid sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of both the heavy and light chains of the antibody of the present invention. [Figure 2] FIG. 1 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) of the heavy chain of an antibody of the present invention. [Figure 3] FIG. 1 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) of the light chain of an antibody of the present invention. [Figure 4] FIG. 1 shows the consensus amino acid sequences of the light chain and heavy chain of the antibody of the present invention. [Figure 5] FIG. 1 shows the nucleic acid sequence of a chimeric antigen receptor of the present invention. [Figure 6] FIG. 1 shows results for the anti-CD123 CAR-T of the present invention on different blood samples. DETAILED DESCRIPTION OF THE INVENTION

[0021] The drawings and descriptions in this specification generally contain elements of a definite nature, and therefore the descriptions and drawings are not only used to better understand the present invention, but also to illustrate its definitions as needed.

[0022] As used herein, the term "and / or" should be interpreted to encompass one or more of the cases to which it connects. For example, the phrase "a protein or protein sequence can be prepared using standard recombinant and / or synthetic methods" indicates that the protein or protein sequence can be prepared using both standard recombinant and synthetic methods, or that the protein or protein sequence can be prepared using standard recombinant methods, or that the protein or protein sequence can be prepared using synthetic methods. Generally, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" when used in phrases such as "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).

[0023] The term "comprising" should be interpreted to include all specifically mentioned features as well as any, additional, or unspecified features. In general, the terms "comprise," "comprises," and "comprising" should be understood to mean the inclusion of a stated step or element or group of steps or elements, and not the exclusion of any other step or element or group of steps or elements. The term "consisting of" should be interpreted as meaning that no features other than those specifically mentioned are present. Furthermore, the indefinite articles "a" or "an" do not exclude a plurality.

[0024] Throughout this specification, when reference is made to "one embodiment," "an embodiment," "particular embodiment," "an embodiment," "additional embodiment," "another embodiment," or "further embodiment," or to "one aspect," "an aspect," "particular aspect," "one aspect," "additional aspect," "another aspect," or "further aspect," these phrases mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. As used herein, the articles "a," "an," and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0025] The term "gene" refers to a DNA sequence (nucleic acid sequence or nucleotide sequence) that codes for a specific sequence of amino acids. A gene may contain all or part of one or more proteins or enzymes and may include regulatory DNA sequences (such as promoter sequences) that at least partially determine the conditions under which the gene is expressed. Some genes that are not structural genes can be transcribed from DNA into RNA but are not translated into amino acid sequences. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may refer to a genomic sequence that codes for a protein, i.e., a sequence that includes regulatory sequences, promoter sequences, intron sequences, and exon sequences.

[0026] The term "identical" or percent (%) "identity" in the context of two or more nucleic acid or amino acid sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned (with gaps introduced, if necessary) for maximum correspondence, without considering conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. A variety of algorithms and software are known in the art that can be used to align amino acid or nucleotide sequences. A non-limiting example of such a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990 (adapted from the article 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, as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997, can also be used. BLAST-2, WU-BLAST-2 (see Altschul et al., Methods in Enzymology 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco, CA), or Megalign (DNASTAR) are other publicly available software programs that can be used to align sequences.Furthermore, the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453, 1970) can be used to measure the percent identity between two amino acid sequences (e.g., using either a Blossum62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, or 5). Alternatively, the Myers and Miller algorithm (CABIOS, 4: 11-17, 1989) can be used to measure the percent identity between nucleotide or amino acid sequences. For example, the ALIGN program (version 2.0) can be used to measure percent identity using PAM120 based on a residue table, with a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for optimal alignment using a particular alignment software can be determined by those skilled in the art. Typically, the default parameters of the alignment software are used.

[0027] A sequence that is "at least 85% identical to a reference sequence" or "has at least 85% identity to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length based on the entire length of the reference sequence. In this context, "percent identity" as used herein is calculated by pairwise alignment (i.e., two sequences are compared over their entire length). As mentioned above, methods for comparing the identity of two or more sequences are well known in the art. For example, bioinformatics tools or programs such as EMBOSS Needle, available on the ebi.ac.uk website, can be used to generate pairwise sequence alignments of the present invention. Generally, many bioinformatics tools and programs use the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970) to find the optimal alignment (including gaps) of two sequences considering their total length, although other algorithms may be implemented in various bioinformatics tools or programs.

[0028] Generally, for purposes of the present invention, "sequence identity" or "sequence homology" is calculated by comparing two aligned sequences within a comparison window. Sequence alignment can determine the number of positions (nucleotides or amino acids) common to both of the two sequences within the comparison window. The number of common positions is then divided by the total number of positions within the comparison window and multiplied by 100 to obtain the percentage of homology. The measurement of the percentage of sequence identity can be performed manually or by using well-known bioinformatics computer programs.

[0029] According to the present invention, the percent identity between two polypeptides can be calculated using the EMBOSS:needle(global) program with a "gap open" parameter of 10.0 and a "gap extension" parameter of 0.5, using a Blosum62 matrix.

[0030] A protein or portion thereof 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 may contain mutations, such as deletions, insertions, and / or substitutions, compared to the reference sequence.

[0031] In the case of substitutions, a protein consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence may correspond to a homologous sequence from another species other than the reference sequence (i.e., there is "homologous"). Amino acid substitutions can be conservative or non-conservative. A conservative substitution is one in which one amino acid is replaced with another amino acid having a similar side chain, i.e., similar structural and / or chemical properties. In this regard, Amino acids with non-polar side chains, namely, glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), tryptophan (Trp), Amino acids with uncharged polar side chains, i.e., asparagine (Asn), glutamine (Gln), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys), Amino acids with acidic side chains, i.e., aspartic acid (Asp), glutamic acid (Glu), Amino acids with basic side chains, namely, lysine (Lys), arginine (Arg), histidine (His), Amino acids with beta-branched side chains, namely threonine (Thr), valine (Val), isoleucine (Ile), Conservative substitutions may be made among amino acids having aromatic side chains, namely, tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), and histidine (His).

[0032] Non-conservative substitutions may occur randomly among the above. Conservative or non-conservative substitutions may otherwise be defined depending on the position of the individual amino acid within the three-dimensional shape of the protein.

[0033] The techniques of the present invention employ, unless otherwise specified, chemical, biochemical, organic chemistry, molecular biological, microbiological, recombinant DNA, genetic, genetic engineering, and / or biotechnological cell engineering, immunological, and cell biological methods known to those skilled in the art. Where appropriate, such methods are described for illustrative purposes. Most of these methods and other techniques are described in the literature, 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, revised July 2008); and "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" (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, 1991), and "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 of the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred embodiments of the compositions, methods, and materials are described herein.

[0034] As will be understood by those skilled in the art and as described herein, an "antibody," also called an "immunoglobulin," comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, and each light chain consists of a variable region and a constant region. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond.

[0035] In mammals, there are two types of light chains, lambda (I) and kappa (k), and five major heavy chain classes (or isotypes), IgA, IgD, IgE, IgG, and IgM, classified as a, d, e, y, and m, which determine the functional activity of the antibody molecule.

[0036] Each chain contains distinct sequence domains. Light chains contain two domains or regions, a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). More precisely, the antibody forms a "Y" shape. The base or stem of the Y consists of the second and third constant regions of the two heavy chains (plus a fourth constant region in IgE and IgM) connected to each other by disulfide bonds, sometimes called interchain disulfide bonds, forming a hinge. Heavy chains y and d have a constant region consisting of three Ig domains in tandem (in series) and a hinge region for added flexibility. Heavy chains m and e have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm, or branch, of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. Generally, the variable regions of the light chain (VL) and the variable regions of the heavy chain (VH) together determine antigen binding recognition and specificity. The constant region domains of the light chain (CL) and the constant region domains of the heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, placental transfer, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable regions of one light chain and one heavy chain. Antibody specificity is based on the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is primarily composed of residues from the hypervariable regions, or complementarity-determining regions (CDRs). Residues in regions other than the hypervariable regions, i.e., framework regions (FR), may influence the overall domain structure and thus the binding site. Complementarity-determining regions (CDRs) refer to amino acid sequences which together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site.The light and heavy chains of immunoglobulins each have three CDRs, commonly referred to as CDR1, CDR2, and CDR3, or more precisely as CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H, respectively. Other nomenclature schemes are found in the literature. Thus, the CDRs located in the variable domain of an antibody's heavy chain can be referred to as CDRH1, CDRH2, and CDRH3, and the CDRs located in the variable domain of an antibody's light chain can be referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Thus, a conventional antibody antigen-binding site contains six CDRs, including the CDRs of the heavy chain V region and the light chain V region, respectively.

[0037] As mentioned above, the light chain variable region and the heavy chain variable region comprise so-called "framework" regions separated by hypervariable CDR regions. CDRs can be defined or identified by, for example, the sequence-based method described in Kabat et al., T.T. Wu and E.A. Kabat (J Exp Med, 132(2):211-50, 1970), P. Borden and E.A. Kabat (PNAS, 84:2440-2443, 1987), Kabat et al., "Sequences of Proteins of Immunological Interest" (US Department of Health and Human Services, 1991), or the structure-based method described in Chothia et al. (C. Choithia and A.L. Lesk, J Mol. Biol., 196(4):901-917, 1987), C. Choithia et al. (Nature, 342:877-883, 1989).

[0038] The sequences of framework regions of different light or heavy chains are relatively conserved within a species, such as humans. Generally, "framework regions" (FRs) are relatively conserved among different immunoglobulins of a single species. The framework region of an antibody is a combination of some of the framework regions of all the constituent light and heavy chains, and contributes to the positioning and orientation of the CDRs when the antibody assumes a three-dimensional structure, i.e., when the antibody is folded and active. The CDRs are primarily responsible for binding to an epitope of an antigen. As mentioned above, the CDRs of each chain are called CDR1, CDR2, and CDR3. That is, the CDRs are numbered sequentially from the N-terminus. The light and heavy chains of immunoglobulins each have four FRs called FR1, FR2, FR3, and FR4, and more precisely, they are called FR1-L, FR2-L, FR3-L, and FR4-L for the light chain, and FR1-H, FR2-H, FR3-H, and FR4-H for the light chain, respectively. Thus, the light chain variable domain may be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain may be designated as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Knowing the amino acid sequences of the CDRs, one skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0039] In this specification, "V H References to "V" or "VH" refer to the variable region of an immunoglobulin heavy chain, including that of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein. L References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including that of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein.

[0040] As used herein, the term "antibody" or "immunoglobulin" refers primarily to conventional antibodies, particularly monoclonal antibodies and fragments thereof, and single domain antibodies and fragments thereof, particularly the variable heavy chains of single domain antibodies, and also to chimeric, humanized, bispecific, or multispecific antibodies. The antibodies are preferably human, murine, or humanized.

[0041] A "monoclonal antibody" or "mAb" is an antibody produced by a single clone of B lymphocytes or by a cell 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 creating hybrid antibody-forming cells from the fusion of myeloma cells and splenic immune cells, or by recombinant technology, i.e., protein engineering. Monoclonal antibodies include humanized monoclonal antibodies. Generally, monoclonal antibodies are molecules of a single amino acid composition that target a specific antigen, and should not be construed as requiring production of the antibody by any particular method other than those known in the art.

[0042] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, a chimeric antibody contains the VH and VL domains of an antibody derived from a non-human animal in combination with the CH and CL domains of another antibody, particularly a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, or rabbit. A chimeric antibody can also represent a multispecific antibody, having specificity for at least two different antigens.

[0043] The term "antibody" or "immunoglobulin" also includes the more recently described and developed "single domain antibodies." Single domain antibodies are antibodies whose complementarity-determining regions (CDRs) are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies that do not naturally have light chains, single domain antibodies derived from traditional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be derived from any species, particularly mouse, human, or rabbit. Single domain antibodies can be naturally occurring single domain antibodies known as heavy chain antibodies that do not have light chains. In particular, Camelidae species (e.g., camels, dromedaries, llamas, alpacas, and guanacos) produce heavy chain antibodies that do not naturally have light chains.

[0044] The variable heavy chains of these single-domain antibodies, which lack light chains, are known in the art as "VHHs" or "nanobodies." Like conventional VH domains, VHHs contain four framework regions (FRs) and three complementarity-determining regions (CDRs). One particular advantage of nanobodies over conventional antibodies is that they are approximately one-tenth the size of IgG molecules. Therefore, properly folded, functional nanobodies can be produced in high yields by in vitro expression. Furthermore, the art has shown that nanobodies are highly stable and resistant to protease action. For the properties and production of nanobodies, see, for example, the article by Harmsen and HJ De Haard (Appl. Microbiol. Biotechnol., 77(1):13-22, November 2007).

[0045] The term "antibody" also includes antigen-binding fragments thereof. Such "fragments" include portions of intact antibodies, particularly the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab' fragments, F(ab)' fragments, Fv, single-chain Fv proteins ("single chain Fv:scFv"), as well as portions of full-length antibodies responsible for antigen binding, diabodies, and bispecific and multispecific antibodies formed from antibody fragments. Conventional antibody fragments may also be heavy-chain antibodies or single-domain antibodies such as VHHs. The term "Fab" refers to an antibody fragment with a molecular weight of approximately 50,000 that has antigen-binding activity and is obtained by treating IgG with protease (papain), in which approximately the N-terminal half of the H chain and the entire L chain are linked together by disulfide bonds. The term "F(ab')2" refers to an antibody fragment with antigen-binding activity having a molecular weight of approximately 100,000, obtained by treating IgG with a protease (pepsin), and is slightly larger than the Fab fragments linked by the disulfide bonds in the hinge region. "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of an antibody, with these domains present in either orientation (e.g., VL-VH or VH-VL) in a single polypeptide chain. The single chain may be cloned to form the V region genes of a hybridoma specific for a desired target. The production of such hybridomas has become commonplace. Techniques for cloning the variable heavy (VH) and variable light (VL) chains are described, for example, in Orlandi et al. (PNAS, 86:3833-3837, 1989). Typically, scFv polypeptides further contain 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, and are typically expressed from a gene fusion comprising genes encoding VH and VL linked by a peptide-encoding linker.In some embodiments, human scFv fragments contain CDRs that are maintained in a suitable conformation, particularly through the use of genetic engineering techniques. Bivalent and multivalent antibody fragments can be formed spontaneously by the association of monovalent scFvs or can be generated by linking monovalent scFvs with a peptide linker, such as a bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. A "(dsFv)2" refers to two dsFvs linked by a peptide linker. The term "bispecific antibody" or "BsAb" typically refers to an antibody that combines the antigen-binding sites of two antibodies in a single molecule. Thus, BsAbs can simultaneously bind to two different antigens. Genetic engineering is being applied with increasing frequency to design, modify, and generate antibodies or antibody derivatives with desired combinations of binding properties and effector functions, as described, for example, in EP 2050764. The term "multispecific antibody" typically refers to an antibody that combines the antigen-binding sites of two or more antibodies in a single molecule. The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). A linker too short to allow pairing between the two domains on the same chain is used to force pairing of the domains with complementary domains on another chain, creating two antigen-binding sites.

[0046] The present invention provides immune effector cells engineered with vectors designed to express chimeric antigen receptors (CARs), which redirect cytotoxicity to cells and / or molecules involved in autoimmune disease. CARs are molecules that combine antibody-based specificity for a target antigen (e.g., an autoimmune disease-associated antigen) with a T cell receptor activating intracellular domain, resulting in a chimeric protein that exhibits specific cellular immune activity. As used herein, the term "chimeric" refers to a protein composed of different proteins or DNA fragments from different sources. A key feature of CARs is their ability to harness the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors to redirect the specificity of immune effector cells to induce proliferation, cytokine production, phagocytosis, or the production of molecules capable of mediating cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0047] As used herein, the terms "binding domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and confer the CAR the ability to specifically bind to a target antigen of interest. A binding domain can include any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biomolecule, such as a cell surface receptor or a protein, lipid, polysaccharide, or other cell surface target molecule involved in autoimmune disease, or a component thereof. A binding domain includes any naturally occurring binding partner, or any synthetic, semi-synthetic, or recombinantly produced binding partner for a biomolecule of interest. As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" refer to the binding of a molecule to another molecule with higher binding affinity than background binding. For example, a binding affinity of about 10 5 M -1A binding domain (or a CAR comprising a binding domain or a fusion protein comprising a binding domain) "specifically binds" to a target molecule if it binds or associates with the target molecule with an affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction, expressed in units of 1 / M) or greater. The affinity of binding domain polypeptides and CAR proteins according to the present disclosure can be readily measured using conventional techniques such as competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, affinity-related binding can be measured, quantified, and assessed to provide a so-called K D It can be expressed as a value of K D and K correlate with the affinity and sensitivity of the antibody. D is the equilibrium dissociation constant, i.e., the k off / k on It is the ratio of K D There is an inverse correlation between affinity and K D The K value is related to the antibody concentration (the amount of antibody needed for a particular experiment). D The lower the value (lower concentration), the higher the affinity of the antibody. D is the antibody dissociation rate (k off ) (how quickly the antibody dissociates from its antigen) and the antibody association rate (k on ) (how quickly it binds to its antigen). D The k value is the k of a specific antibody / antigen interaction. on and k off The ratio of these values ​​is then used to calculate K D Generally, the affinity of an antibody is the strength with which a single molecule binds to its ligand. This is typically measured by the equilibrium dissociation constant (K ) which is used to assess and rank the strength of bimolecular interactions. D ) is measured and reported as the affinity constant (1 / K). The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate of formation of the [antibody]|[antigen] complex equals the rate of dissociation into its components [antibody]+[antigen]. The measured rate constant is the equilibrium constant or affinity constant (1 / K D) can be used to define K D The lower the value, the greater the affinity of the antibody for its target.

[0048] Generally, the binding domain of CAR is followed by one or more "hinge regions". The hinge region separates the antigen binding domain from the effector cell surface, allowing proper cell-cell contact, antigen binding, and activation. CAR can have one or more hinge regions between the binding domain and the transmembrane domain. As described in the art, the hinge region can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0049] The "transmembrane domain" is the part of CAR that connects the extracellular binding portion and the intracellular signaling domain. The transmembrane domain anchors the CAR to the cell membrane of immune effector cells. As described in the art, the transmembrane region can be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0050] In a preferred embodiment, the CAR of the present invention comprises an intracellular signaling domain. The term "intracellular signaling domain" refers to a portion of a CAR that is involved in transmitting a signal of effective CAR binding to a target antigen to the interior of an immune effector cell, thereby inducing effector cell function, such as cytotoxic activity, including activation, cytokine production, proliferation, and release of cytotoxic factors to CAR-bound target cells, or other cellular responses induced by antigen binding to the extracellular CAR domain. "Effector function" refers to a specialized function of a cell. The effector function of a T cell can be cytolytic activity or cytokine secretion. Therefore, as used herein, the term "intracellular signaling domain" refers to a portion of a protein that transmits an "effector function" signal that causes a cell to perform a specialized function. As described in the art, either the complete intracellular signaling domain or a truncated portion of the intracellular signaling domain can be used. As will be understood by those skilled in the art, an effective truncated portion will transmit an effector function signal substantially similarly to the complete domain. As used herein, the term "intracellular signaling domain" is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0051] In a preferred embodiment of the present invention, the CAR has a hinge region IgG1-CH2-CH3 (the first part of IgG1 is located in the membrane, and the second part of IgG1 is located extracellularly, on the outside of the cell) and CD28 as a transmembrane domain (the first part of CD28 is located in the membrane, and the second part of CD28 is located intracellularly, in the cytoplasm). CD8α can also be used as the transmembrane domain and hinge region. Sequences derived from IgG4 can also be used.

[0052] As is known in the art, signals generated solely through the T-cell receptor (TCR) are insufficient for the complete activation of T cells; secondary or costimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains (e.g., TCR / CD3 complexes) that initiate antigen-dependent primary activation via the TCR, and costimulatory signaling domains that act antigen-independently to confer secondary or optional costimulatory signals. In a preferred embodiment, the CAR of the present invention comprises an intracellular signaling domain comprising one or more "costimulatory signaling domains." Thus, in another preferred embodiment, the isolated nucleic acid molecule of the present invention encodes an intracellular signaling domain comprising at least one costimulatory domain, and the resulting intracellular signaling domain comprises at least one costimulatory domain.

[0053] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, confers a second signal required for efficient T cell activation / function.

[0054] As used herein, the terms "polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably and, unless otherwise specified, have their conventional meaning, i.e., amino acid sequence. A polypeptide is not limited to a specific length and can include a full-length protein sequence or a fragment thereof. A polymer can be linear or branched, can contain modified amino acids, or can be separated by non-amino acids. Furthermore, polypeptides can be subjected to any other manipulation or modification, such as post-translational modifications, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component, generally any naturally occurring and / or non-naturally occurring modification. Given that the polypeptides of the present invention are based on antibodies, in some embodiments, the polypeptides can exist as single chains or associated chains. Polypeptides can be prepared using any technique known in the art, i.e., recombinant and / or synthetic techniques. More specifically, the polypeptides described herein encompass the CARs of the present disclosure or sequences of the CARs disclosed herein with one or more amino acid deletions, additions, and / or substitutions.

[0055] As used herein, "isolated peptide" or "isolated polypeptide" or the like refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and that is substantially free of extracellular matrix.

[0056] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying or transporting another nucleic acid molecule. Generally, a "vector" refers to a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. The nucleic acid to be transported or delivered is linked to the vector nucleic acid molecule by any method known in the art, for example, by insertion. The vector may contain a sequence that induces autonomous replication in the cell or may contain a sequence sufficient to allow integration into host cell DNA. The present invention also provides a vector comprising a nucleic acid molecule encoding a CAR of the present invention. The vector may be selected from DNA, RNA, a phage vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. The vector of the present invention preferably comprises a promoter, such as the EF-1 alpha promoter. As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which RNA polymerase binds. As known to those skilled in the art, RNA polymerase initiates transcription of a polynucleotide operably linked to the promoter. In certain embodiments, it may be desirable to express a polynucleotide comprising a CAR from a promoter that confers stable, long-term expression of the CAR in T cells. Expressing the CAR under the control of a promoter further ensures that expression is at a level sufficient for T cells to target cells expressing the target antigen.

[0057] Retroviruses are common tools for gene delivery. In some embodiments, retroviruses are used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to cells, preferably immune cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA to synthesize a linear double-stranded DNA copy, which is then covalently integrated into the host genome. Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase and induces the expression of RNA molecules encoding structural proteins and enzymes necessary to produce new viral particles. As a result, T cells transduced with the vectors of the present invention can generate stable, long-term, and sustained CAR-mediated T cell responses. In certain embodiments, T cells are transduced with a lentiviral vector, i.e., a lentivirus, encoding a CAR according to the present invention. The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, primarily comprising long terminal repeats (LTRs) derived from lentiviruses. 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 anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).The term "self-inactivating" (SIN) vector refers to a replication-deficient vector, e.g., a retroviral or lentiviral vector in which the 3' LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the initial stages of viral replication.

[0058] The terms "purified" and "isolated," when referring to a molecule such as a polypeptide or antibody or nucleotide sequence of the present invention, mean that the molecule is present in the absence of other biological macromolecules of the same type. More specifically, the term "purified" as used herein means that the molecule is present in an amount of at least 85%, 90%, 95%, or 98% by weight of biological macromolecules of the same type. More specifically, the term "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 of interest. However, the molecule may contain some additional bases or moieties that do not adversely affect the basic properties of the composition.

[0059] The terms "antigen," "target," or "target antigen" refer to a molecule or portion of a molecule that can be bound by an antibody or antibody-like binding protein. The term also refers to a molecule or portion of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of that antigen. A target antigen can have one or more epitopes. For each target antigen recognized by an antibody or antibody-like binding protein, the antibody-like binding protein can compete with the intact antibody to recognize the target antigen.

[0060] The terms "CD123," "IL-3Ra" or "IL-3Rα," and "interleukin-3 receptor alpha" or "interleukin-3 receptor α" are used interchangeably herein and, unless otherwise specified, refer to any native (human) IL-3Ra or CD123. The CD123 protein is the interleukin-3-specific subunit of the heterodimeric cytokine receptor (i.e., the IL-3 receptor or IL-3R). IL-3R is composed of a ligand-specific alpha subunit and a signaling common beta subunit (also known as CD131) shared by the receptors for interleukin 3 (IL3), colony-stimulating factor 2 (CSF2 / GM-CSF), and interleukin 5 (IL5). Binding of CD123 / IL-3Ra to IL3 is dependent on the beta subunit, which is activated by ligand binding and is required for IL3 biological activity. All of these aforementioned terms for CD123 may refer to any of the protein or nucleic acid sequences set forth herein. The term "CD123 / IL-3Ra" encompasses unprocessed "full-length" CD123 / IL-3Ra and any form of CD123 / IL-3Ra resulting from processing within a cell. The term also encompasses naturally occurring variants of CD123 / IL-3Ra proteins or nucleic acids, such as splice variants, allelic variants, and isoforms. The CD123 / IL-3Ra polypeptides and polynucleotides described herein may be isolated from various sources, such as human tissue types or another source, or prepared by recombinant or synthetic methods. Examples of CD123 / IL-3Ra sequences include, but are not limited to, NCBI reference numbers NP_002174 and NM_002183 (protein and nucleic acid sequences for human CD123 variant 1) and NP_001254642 and NM_001267713 (protein and nucleic acid sequences for human CD123 variant 2).

[0061] The terms "anti-CD123 antibody," "anti-IL-3Ra antibody," or "anti-IL-3Rα antibody," and "antibody that specifically binds to CD123," or "antibody that specifically binds to IL-3Ra / IL-3Rα," refer to an antibody that can bind to CD123 with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD123. Unless otherwise specified, the extent of binding of an anti-CD123 antibody to unrelated, non-CD123 proteins is less than about 10% of the measured binding of the antibody to CD123. Generally, the term "specifically binds" refers to an antibody that binds to an epitope via its antigen-binding domain and that the 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 if it binds to that epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope. Herein, the term "specificity" is used to define relative affinity by the extent to which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," and antibody "A" may be said to bind epitope "C" with greater specificity than it has for the related epitope "D." Thus, antibodies or antigen-binding fragments of the invention specifically bind to the CD123 antigen by virtue of having greater binding specificity for the CD123 antigen (from any species) than for non-CD123 antigens. More specifically, antibodies or antigen-binding fragments of the invention specifically bind to the human CD123 antigen by virtue of having greater binding specificity for the human CD123 antigen than for non-human CD123 antigens (e.g., mouse or rat CD123). The term "preferentially binds" may also indicate that an antibody specifically binds to an epitope more readily than it binds to a related, similar, homologous, or analogous epitope. Thus, an antibody that binds preferentially to a given epitope will more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.For example, an antibody or antigen-binding fragment of the invention may preferentially bind to the human CD123 antigen over mouse CD123.

[0062] Based on the above, the terms related to the antibody, CAR, vector, or cell of the present invention can be easily derived. In fact, in the context of the entire specification, it is not necessary to follow the exact same expressions as the above definitions. Some deviations may occur. For example, when referring to a CAR of a T cell, it is not necessary that the CAR contain a portion that targets a specific antigen, i.e., to specify "anti-something." Therefore, generally, a CAR of the present invention that contains a portion that specifically binds to CD123 can be referred to not only as an anti-CD123 CAR, but also as a CD123 CAR. Similarly, a T cell transduced with a CAR that specifically binds to CD123 according to the present invention can be referred to as a CD123 CAR T cell.

[0063] As used herein, "treatment" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include any decrease in one or more measurable markers of a disease or condition, e.g., an autoimmune disease, during treatment. Treatment may include either alleviation of symptoms or any improvement with respect to the symptoms, disease, or condition. Treatment may also slow the progression of a disease or condition. Thus, the term "treatment" does not necessarily imply complete eradication or cure of a disease or condition, or its associated symptoms. With this in mind, the present disclosure provides for the treatment or prevention of BPDCN, comprising administering a therapeutically effective amount of the T cells of the present invention to a subject in need thereof.

[0064] In this regard, the T cells described herein can be administered alone or as a pharmaceutical composition (also referred to as a pharmaceutical formulation). The terms "pharmaceutical composition" or "pharmaceutical formulation" refer to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the composition / formulation is administered. Such formulations can be sterile. Pharmaceutical compositions may include T cells according to the present invention alone or in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, sugars such as glucose, mannose, sucrose, dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants, and stabilizers. As used herein, the terms "pharmaceutically acceptable" or "physiologically acceptable" refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. The compositions of the present invention are preferably formulated for parenteral administration. "Parenteral administration" can refer to modes of administration other than enteral and topical administration. Parenteral administration is typically performed by injection, such as intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and / or infusion. Preferably, the CAR-modified T cells or compositions of the present invention are administered to a subject by direct injection into a site of inflammation, a lymph node, the systemic circulation, or a site of infection. Generally, the present invention is useful for treating subjects diagnosed with a disease that overexpresses CD123, particularly patients with BPDCN.The treatment involves removing immune effector cells from a subject, genetically modifying the immune effector cells with a vector containing a nucleic acid encoding a CAR contemplated herein, thereby generating a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells are T cells. The dosage and frequency of administration are determined by the patient's physical condition (patient's condition, the type and severity of the patient's disease). Appropriate doses can also be determined through clinical trials using animal models. When referring to doses, the term "effective amount" refers to an amount sufficient to achieve a specifically stated purpose. The so-called "therapeutically effective amount" of an active ingredient, such as genetically modified T cells, may depend on factors such as the disease and its condition, the patient's age, sex, and weight, and may further depend on the ability of the cells to induce a desired response in the patient. A therapeutically effective amount is an amount in which the therapeutically beneficial effects outweigh any toxic or adverse effects of the virus or transduced therapeutic cells. The pharmaceutical compositions comprising the T cells described herein include all integer values ​​within these ranges. 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 It can generally be stated that the amount of the antibody or agent administered may be in the form of a dose of cells / kg body weight. Generally, the term "therapeutically effective amount" refers to an amount of antibody, active ingredient, or other effective drug effective to treat a disease or disorder of interest.

[0065] In the case of autoimmune diseases, a therapeutically effective amount of a drug may reduce the number of cells involved in the pathophysiology of the disease, resulting in a decrease in the amount / concentration of inflammatory molecules, a decrease in the size of inflammation, suppress the disease (i.e., delay or halt the disease to some extent), alleviate to some extent one or more symptoms associated with the autoimmune disease, and / or induce a favorable response, such as an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), a complete response (CR), a partial response (PR), or in some cases stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), or any combination thereof. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve the desired preventive result. Because a prophylactic dose is administered to a subject before the onset or at an early stage of disease, the prophylactically effective amount is usually less than the therapeutically effective amount.

[0066] In certain embodiments, a cell source is obtained from a subject prior to in vitro manipulation or genetic modification of immune effector cells as described herein. In certain embodiments, immune effector cells expressing a CAR of the present invention on their membrane comprise T cells. T cells can be obtained from many sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, and spleen tissue. In certain embodiments, T cells can be obtained from a single unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In another embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes 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 may be washed to remove the plasma fraction, and the cells may be placed in an appropriate buffer or medium for further processing. In other embodiments, T cells are isolated from peripheral blood mononuclear cells by lysing red blood cells and removing monocytes, for example, by centrifugation through a Percoll™ gradient. 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, T cell populations can be enriched by negative selection using a combination of antibodies that target surface markers not expressed on the negatively selected cells.

[0067] Generally, this document features cell therapy in which T cells are genetically engineered (or modified) ex vivo to express a CAR and then infused into a patient, preferably a human, in need of CAR T cells.

[0068] The infused cells can kill the patient's own cells given their expression of specific molecules (e.g., CD123). As previously explained, unlike antibody therapy, CAR-containing T cells can replicate in vivo, resulting in long-term persistence that can induce sustained control of autoimmune diseases. Furthermore, CARs allow effector T cells to be redirected and activated by any cell surface molecule through antibody-derived receptor binding, independent of MHC restriction (MHC-restricted antigen recognition, where MHC stands for major histocompatibility complex). As described above, the engineered T cells of the present invention can be constructed starting from T cells harvested from the patient themselves (autologous), but can also be derived from other allogeneic donors to provide allogeneic engineered T cells in bone marrow or peripheral hematopoietic stem cell allografts (donor lymphocyte infusion). These T cells expressing the CAR molecules of the present invention are useful for treating autoimmune diseases in mammals, preferably humans. [Example]

[0069] The present invention will now be described in further detail with reference to experimental data and specific examples, which are presented for purposes of illustration only and are not intended to be limiting unless otherwise specified.

[0070] The present invention may generally provide an improvement and / or solution to autoimmune diseases in which CD123 overexpression occurs.

[0071] Thus, a primary focus of the present invention is to provide treatments for autoimmune diseases, specifically cutaneous lupus erythematosus, dermatomyositis, psoriasis, and systemic sclerosis.

[0072] However, developing treatments or drugs presents obstacles for those skilled in the art: for example, some drugs may prove too toxic to treat non-fatal diseases, and development is often so expensive that it generally shuns those skilled in the art for less prominent diseases.

[0073] Furthermore, therapies such as those involving CARs are difficult to develop. For example, constructing CARs is an extremely challenging task. This is because CARs are often constructed from the patient's own cells, or at least by using specific cells extracted from the patient, such as T lymphocytes. However, these cells are often abnormal in patients with autoimmune diseases. This is particularly true for T lymphocytes in autoimmune diseases, since these cells themselves are autoimmune.

[0074] Many other obstacles must be overcome before CAR therapy can be considered. For another example, some diseases require CAR to diffuse into the skin, which may present several additional obstacles.

[0075] According to one embodiment of the present invention, CARs are engineered to be constructed with single-chain variable fragments (ScFv) of the heavy and light chains of a monoclonal antibody (MAb) linked to the intracellular signaling chain of a T cell receptor (TCR). Preferably, the intracellular portion of the CAR contains CD3ζ and costimulatory domains such as CD28 and 4-1BB, which enable better activation and cell signaling. Such CARs may be defined as third-generation CARs. 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, September 2016). ScFvs enable antigen recognition independent of major histocompatibility complex (MHC) presentation.

[0076] Increasing clinical trials have shown that patients undergoing CAR therapy experience toxicity. This is a major challenge in the development of efficient CARs. The present applicant has developed a specific CAR that overcomes the problems of the prior art (see European Patent No. 3753954). More specifically, the present applicant has engineered both third-generation retroviral and lentiviral CARs. The CARs of the present invention overcome the problems of the prior art, particularly those related to cytotoxicity. The CARs of the present invention have improved functional activity against BPDCN cells (BPDCN cell lines, PDX cells, and primary BPDCN cells) in vitro as well as in vivo in several mouse models of BPDCN. Evaluation of their potential cytotoxicity against CD123 weakly positive cells has been surprisingly promising.

[0077] According to the present invention, a monoclonal antibody (Mab) targeting human CD123 was produced. The monoclonal antibody used in the present invention can be used both allogeneic and autologous.

[0078] The method for generating anti-CD123 monoclonal antibodies involved immunizing mice with recombinant CD123 protein. More specifically, five mice were immunized with recombinant CD123, reference number 301-R3-025, available from R&D Systems (Minnesota, USA). Immunization was performed via footpad and / or intraperitoneal administration. B cells from lymph nodes and / or spleens were used to generate hybridomas secreting MAbs. CD123 + and CD123 - The cell lines were used to select hybridomas based on MAb affinity and specificity.

[0079] For the present invention, a specific antibody was selected, designated herein as 18B4D5 (also referred to herein as AB1 or abbreviated as B4D5). Molecular characterization and DNA sequencing were performed according to the SANGER method.

[0080] VDJ and VJ gene rearrangements were sequenced and identified after aligning consensus nucleotide sequences against the IMGT® database using the VQUEST online tool. See X. Brochet, M.P. Lefranc, and V. Giudicelli, "IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis," Nucleic Acids Research, 36, Web Server Distribution, W503-8, 2008. The sequences (nucleotide and amino acid sequences) of antibody 18B4D5 are listed in Figures 1-5.

[0081] FIG. 1 shows the nucleotide and amino acid sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3) of both the heavy and light chains of antibody 18B4D5 used in the present invention.

[0082] Figure 2 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) of the heavy chain of antibody 18B4D5 used in the present invention.

[0083] Figure 3 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3), and framework region 4 (FR4) of the light chain of antibody 18B4D5 used in the present invention.

[0084] Figure 4 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of antibody AB1 used in the present invention, designated 18B4D5 or B4D5. The amino acids corresponding to CDR1, CDR2, and CDR3, respectively, are highlighted in reading order from left to right.

[0085] Table 1 shows the sequence homologies of the heavy chain VH and JH and light chain VK and JK of the antibodies used in the present invention based on the VQUEST online tool described above.

[0086] [Table 1]

[0087] By selecting this specific antibody, the applicant has engineered specific chimeric antigen receptor T (CAR-T) cells that are highly effective in treating autoimmune diseases.

[0088] To do so, we first based our research on the findings of CAR-T therapy. CAR-T therapy is one of the most promising cell-based therapies, based on over 800 clinical studies conducted worldwide to evaluate many targets and indications. See "CAR T cells: continuation in a revolution of immunotherapy" by A.K. Singh and J.P. McGuirk (Lancet Oncol. 21, e168-e178, 2020). It has been primarily developed in the field of oncology, particularly hematology. It has produced promising results, enabling high remission rates in patients who have relapsed or are refractory to other treatments. See "Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia" by S.L. Maude et al. (N. Engl. J. Med. 378, 439-448, 2018).

[0089] More recently, the application of CAR-T therapy has expanded to other areas, such as infectious diseases, myocardial fibrosis, and autoimmune diseases (AIDs). See "CAR-based therapies: opportunities for immunomedicine beyond cancer" by H. Aghajanian, J. G. Rik, and J. A. Epstein (Nat. Metab. 4, 163-169, 2022). Rapid and durable remissions were also recently reported in patients with severe lupus erythematosus treated with CD19-targeted CAR-T. See "CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus" by D. Mougiakakos et al. (N. Engl. J. Med. 385, 567-569, 2021).

[0090] Therefore, the applicant has focused on the application of specific autologous CAR-T in autoimmune diseases.However, a major difficulty is associated with identifying an effective CAR-T that also exhibits cytotoxicity suitable for patient treatment.This identification is particularly difficult because, among other difficulties, many CAR-Ts are available in the art.

[0091] Another study showed that anti-CD19 CAR-T applied to refractory lupus erythematosus in other patients was successful with satisfactory clinical results. See A. Mackensen et al., "Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus" (Nat. Med. 28, 2124-2132, 2022). Patients did not appear to experience significant adverse effects from the infused CAR-T. Various preclinical studies are currently underway in the field of anti-CD19 CAR-T. Some CAR-Ts, such as anti-CD19 (NCT03030976, lupus erythematosus), anti-CD19 / BCMA (NCT05030779, lupus erythematosus), anti-BCMA (NCT0414051, generalized myasthenia gravis), and anti-DSG34.6 CAAR-T (NCT04422912, pemphigus vulgaris) (see CT Elebrecht et al., “Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease,” Science 353, 179–184, 2016), have shown preclinical efficacy in autoimmune diseases and are currently in clinical trials.

[0092] The present applicant has developed its research in a different direction from the above-mentioned CAR-T in order to identify a new generation of CAR-T active against autoimmune diseases. By doing so, the present applicant has generally confirmed the feasibility of CAR-T transfer and further succeeded in identifying a CAR-T that is not only well tolerated by patients but also highly effective against systemic lupus erythematosus.

[0093] However, the development of CAR-T therapy depends heavily on the selection of its antigen target, and a challenging aspect is that the target must be as specific as possible to the cells to be eliminated in order to reduce so-called "off-target off-tumor" adverse effects.

[0094] The applicant has surprisingly discovered that anti-CD123 CAR-T meets the above challenges and requirements.

[0095] The CD123 marker is the α subunit of the IL-3 heterodimeric receptor. It is strongly expressed on pDCs and basophils and weakly expressed on other cell types, such as monocytes, myeloid progenitors, and endothelial cells. IL-3 signaling is essential for pDC survival. See E. Bole-Richard et al., "CD28 / 4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasms" (Leukemia 34, 3228-3241, 2020) and S. Oon et al., "A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus" (JCI Insight 1, 2016).

[0096] The present applicants have proposed that pDCs account for 0.1-0.5% of circulating mononuclear cells and are part of the innate immune system. See C. Bode 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, the present applicants have proposed that pDCs have a significant ability to secrete type I interferon (IFN-I). IFN-I is a key cytokine of the innate immune system produced through stimulation of TLR7 and TLR9. TLR7 and TLR9 are cytoplasmic receptors of pDCs that are activated primarily by RNA and DNA fragments. See MDAh Kioon et al., "Plasmacytoid dendritic cells promote systemic sclerosis with a key role for TLR8," Sci. Transl. Med. 10, eaam8458, 2018; S. Kafaja et al., "pDCs in lung and skin fibrosis in a bleomycin-induced model and patients with systemic sclerosis," JCI Insight 3, 98380, 2018; and D. Ganguly et al., "Self-RNA-antimicrobial peptide complexes activate human dendritic cells through TLR7 and TLR8," J. Exp. Med. 206, 1983-1994, 2009.

[0097] Some studies suggest that pDCs may be involved in the development of autoimmune and / or autoinflammatory diseases associated with the overproduction of IFN-I. See S. Li, J. Wu, S. Zhu, Y. J. Liu, and J. Chen, "Disease-Associated Plasmacytoid Dendritic Cells," Front. Immunol. 8, 1268, 2017.

[0098] The present applicants also believe that pDCs have the functions of antigen presentation and secretion of inflammatory cytokines (IFN-I, IL-6, IL-12, CXCL8, CXCL10, CCl3, and CCL4). Furthermore, the present applicants' research covered data showing that the circulating pDC rate in patients with certain autoimmune diseases is lower than that in healthy individuals. However, in these patients, pDCs infiltrate damaged and inflamed tissues. Therefore, pDCs appear to have a strong tissue infiltration ability, particularly in the skin of patients with systemic scleroderma (SSc) or the kidneys of patients with lupus erythematosus."A simple one-step method for isolating highly purified plasmacytoid dendritic cells from human peripheral blood (78.33)" by AI Kokaji, S. Holland, MA Fairhurst, TE Thomas and BG Guilbault (J. Immunol. 182, 78.33-78.33, 2009); "Cutaneous distribution of plasmacytoid dendritic cells in lupus erythematosus" by W. Vermi et al. (Selective tropism at the site of epithelial apoptotic damage - Immunobiology 214, 877-886, 2009); "Immune functions and recruitment of plasmacytoid dendritic cells in psoriasis" by C. Albanesi, C. Scarponi, D. Bosisio, S. Sozzani and G. Girolomoni (Autoimmune 43, 215-219, 2010), F. O. Nestle et al., "Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production," J. Exp. Med. 202, 135-143, 2005, and E. Bell, "Plasmacytoid dendritic cells in psoriasis," Nat. Rev. Immunol. 7, 839-839, 2007.

[0099] Other autoimmune diseases, such as psoriasis and dermatomyositis, appear to be associated with pDC activity producing IFN-I. See C. Albanesi et al., "Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment," J. Exp. Med. 206, 249-258, 2009, and Z. Tezak et al., "Gene expression profiling in DQA1*0501+ children with untreated dermatomyositis: A novel model of pathogenesis," J. Immunol. 168, 4154-4163, 2002.

[0100] In humans and mice, pDC infiltration in the lungs of SSc, accompanied by secretion of IFN-α and CXCL4, is thought to be associated with poor prognosis, skin and lung fibrosis, and a more severe clinical profile. See L. van Bon 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 / chemokines (IFN-α and CXCL4) identified in patients are associated with skin and lung fibrosis and a more severe clinical profile. This indicates that pDCs secreting these cytokines play an unfavorable role in this pathology. This is also observed in mouse models. Therefore, pDC infiltration is a poor prognostic factor.

[0101] FIG. 5 shows the nucleic acid sequence of a CAR according to the present invention.

[0102] This CAR is constructed based on antibody AB1. The nucleic acids corresponding to signal peptide, Tag HA, heavy chain, Hinge1, light chain, Hinge2, CD28, 4.1BB and CD3z are highlighted in different colors, respectively, in left-to-right reading order. Further details about the construction of CAR123 of the present invention can be found in European Patent No. 3753954, which the reader can refer to.

[0103] Thus, an object of the present invention is an isolated nucleic acid molecule for use in the treatment of autoimmune diseases, which consists of a sequence having at least 85%, preferably 90%, more preferably 95% identity to SEQ ID NO: 6 and which encodes a chimeric antigen receptor (CAR).

[0104] Another object of the present invention is an isolated nucleic acid molecule for use in treating an autoimmune disease, wherein the nucleic acid consists of SEQ ID NO: 6 and encodes a chimeric antigen receptor (CAR).

[0105] The affinity of the antibody to CD123 is tested using a streptavidin biosensor. D To measure the values, seven different concentrations of CD123 were used with the immobilized antibody of the present invention. The results were analyzed based on the Octet system available from ForteBio.

[0106] The antibody affinity results are shown in Table 2.

[0107] [Table 2]

[0108] Applicant isolated mononuclear cells using Ficoll. T lymphocytes were isolated from the mononuclear cells and then activated by magnetic sorting (anti-CD3 / CD28 magnetic beads, Gibco™). Activated T lymphocytes were cultured in RPMI 10% (supplemented with human serum) and IL-2. pDCs were isolated by negative sorting (EasySep™ Human pDC Isolation kit, StemCell) and phenotyped both before and after sorting to determine sort purity before freezing at -80°C.

[0109] Two days after activation, T lymphocytes were transduced with lentiviral vectors. Transduction efficiency was assessed 7 days after transduction (CD3+ / CD19+). Non-transduced T lymphocytes (C0) served as a control for all experiments.

[0110] The functionality of CAR123 was examined after co-culture of T lymphocytes (C0 or CAR123) with target cells (sorted pDC or CAL-1) for 6 hours, and the percentage of cytotoxicity was then assessed by flow cytometry.

[0111] FIG. 6 shows the results of the above-described analysis of the CAR123 of the present invention using different blood samples (healthy donors (HD), patients with systemic lupus (SLE) or cutaneous-articular lupus (CLE), patients with dermatomyositis (DM), patients with psoriasis (PSO), and patients with systemic sclerosis (SSc)).

[0112] Figure 6A shows the T cell transduction efficiency measured by CD3+ / CD19+ expression 7 days after transduction, which corresponds to the percentage of transduced T cells, i.e., expressing the CAR of the present invention on their surface. No significant difference was observed between HD and patients.

[0113] Figure 6B shows the fold expansion of T cells measured after 9 days of culture in RPMI medium supplemented with human serum and IL-2, demonstrating the efficient activation and expansion of patient-derived T cells.

[0114] Figure 6C shows that the proportion of pDCs measured by flow cytometry is identical in HD compared to patients, and pDCs express CD123 and BDCA2.

[0115] FIG. 6D shows the ability to sort pDCs using the EasyStem™ Human pDC Isolation Kit (StemCell).

[0116] Figure 6E shows the percentage of target cell killing. The percentage of cytotoxicity was assessed by the percentage of lysed target cells (pDC or CAL-1) 6 hours after co-culture with effector (CO CAR-T) at an effector / target ratio of 5 / 1. CAL-1 was used as a positive control (CD123+ BPDCN cell line). We observed strong cytotoxicity against CAL-1 or pDC with patient-derived CAR-T, with no difference between patient-derived and HD-derived CAR-T. These results demonstrate that we can obtain functional CAR-T from T lymphocytes derived from patients and healthy individuals.

[0117] For statistical testing, statistical analysis was performed using the non-parametric t-test (Wilcoxon). Data are expressed as mean and standard deviation. A p-value of <0.05 was considered statistically significant (***p<0.001).

[0118] The results demonstrate the robust ability of the autologous CAR123 of the present invention, generated from samples of patients with pDC-mediated autoimmune diseases, to eliminate circulating autologous pDCs in vitro. As known to those skilled in the art, these results also apply to in vivo models.

[0119] Other cells express CD123 molecule on their surface.For example, this is the case of polynuclear basophils (high expression) and monocytes (low expression).Therefore, these cells are also the target of CAR123 of the present invention.Therefore, the applicant has studied CAR123 of the present invention in relation to autoimmune disease.

[0120] More specifically, in lupus erythematosus, the applicant has observed significant infiltration of monocytes and macrophages in renal inflammatory lesions of patients with lupus nephritis. Furthermore, the presence of monocytes (CD16+ subpopulation) in the lesions leads to increased activation of autoreactive T and B lymphocytes, enhanced antigen presentation, and inefficient clearance of apoptotic bodies and immune complexes. Furthermore, they secrete large amounts of proinflammatory cytokines. See "Aberrant expression of the costimulatory molecule CD40 ligand on monocytes from patients with systemic lupus erythematosus" by C.G. Katsiari 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); "Macrophages in Lupus Nephritis: Exploring a potential new therapeutic avenue" by L.E.Kwant et al., "Autoimmun. Rev. 21, 103211, 2022"; and "Inflammatory monocyte-derived dendritic cells mediated autoimmunity in a murine model of systemic lupus erythematosus" by F. Miyagawa, Y. Tagaya, K. Ozato, K. Horie, and H. Asada, "Inflammatory monocyte-derived dendritic cells mediated autoimmunity in a murine model of systemic lupus erythematosus" (J. Transl. Autoimmun. 3, 100060, 2020).

[0121] With regard to SSc, the present applicant has considered the importance of monocyte / macrophage involvement. Specifically, the proportion of circulating monocytes is significantly higher in SSc compared with healthy donors, and the CD16-expressing monocyte subpopulation is higher in diffuse SSc compared with limited SSc. This subpopulation is also associated with more severe skin fibrosis, pulmonary fibrosis, and impaired pulmonary function, suggesting a link between these monocytes and the pathogenesis of fibrosis in SSc. See A. Lescoat et al., "CD16-positive circulating monocytes and fibrotic manifestations of systemic sclerosis," Clin. Rheumatol. 36, 1649-1654, 2017, and P. Laurent et al., "Innate Immunity in Systemic Sclerosis Fibrosis: Recent Advances," Front. Immunol. 9, 2018. Furthermore, the present applicant has hypothesized that profibrotic cells derived from circulating CD14+ monocytes in SSc are associated with interstitial lung disease and contribute to the pathogenesis of SSc, and furthermore, that there is a correlation between the presence of monocytes and the development of fibrosis accompanied by CD14+ monocyte / macrophage infiltration in lung tissue. See N. Higashi-Kuwata 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.

[0122] Polynuclear basophils have been shown to play a detrimental role in lupus erythematosus. The presence of autoreactive IgE has been found in the serum of lupus erythematosus patients, and this presence correlated with clinical severity scores, with a higher rate in patients with lupus nephritis. Polynuclear basophils have been shown to contribute to the maintenance of a pathogenic Th2 environment in lupus erythematosus, inducing the maturation of autoreactive B lymphocytes and the production of autoreactive IgE. Another study showed that polynuclear basophils and the Th2 environment are associated with the development of lupus nephritis. See "Basophils and the T helper 2 environment can promote the development of lupus nephritis" by N. Charles, D. Hardwick, E. Daugas, G. G. Illei, and J. Rivera (Nat. Med. 16, 701-707, 2010) and "The deleterious role of basophils in systemic lupus erythematosus" by C. Pellefigues and N. Charles (Curr. Opin. Immunol. 25, 10.1016 / j.coi.2013.10.003, 2013). In SSc, peripheral polynuclear basophils are involved in fibrosis by stimulating LBs and fibroblasts, suggesting an important role for polynuclear basophils 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 / ).

[0123] The CAR123 of the present invention acts on different immune cells, which is an advantage in the above-mentioned diseases in which different immune cells are involved depending on the etiology.

Claims

1. 1. An isolated chimeric antigen receptor (CAR) molecule for use in treating an autoimmune disease, comprising an antibody or antibody fragment comprising an anti-CD123-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, 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 comprising a complementarity-determining region 1 (CDR1) having at least 90% identity to the amino acid sequence SEQ ID NO:4, 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.

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

3. 3. The isolated chimeric antigen receptor of claim 1, wherein the anti-CD123 binding domain is selected from the group consisting of an antibody, an Fv, an scFv, an Fab, or another antibody fragment, preferably an scFv.

4. 4. The isolated chimeric antigen receptor of claim 1, wherein the intracellular signaling domain is CD3 zeta (CD3ζ) and optionally comprises a costimulatory domain selected from the group consisting of CD28, 4.1BB, inducible T cell costimulatory factor (ICOS), OX-40, or a combination thereof.

5. 2. The isolated chimeric antigen receptor of claim 1, having at least 90% identity, preferably 100% identity, to the nucleic acid sequence SEQ ID NO:

6.

6. 10. An expression vector for use in treating an autoimmune disease, comprising a nucleic acid molecule as defined in claim 5, selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

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

8. 7. An engineered immune cell for use in treating an autoimmune disease, comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

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

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

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

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