Immunotherapy for skeletal myopathy using anti-FAP CAR-T cells

Abstract

The present invention relates to immune cells engineered to express a chimeric antigen receptor (CAR) that specifically binds fibroblast activation protein (FAP) for use in the treatment of skeletal muscle fibrosis in muscular dystrophies.

Description

[Technical Field]

[0001] The present invention relates to the field of immunotherapy. The present invention relates to the use of chimeric antigen receptor (CAR) immune cells specific for fibroblast activation protein for the treatment of fibrosis in skeletal muscle myopathy, particularly for the treatment of skeletal muscle fibrosis associated with muscular dystrophy. The present invention also relates to the use of said CAR immune cells in combination with gene therapy for the treatment of muscular dystrophy. [Background technology]

[0002] Skeletal muscle fibrosis is a common feature of chronic skeletal muscle degenerative disorders, most notably associated with muscular dystrophies and aging. In muscle, fibrosis manifests as the replacement of myofibrils with fibroblasts and extracellular matrix components. Excessive accumulation of fibrous tissue within muscle fibers leads to decreased muscle contractility and ultimately to a decrease in its regenerative capacity and function.

[0003] In addition to worsening the patient's overall condition, fibrosis can also hinder the effectiveness of gene therapy approaches, particularly those involving the delivery of functional genes via viral vectors. Reducing fibrosis could improve the phenotype of muscular dystrophy patients and render muscle more permissive to viral vectors, which should improve the efficacy of gene therapy.

[0004] To date, there are no approved therapies for fibrosis in skeletal muscle myopathies, particularly muscular dystrophies.

[0005] Fibroblast activation protein (FAP) is highly expressed in stromal fibroblasts of the majority of primary and metastatic epithelial tumors, while generally absent from normal adult tissues, and has therefore been identified as a target for the diagnosis and treatment of epithelial cancers. Several monoclonal antibodies have been raised against FAP for research, diagnostic, and therapeutic purposes (WO 2012 / 02006). FAP-specific chimeric antigen receptor (CAR) T cells have also been generated and shown to deplete FAP+ stromal cells and reduce tumor growth in mice (WO 2014 / 055442).

[0006] Recently, FAP-specific CAR-T cells have been shown to reduce cardiac fibrosis in an angiotensin II-induced model of cardiac fibrosis (Aghajanian et al., Nature, 2019, 573, 430-433; WO 2019 / 067425).

[0007] Although the presence of FAP proteins in cancer and cardiac fibrosis is well described, the relative levels of FAP expression in muscular dystrophies and comparisons with other markers are unknown, and therefore it is unclear whether FAP could be a target that could be used for immunotherapy of muscular dystrophies.

[0008] Furthermore, to the best of our knowledge, the use of CAR-T cell approaches to reduce fibrosis in skeletal muscle diseases has not previously been reported.

[0009] Additionally, the efficacy of CAR-T cells in vivo in both humans and mice is highly dependent on the tissue microenvironment and accessibility of the target tissue, regardless of whether solid or liquid targets (tumors or otherwise) are being treated. In the case of tumors, the efficacy of CAR-T has been shown to be limited by the immunosuppressive microenvironment surrounding the tumor, particularly the presence of regulatory T lymphocytes and macrophages, which inhibit the cytolytic activity of CAR-T (Sterner and Sterner, Blood, Cancer Journal, 2021, 11, 69). In muscular dystrophies, such as Duchenne muscular dystrophy, degeneration of muscle fibers creates a significant inflammatory environment due to the presence of macrophage cells secreting TGF-beta (an immunosuppressive cytokine) and other cytokines, as well as regulatory T lymphocytes (Li et al., Frontiers in Immunology, March 2018, doi: 10.3389 / fimmu.2018.00585; Juban et al., Cell Reports, 2018, 25, pp. 2163-2176). This immunosuppressive microenvironment has not been reported in induced cardiac fibrosis and is likely absent given the short timeframe for induction of cardiac fibrosis by pharmacological agents in the studies of Aghajanian et al., Nature, 2019, 573, pp. 430-433 and WO 2019 / 067425. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2012 / 02006 [Patent Document 2] International Publication No. 2014 / 055442 [Patent Document 3] International Publication No. 2019 / 067425 [Patent Document 4] International Publication No. 93 / 05804 [Patent Document 5] International Publication No. 99 / 57151 [Patent Document 6] International Publication No. 01 / 68708 [Patent Document 7] International Publication No. 2007 / 077173 [Patent Document 8] International Publication No. 2012 / 020006 [Patent Document 9] International Publication No. 2019 / 193119 [Patent Document 10] International Publication No. 2020 / 216861 [Patent Document 11] International Publication No. 2021 / 219762 [Patent Document 12] International Publication No. 2022 / 003211 [Patent Document 13] International Publication No. 2022 / 053630 [Non-patent literature]

[0011] [Non-Patent Document 1] Aghajanian et al., Nature, 2019, 573, pp. 430-433 [Non-patent document 2] Sterner and Sterner, Blood,Cancer Journal, 2021, 11, 69 [Non-patent document 3] Li et al., Frontiers in Immunology, March 2018, doi: 10.3389 / fimmu.2018.00585 [Non-patent document 4] Juban et al., Cell Reports, 2018, 25, 2163-2176 [Non-Patent Document 5] Mehrabagi et al., Biomedicine and Pharmacotherapy, 2022, p. 146 [Non-patent document 6] Rafiq et al., Nat. Rev. Clinical Oncology, 2020, 17, 150 [Non-Patent Document 7] Sadelain et al., Nat. Rev. Cancer, 2003, 3, 35-45

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[0012] We generated mRNA expression data in a mouse model of Duchenne muscular dystrophy (the severe DBA2-mdx model) to compare the expression of several fibrosis-related genes, including FAP. Dystrophic mice were compared with age-matched littermates (WT) over time (at 2, 3, or 4 months of age). Results show that the FAP gene is overexpressed in the skeletal muscle of dystrophic mice at all time points compared with WT controls. We tested the therapeutic efficacy of anti-FAP CAR-T cells in muscular dystrophy. We showed that anti-FAP CAR-T cells not only reduced skeletal muscle fibrosis in the DBA2-mdx mouse model of Duchenne muscular dystrophy, but also improved the structure of skeletal muscle fibers at the histological level.

[0013] The inventors also tested the combination of FAP-CAR-T treatment and AAV-microdystrophin gene therapy in a DMD mouse model. The results show that FAP-CAR-T cell-mediated fibrosis reduction can improve AAV vector copy levels in the skeletal muscle of DBA2-mdx mice and improve microdystrophin expression levels in muscle fibers. DBA2-mdx mice treated with the combination therapy exhibit reduced collagen in skeletal muscle and improved structural organization of skeletal muscle fibers. These results provide a novel therapeutic option for treating skeletal muscle fibrosis in muscular dystrophy. The results also show that FAP-CAR-T cell treatment can alleviate the blockage of AAV transduction and provide higher levels of transduction in muscle, potentially reducing the dose required for similar effects.

[0014] Unlike induced cardiac fibrosis, fibrosis in muscular dystrophies is characterized by an inflammatory microenvironment that is known to reduce the antitumor efficacy of CAR-T cells, so it was unclear whether anti-FAP CAR-T cells would act to significantly reduce fibrosis in muscular dystrophies.

[0015] Accordingly, the present invention relates to immune cells engineered to express a chimeric antigen receptor (CAR) that specifically binds fibroblast activation protein (FAP) for use in the treatment of skeletal muscle fibrosis in muscular dystrophy. [Means for solving the problem]

[0016] In some embodiments of immune cells for use according to the invention, the CAR comprises: (i) an extracellular domain comprising at least one antigen-binding domain that specifically binds a FAP or said other protein, (ii) a transmembrane domain, and (iii) an intracellular domain comprising an intracellular signaling domain capable of activating the immune cell, and optionally comprising one or more costimulatory signaling domains.

[0017] In some particular embodiments of the immune cells for use according to the invention, the antigen-binding domain is a single-chain variable fragment (scFv) of a monoclonal antibody that specifically binds a FAP. In some more particular embodiments, the single-chain variable fragment (scFv) that binds the FAP comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2; preferably, it comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8.

[0018] In some particular embodiments of the immune cells for use according to the invention, the extracellular domain further comprises a hinge domain, preferably from an IgG4 heavy chain.

[0019] In some particular embodiments of the immune cells for use according to the present invention, the transmembrane domain is from CD28.

[0020] In some particular embodiments of immune cells for use according to the invention, the intracellular signaling domain is a CD3 zeta signaling domain.

[0021] In some particular embodiments of the immune cells for use according to the invention, the intracellular domain further comprises one or more costimulatory signaling domains, preferably the costimulatory signaling domains of both CD28 and 4-1BB.

[0022] In some preferred embodiments of immune cells for use according to the invention, the CAR comprises, from its N-terminus to C-terminus: a signal peptide derived from a mouse Ig kappa light chain; an scFv fragment derived from an anti-FAP monoclonal antibody, a modified hinge domain derived from an IgG4 heavy chain; a transmembrane domain derived from human CD28, a first costimulatory domain derived from human CD28, a second costimulatory domain derived from human 4-1BB, and an intracellular signaling domain derived from a human CD3 zeta chain; preferably, the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0023] In certain embodiments, immune cells for use according to the invention are lymphocytes such as T cells and / or NK cells, preferably cytolytic lymphocytes such as cytolytic T cells.

[0024] In certain embodiments, immune cells for use according to the invention are modified with a vector selected from an expression vector comprising a nucleic acid construct encoding a CAR, preferably lipid nanoparticles packaging an RNA molecule, and a lentiviral vector, more preferably a self-inactivating and / or VSVG-pseudotyped lentiviral vector.

[0025] In certain embodiments, the immune cells for use according to the present invention reduce the expression level of at least one biomarker of fibrosis, preferably type III collagen.

[0026] In certain embodiments, the immune cells for use according to the present invention are for use in combination with a gene therapy vector for muscular dystrophy, preferably a recombinant AAV vector. In some more specific embodiments, the combination therapy comprises administration of a reduced dose of the gene therapy vector compared to use of the gene therapy vector without immune cells.

[0027] In certain embodiments, the immune cells for use according to the present invention are for the treatment of a muscular dystrophy selected from dystrophinopathies, limb-girdle muscular dystrophies, and congenital muscular dystrophies; preferably Duchenne muscular dystrophy. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides an immunotherapeutic approach to treating skeletal muscle fibrosis in muscular dystrophies using chimeric antigen receptor (CAR) immune cells that are specific for fibroblast activation protein (FAP) and can recognize and deplete fibrotic cells in skeletal muscle.

[0029] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) are well known to those skilled in the art (see, for example, Mehrabagi et al., Biomedicine and Pharmacotherapy, 2022, p. 146; Rafiq et al., Nat. Rev. Clinical Oncology, 2020, 17, pp. 150-159). CARs are synthetic antigen receptors that generally combine an extracellular antigen-binding domain with an intracellular signaling motif involved in lymphocyte activation (Sadelain et al., Nat. Rev. Cancer, 2003, 3, pp. 35-45). CARs comprise three major domains: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains the target antigen-binding region (the FAP-binding region in the present invention) and is involved in antigen recognition. The extracellular domain optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored to the cell membrane of immune effector cells via the transmembrane domain. The optional signal peptide is located at the N-terminus of the extracellular domain. The transmembrane domain connects the extracellular domain to the intracellular domain and resides within the cell membrane when expressed by a cell. An optional hinge domain consisting of a flexible linker that provides flexibility to the binding domain can be inserted between the extracellular domain and the transmembrane domain. The intracellular domain of a CAR transmits an activation signal to immune effector cells after antigen recognition and typically includes an intracellular signaling domain (activation domain) and, optionally, one or more costimulatory signaling domains (costimulatory domains). The intracellular signaling domain generally contains an immunoreceptor tyrosine-based activation motif (ITAM), which activates a signal transduction cascade when the ITAM is phosphorylated. The term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory protein receptor, such as CD28, 4-1BB, ICOS, etc., which can enhance T cell activation via a T cell receptor. The different domains are linked to each other by peptide bonds or linkers.

[0030] CARs have been classified into five distinct generations based on the structure of their intracellular domains. While the first generation of CARs contains only the CD3 chain (CD3 zeta or CD3ζ) or Fc receptor chain (FcR) derived from the CD3 TCR, the second generation includes additional costimulatory signaling domains (CD28 or 4-1BB). Third-generation CARs were generated by combining multiple costimulatory domains, such as CD28-41BB or CD28-OX40, to enhance the potency of CAR-T cells. The fourth generation, also known as T cell redirected for universal cytokine-mediated killing (TRUCK), was generated by further genetic modifications to the second-generation construct, including additional transgenes for cytokine (e.g., IL-2, IL-5, IL-12) secretion or additional costimulatory ligands. Fifth-generation CAR-T cells contain more intracellular domains than their predecessors. CARs contain a truncated intracellular domain of a cytokine (e.g., an IL-2R chain fragment) with motifs that bind transcription factors such as STAT-3 / 5.

[0031] Fibrosis, also known as fibrous scarring, is a pathological process generally resulting from chronic inflammation, in which connective tissue replaces normal parenchymal tissue, leading to extensive tissue remodeling and the formation of permanent scar tissue. Fibrosis is defined as the deposition of excess extracellular matrix, resulting in tissue destruction and impaired organ function. Fibrosis is characterized by fibroblast activation and proliferation and the accumulation of extracellular matrix components, including collagen and glycosaminoglycans, in surrounding connective tissue in response to injury, infection, or other known or unknown disease processes. In fibrosis, the accidental excessive accumulation of extracellular matrix components, such as collagen, produced by fibroblasts leads to the formation of permanent fibrous scarring.

[0032] As used herein, the term "overexpressed in muscular dystrophy" refers to a FAP protein encoded by a FAP gene whose expression level is increased in the skeletal muscle of subjects with muscular dystrophy compared to normal subjects. The gene expression level is preferably increased by at least 1.5-fold, particularly 1.5-fold, 2-fold, 2.5-fold, or 3-fold to 20-fold in the skeletal muscle of subjects with muscular dystrophy compared to normal subjects. Gene expression levels can be measured in mouse models of muscular dystrophy well known to those skilled in the art, such as the DBA2-mdx mouse disclosed in the Examples.

[0033] As used herein, the term "gene expression level" refers to the amount or concentration of a transcription product (or transcript), such as mRNA, or a translation product, such as a protein or polypeptide. Generally, the level of mRNA expression can be expressed in units such as nanograms per cell or microgram of tissue. Polypeptide levels can be expressed, for example, as nanograms per microgram of tissue. Alternatively, relative units can be used to describe gene expression levels. Generally, gene expression levels can be determined by routine techniques well known to those skilled in the art and disclosed in the examples of the present application. Methods for determining the amount of mRNA are well known to those skilled in the art. For example, mRNA contained in a sample is first extracted by standard methods, for example, using lytic enzymes or chemical solutions, or extracted by nucleic acid-binding resins following the manufacturer's instructions. The presence of the extracted mRNA in the sample is then detected by any suitable method, including, but not limited to, spectrophotometry; hybridization such as Northern blot, in situ hybridization such as microarray, RNAscope; sequencing methods such as next-generation sequencing (NGS) and single molecule sequencing; electrochemical, electrical, mechanical, or optical detection based on micro- and nanosensors, and nucleic acid amplification techniques. Nucleic acid amplification methods include isothermal and polymerase chain reaction (PCR)-based techniques, such as reverse transcription PCR (RT-PCR), quantitative PCR (Q-PCR), particularly real-time Q-PCR, RT-qPCR, droplet digital PCR (ddPCR), PCR-HM (high-sensitivity DNA melting), and PCR coupled with a ligase detection reaction based on fluorescent microspheres (Luminex® microspheres). RNA-Seq, also known as whole transcriptome shotgun sequencing (WTSS), is a technique that uses next-generation sequencing (NGS) to determine the amount and sequence of RNA in a sample (reviewed in Wang et al., Nat. Rev. Genet. 2009, 10, 57-63). The method analyzes the transcriptome for gene expression patterns encoded within RNA.The level of a protein can be determined by any suitable method known to those skilled in the art. Typically, these methods involve contacting a cell sample, preferably a cell lysate, with a binding partner capable of selectively interacting with a protein present in the sample. The binding partner is generally a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The method generally involves the use of an appropriate label, such as a fluorescent, chemiluminescent, radioactive, enzyme-labeled, or dye molecule, or other method for detecting the amount of complex formed between the protein and the antibody or antibodies reacted therewith. The amount of a protein can be measured, for example, by semi-quantitative Western blotting, enzyme-labeled and mediated immunoassays such as ELISA, biotin / avidin-type assays, radioimmunoassays, immunoelectrophoresis, mass spectrometry, immunoprecipitation, or protein or antibody arrays.

[0034] As used herein, the term "muscular dystrophy" refers to muscular dystrophies that affect skeletal muscle, particularly muscular dystrophies that primarily affect skeletal muscle, including muscular dystrophies that primarily affect skeletal muscle, muscular dystrophies that affect only skeletal muscle, and muscular dystrophies that do not affect the myocardium (heart) and / or muscular dystrophies that are not associated with heart disease (cardiomyopathies).

[0035] The term "fibroblast activation protein" (fibroblast activation protein alpha, prolyl endopeptidase FAP, or FAP), also known as seprase, refers to a protein encoded by the FAP gene in the mammalian genome. FAP is a homodimeric cell surface glycoprotein belonging to the serine protease family. Representative examples of FAP include, but are not limited to, human (NCBI Gene ID: 2191), mouse (NCBI Gene ID: 14089), and other functional orthologs. Human FAP has the 760 amino acid sequence UniProtKB / Swiss-Prot Q12884. Mouse FAP has the 761 amino acid sequence UniProtKB / Swiss-Prot P97321.

[0036] As used herein, the term "functional ortholog" refers to a gene from another species that encodes a protein with substantially the same activity as the original gene. In the following description, amino acid residues are designated by the standard single-letter amino acid code.

[0037] Unless the context clearly dictates otherwise, the terms "a," "an," and "the" include plural referents. Thus, the terms "a" (or "an"), "one or more," or "at least one" can be used interchangeably herein; "or" means "and / or" unless otherwise specified.

[0038] Like other CARs, the CARs used in the present invention comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises an antigen-binding domain capable of specifically binding a FAP. The antigen-binding domain can be derived from any ligand of a FAP that binds the FAP on a cell and / or binds the extracellular region of the FAP. The ligand (anti-FAP binder) can be a natural ligand, a synthetic ligand, or a variant and / or fragment thereof capable of specifically binding a FAP. Natural ligands include antibodies specific for a FAP (anti-FAP antibodies) and antigen-binding fragments thereof. Synthetic ligands include, for example, oligonucleotides and peptides such as aptamers and DARPins (designed ankyrin repeat proteins), which can be selected by screening libraries of random oligonucleotide or peptide sequences for the ability to specifically bind a FAP using standard assays well known to those skilled in the art.

[0039] A designated ligand specifically binds (or is specific for) a particular target (FAP) if it detectably binds to its target but does not bind (detectably bind) in significant amounts to other proteins present in the sample or to other proteins with which the ligand may come into contact in an organism. Specific binding of a ligand to its target can be determined by standard assays, such as immunoassays, in the case of antibodies. A ligand specifically binds to its target if it has a dissociation constant (KD) of 1 μM or less for the target in a standard KD determination assay. KD values ​​are expressed as molar concentrations (M); KD for antibodies is typically determined by surface plasmon resonance using a Biacore assay.

[0040] As used herein, the term "specifically binds" in reference to an antibody refers to an antibody or antibody fragment that recognizes and binds to a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such cross-species reactivity, in and of itself, does not modify the antibody's classification as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity, in and of itself, does not modify the antibody's classification as specific. In some cases, the terms "specific binding" or "specifically binding" or "specifically binds" in reference to the interaction of an antibody, protein, or peptide with a second chemical species can be used to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies generally recognize and bind to specific protein structures, rather than proteins. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that becomes bound to the antibody.

[0041] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and antibody variants (including derivatives).

[0042] As used herein, the term "antibody fragment" refers to a fragment of an antibody that retains the ability to specifically bind to an antigen (e.g., a FAP). Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single antibody arm; a dAb fragment consisting of the VH domain (Ward et al., 1989, Nature, 341:544-546); nanobodies, VHH single-domain antibody fragments of camelids, and other fragments and any fusion proteins comprising such antigen-binding fragments. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be connected by a synthetic linker, which allows the VL and VH regions to pair using recombinant methods to produce a single-chain protein to form a monovalent molecule (known as a single-chain variable fragment or scFv; see, e.g., Bird et al., 1988, Science 242:423-426; and Huston et al., 1988, Proc. Natl. Acad. Sci. 85:5879-5883). Suitable linkers for scFvs are known to those of skill in the art and include, for example, linkers containing glycine and serine residues. Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained using conventional techniques known to those of skill in the art.

[0043] As used herein, the term "monoclonal antibody" refers to a preparation of monospecific antibody molecules. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from or based on human germline immunoglobulin sequences, or derived from entirely synthetic sequences. The method by which the monoclonal antibody is prepared is not related to the binding specificity.

[0044] As used herein, the term "recombinant antibody" refers to antibodies that are made, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g., a mouse) that is transgenic with human immunoglobulin genes; or antibodies made, expressed, produced, or isolated in any other manner in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

[0045] Various anti-FAP antibodies and derived CARs have been described in the art and can be used in the CARs of the present invention. Non-limiting examples of known anti-FAP antibodies and derived CARs, including anti-human or murine FAP antibodies and chimeric, fully human, and humanized versions thereof, are disclosed in the following references: The well-characterized F19 anti-human FAP monoclonal antibody (ATCC Accession No. HB 8269) is described in WO 93 / 05804. Sibrotuzumab (BIBHB1) is a humanized version of the F19 antibody that specifically binds human FAP described in WO 99 / 57151. WO 99 / 57151 discloses a humanized version of the F19 antibody that specifically binds human FAP and comprises a light chain variable region (VL) of SEQ ID NO: 2 or 20 and a heavy chain variable region (VH) of SEQ ID NO: 8, 10, 12, 14, or 22 (see also Figures 24 and 25). Additional humanized or fully human antibodies against the FAP antigen with F19 epitope specificity have been developed (Mersmann et al., Int. J. Cancer, 2001, 92, 240-248; Schmidt et al., Eur. J. Biochem., 2001, 268, 1730-1738; described in WO 01 / 68708). The OS4 antibody is another humanized (CDR-grafted) version of the F19 antibody (Wuest et al., J. Biotech., 2001, 92, 159-168). The scFv MO33 and scFv MO36 have binding specificities distinct from F19 and are cross-reactive with human and mouse FAP proteins (Brocks et al., Mol. Med., 2001, 7, 461-469). The VH, VL and six CDR sequences of scFv MO33 and scFv MO36 are disclosed in Figure 2 of Brocks et al. (Mol. Med;, 2001, 7, 461-469). A second generation CAR derived from scFv MO36 is disclosed in Kakarla, Mol. Ther., 2013, 21, 1611-1620.Other anti-human or mouse FAP antibodies and chimeric, fully human, and humanized versions thereof are described in WO 2007 / 077173; Ostermann et al., Clin. Cancer Res. 2008, 14, 4584-4592; WO 2012 / 020006; WO 2014 / 055442; Wang et al., Cancer Immunol. Res. 2014, 2, 154-166). In particular, Ostermann et al., Clin. Cancer Res. 2008 discloses the monoclonal antibody FAP5-DM1, which targets a shared epitope in human, mouse, and cynomolgus monkey FAP alpha. Third-generation CAR-T cells derived from mAb FAP5 or sibrotuzumab are disclosed in Tran et al., J. Exp. Med., 2013, 210, 1125-1135. An anti-mouse FAP antibody (clone 73.3) is described in WO 2014 / 055442 and Wang et al., Cancer Immunol. Res., 2014, 2, 154-166. A review of CAR-T targeted to FAP is provided in Bughda et al., Immunotargets Ther., 2021, 313-323.

[0046] Alternatively, new anti-FAP antibodies can be obtained by standard methods well known to those skilled in the art. The antibodies of the present disclosure can be produced by immunizing an experimental animal with an antigen (FAP protein or a fragment thereof, ultimately coupled to a carrier) to induce the production of anti-FAP antibodies by B cells of the mammal; and recovering antibodies from the serum of the immunized animal. To obtain monoclonal antibodies, B cells are isolated from the spleen of the immunized animal and immortalized according to standard hybridoma production techniques. The antibodies of the present disclosure can also be obtained by screening a phage display library. In particular, VH and VL fragments of anti-FAP antibodies can be screened from a phage display library using peptide antigens, and recombinant antibodies can be produced according to standard techniques well known to those skilled in the art. Antibody VH and VL sequences can be obtained by sequencing the receptor of single-cell B cells using the high-throughput sequencing technology disclosed in Goldstein et al., Communications Biology, 2019, 2, 304. The antibodies of the present disclosure can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods well known to those of skill in the art (Morrison, Science, 1985, 229, 1202-1207). For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells (e.g., electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc.). When a recombinant expression vector encoding an antibody gene is introduced into a host cell, particularly a eukaryotic cell such as a mammalian cell, the antibody is produced by culturing the host cell for a period of time sufficient for expression of the antibody in the host cell and, optionally, secretion of the antibody into the culture medium in which the host cell is grown. After secretion, the antibodies may be recovered, for example, from the culture medium and purified using standard protein purification methods (Shukla et al., Journal of Chromatography, 2007, 848, 28-39).In some embodiments, the antigen-binding domain is an antibody fragment, particularly a Fab, nanobody, or single-chain variable fragment (scFv), that specifically binds a FAP. The antibody or antibody fragment can specifically bind a human FAP; can specifically bind a mouse FAP; or can be cross-reactive with human and mouse FAP proteins. In some embodiments, the antibody or antibody fragment specifically binds a human FAP or is cross-reactive with human and mouse FAP proteins.

[0047] In certain embodiments, the antigen-binding domain is an scFv comprising a variable heavy (VH) domain and a variable light (VL) domain having one to six CDR sequences (VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3) of the scFv comprising a variable heavy (VH) domain comprising the amino acid sequence of SEQ ID NO: 1 and a variable light (VL) domain comprising the amino acid sequence of SEQ ID NO: 2. The scFv comprising a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 2 corresponds to clone 73.3 described in WO 2014 / 055442, and has a VH-CDR1 sequence of SEQ ID NO: 3, a VH-CDR2 sequence of SEQ ID NO: 4, a VL-CDR1 sequence of SEQ ID NO: 5, a VL-CDR2 sequence of SEQ ID NO: 6, and a VL-CDR3 sequence of SEQ ID NO: 7. In some preferred embodiments, the scFv comprises a variable heavy (VH) domain comprising the amino acid sequence of SEQ ID NO: 1 and a variable light (VL) domain comprising the amino acid sequence of SEQ ID NO: 2, or a functional variant of said VH and VL sequences. In some preferred embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof.

[0048] In certain embodiments, the antigen-binding domain is derived from a monoclonal antibody that binds to human and mouse FAP. The antibody or antibody fragment may be any of the sequences disclosed in WO 2012 / 020006, including SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 15 It may comprise 1 to 6 CDRs selected from 99, 101, 103, 105, 107, 109, 11, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177. In certain embodiments, the antibodies comprise the following sequences disclosed in WO 2012 / 020006: SEQ ID NOs: 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 32 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309 and 311, in particular comprising both a VH and a VL.

[0049] As used herein, the term "variant" refers to a polypeptide comprising an amino acid sequence having at least 70% sequence identity with a native sequence. The term "variant" refers to a functional variant that retains the activity of the native sequence. The activity of a variant or fragment can be assessed using methods well known to those skilled in the art, such as immunoassays using a monoclonal antibody or antigen-binding fragment thereof against a FAP protein.

[0050] In particular, it is contemplated that a monoclonal antibody or antigen-binding fragment thereof may have one, two, three, four, five, six, or more modifications (i.e., mutations) within the amino acid sequence of one, two, three, four, five, or six CDRs of a monoclonal antibody provided herein. The amino acid mutations may be conservative or non-conservative amino acid insertions, deletions, or substitutions. In certain embodiments, a monoclonal antibody or antigen-binding fragment has one or two conservative substitutions within the amino acid sequence of one, two, three, four, five, or six CDRs of a monoclonal antibody provided herein. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, and threonine).

[0051] The percent amino acid or nucleotide sequence identity is defined as the percent of amino acid residues or nucleotides in a comparison sequence that are identical to a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity; any conservative substitutions in the amino acid sequence are not considered part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as the GCG (Genetics Computer Group, Inc., Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any sequence comparison algorithm such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-), FASTA, or CLUSTALW. When using such software, default parameters are preferably used.

[0052] The extracellular domain may comprise or consist of a FAP antigen-binding domain, particularly an anti-FAP antibody scFv fragment as described herein.

[0053] In some embodiments, the extracellular domain further comprises a hinge domain. The hinge domain consists of a flexible linker that provides flexibility to the antigen-binding domain. The hinge domain may be a natural or synthetic sequence; it can be obtained from any suitable protein. The hinge domain is inserted between the extracellular domain and the transmembrane domain. Examples of hinge domains include those derived from proteins of the immunoglobulin superfamily, such as Ig (heavy chain), particularly IgG1 and IgG4; CD3, CD4, CD28, and CD8 alpha, as well as modified hinge domains thereof (Fujiwara et al., Cells, 2020, 9, pp. 1182-; Peters et al., J. Biol. Chem., 2012, 287, pp. 24525-24533). A specific IgG4 hinge domain is a modified human IgG4 hinge domain containing the S225P mutation corresponding to the sequence of SEQ ID NO: 9. In certain embodiments, the hinge domain is derived from a molecule selected from the group consisting of proteins of the immunoglobulin superfamily, such as: IgG4, IgG1, CD28, CD3, and CD8, etc. In some more particular embodiments, the hinge domain is derived from an IgG4 heavy chain; preferably comprising the sequence of SEQ ID NO:9.

[0054] The extracellular domain can optionally include a signal peptide at its N-terminus. Non-limiting examples of signal peptides include signal peptides from immunoglobulin molecules, particularly Ig kappa light chains, more specifically the signal peptide of SEQ ID NO: 10. The extracellular domain can also optionally include one or more additional antigen-binding domains that specifically bind different antigens. The extracellular domain can include at least two antigen-binding domains that specifically bind different FAP proteins according to the present disclosure. Alternatively, the extracellular domain can include at least one first antigen-binding domain that specifically binds a FAP protein according to the present disclosure. The additional antigen-binding domain can be an antibody scFv fragment or another ligand, such as a cytokine that binds a cytokine receptor present on a target cell. For example, a tandem CAR (TanCAR) system consists of a single CAR polypeptide containing two antigen-binding domains that bind two different antigens from the same target cell, thereby increasing the safety and / or efficacy of CAR-T cells.

[0055] The transmembrane domain may be derived from any transmembrane protein or may be a synthetic sequence. The transmembrane region may be derived from any of the following proteins: the alpha or beta chain of the T cell receptor, CD3 zeta, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha (CD8a), CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 ( CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, I TGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18 , LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96, CEACAM1, CRTAM, Ly9(CD229), CD The CAR may be derived from (i.e., comprise) at least the transmembrane region of 160 (BY55), PSGL1, CD100 (SEMA4D), SLAF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SEPLG ​​(CD162), LTBR, ​​PAG / Cpb, and modified versions thereof (e.g., functional variants thereof). Alternatively, the transmembrane domain may be synthetic and comprise primarily hydrophobic residues such as leucine and valine. A CAR may have two or more transmembrane domains, which may be repeats of the same transmembrane domain or different transmembrane domains. The transmembrane domain may be linked to the intracellular domain via a short sequence, preferably up to 10 amino acids in length. Examples of transmembrane domains include transmembrane domains derived from the CD3 zeta chain, CD8a, or CD28 molecules, particularly human CD3 zeta, CD8a, or CD28 molecules.A particular example of a transmembrane domain derived from human CD28 comprises the sequence of SEQ ID NO: 11. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of human CD28 and human CD8a, and in particular is a human CD28 transmembrane domain comprising the sequence of SEQ ID NO: 11.

[0056] Like other CARs, the CAR used in the present invention comprises a transmembrane domain and an intracellular domain capable of activating immune effector cells. The intracellular domain is the functional end of the CAR that transmits a signal to immune effector cells after antigen recognition and activates at least one of the normal effector functions of immune effector cells. T cell effector function can be, for example, cytolytic activity or helper activity, including cytokine secretion. Immune effector cell activity can be assessed by standard assays well known to those skilled in the art, such as proliferation assays, cytokine assays, and cytotoxicity assays. The intracellular domain can comprise the intracellular signaling domain of a T cell receptor (TCR), optionally a coreceptor. The term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule (protein receptor) other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen.

[0057] Cytoplasmic signaling sequences that regulate the primary activation of TCR complexes acting in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM-containing cytoplasmic signaling sequences include those obtained from CD8, CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD32 (Fc gamma RIIa), DAP10, DAP12, CD79a, CD79b, Fc gamma RI gamma (FcγRIγ), Fc gamma RIII gamma (FcγRIIIγ), FcR epsilon RI beta (FcεRIβ or FCERIB), and Fc epsilon RI gamma (FcεRIγ or FCERIG). Examples of costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, KIR2DS2, and ligands that specifically bind CD123, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MYD88, BTNL3, and NKG2D.

[0058] The T cell surface glycoprotein CD3 zeta (CD3ζ) chain, also known as the T cell receptor T3 zeta chain, TCR zeta chain, or CD247, is encoded by the CD247 gene (gene ID 919 in humans). The human CD3 zeta protein has the amino acid sequence UniProtKB / Swiss-Prot P20963.

[0059] The T cell-specific surface glycoprotein CD28 is encoded by the CD28 gene (gene ID 940 in humans). Human CD28 protein has the amino acid sequence UniProtKB / Swiss-Prot P10747. 4-1BB (CD137), also known as TNF receptor superfamily member 9 or TNFR9, is expressed on the surface of activated T cells and is encoded by the TNFRSF9 gene (gene ID 3604 in humans). Human 4-1BB protein has the amino acid sequence UniProtKB / Swiss-Prot Q07011.

[0060] The intracellular domain comprises an intracellular signaling domain (activation domain) and optionally one or more costimulatory signaling domains (costimulatory domains). The costimulatory signaling region can contain one, two, three, four, or more cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. The costimulatory signaling region can contain one or more mutations in the cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules that enhance signal transduction. Although the entire signaling domain is typically used, a truncated signaling domain may alternatively be used as long as it transmits a signal for effector function. The intracellular signaling domain is typically C-terminal to the intracellular signaling domain, and the CAR and costimulatory signaling domains are N-terminal to the intracellular signaling domain.

[0061] In some embodiments, the intracellular signaling domain is a CD3 zeta signaling domain; particularly a human CD3 zeta signaling domain comprising the sequence of SEQ ID NO:12.

[0062] In some embodiments, the intracellular domain further comprises one or more costimulatory signaling domains from a molecule selected from the group consisting of CD28 and 4-1BB, preferably a human CD28 or 4-1BB molecule. A particular example of a costimulatory signaling domain from human CD28 comprises the sequence of SEQ ID NO: 13. A particular example of a costimulatory signaling domain from human 4-1BB comprises the sequence of SEQ ID NO: 14. The intracellular domain preferably comprises costimulatory signaling domains from both CD28 and 4-1BB, more preferably from human CD28 and 4-1BB.

[0063] The CAR can be preferably a CAR of any generation other than the first generation, and more preferably a CAR of the third or fourth generation.

[0064] The CAR may be any one of a variety of CAR constructions, such as, for example, a multi-chain CAR, a TRUCK, a universal CAR, a self-driving CAR, a weaponized CAR, a self-destruct CAR, a conditional CAR, a marked CAR, a tandem CAR (TanCAR), a dual target CAR, a safety CAR (sCAR), etc.

[0065] A multi-chain CAR contains separate extracellular ligand-binding and signaling domains in different transmembrane polypeptides. The signaling domains can be designed to assemble in a juxtamembrane location. For example, a multi-chain CAR can contain a portion of the FCERI alpha chain and a portion of the FCERI beta chain, and the FCERI chains spontaneously dimerize together to form a CAR.

[0066] Armed CARs are CAR T cells engineered to be resistant to immunosuppression and may be genetically modified to no longer express various immune checkpoint molecules (e.g., cytotoxic T-lymphocyte-associated antigen 4 (CTLA-A4) or programmed cell death protein 1 (PD-1), which contains an immune checkpoint switch receptor), or may be administered with monoclonal antibodies that block immune checkpoint signaling.

[0067] Self-destructive CARs can be designed using RNA delivered by electroporation to encode the CAR. Alternatively, inducible apoptosis of T cells can be achieved based on the binding of ganciclovir to lymphocytes modified with the thymidine kinase gene or the activation of human caspase 9 by a small molecule dimerizer.

[0068] Safety CARs (sCARs) consist of an extracellular scFv fused to an intracellular inhibitory domain. sCAR T cells co-expressing a standard CAR become activated only when they encounter target cells that bear the standard CAR target but lack the sCAR target.

[0069] Dual-targeted CAR T cells express two separate CARs with different ligand binding targets; one CAR contains only the CD3 zeta domain, and the other CAR contains only the costimulatory domain. Dual-targeted CAR T cell activation requires the simultaneous expression of both targets.

[0070] Tandem CAR (TanCAR T) T cells express a single CAR consisting of two linked single-chain variable fragments (scFvs) with different antigen specificities fused to an intracellular costimulatory domain and a CD3 zeta domain; TanCAR T cell activation is achieved only when the target cell co-expresses both targets.

[0071] Conditional CAR Ts are initially unresponsive or switched off until a small molecule is added to complete the cycle, thereby enabling transduction of both signal 1 and signal 2, which activates the CAR T cell. Alternatively, T cells may be engineered to express an adaptor-specific receptor with affinity for a subsequently administered secondary antibody directed against the target antigen.

[0072] In some embodiments, the CAR comprises a human sequence, e.g., a sequence derived from a human molecule or a humanized molecule.

[0073] As with other CARs, the CARs used in the present invention can include additional sequences.

[0074] In certain embodiments, the CAR comprises, from its N-terminus to C-terminus: a signal peptide derived from a mouse Ig kappa light chain (SEQ ID NO: 10); an scFv fragment derived from an anti-FAP monoclonal antibody (anti-FAP scFv of SEQ ID NO: 8 comprising a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 2 connected by a linker of SEQ ID NO: 15), a modified hinge domain derived from a human IgG4 heavy chain (SEQ ID NO: 9); a transmembrane domain derived from human CD28 (SEQ ID NO: 11), a first costimulatory domain derived from human CD28 (SEQ ID NO: 13), a second costimulatory domain derived from human 4-1BB (SEQ ID NO: 14), and an intracellular signaling domain derived from a human CD3 zeta chain (SEQ ID NO: 12); preferably, the CAR comprises the amino acid sequence of SEQ ID NO: 16.

[0075] The CAR is generally delivered to immune cells in the form of a nucleic acid construct comprising a nucleotide sequence encoding the CAR described herein. The nucleic acid construct can comprise or consist of DNA, RNA, or a synthetic or semi-synthetic nucleic acid capable of expression in the target cells (immune cells) of an individual. The sequence encoding the CAR may be codon-optimized for expression in the immune cells of the individual, preferably a human individual. Suitable software for codon optimization in a desired individual is well known to those skilled in the art and is publicly available. In some embodiments, the nucleotide sequence encoding the CAR comprises the sequence of SEQ ID NO: 17. SEQ ID NO: 17 is the nucleotide sequence encoding the CAR amino acid sequence of SEQ ID NO: 16.

[0076] The nucleic acid construct comprises an expression cassette, in which the coding sequence is operably linked to a suitable regulatory sequence for expressing the transgene in target cells (immune cells) of an individual. Such sequences are well known to those skilled in the art, and include, in particular, promoters, as well as additional regulatory sequences that can further control the expression of the transgene, including, but not limited to, enhancers, terminators, introns, silencers, especially tissue-specific silencers such as miRNAs, and post-translational regulatory elements.

[0077] The promoter may be a tissue-specific, ubiquitous, constitutive, or inducible promoter that functions in the target cells of an individual. Examples of constitutive promoters that can be used in the present invention include, but are not limited to, the phosphoglycerate kinase promoter (PGK); promoters including the short form (EFS) of the elongation factor-1 alpha (EF-1 alpha) promoter; the dihydrofolate reductase promoter; the β-actin promoter; and viral promoters such as the cytomegalovirus (CMV) immediate-early enhancer and promoter, the cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, the SV40 early promoter, and retroviral 5' and 3' LTR promoters, including hybrid LTR promoters. A preferred ubiquitous promoter is the EF-1 alpha promoter. An example of an inducible promoter that can be used in the present invention is a tetracycline-regulated promoter. The promoter is preferably a human promoter, i.e., a promoter derived from human cells or human viruses. Such promoters are well known to those skilled in the art, and their sequences are available in public sequence databases.

[0078] Furthermore, transgene expression levels can be improved by including post-transcriptional regulatory elements, such as the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE), in the transgene expression cassette, which can increase transcript levels and / or stability.

[0079] Engineered immune cells Immune cells include any white blood cells involved in the body's defense against foreign insults. Immune cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. As used herein, immune cells include immune effector cells and their precursors. Immune effector cell precursors have a higher proliferative potential. Thus, the engineered immune cell population preferably contains effector cells and less differentiated cell subsets with a higher proliferative potential.

[0080] In some embodiments, the immune cells are lymphocytes, particularly T cells and / or NK cells. Lymphocytes include T lymphocytes such as alpha-beta T cells and gamma-delta T cells; natural killer (NK) cells; natural killer T (NKT) cells; cytokine-induced killer cells; cytotoxic T lymphocytes (CTL); and lymphokine-activated killer (LAK) cells. The effector function of lymphocytes, such as T cells or NK cells, can be cytolytic activity. The effector function of T cells can also be helper activity. The effector function (cytolytic or helper) of T cells includes the secretion of cytokines. T cells are CD3+ cells. NK cells are CD56+CD3- cells. Cytotoxic T cells (or CTL) are CD8+ T cells. Helper T cells are CD4+ T cells. T cell precursors include naive T cells, stem cell memory T cells (T SCM ) cells and central memory T(T CM ) cells.

[0081] In certain embodiments, the immune effector cells are cytolytic lymphocytes, more specifically cytolytic T cells. The immune cells can include CD8+ T cells or a mixture of CD4+ and CD8+ T cells, for example, a 1:1 ratio of CD4+ to CD8+ T cells. The T cells can include naive T cells, stem cell memory T cells (T SCM ) cells and central memory T(T CM The T cells preferably contain a high percentage of more proliferative, less differentiated T cell subsets, such as T cell subsets with higher proliferation potential, such as T cell subsets with higher proliferation potential, such as T cell subsets with lower differentiation ... lower differentiation potential, such as T cell subsets with higher proliferation potential, such as T cell subsets with lower differentiation potential, such as T cell subsets with lower differentiation potential, such as T cell subsets with lower differentiation potential, such as T cell subsets with lower differentiation potential, such as T cell subsets with lower differentiation potential, such as T cell subsets with lower differentiation potential, such as

[0082] Immune cells are preferably obtained from the subject to be treated (autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) may be used. Allogeneic refers to histocompatible (HLA-matched) cells.

[0083] As used herein, the terms "individual," "subject," or "patient" include human and other mammalian subjects. Preferably, a patient, individual, or subject according to the present invention is a human.

[0084] Immune cells (including effector cells and / or their precursors) can be obtained from several sources, such as blood or other body fluids and lymphoid tissues or organs (bone marrow, spleen, lymph nodes, umbilical cord blood, thymus), as well as others known to those skilled in the art. In some embodiments, immune cells can be obtained from blood collected from a subject, particularly the subject to be treated.

[0085] Immune cells are isolated and cultured using standard methods well known to those skilled in the art. Immune cells can be isolated by leukapheresis, which typically uses differential centrifugation techniques to separate leukocytes from whole blood. Specific subpopulations of immune cells, particularly T cells, can be further isolated by positive or negative selection techniques, for example, using flow cytometry-assisted cell sorting or magnetic-activated cell separation. For example, immune cells, particularly T cells, can be isolated using a combination of antibodies directed against surface markers specific to the positively selected cells, for example, by incubation with antibody-conjugated beads for a time sufficient for positive selection of the desired immune cells. Alternatively, enrichment of immune cells, particularly T cells, can be performed by negative selection using a combination of antibodies directed against surface markers specific to the negatively selected cells. Isolated immune cells, particularly T cells, can be selected from a wide variety of immune cell types, including naive T cells, stem cell memory T cells (T cells), and pluripotent stem cells (T cells). SCM ) cells and central memory T(T CM The cells preferably contain a high percentage of more proliferative, less differentiated cell subsets, such as .

[0086] The isolated immune cells, particularly T cells, are cultured and optionally activated in an appropriate culture medium, e.g., supplemented with cytokines for T cells, such as IL-7 and IL-15. The isolated immune cells, particularly T cells, are then genetically modified with a CAR construct to express the CAR on their surface. The genetically modified (i.e., engineered) immune cells expressing the CAR can be expanded in an appropriate culture medium, e.g., supplemented with cytokines for T cells, such as IL-7 and IL-15, before administration to a patient. The immune cells can be engineered in the presence of kinase inhibitors to generate CAR T cells with a less differentiated phenotype, to improve engraftment, in vivo expansion, and effector activity of these cells.

[0087] Immune cells are genetically modified using standard nucleic acid delivery agents or systems suitable for delivery and expression of nucleic acids to immune effector cells of an individual, particularly suitable for gene therapy. The present invention can use chemical, biological, or physical delivery methods, or a combination thereof. Such vectors, which are well known to those skilled in the art, include viral and non-viral vectors, which may be integrating or non-integrating; and may be replicating or non-replicating.

[0088] The vector comprises a nucleic acid construct comprising the CAR in an expressible form as described above. To assess the expression of the CAR polypeptide, the expression vector introduced into cells can also contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells attempted to be transfected or infected with the expression vector. The selectable marker or reporter gene can be expressed from the same promoter as the CAR or from different promoters. To express the CAR and the selectable marker or reporter gene from the same promoter, an internal ribosome entry site (IRES) or a viral 2A peptide (T2A) is inserted between the two coding sequences. The viral 2A peptide is a self-cleavable peptide. For example, the vector can further comprise CD19 or a detectable fragment thereof (e.g., detectable with a CD19-specific antibody).

[0089] Non-viral vectors include various (non-viral) agents commonly used to either introduce or maintain nucleic acids in the cells of an individual. Agents used to introduce nucleic acids into the cells of an individual by various means include, but are not limited to, nanoparticles, including cationic polymers, dendrimers, micelles, liposomes, exosomes, microparticles, and lipid nanoparticles (LNPs); and cell-penetrating peptides (CPPs), such as polymer-based, particle-based, lipid-based, and peptide-based delivery vehicles, or combinations thereof. CPPs are, in particular, cationic peptides, such as poly-L-lysine (PLL), oligoarginine, Tat peptide, penetratin, or transportan peptide, and derivatives thereof, such as Pip. Agents used to maintain nucleic acids in the cells of an individual (either chromosomally integrated or in an extrachromosomal form) include, in particular, naked nucleic acid vectors, such as plasmids, transposons, and minicircles, as well as gene editing and RNA editing systems. Transposons include, in particular, the hyperactive Sleeping Beauty (SB100X) transposon system (Mates et al., 2009). Gene editing and RNA editing systems can use any site-specific endonuclease, such as Cas nucleases, TALENs, meganucleases, zinc finger nucleases, etc. Nucleic acids can also be introduced into an individual's cells by physical means, such as particle bombardment, microinjection, electroporation, etc. In addition, these techniques can be advantageously combined to introduce and maintain the nucleic acids of the present invention in an individual's cells.

[0090] Viral vectors are naturally capable of entering cells and delivering nucleic acids of interest into cells through a process termed viral transduction. As used herein, the term "viral vector" refers to a non-replicating, non-pathogenic virus engineered to deliver genetic material into cells. In viral vectors, viral genes essential for replication and pathogenicity are replaced with an expression cassette for a transgene of interest. Thus, the viral vector genome contains the transgene expression cassette flanked by viral sequences required for viral vector production. As used herein, the term "recombinant virus" refers to viruses, particularly viral vectors, produced by standard recombinant DNA engineering techniques known to those skilled in the art. As used herein, the term "virus particle" or "viral particle" refers to the extracellular form of a non-pathogenic virus, particularly a viral vector composed of genetic material made from either DNA or RNA surrounded by a protein coat called a capsid, optionally derived from a portion of the host cell membrane and containing viral glycoproteins. As used herein, viral vector refers to a viral vector particle.

[0091] The first type of vector for delivering the nucleic acid (nucleic acid construct) of the present invention is a viral vector, particularly suitable for gene therapy of an individual's immune cells. In particular, viral vectors can be derived from non-pathogenic parvoviruses such as adeno-associated viruses (AAVs), retroviruses such as gammaretroviruses, spumaviruses, and lentiviruses, and adenoviruses. The viral vector is preferably an integrating vector, such as an AAV or lentiviral vector, preferably a lentiviral vector. The vector contains viral sequences required for viral vector production, such as lentiviral LTR sequences or AAV ITR sequences, adjacent to the expression cassette. Lentiviral vectors can be pseudotyped with other viral envelope glycoproteins, preferably VSV-G. Pseudotyped lentiviral vectors improve the transduction of an individual's immune cells. Advantageously, the lentiviral vector is a self-inactivating (SIN) lentiviral vector (third generation), as originally described by Zufferey et al. (J. Virol., 1998, 72, 9873-9880). In certain embodiments, the lentiviral vector is derived from the lentiviral transfer plasmid of SEQ ID NO: 19 or 20.

[0092] Another type of vector for delivering the nucleic acid (nucleic acid construct) of the invention is a particle or vesicle, a macromolecule complex, a nanocapsule, a microsphere, a bead, and a lipid-based system, including an oil-in-water emulsion, a micelle, a mixed micelle, and in particular a lipid-based micro- or nano-vesicle or particle, such as a liposome or a lipid nanoparticle (LNP). In a more particular embodiment, the nucleic acid is RNA and the vector is a particle or vesicle as described above.

[0093] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure as micelles or in a "collapsed" structure. They can also simply be dispersed in solution, optionally forming aggregates of varying size or shape. Lipids are fatty substances that can occur in nature or are synthetic. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0094] Lipids suitable for use can be obtained commercially. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories, Inc. (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991, Glycobiology 5:505-10). However, compositions with structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids can assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine nucleic acid complexes are also contemplated.

[0095] therapeutic use The engineered immune cells according to the present disclosure are used in adoptive cell therapy (ACT) of skeletal muscle fibrosis associated with muscular dystrophy.

[0096] Adoptive cell therapy (ACT), also known as adoptive T cell therapy, adoptive cell transfer, cellular adoptive immunotherapy, or T cell transfer therapy, is a type of immunotherapy in which immune cells, specifically T cells, are engineered to recognize an antigen of interest for administration to the patient (redirected T cell immunotherapy, CAR T cell therapy) to help the immune system fight disease.

[0097] Immune cells used in adoptive cell therapy can be autologous or allogeneic immune cells. Allogeneic refers to histocompatible (HLA-matched) cells. For example, immune cells obtained from a subject's sample, particularly a population of lymphocytes such as T cells, are engineered to express a CAR specific for a FAP according to the present disclosure and are re-administered to a subject with skeletal fibrosis associated with muscular dystrophy. Alternatively, allogeneic immune cells, particularly a population of T cells obtained from a donor's sample, are engineered to express a CAR specific for a FAP according to the present disclosure and are administered to a recipient subject with skeletal fibrosis associated with muscular dystrophy.

[0098] The engineered immune cells may be delivered to an individual in need thereof by any suitable means, such as, for example, intravenous injection (infusion or perfusion) or injection in the tissue of interest (transplantation).

[0099] Skeletal muscle fibrosis is a common feature of chronic skeletal muscle degenerative disorders such as muscular dystrophies. Muscular dystrophies are a group of genetically and clinically diverse neuromuscular diseases that gradually cause progressive weakness and breakdown of skeletal muscles. Despite the variability of clinical symptoms, dystrophies share a common histological finding characterized by variations in muscle fiber size and myofibril necrosis. Muscular dystrophies are caused by spontaneous or genetic mutations in various genes involved in muscle development or function. Muscular dystrophies can be X-linked recessive, autosomal recessive, or autosomal dominant.

[0100] More than 30 different disorders are classified as muscular dystrophies. Of these, Duchenne muscular dystrophy (DMD) accounts for approximately 50% of cases and affects males, starting around age 4. Other relatively common muscular dystrophies include Becker muscular dystrophy (BMD); facioscapulohumeral muscular dystrophy and tonic muscular dystrophy, while limb-girdle muscular dystrophies and congenital muscular dystrophies are themselves groups of several genetic disorders that are usually quite rare. Dystrophinopathies are a range of X-linked muscle disorders caused by pathogenic variants in the DMD gene, which encodes the protein dystrophin. Dystrophinopathies include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Limb-girdle muscular dystrophies (LGMDs) are a group of disorders that are clinically similar to DMD but occur in both sexes as a result of autosomal recessive and autosomal dominant inheritance. Limb-girdle dystrophies result from mutations in genes encoding sarcoglycans and other proteins associated with muscle cell membranes that interact with dystrophin. The term LGMD1 refers to the dominantly inherited (autosomal dominant) genotype, while LGMD2 refers to the autosomal recessive genotype. Pathogenic variants have been reported at more than 50 loci (LGMD1A-LGMD1G; LGMD2A-LGMD2W). Calpainopathy (LGMD2A) results from mutations in the CAPN3 gene, and more than 450 pathogenic variants have been described.Genes that contribute to the LGMD phenotype include: anoctamin 5 (ANO5), vascular epicardial substance (BVES), calpain 3 (CAPN3), caveolin 3 (CAV3), CDP-L-ribitol pyrophosphorylase A (CRPPA), dystroglycan 1 (DAG1), desmin (DES), DnaJ heat shock protein family (Hsp40) homolog, subfamily B, member 6 (DNAJB), dysferlin (DYSF), fukutin-related protein (FKRP), fukutin (FKTN), GDP mannose pyrophosphorylase B (GMPPB), heterogeneous nuclear ribonucleoprotein D-like (HNRNPDL), LIM zinc finger domain-containing 2 (LIMS2), laminin A / C (LMNA), myotilin (MYOT), plectin (PLEC), and protein Protein O-glucosyltransferase 1 (PLOGLUT1), protein O-linked mannose N-acetylglucosaminyltransferase 1 (beta 1,2-) (POMGNT1), protein O-mannose kinase (POMK), protein O-mannosyltransferase 1 (POMT1), protein O-mannosyltransferase 2 (POMT2), sarcoglycan alpha (SGCA), sarcoglycan beta (SGCB), sarcoglycan delta (SGCD), sarcoglycan gamma (SGCG), titin cap or telethonin (TCAP), transportin 3 (TNPO3), torsin1A-interacting protein (TOR1AIP1), transport protein particle complex 11 (TRAPPC11), E3-ubiquitin ligase or tripartite motif-containing 32 (TRIM 32), and titin (TTN). Major genes contributing to the LGMD phenotype include CAPN3, DYSF, FKRP, and ANO5 (Babi Ramesh Reddy Nallamilli et al., Annals of Clinical and Translational Neurology, 2018, 5, 1574-1587). - Emery-Dreifuss muscular dystrophy (EDMD), which is caused by a defect in one of the genes, including the EMD gene (encoding emerin), the FHL1 gene, and the LMNA gene (encoding lamins A and C). - Nesprin-1 and Nesprin-2 associated muscular dystrophy, caused by defects in the SYNE1 and SYNE2 genes, respectively; LUMA associated muscular dystrophy, caused by defects in the TMEM43 gene; LAP1B associated muscular dystrophy, caused by defects in the TOR1AIP1 gene. Facioscapulohumeral muscular dystrophy, type 1 (FSHD1A), such as those associated with defects in the DUX4 gene (shortening of the D4Z4 macrosatellite repeat in the peritelomeric region of chromosome 4q35) or the FRG1 gene; Facioscapulohumeral muscular dystrophy, type 2 (FSHD1B), caused by defects in the SMCHD1 gene. - Oculopharyngeal muscular dystrophy (autosomal dominant) caused by a defect in the PABPN1 gene, which encodes polyadenate-binding protein nuclear 1. - Tonic muscular dystrophy (autosomal dominant) caused by a deficiency in the DMPK gene (type 1) encoding DM1 protein kinase or the CNBP gene (type 2) encoding CCHC-type zinc finger nucleic acid binding protein. - Congenital muscular dystrophies (CMDs) are a diverse group of disorders characterized by muscle weakness present at or shortly after birth and various clinical manifestations of the eye and central nervous system. With some exceptions, CMDs are inherited in an autosomal recessive manner. At least 35 genes have been implicated in CMDs that are abnormal at different cellular levels: - extracellular matrix MDC1A: LAMA2 gene; Collagen 6-related dystrophies (UCMD, BM): COL6A1, COL6A2, COL6A3 genes; Integrinopathies: ITGA7, ITGA9 genes; CMD with hyperlaxity: ITGA9, LAMR1, ACVR2B genes. - Dystrophin-related glycoprotein complex: alpha-dystroglycanopathy (DGP): DAG1, POMPT1, POMPT2, POMGNT1, POMGNT2 / GTDC2, FKTN, FKRP, LARGE, ISPD, TMEM5, B3GALNT2; B4GAT1 / B3GNT1, DPM1, DPM2, DPM3, POMK / SKG196, GMPPB, DOLK, DAG1 and INPP5K genes. - ER: Ankylosing spine syndrome [RSMD1]: SEPN1 gene - nuclear envelope: LMNA-related CMD; [L-CMD]: LMNA gene; nesprin-1-related CMD: SYNE1 gene, - ER to Golgi transport: CMD associated with fatty liver and childhood-onset cataract: TRAPCC11, GOSR2 genes. - Other CMDs: RYR1, CHKB, MSTO1, MICU1, INPP5K, SIL-1, MCOLN1, GGPS1, FHFL1, ALG13, ACTA1 genes. The most common forms of CMD are COL6-associated disease, dystroglycanopathy (DGP), laminin alpha 2 (LAMA2)-associated dystrophy, and selenium-containing protein N-associated myopathy (SEPN1). - Other genes: polymerase I and transcript release factor (PTRF), heterogeneous nuclear ribonucleoprotein D-like (HNRNPDL), plectin (PLEC), valosin-containing protein (VCP), LIM and senescent cell antigen-like domain 2 (LIMS2).

[0101] The engineered immune cells of the present disclosure are used to treat skeletal fibrosis in muscular dystrophy, which means treating skeletal muscle fibrosis associated with muscular dystrophy.The engineered immune cells of the present disclosure can immunologically target and deplete fibrotic cells in the skeletal muscle of a subject suffering from muscular dystrophy.The engineered immune cells of the present disclosure provide a treatment for muscular dystrophy, particularly in combination with gene therapy.

[0102] Administration of engineered immune cells according to the present disclosure reduces skeletal muscle fibrosis in subjects suffering from muscular dystrophy. Reduction of skeletal muscle fibrosis can be determined using standard methods well known to those skilled in the art and disclosed in the Examples. These methods include, inter alia, measuring the expression levels of fibrosis biomarkers, particularly type III collagen (type III collagen alpha 1 chain; COL3A1 gene) or other biomarkers; histological or immunohistological analysis of skeletal muscle samples; and combinations thereof. In some embodiments, administration of engineered immune cells according to the present disclosure to subjects suffering from muscular dystrophy reduces the level of type III collagen (Col3) in skeletal muscle compared to untreated controls.

[0103] In some embodiments, the engineered immune cells are used for the treatment of muscular dystrophies selected from: dystrophinopathies, limb-girdle muscular dystrophies and congenital muscular dystrophies, particularly dystrophinopathies associated with mutations in the DMD gene; limb-girdle muscular dystrophies (LGMD) associated with mutations in the CAPN3, DYSF, FKRP, ANO5, DNAJB6, SGCA, SGCB or SGCG genes, and congenital muscular dystrophies associated with mutations in the COL6A1, COL6A2, COL6A3, LAMA2 or SEPN1 genes; preferably Duchenne muscular dystrophy (DMD gene).

[0104] In some embodiments, engineered immune cells, preferably in combination with gene therapy, are used to treat skeletal muscle disease or injury in patients suffering from muscular dystrophy. This means that immunotherapy using engineered immune cells according to the present disclosure, or immunotherapy in combination with gene therapy, is used to treat the skeletal muscle damage component of muscular dystrophy. The treatment is particularly used to improve skeletal muscle function, thereby reversing symptoms associated with skeletal muscle disease, such as muscle weakness, muscle stiffness, difficulty walking, climbing, running, or rising from a sitting or lying position, frequent falls, etc.

[0105] In some embodiments, engineered immune cells are used to treat forms of muscular dystrophy that primarily or exclusively affect skeletal muscle (skeletal muscular dystrophy). In some embodiments, engineered immune cells are used to treat forms of muscular dystrophy that do not affect the myocardium (heart) and / or are not associated with heart disease (cardiomyopathy). In some embodiments, engineered immune cells are used to treat muscular dystrophy in subjects who do not have cardiomyopathy.

[0106] As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent or combination of therapeutic agents to a patient, or to an isolated tissue or cell line from a diseased patient for the purpose of curing, ameliorating, alleviating, mitigating, altering, correcting, ameliorating, improving, or affecting the disease or any symptom of the disease. In particular, the term "treat" or "treatment" refers to the reduction or alleviation of at least one adverse clinical symptom associated with the disease.

[0107] The terms "treatment" or "treating" are also used herein in the context of prophylactic administration of a therapeutic agent.

[0108] In various embodiments of the invention, the pharmaceutical composition comprises a therapeutically effective amount of engineered immune cells. A therapeutically effective amount, in the context of the present invention, refers to a dose sufficient to reverse, alleviate, or inhibit the progression of the disorder or condition to which such term applies, or to reverse, alleviate, or inhibit the progression of one or more symptoms of the disorder or condition to which such term applies. The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0109] The effective dose will be determined and adjusted depending on factors that will be recognized by those skilled in the medical arts, such as the composition used, the route of administration, the physical characteristics of the individual under consideration, such as sex, age, and weight, concurrent medications, and other factors. Effective doses can be determined by standard clinical techniques. Additionally, in vivo and / or in vitro assays can optionally be used to predict optimal dosage ranges.

[0110] The exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, and symptoms of the patient (subject). The pharmaceutical compositions comprising T cells described herein can be administered in amounts up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 It can generally be stated that T cell compositions can be administered at a dosage of 1000 cells / kg body weight. The T cell compositions can be administered multiple times at these dosages. The cells can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med., 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0111] The dosages of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to the routine of one of ordinary skill in the art. Strategies for dosing and scheduling of CAR T cells have been reviewed (Ertl et al., 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

[0112] In certain embodiments, it may be desirable to administer activated T cells to a subject, then subsequently draw blood again (or perform apheresis), activate T cells therefrom according to the present invention, and reinfuse these activated, expanded T cells into the patient. This process may be performed multiple times, every few weeks. In certain embodiments, T cells may be activated from a blood draw of 20 cc to 400 cc. In certain embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Without being bound by theory, the use of this multiple blood draw / multiple reinfusion protocol may be useful for selecting specific populations of T cells.

[0113] In various embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or vehicle.

[0114] A "pharmaceutically acceptable carrier" refers to a vehicle in which a therapeutic is administered that does not produce adverse, allergic, or other untoward reactions when properly administered to a mammal, especially a human. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0115] Preferably, the pharmaceutical composition contains a vehicle that is pharmaceutically acceptable for injectable preparations, and can in particular be isotonic sterile saline (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or a dried, in particular lyophilized, composition that can be made into an injectable solution by addition of sterile water or physiological saline, as the case may be.

[0116] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions. The solutions or suspensions may contain additives that are compatible with the viral vector and do not interfere with the entry of the viral vector particles into target cells. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Examples of suitable solutions are buffer solutions such as phosphate-buffered saline (PBS) or Ringer's lactate.

[0117] The pharmaceutical compositions may also include additional therapeutic agents, particularly agents useful in the treatment of diseases according to the present disclosure.

[0118] The pharmaceutical compositions of the present invention are generally administered according to known procedures at dosages and for periods effective to induce a therapeutic effect in patients. The pharmaceutical compositions may be administered by any convenient route, including but not limited to, by injection, perfusion, or implantation. Administration may be systemic, local, or a combination of systemic and local. Systemic administration is preferably intravascular, such as intravenous (IV) or intraarterial; intraperitoneal (IP); intradermal (ID), epidural, etc. In some preferred embodiments, administration is parenteral, preferably intravascular, such as intravenous (IV) or intraarterial. Parenteral administration is advantageously by injection or perfusion.

[0119] The engineered immune cells or pharmaceutical compositions described herein can be used in combination with other biologically active agents, and the combined use can be simultaneous, separate or sequential administration.

[0120] In some embodiments, immunotherapy according to the present invention is combined with gene therapy for muscular dystrophy. Gene therapy is advantageously carried out using recombinant AAV (rAAV) vectors, particularly those disclosed in WO 2019 / 193119, WO 2020 / 216861, WO 2021 / 219762, WO 2022 / 003211, and WO 2022 / 053630. The recombinant AAV vector contains a transgene intended for use in gene replacement or gene editing of a mutant gene associated with the muscular dystrophy being treated in a subject. In certain embodiments, immunotherapy according to the present invention is combined with gene therapy for muscular dystrophies selected from dystrophinopathy, limb-girdle muscular dystrophies, and congenital muscular dystrophies; more specifically, dystrophinopathy (DMD gene), limb-girdle muscular dystrophies (LGMD) (CAPN3, DYSF, FKRP, ANO5, DNAJB6 SGCA, SGCB, or SGCG genes), and congenital muscular dystrophies (COL6A1, COL6A2, COL6A3, LAMA2, or SEPN1 genes); preferably, dystrophinopathy (DMD gene). In some more specific embodiments, the combination therapy involves administration of a gene therapy vector at a reduced dose compared to the use of a gene therapy vector without immune cells. The possibility of reducing the dose of the administered gene therapy vector potentially means higher functional efficacy and a lower risk of toxicity. In certain embodiments, gene therapy for the disease is performed after immunotherapy according to the present invention.

[0121] The present invention provides a pharmaceutical combination for use in the treatment of skeletal fibrosis in a subject in need thereof, comprising a therapeutically effective amount of: (i) an immune cell engineered according to the present disclosure; and (ii) A pharmaceutical combination comprising a gene therapy vector, preferably a recombinant AAV vector according to the present disclosure.

[0122] In some embodiments, the combination pharmaceutical is for sequential use and the treatment includes at least one initial administration of the engineered immune cells prior to administration of the gene therapy vector, preferably an rAAV vector.

[0123] The present invention provides a method for treating skeletal fibrosis in the above-mentioned muscular dystrophies, comprising: administering to a patient in need thereof a therapeutically effective amount of the above-mentioned pharmaceutical composition; and preferably further comprising administration of a therapeutically effective amount of a gene therapy vector, particularly an rAAV vector, according to the present disclosure.

[0124] A further aspect of the present invention relates to the use of engineered immune cells according to the present disclosure in the manufacture of a medicament for treating skeletal fibrosis in muscular dystrophy according to the present disclosure.

[0125] Another aspect of the present invention relates to the use of engineered immune cells according to the present disclosure, preferably in combination with a gene therapy vector according to the present disclosure, more preferably an rAAV vector, to treat skeletal fibrosis in muscular dystrophies according to the present disclosure.

[0126] A further aspect of the present invention relates to a pharmaceutical composition comprising engineered immune cells according to the present disclosure as an active ingredient for treating skeletal fibrosis in muscular dystrophies according to the present disclosure.

[0127] The present invention also relates to the use of engineered immune cells according to the present disclosure in therapies to reduce fibrosis and enhance the efficacy of AAV gene transfer.

[0128] The various embodiments of the present disclosure may be combined with each other, and the present disclosure encompasses various combinations of the embodiments of the present disclosure.

[0129] The practice of the present invention will employ, unless otherwise indicated, conventional techniques that are within the skill of those skilled in the art, such techniques being explained more fully in the literature.

[0130] The present invention will now be illustrated by the following non-limiting examples with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0131] [Figure 1] Schematic representation of third-generation lentiviral vectors (A) pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE and (B) pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-WPRE, in which the scFv against mFAP is fused to the T cell signaling domains CD28, 4.1BB, and CD3ζ. The selection marker ΔCD19 is also shown (A only). [Figure 2] Figure 1 shows multicolor flow cytometry analysis of the phenotype of untransduced (left panel) or transduced (right panel) T lymphocytes (LT) after 7 days of activation, including 5 days of transduction (PT). Cells were immunolabeled with mouse anti-CD3, anti-CD4, and anti-CD8, and human anti-CD19, prior to analysis, and analysis was performed gated on live cells. [Figure 3] Figures showing mouse FAP expression in 3T3 and 3T3-FAP cells. (A) mRNA FAP expression measured by ddPCR in 3T3, 3T3-FAP, and a mixed (50:50) population of 3T3 and 3T3-FAP cells. FAP expression was confirmed to be absent in 3T3 and specifically expressed in the stable cell line 3T3-FAP. (B) Mouse FAP expression was revealed by immunocytochemistry using a mouse anti-FAP antibody. [Figure 4] Figure 1 shows luciferase assays after 24 hours of co-culture of 3T3-Luc2 or 3T3-FAP-Luc2 with T cells or FAP-CAR-T at ratios of 1:7.5 and 1:15 (7.5 or 15 T to 1 3T3). The results correspond to two experiments performed in triplicate (one point corresponds to one value). p values ​​were calculated using Student's T-test. [Figure 5]Figure 1 shows degranulation assay after co-culture of 3T3-Luc2 or 3T3-FAP-Luc2 with T cells or FAP-CAR-T at a ratio of 1:15 for 6 hours. [Figure 6] Dynamics of FAP (top) and Col3 (bottom) expression in the tibialis (TA), gastrocnemius (GA), and extensor digitorum longus (EDL) muscles of DBA2 and DBA2-MDX mice aged 2-4 months compared to P0. Statistical analysis: Unpaired Student's T-test. [Figure 7] Figure 1 shows a schematic representation of FAP-CAR-T treatment and details of each study. DBA2-MDX mice aged 2-3 months were administered two consecutive doses (low or high) of FAP-CAR-T cells, and skeletal muscle was harvested for molecular and histological analysis 2 weeks after the second injection. The number of injected T cells (Nb) represents the total number of cells in the CAR-T cell preparation. The input dose was calculated from the vector copy number (VCN) in the CAR-T cell preparation and represents the active ingredient. These input numbers varied depending on the specific preparation of CAR-T cells produced and are reported in the table for each injection. [Figure 8] FAP (top) and Col3 (bottom) mRNA expression in the tibialis (TA), extensor digitorum longus (EDL), and heart of DBA2 (WT) and DBA2-MDX mice control (white bars) or treated with LT (darker gray bars) or high doses of FAP-CAR-T cells (1 x 10 cells, light gray bars) (n = 6 mice per group). Statistical analysis: Mann-Whitney unpaired t-test. [Figure 9A] 1 shows Sirius red sections of the TA, EDL and heart of DBA2 mice, untreated DBA2-MDX control and treated DBA2-MDX mice. Dark staining intensity represents collagen fibers (Obf: 10×). [Figure 9B] 1 shows Sirius red sections of the TA, EDL and heart of DBA2 mice, untreated DBA2-MDX control and treated DBA2-MDX mice.Collagen deposition was quantified on muscle sections. [Figure 10]Schematic representation of combined FAP-CAR-T treatment and AAV-microdystrophin gene therapy in the MDX mouse model of DMD. [Figure 11] Figure 10 shows increased microdystrophin mRNA expression in the TA and EDL after FAP-CAR-T treatment. MDX mice were treated as shown in Figure 10. MDX mice treated with a 10-fold higher dose of AAV-MD1 (4 x 10 vg / kg) alone served as a positive control. Microdystrophin mRNA levels were determined by digital droplet PCR using primers in Table 4 and normalized to mouse Mpz (myelin protein zero or P0) mRNA levels. [Figure 12] Figure 10 shows the increase in AAV viral genome copies in the TA and EDL after FAP-CAR-T treatment. MDX mice were treated as shown in Figure 10. MDX mice treated with a 10-fold higher dose of AAV-MD1 (4 x 10 vg / kg) alone served as a positive control. Viral genome copies were determined by digital droplet PCR using the primers in Table 4. [Figure 13] Figure 10 shows an increase in microdystrophin-positive fibers in the TA and EDL after FAP-CAR-T treatment. MDX mice were treated as shown in Figure 10. MDX mice treated with a 10-fold higher dose of AAV-MD1 (4 x 10 vg / kg) alone served as a positive control. Microdystrophin-positive fibers were quantified after immunostaining on muscle sections. [Figure 14] Figure 10 shows increased microdystrophin protein expression in the TA and EDL after FAP-CAR-T treatment. MDX mice were treated as shown in Figure 10. MDX mice treated with a 10-fold higher dose of AAV-MD1 (4 x 10 vg / kg) alone served as a positive control. Microdystrophin protein expression was quantified by simple Western blotting. [Figure 15]Figure 10 shows the reduction of collagen deposition area in the TA and EDL after FAP-CAR-T treatment. MDX mice were treated as shown in Figure 10. MDX mice treated with a 10-fold higher dose of AAV-MD1 (4 x 10 vg / kg) alone served as a positive control. Collagen deposition was quantified on muscle sections. [Example]

[0132] Materials and Methods 1. Plasmid Construction 1.1 Construction of pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE The construct pCCL-EF1a-scFvCD123-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE, previously described in Bole-Richard et al., Leukemia, 2020, 34, 3228-3241, was used to remove the fragment scFvCD123 using the restriction enzymes PspXI and BstEII to generate the acceptor backbone for the scFv FAP fragment. The fragment scFv FAP was synthesized from the sequence of the anti-mouse FAP monoclonal antibody described in WO 2014 / 055442 as SEQ ID NO: 3 and listed below as SEQ ID NO: 18, with the restriction enzymes PspXI and BstEII appended to each end. The scFv FAP fragment was digested with the restriction enzymes PspXI and BstEII and ligated into the acceptor pCCL backbone digested with PspXI and BstEII, and the product was used to transform XL10 competent cells. Colonies were screened by digestion and correct clones were confirmed by sequencing. pCCL-EF1a-scFvCD123-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE corresponds to the nucleotide sequence of SEQ ID NO: 19.

[0133] 1.2 Construction of pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-WPRE From the pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE plasmid, (a) a PCR reaction was performed to generate the fragment EcoR1-scFv-FAP-T2A-Sal1-EcoR1, (b) the fragment scfvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19 was removed using the restriction enzyme EcoRI, and (c) the PCR fragment generated in (a) that no longer contained ΔCD19 was ligated into the EcoR1-digested pCCL backbone obtained in (b) to generate the pCCL-EF1a-scfvFAP-CD28-4.1BB-CD3ζ-WPRE construct (SEQ ID NO: 20). The product was used to transform XL10 competent cells. Colonies were screened by digestion, and correct clones were confirmed by sequencing.

[0134] 1.3 Construction of pCCL-SFFV-mFAP-WPRE The luciferase 2 fragment was removed from the construct pCCL-SFFV-Luc2-WPRE available in the laboratory using the restriction enzymes BamHI and SalI. A plasmid containing the sequence encoding the mouse FAP protein was purchased from R&D Systems (RDC2905), and the mFAP cDNA fragment was extracted using the restriction enzymes BamHI and SalI before ligation into the pCCL backbone to obtain pCCL-SFFV-mFAP-WPRE. The new product was used to transform XL10 competent cells. Colonies were screened by digestion, and correct clones were confirmed by sequencing.

[0135] 2. Production of lentivirus and generation of stably transduced target and control cell lines Lentiviral vectors were generated by calcium phosphate transient transfection of HEK293T cells using four plasmids, including a transfer plasmid and three accessory plasmids (HIV-1 gagpol, HIV-Rev, and VSV-G). The following transfer plasmids were used: pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE, pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-WPRE, pCCL-SFFV-mFAP-WPRE, and pCCL-SFFV-Luc2-WPRE. The collected particles were concentrated approximately 500-fold by ultracentrifugation (50,000 × g, 2 h, 12°C), suspended in phosphate-buffered saline, and stored frozen at -80°C. Infectious titers of vectors were determined as infectious genomes (IG) / mL titer in HCT116 cells using ddPCR proviral primers normalized to albumin (see primer and probe sequences below):

[0136] [Table 1]

[0137] The following stable cell lines expressing FAP, Luc2, or both transgenes: 3T3-FAP, 3T3-Luc2, 3T3-FAP-Luc2 were generated by lentiviral transduction of NIH-3T3 fibroblast cell lines (1 × 10 cells) with the above vectors in 1 mL of DMEM complete medium (1% glutamine, 1% Pen / Strep, 10% FBS). 5 Individual cells, 2×10 6 1g / mL). Expression of the transgene was confirmed by immunochemical or bioluminescence assays.

[0138] 3. Preparation of FAP-CAR-T cells DBA2 mouse primary splenic T cells were isolated using the "Pan T cell negative selection" kit as recommended by the manufacturer (Miltenyi Biotec) and transduced with the indicated LV. Cells (1 × 10 6Cells (1 x 10 cells / well) were incubated in 1 mL of complete medium in a 24-well plate (complete medium = RPMI-1640 + 1% glutamine + 1% Pen / Strep + 10% FBS + 50 μM beta-mercaptoethanol + 50 μg / mL IL-2 + freshly prepared CD3 / CD28 activation beads (Gibco) at a 1:1 ratio). After 48 h, cells (1 x 10 cells / well in 1 mL of complete medium) were incubated in 1 mL of complete medium. 6 cells / well) in a 24-well plate with FAP-CAR-T lentivirus (2 × 10 7 The LV pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE vector was mixed with 0.1 mg / mL of LV-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE and Lentiboost (0.5 mg / mL). After overnight incubation, cells were expanded for an additional 4 days in complete medium. T cell transduction efficacy was determined either by flow cytometry using anti-human CD19 (see below) in the case of LV pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-WPRE, or by ddPCR to determine vector copy number per cell in the case of LV pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-WPRE (using proviral ddPCR primers referenced to mouse titin).

[0139] [Table 2]

[0140] 4. FAP-CAR-T cell phenotyping After 7 days of culture, LT and FAP-CAR-T cells were incubated with the following conjugated antibodies: anti-mouse CD3, anti-mouse CD8, and anti-mouse CD4 for 1 hour before FACS analysis, and, when using LV pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE, anti-human CD19. After washing and adding the viability marker 7AAD, the cells were analyzed by flow cytometry.

[0141] 5. Luciferase assay for the recent cytotoxic activity of FAP-CAR-T cells Five thousand 3T3-Luc2 control cells and 3T3-FAP-Luc2 target cells were seeded in 100 μL of DMEM complete medium (1% glutamine, 1% Pen / Strep, 10% FBS) in a 96-well plate (ViewPlate, Perkin Elmer, 6005181). The next day, FAP-CAR-T cells or control nonspecific T lymphocytes (TLs) were added at two different concentrations (37,500 or 75,000 cells per 100 μL of medium), and the cells and targets were co-cultured for 24 hours. At the end of the co-culture, 100 μL of supernatant medium was removed from each well and mixed with 100 μL of luciferin solution (Bright-Glo Luciferase assay, Promega). Bioluminescence levels were immediately measured using a luminometer (560 nm).

[0142] 6. Degranulation assay to measure FAP-specific cytotoxicity The functional cytotoxic activity of FAP-CAR T cells was measured by detecting CD107 lysosomal-associated membrane protein (LAMP) on the surface of recently degranulated cells. Five thousand control 3T3 cells and 3T3-FAP target cells were seeded in 100 μL of DMEM complete medium in a 96-well plate. The next day, 100 μL containing 75,000 FAP-CAR T cells or control nonspecific T lymphocytes (TLs) was added to the corresponding wells along with brefeldin A (1 / 2000) and 20 μL / mL anti-CD107a. After 6 hours of incubation at 37°C, the cells were washed with PBS-1X and stained with anti-CD8 antibody. After adding the viability marker 7AAD, the cells were analyzed by flow cytometry.

[0143] 7. Mice For all studies, only males were used. DBA2 control was purchased directly from Charles River Laboratories (DBA / 2J, Ref: 625), while DBA2 / MDX was bred and obtained at the Centre d'Exploration et de Recherche Fonctionnelle Experimentale (CERFE; Evry, France). DBA2-MDX was originally purchased from Jackson Laboratories (D2.B10-Dmd mdx / J, lineage 013141), which has been bred at CERFE for approximately 8–10 generations.

[0144] 8. Expression of fibrosis marker genes The levels of mouse collagen 3a1 and mouse FAP gene mRNA were measured by ddPCR in different muscle tissues and normalized to the level of mouse Mpz (myelin protein zero or P0) mRNA. The tibialis anterior (TA), gastrocnemius (GA), extensor digitorum longus (EDL), and heart were isolated from euthanized DBA2 and DBA2-MDX mice and directly stored in RNAlater. RNA was extracted from these tissues using the RNeasy fibrous tissue kit (Qiagen) following the manufacturer's recommendations. RNA was then reverse transcribed using the Verso cDNA Synthesis kit (ThermoFisher Scientific). For ddPCR, 1x ddPCR Supermix for Probes no dUTP (Biorad), 16 ng of complementary DNA, and the following primer sets were used: ddPCR Gene Expression Assay: Fap, Mouse (Biorad, 10031252), ddPCR Gene Expression Assay: Col3a1, Mouse (Biorad, 10031252), and ddPCR Gene Expression Assay: Mpz, Mouse (Biorad, 10031255).

[0145] [Table 3]

[0146] After PCR [95°C for 10 min + 40x (94°C for 30 sec, 60°C for 1 min) + 98°C for 10 min], droplets were generated using a droplet generator QX200 (Biorad), and the reactions were read and analyzed using QuantaLife software (Biorad).

[0147] 9. Mouse and in vivo studies Anesthetized DBA2-MDX mice aged 2–3 months were inoculated with 5 × 10 control LT or specific FAP-CAR-T cells resuspended in 100 μL of PBS-1X. 5 (low dose) or 1 x 10 6 Mice were intravenously injected with either one of the two (high dose) or two (high dose) cells into the retroorbital vein. One week later, the mice were re-injected with the same dose of the same cells. Two weeks later, age-matched control mice (DBA2) and treated mice were euthanized, and skeletal muscle was collected for molecular and histological analysis.

[0148] For sequential gene therapy, 2-3 month old DBA2-MDX mice were administered two consecutive doses (1 × 10 6 ) of FAP-CAR-T cells, and two weeks after treatment, mice received one dose (5.10 12 For simultaneous gene therapy, DBA2-MDX mice were injected with the first dose (1 × 10 vg / kg) of rAAV9-microdystrophin (MD1) vector. 6 ) of FAP-CAR-T cells, and 2 weeks after treatment, mice received a second dose (1 × 10 6 ) FAP-CAR-T cells and 1 dose (5.10 12 Control DBA2-MDX mice were simultaneously injected with two consecutive doses (1 × 10 6 1 dose (5.10 12The mice were injected with 1000 mg / kg of rAAV9-microdystrophin (MD1) vector (Bourg et al., Int. J. Mol. Sci., 2022, 23, 2016; doi: 10.3390 / ijms23042016). After 6 weeks, blood was collected and force tests (escape test dTA in situ) were performed on control and treated mice (DBA2-MDX). After 5 weeks, control and treated mice (DBA2-MDX) were euthanized, and heart and skeletal muscle (TA, ED) were collected for molecular and histological analysis.

[0149] 10. Histological analysis The TA, GA, and EDL collected from the study mice were immediately snap-frozen in liquid nitrogen. The frozen muscles were then sectioned (8 μm) using a cryostat and stained with Sirius Red dye using standard procedures. Images were then captured using an Axioscan microscope (10× objective, Zeiss).

[0150] Quantification of microdystrophin-positive fibers Fiber cytoplasmic regions surrounded by membrane staining (laminin) are segmented by morphological segmentation after contrast enhancement and artifact sorting (FiJi software 2.0.0-rc / 1.52p, Morpholib plugin version 1.4.1). Fibers are magnified by magnification to capture membrane regions. Nuclei are detected in the DAPI channel using local maximum detection. The fluorescence intensity of each object (fiber, fiber membrane, and nucleus) is measured for each channel along with fiber shape and size.

[0151] Nuclei were associated with their parent fibers using R software, and all fluorescence and shape data were integrated together. Non-fiber objects were sorted based on shape, size, and fluorescence criteria. Positive fibers in every channel (laminin, microdystrophin, and nuclei) were detected based on the fluorescence distribution of negative control slices or slices with known negative conditions, and the percentage of microdystrophin fibers was determined.

[0152] Quantification of collagen on muscle sections Collagen deposition on muscle sections was quantified using Qupath software, an open-source software for bioimage analysis. To do so, a two-pixel classifier was created and trained on three muscle sections to identify representative areas (collagen regions) by enclosing the sections. The first pixel was used to define the tissue being analyzed, and the second pixel was used to identify the areas of collagen deposition. The surface area occupied by collagen was quantified relative to the total surface area of ​​the muscle section.

[0153] 11. Molecular analysis Micro-dystrophin mRNA expression levels and AAV vector copy numbers in the muscles of DBA2-MDX mice receiving combination therapy were analyzed by digital droplet PCR on cDNA or gDNA using the oligonucleotide primers and probes described below.

[0154] [Table 4]

[0155] Quantification of microdystrophin expression by simple Western blotting Muscle proteins were extracted by homogenization in RIPA buffer supplemented with EDTA-free protease inhibitor cocktail (Roche) and benzonase. Total protein was then quantified by the BCA method using the Pierce BCA protein assay kit (Invitrogen) according to the manufacturer's instructions. Protein detection was performed by capillary Western blotting using JESS protein simple (Bio-Techne) according to the manufacturer's instructions. Microdystrophin detection was performed using the antibody DysB (NCL-DYSB, Leica, 1:20), and its expression was quantified using Compass software.

[0156] 12. Statistical analysis Differences between two groups were compared using Student's T-test. Differences between multiple groups were compared using one-way analysis of variance (ANOVA). Statistics were calculated using GraphPad Prism 7 software.

[0157] result 1. Construction of lentiviral gene transfer plasmids for generating mFAP-directed CAR T cells To express third-generation chimeric antigen receptor (CAR) constructs directed against murine fibroblast activation protein (FAP), we generated advanced-generation HIV-1-derived lentiviral gene transfer plasmids containing human CD28, 4.1BB, and CD3 zeta chain modules for costimulation, survival, and signal transduction, combined with a final truncated human CD19 marker (Figure 1A) or not (Figure 1B). These constructs were obtained by replacing the single-chain variable fragment (ScFv) immunoglobulin fusion protein sequence of another CAR previously described in Bole-Richard et al., Leukemia, 2020, 34, 3228-3241, with an scFv specific for mFAP synthesized from the sequence published in WO 2014 / 055442. Both lentiviral plasmids shown in Figure 1 were used to generate lentiviral vectors for this study. The vectors were pseudotyped with VSVG and titered according to standard laboratory techniques (Corre et al., Gene Therapy, 2022, 29, 536-543).

[0158] 2. Generation and characterization of murine FAP-CAR-T cells T lymphocytes (LTs) isolated from the spleens of 2-3 month-old DBA2 mice were cultured and activated with CD28 / CD3 beads and interleukin-2 for 48 hours before lentiviral transduction. Five days after transduction, the cell surface phenotype of the cells was characterized by flow cytometry (Figure 2). Results showed that 7 days after activation, the cells were predominantly CD8+, and it can be estimated that approximately 30% of the cells were effectively transduced (CD19+).

[0159] 3. Generation of FAP-CAR T cells and in vitro functional characterization 3.1 Generation of cells expressing mouse FAP To test the specificity and efficacy of FAP-CAR-T in vitro, NIH-3T3 cells, a mouse fibroblast cell line known to lack mouse FAP, were used as a negative control to generate specific target cells. A lentiviral construct encoding mouse FAP was generated and used to transduce NIH-3T3 cells, thereby generating the stable cell line 3T3-FAP, which expresses high levels of mFAP mRNA (Figure 3A) and FAP protein (Figure 3B).

[0160] 3.2 Generation of cells for cytotoxicity assay and verification of FAP-CAR-T cell-specific cytotoxic activity To measure FAP-CAR-T cell-specific killing of FAP-bearing cells in vitro, two new stable cell lines were generated using lentiviral vectors encoding the bioluminescent marker luciferin 2: 3T3-Luc2 and 3T3-FAP-Luc2 (Figure 4). The results show that cell death (indicated by a decrease in bioluminescence compared to control) occurred in a statistically significant manner only when FAP-CAR-T cells were cocultured with cells expressing the FAP target.

[0161] 3.3 Degranulation assay The functional properties of FAP-CAR-T cells were also tested in vitro using a CD107a degranulation assay after co-culture of 3T3 or 3T3-FAP with T cells or FAP-CAR-T (Figure 5). The results show that FAP-CAR-T cells degranulate only in the presence of murine FAP targets.

[0162] 4. Skeletal muscle fibrosis in the DBA2-MDX model of Duchenne muscular dystrophy Fibrosis biomarkers, such as FAP expression and another fibrosis marker, collagen III, were quantified over time and at steady state using ddPCR in skeletal muscles of interest (TA = tibialis anterior; GA = gastrocnemius; EDL = extensor digitorum longus) in the DBA2-MDX mouse model of Duchenne muscular dystrophy (Figure 6). Results show that, independent of age, these two fibrosis markers are consistently overexpressed in skeletal muscle of DBA2-MDX mice compared with age-matched DBA2 controls. Significant biomarker differences were identified in the TA at 3 and 4 months and in the GA at 2 or 3 months. A similar trend was seen in the EDL, although statistical significance was not reached. The lack of significance for some groups may be due to the small number of mice per group (n = 3).

[0163] 5. In vivo effects of FAP-CAR-T cells on DBA2-MDX-associated fibrosis To test the efficacy of FAP-CAR-T treatment on DBA2-MDX-associated fibrosis in vivo, 2- to 3-month-old DBA2-MDX mice were injected with two consecutive doses of FAP-CAR-T cells, prepared and characterized as previously described (see Figure 7 for a schematic representation of the protocol). To evaluate the effect of CAR-T cells on fibrosis, mRNA was extracted from the skeletal muscle of control or treated mice, and the expression of FAP and collagen III was determined by ddPCR after reverse transcription (Figure 8). The results show that systemically injected FAP-CAR-T cells can reduce fibrosis biomarkers in skeletal muscle in DBA2-MDX mice. The reduction of biomarkers by FAP-CAR T cells is more pronounced in the TA and EDL, where these two biomarkers are expressed at higher levels compared to the GA. The results also show that injection of FAP-CAR-T cells does not significantly affect the reduction of fibrosis biomarkers in the hearts of DBA2-MDX mice, as FAP is not expressed there (Figure 8).

[0164] To confirm the effect on fibrosis, histological sections of skeletal muscle from control and treated mice were stained with Sirius Red to visualize the amount of collagen fibers present in the tissue. The results show a decrease in collagen and an improvement in the structural organization of muscle fibers in the skeletal muscle of DBA-MDX mice treated with FAP-CAR-T (Figures 9A and 9B).

[0165] 6. Effect of gene therapy on FAP-CAR-T cell-mediated fibrosis reduction in a DMD mouse model To assess the effect of FAP-CAR-T-mediated fibrosis reduction on gene therapy for Duchenne muscular dystrophy, we tested two treatment combinations: sequential and simultaneous injection of FAP-CAR-T and rAAV vectors expressing microdystrophin. For sequential gene therapy, 2-3 month-old DBA2-MDX mice were injected with two consecutive doses of FAP-CAR-T cells. Two weeks after treatment, the mice were injected with one dose of rAAV9-microdystrophin (MD1) vector. For simultaneous gene therapy, 2-3 month-old DBA2-MDX mice were injected with one dose of FAP-CAR-T cells. Two weeks after treatment, the mice were injected with a second dose of FAP-CAR-T cells and one dose of rAAV9-microdystrophin (MD1) vector simultaneously (Figure 10). Different studies were performed to evaluate microdystrophin mRNA and protein expression, evaluate AAV vector copy dose in muscle, and assess muscle integrity in control or treated mice.

[0166] The results of sequential gene therapy demonstrate that FAP-CAR-T cell-mediated fibrosis reduction leads to higher microdystrophin expression levels systemically in the myofibers of the skeletal muscle of DBA2-MDX mice (Figures 11 and 14). There is an increase in AAV vector copies in the muscle and an increase in microdystrophin-positive fibers (Figures 12 and 13). These effects appear to be more pronounced in the TA and EDL, where fibrosis is reduced. To confirm the effect on muscle integrity, histological sections of skeletal muscle from control and treated mice were stained with Sirius red and hematoxylin-eosin to visualize the amount of collagen fibers present in the tissue. The results demonstrate a reduction in collagen in the skeletal muscle of DBA-MDX mice treated with FAP-CAR-T (Figure 15) and an improvement in the structural organization of myofibers. Functional analysis of skeletal muscle is performed to confirm the effectiveness of the combined therapy approach: FAP-CAR-T cells and AAV-MD1 to improve gene therapy for Duchenne muscular dystrophy.

[0167] In the case of simultaneous injection of FAP-CAR-T cells and AAV-MD1, the reduction in dystrophin expression levels and collagen deposition is expected to be less significant than in the case of sequential injection of FAP-CAR-T cells and AAV-MD1.

[0168] Array Description SEQ ID NO: 1: Anti-FAP scFv VH

[0169] [ka]

[0170] SEQ ID NO: 2: Anti-FAP scFv VL

[0171] [ka]

[0172] SEQ ID NO: 3: Anti-FAP scFv VH-CDR1 SYGMS SEQ ID NO: 4: Anti-FAP scFv VH-CDR2 TNNNGGVTYYPDSVKG SEQ ID NO: 5: Anti-FAP scFv VL-CDR1 RSSQSIVHSNGNTYLE SEQ ID NO: 6: Anti-FAP scFv VL-CDR2 KVSNRFS SEQ ID NO: 7: Anti-FAP scFv VL-CDR3 FQGSHVPYT SEQ ID NO: 8: Anti-FAP scFv

[0173] [ka]

[0174] SEQ ID NO: 9: Modified human IgG4 hinge domain ESKYGPPCPP SEQ ID NO: 10: Signal peptide (mouse Ig kappa) MKLPVRLLVLMFWIPASSS SEQ ID NO: 11: hCD28 TM domain FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 12: hCD3-zeta activation domain MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS SEQ ID NO: 13: hCD28 costimulatory domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 14: h4-1BB costimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 15: VH-VL linker SSGGGGSGGGGSSGGG SEQ ID NO: 16: anti-FAP CAR

[0175] [ka]

[0176] SEQ ID NO: 17: anti-FAP CAR

[0177] [ka]

[0178] SEQ ID NO: 18: Anti-FAP scFv

[0179] [ka]

[0180] SEQ ID NO: 19: pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-T2A-ΔCD19-WPRE

[0181] [ka]

[0182] [ka]

[0183] [ka]

[0184] SEQ ID NO: 20: pCCL-EF1a-scFvFAP-CD28-4.1BB-CD3ζ-WPRE

[0185] [ka]

[0186] [ka]

[0187] [ka]

[0188] SEQ ID NO: 21: Alb.fw GCTGTCATCTCTTGTGGGCTGT SEQ ID NO: 22: Alb.rv ACTCATGGGAGCTGCTGGTTC SEQ ID NO: 23: Alb.pr CGCACGGCAAGAGGCGAGG Sequence number 24: PRO.fw CACTCCCAACGAAGACAAGA SEQ ID NO: 25: PRO.rv TCTGGTTTCCCTTTCGCTTT SEQ ID NO: 26: PRO.pr TCTCTAGCAGTGGCGCCCGAACAGG SEQ ID NO: 27: mTitin-F AAAACGAGCAGTGACGTGAGC SEQ ID NO: 28: mTitin-R TTCAGTCATGCTGCTAGCGC SEQ ID NO: 29: mTitin-P TGCACGGAAGCGTCTCGTCTCAGTC SEQ ID NO: 30: Amplicon Mpz

[0189] [ka]

[0190] SEQ ID NO: 31: Amplicon FAP

[0191] [ka]

[0192] SEQ ID NO: 32: Amplicon Col3al

[0193] [ka]

[0194] SEQ ID NO: 33: AAV22mers.F CTCCATCACTAGGGGTTCCTTG SEQ ID NO: 34: AAV18mers.R GTAGATAAGTAGCATGGC SEQ ID NO: 35: AAV_MGB.P TAGTTAATGATTAACCC SEQ ID NO: 36: μdys.R GGTTGTGCTGGTCCAGGGCGT SEQ ID NO: 37: μdys.F CCAACAAAGTGCCCTACTACATC SEQ ID NO: 38: μdys.P CCGAGCTGTACCAGAGCCTGGCC SEQ ID NO: 39: MH181P0.F CTCCAAGCAGATGCAGCAGA SEQ ID NO: 40: M267PO.R ATAGCCTTGCGCATCATGGT SEQ ID NO: 41: M225PO.P CCGTGGTGCTGATGGGCAAGAA

Claims

1. Immune cells engineered to express a chimeric antigen receptor (CAR) that specifically binds fibroblast activation protein (FAP) for use in treating skeletal muscle fibrosis in muscular dystrophy.

2. 2. The immune cell for use of claim 1, wherein the CAR comprises: (i) an extracellular domain comprising at least one antigen-binding domain that specifically binds a FAP, (ii) a transmembrane domain, and (iii) an intracellular domain comprising an intracellular signaling domain capable of activating the immune cell, and optionally comprising one or more costimulatory signaling domains.

3. 3. The immune cell for use according to claim 1 or claim 2, wherein the antigen-binding domain is a single-chain variable fragment (scFv) of a monoclonal antibody that specifically binds a FAP.

4. 4. The immune cell for use according to claim 3, wherein the single chain variable fragment (scFv) that binds FAP comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2; preferably a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

8.

5. 5. The immune cell for use according to any one of claims 2 to 4, wherein the extracellular domain further comprises a hinge domain, preferably from an IgG4 heavy chain.

6. 6. The immune cell for use according to any one of claims 2 to 5, wherein the transmembrane domain is derived from CD28.

7. 7. The immune cell for use according to any one of claims 2 to 6, wherein the intracellular signaling domain is a CD3 zeta signaling domain.

8. 8. The immune cell for use according to any one of claims 2 to 7, wherein the intracellular domain further comprises one or more costimulatory signalling domains from CD28 or 4-1BB, preferably the costimulatory signalling domains of both CD28 and 4-1BB.

9. 9. The immune cell for use according to any one of claims 2 to 8, wherein the CAR comprises, from its N-terminus to C-terminus: a signal peptide derived from a mouse Ig kappa light chain; an scFv fragment derived from an anti-FAP monoclonal antibody, a modified hinge domain derived from a human IgG4 heavy chain; a transmembrane domain derived from human CD28, a first costimulatory domain derived from human CD28, a second costimulatory domain derived from human 4-1BB, and an intracellular signaling domain derived from a human CD3 zeta chain; preferably, the CAR comprises the amino acid sequence of SEQ ID NO:

16.

10. 10. Immune cells for use according to any one of claims 1 to 9, which are lymphocytes such as T cells and / or NK cells, preferably cytolytic lymphocytes such as cytolytic T cells.

11. 11. An immune cell for use according to any one of claims 1 to 10, modified with a vector selected from an expression vector comprising a nucleic acid construct encoding the CAR, preferably lipid nanoparticles packaging an RNA molecule, and a lentiviral vector, more preferably a self-inactivating and / or VSVG-pseudotyped lentiviral vector.

12. 12. Immune cells for use according to any one of claims 1 to 11, which reduce the expression level of at least one biomarker of fibrosis, preferably type III collagen.

13. 13. Immune cells for use according to any one of claims 1 to 12, for use in combination with a vector for gene therapy of muscular dystrophy, preferably a recombinant AAV vector.

14. 14. Immune cells for use according to any one of claims 1 to 13, wherein the muscular dystrophy is selected from dystrophinopathies, limb-girdle muscular dystrophies and congenital muscular dystrophies; preferably Duchenne muscular dystrophy.

15. 15. The immune cell for use according to claim 13 or 14, wherein the combination therapy comprises the administration of a gene therapy vector at a reduced dose compared to the use of the gene therapy vector without the immune cell.

Images