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EP4583905A1 - Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives - Google Patents

Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives

Info

Publication number
EP4583905A1
EP4583905A1 EP23765243.3A EP23765243A EP4583905A1 EP 4583905 A1 EP4583905 A1 EP 4583905A1 EP 23765243 A EP23765243 A EP 23765243A EP 4583905 A1 EP4583905 A1 EP 4583905A1
Authority
EP
European Patent Office
Prior art keywords
cells
host immune
immune cell
car
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23765243.3A
Other languages
German (de)
English (en)
Inventor
Jean-Christophe BORIES
Bertrand ARNULF
Jean-Paul Fermand
Nathalie RODERS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ifm Sas
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Cite
Original Assignee
Ifm Sas
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Cite
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ifm Sas, Assistance Publique Hopitaux de Paris APHP, Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Cite filed Critical Ifm Sas
Publication of EP4583905A1 publication Critical patent/EP4583905A1/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4222—CD38 not IgG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • PI proteasome inhibitors
  • IMIDs immunomodulators
  • R/R refractory/resistant diseases
  • New strategies based on the combination of PI, IMIDs and monoclonal antibodies (mAb) targeting the CD38 antigen, have significantly improve the prognosis (Dimopoulos et al., 2016). Nevertheless, most patients still relapse and MM remains an incurable disease.
  • Chimeric Antigen Receptors are hybrid molecules associating an extracellular portion, involved in antigen-recognition, with a transmembrane region fused with signalling domains including (in most cases) the CD3z and CD28 and/or the CD137 receptor, also referred to as 4- 1BB (Sadelain et al., 2013).
  • the CD3z provides an activation signal and the 4- 1BB region provides a co-stimulation signal, both of which are mandatory to mimic the physiological T cell mechanisms required for cytotoxicity, differentiation and persistence of T cells in vivo.
  • CAR T cells targeting the B cell maturation antigen (BCMA) have brought up to 80% response rates in MM patients (depending on studies), however, the median overall survival remains below 25 months (Munshi et al., 2021).
  • BCMA is detected on most post germinal centre B lineage cells, expression on plasma cells can vary through several mechanisms, including antigen loss or expression shedding, potentially leading to variable responses (Da Via et al., 2021; Laurent et al., 2015).
  • CD38 is a glycoprotein with cyclic ADP ribose hydrolase activities, which is expressed on tumor plasma cells (and normal plasma cells) as well as on other lymphoid and myeloid cell populations. It was originally identified as a T and B lymphocyte activation marker and was later shown to be expressed on multiple haematopoietic cells (HSC), including subsets of haematopoietic stem cells, NK cells and monocytes.
  • HSC haematopoietic cells
  • anti-CD38 CAR T cells have shown good anti-MM activities in pre-clinical models (Drent et al. Mol Ther.2017).
  • anti-CD38 CAR-T cell therapy might trigger side effects such as fratricide killing of activated CAR-T cells, as well as toxicity against myeloid cells, HSC or non-hematopoietic CD38 expressing cells (ie, endothelial cells).
  • CD38 represents a validated target for immune therapy in MM
  • improvements are needed to deliver safer and more efficient anti-CD38 CAR-T.
  • CAR-T cells targeting CD38 represent a potential alternative, but expression of CD38 on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such therapy.
  • the inventors developed DCAR, a double CAR system targeting CD38 and SLAMF7 (CS1) through split activation and co-stimulation receptors, respectively.
  • the inventors show that CRISPR/Cas9 inactivation of the CD38 gene enhances the anti-MM activity of DCAR-T in vitro. Edited DCAR-T developed strong responses specifically against MM cells expressing both the CD38 and the CS1 antigens in vitro and in vivo.
  • DCAR-T provides a safe and efficient alternative to treat MM patients.
  • the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • engineered cell refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated.
  • the manipulation is or comprises a genetic manipulation.
  • a genetic manipulation is or comprises one or more of (i) introduction of a nucleic acid not present in the cell prior to the manipulation (i.e., of a heterologous nucleic acid); (ii) removal of a nucleic acid, or portion thereof, present in the cell prior to the manipulation; and/or (iii) alteration (e.g., by sequence substitution) of a nucleic acid, or portion thereof, present in the cell prior to the manipulation.
  • the term “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • a "polynucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • CD38 has its general meaning in the art and refers to the ADP- ribosyl cyclase/cyclic ADP-ribose hydrolase 1.
  • An exemplary amino acid sequence for CD38 is represented by SEQ ID NO:1.
  • the extracellular domain of CD38 ranges from the amino acid residue at position 43 to the amino acid residue at position 300 in SEQ ID NO:1.
  • SEQ ID NO:1 >sp
  • OS Homo sapiens
  • GN CD38
  • SLAMF7 is also known as CD2 subset 1, CD2-like receptor-activating cytotoxic cells (CRACC), Membrane protein FOAP-12, Novel Ly9, Protein 19A, and CD319.
  • An exemplary amino acid sequence for SLAMF7 is represented by SEQ ID NO:2.
  • the extracellular domain of SLAMF7 ranges from the amino acid residue at position 23 to the amino acid residue at position 226 in SEQ ID NO:2.
  • the term “immune cell” refers to a cell that functions in an immune response or a progenitor, or progeny thereof.
  • the term “population” refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest (e.g. a population of human CAR-host immune cells comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the highly suppressive functions and which express the particular markers of interest).
  • T cell has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity.
  • T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility.
  • TCR T cell receptor for the antigen
  • There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, and gamma delta T cells.
  • Cytotoxic T cells are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • chimeric antigen receptor has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., scFv) linked to T- cell signalling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • TCR T cell receptor
  • the chimeric antigen receptor of the present invention typically comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signalling domain.
  • the term “chimeric co-stimulatory receptor” or “CCR” refers to a specific type of chimeric antigen receptor (CAR) that mediates costimulation independently of activation. When expressed on host immune cells in combination with a CAR, the CCR is targeted to a second antigen.
  • CAR-T cell refers to a T lymphocyte that has been genetically engineered to express a CAR.
  • nucleic cytotoxicity means that the cytotoxicity is reduced by 50, 60, 70, 80, 85, 90, 95, 99 or 100%.
  • antigen has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized and selectively bound by an antibody or by a T cell antigen receptor, resulting in induction of an immune response.
  • Antigens according to the invention are typically, although not exclusively, peptides and proteins. Antigens may be natural or synthetic and generally induce an immune response that is specific for that antigen.
  • antibody and “immunoglobulin” have the same meaning, and will be used equally in the present invention.
  • 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.
  • antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.
  • two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k).
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • treatment according to the present invention may be provided as a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • a daily dosage of a inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2
  • the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients.
  • the pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W.
  • the first object of the present invention relates to a host immune cell engineering to express a) a chimeric antigen receptor (CAR) that binds to CD38, wherein binding of the CAR to CD38 is capable of delivering an activation signal to the host immune cell, and b) a chimeric co- stimulating receptor (CCR) that binds to a second antigen, wherein binding of the CCR to the second antigen is capable of delivering a costimulatory signal to the host immune cell but does not alone deliver an activation signal to the host immune cell, wherein the host immune cell is capable of (i) exhibiting negligible cytotoxicity against cells that are single positive for CD38, and (ii) inducing cytotoxicity against cells that are positive for both CD38 and the second antigen.
  • CAR chimeric antigen receptor
  • CD38 chimeric co- stimulating receptor
  • the host immune cell is a hematopoietic cell from the lymphoid lineage that comprises B, T and natural killer (NK) cells.
  • the host immune cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a human embryonic stem cell, and a pluripotent stem cell from which lymphoid cells may be differentiated.
  • the host cell is a pluripotent stem cell (PSC). PSCs can be indeed be modified by a CAR and then can be used for deriving T cells (e.g. WO 2017100403).
  • PSCs include embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs).
  • iPSCs can be generated directly from adult cells (e.g., somatic cells).
  • iPSCs can be typically derived or generated by introducing a specific set of pluripotency-associated genes, or "reprogramming factors", into a given cell type.
  • Reprogramming factors include, but are not limited to, OCT4 (also known as "POU5FL”), SOX2, cMYC, and KLF4, which are also known as Yamanaka factors. See Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”. Cell 126 (4): 663-76.
  • Hematopoietic stem progenitor cells display a number of phenotypes, such as Lin-CD34+CD38 ⁇ CD90+CD45RA ⁇ , Lin-CD34+CD38 ⁇ CD90 ⁇ CD45RA ⁇ , Lin-CD34+CD38+IL-3aloCD45RA ⁇ , and Lin- CD34+CD38+CD10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. III: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994).
  • the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life.
  • the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.
  • Chimeric antigen receptor (CAR) The chimeric antigen receptor (CAR) typically comprises an extracellular domain and an intracellular domain joined by a transmembrane domain.
  • the extracellular domain, expressed on the surface of the host immune cell comprises an antigen binding domain having binding affinity for CD38.
  • such antigen binding domain is an antibody, preferably a single chain antibody.
  • the antibody is a humanized antibody.
  • such antigen binding domain is an antibody fragment selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFv.
  • such antigen binding domain is selected from a Fab and a scFv.
  • the antigen targeting domain when the antigen targeting domain is a scFv, the scFv can be derived from the variable heavy chain (VH) and variable light chain (VL) regions of an antigen-specific mAb linked by a flexible linker.
  • the scFv retains the same specificity and a similar affinity as the full antibody from which it is derived.
  • the peptide linker connecting scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH–VL paring and antigen- binding sites.
  • Peptide linkers can vary from 10 to 30 amino acids in length.
  • the scFv comprises a VH domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:9. In some embodiments, the scFv comprises a VL domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:10.
  • the scFv comprises an amino acid sequence having 90% of identity with the amino acid sequence as set forth in SEQ NO:11.
  • transmembrane domain is typically a hydrophobic alpha helix that spans across the lipid bilayer of the cell membrane.
  • the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • such antigen binding domain is an antibody fragment selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFv.
  • such antigen binding domain is selected from a Fab and a scFv.
  • the antigen targeting domain is a scFv
  • the scFv is specific for an epitope located in the extracellular domain of SLAMF7.
  • the scFv comprises a VH domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:15.
  • the scFv comprises a VL domain having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:16.
  • transmembrane domain of the CCR is the same nature as for the CAR and is typically a hydrophobic alpha helix that spans across the lipid bilayer of the cell membrane.
  • the transmembrane domain is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some embodiments is derived from any membrane -bound or transmembrane protein. Transmembrane regions include those derived from (i.e.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a transmembrane domain is thermodynamically stable in a membrane. It may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure.
  • a short oligo- or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signalling domain(s) of the CCR.
  • a glycine-serine doublet may provide a suitable linker.
  • a costimulatory molecule can be defined as a cell surface molecule that is required for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
  • the intracellular signalling portion of the above recited co-stimulatory domains can be used alone or in combination with other co-stimulatory domains.
  • the CCR can comprise one or more co-stimulatory domains from the group consisting of CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
  • the intracellular domain of the CCR comprises the co-stimulatory domain of 4-1BB (CD137). In some embodiments, the intracellular domain of the CCR comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:18. SEQ ID NO:18> CD137 costimulating domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL In some embodiments, the CCR of the present invention comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:19.
  • the CAR and CCR are expressed in a single open reading frame (ORF), thereby creating a single polypeptide.
  • ORF open reading frame
  • an amino acid sequence or linker containing a high efficiency cleavage site is disposed between the CAR unit and the CCR unit.
  • “high cleavage efficiency” is defined as more than 50%, more than 70%, more than 80%, or more than 90% of the translated protein is cleaved. Cleavage efficiency may be measured by Western Blot analysis.
  • high efficiency cleavage sites examples include porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thoseaasigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A (BmIFV2A), or a combination thereof.
  • the high efficiency cleavage site is P2A.
  • High efficiency cleavage sites are described in Kim J H, Lee S-R, Li L-H, Park H-J, Park J-H, Lee K Y, et al.
  • Retroviral vectors are able to infect a broad variety of cell types.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
  • Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV).
  • Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference.
  • the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell.
  • the gag, pol and env genes of the vectors of interest also are known in the art.
  • the relevant genes are cloned into the selected vector and then used to transform the target cell of interest.
  • Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No.5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell.
  • nucleic acid sequence is a "promoter” sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
  • the polynucleotide is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell.
  • Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.
  • a variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.
  • a variety of assays may be performed.
  • the host immune cell of the present invention is engineered such that it does not express CD38.
  • the method includes introducing into the host immune cell a genome- editing nuclease designed to edit the CD38 coding region, and culturing the host immune cell under conditions for the genome-editing nuclease to modify the CD38 coding region to inhibit the expression of CD38.
  • introducing the genome-editing nuclease into the host immune cell includes introducing into the host immune cell a polynucleotide that encodes the genome-editing nuclease.
  • the genome-editing nuclease includes a TALEN nuclease, a CRISPR-associated endonuclease, or a megaTAL nuclease.
  • the genome-editing nuclease is a CRISPR-associated endonuclease.
  • CRISPR/Cas systems for gene editing in eukaryotic cells typically involve (1) a guide RNA molecule (gRNA) comprising a targeting sequence (which is capable of hybridizing to the genomic DNA target sequence), and sequence which is capable of encoding for the CRISPR- associated endonuclease.
  • gRNA guide RNA molecule
  • the CRISPR-associated endonuclease is a Cas9 nuclease.
  • the Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence.
  • the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of pX330, pX260 or pMJ920 (Addgene, Cambridge, MA).
  • Artificial CRISPR/Cas systems can be generated, using technology known in the art, e.g., that are described in U.S. Publication No. 20140068797, WO2015/048577, and Cong (2013) Science 339: 819-823.
  • these therapies involve processing the patient's own lymphocytes.
  • the treatments are accomplished by removing the patient's lymphocytes and transforming said cells in the population of DCAR-T cells as above described.
  • DCAR- T cells are prepared with the DCAR of the present invention, these ex vivo cells are reinfused into the patient to enhance the immune system to kill tumor calls.
  • the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount.
  • Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • a treatment-effective amount of cells in the composition is dependent on the relative representation of the host immune cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 103/kg, preferably 5x103/kg; and as high as 107/kg, preferably 108/kg.
  • the number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.
  • the host immune cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a pharmaceutically acceptable carrier) in a treatment- effective amount.
  • a further object of the present invention relates to a pharmaceutical composition comprising a population of host immune cells of the present invention and a pharmaceutically acceptable carrier.
  • Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized.
  • the infusion medium can be supplemented with human serum albumin.
  • a treatment-effective amount of cells in the composition is dependent on the relative representation of the T cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 103/kg, preferably 5x103/kg; and as high as 107/kg, preferably 108/kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells.
  • the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.
  • FIGURES Figure 1: CAR T constructs and expression. (a) Schematic diagram of the CAR construct.
  • First generation anti-CD38 CAR (1G) is comprised of a scFv specific for CD38(Fayon et al., 2021), linked to the human CD8 hinge and transmembrane regions, followed by the human CD3 zeta intracellular signaling domain.
  • the second generation anti-CD38 CAR (2G) is comprised of the anti-CD38 scFv linked to the human transmembrane and co-stimulation domain of CD28 and the activation domain from CD3 zeta.
  • the DCAR was designed as a bicistronic construct.
  • CAR T cytotoxicity was determined by co-culturing MM.1Sluc cells for 4 hours in the presence of 1G, 2G or DCAR CAR-T cells at different effector:target (E:T) ratios. Cell viability was assessed by luciferase activity and normalized to un-Transduced T cells. Each E:T ratio shows the mean ⁇ SEM from triplicate experiments. Significance was determined by a 2-way anova with Sidak’s multiple comparison test. ns ⁇ 0.05 *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 **** ⁇ 0.0001
  • Figure 3 DCAR efficiency In vitro compared to “classic” CD38 targeting CAR-T cells.
  • DCAR-T cells need both CD38 and CS1 for an “optimal/complete” anti-tumor response
  • CAR T cytotoxicity was determined by co-culturing differentially edited MM.1Sluc cells (wt, CD38ko, CS1ko and doubleko) for 24 hours in the presence of DCAR-T cells at different effector:target (E:T) ratios. Cell viability was assessed by luciferase activity and normalized to un-transduced-T cells.
  • E:T ratio shows the mean ⁇ SEM from triplicate experiments Significance was determined by a 2-way anova with Sidak’s multiple comparison test. The table shows the statistical analysis for E:T ratios 0.5 and 0.1.
  • CAR-T cell proliferation capacity was determined by a 14-day co-culture on NIH3T3 cells expressing differentially edited to express either no target (NIHwt), one of the targets (NIH38 and NIHCS1) or both targets (NIH38CS1).25.000 CAR-T cells were seeded on 100.000 NIH cells at day 0, and passed every 2-3 days on a new feeder layer. Cell number was determined using an automated cell counter.
  • the graph represents the mean ⁇ SEM from triplicate experiments, significance was determined using a 2-way anova with Turkey’s multiple comparison test. Significance is indicated for the proliferation at day 14.
  • Cytokine secretion was analyzed by taking supernatant from the CAR-T cells co-cultured on the different NIH3T3 feeder cell lines for 4 days. Cytokine levels of interleukin (IL)-2 and tumour necrosis factor-alpha (TNF-a) were determined by a bead- based immunoassay. The graphs represent the mean ⁇ SEM from four separate experiments. Significance was determined using Mann-Whitney tests.
  • mice within each treated group indicated by bar graphs ⁇ SEM and statistical significance was determined using a 1-way anova with Turkey’s multiple comparison test.
  • f Cell count of B cells or CD38 positive
  • Graphs represent the graphs indicate the mean ⁇ SEM and significance was determined using an unpaired T-test. *p ⁇ 0.05 **p ⁇ 0.01, ***p ⁇ 0.001.
  • EXAMPLE Methods: Construction of CAR Lentiviral vectors DNA sequences encoding for the anti-CD38 CAR (1G and 2G) and the anti-CS1 CCR were chemically synthetized (GeneArt ThermoFisher). Linkage of 1G anti-CD38 CAR to the P2A motif and the anti-CS1 CCR was performed using In-Fusion® snap assembly (Takara Bio) according to the manufacturers protocol.
  • the pSFFV-Kana lentiviral vector was generated by replacing the ampicillin resistance gene of the pRRL–PGK–WPRE (a gift from Dr Hana Raslova, Villejuif, France) with the chemically synthetized Kanamycin resistance gene and by inserting the SFFV promoter in an EcoRV site, upstream of the PGK promoter. Insertion of the CAR and CCR coding sequences into the pSFFV-Kana vector was performed using In-Fusion® snap assembly. Cell lines The MM1.S, KMS11 and NCI-H929 MM cell lines have been previously described (Fayon et al., 2021).
  • MM.1S, KMS11 or NCI-H929 were transduced with either lentiviral particles produced from the pLenti CMV Puro LUC (Addgene #17477) or retroviral particles.
  • MM1.S inactivated for the CD38 and/or SLAMF7 (CS1) genes have been previously described (Fayon et al., 2021). All MM cell lines were cultured in complete RPMI medium: RPMI-1640 supplemented with 10% heat-inactivated foetal bovine serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 2 nM glutamine (all from ThermoFisher) at 37°C + 5% CO2.
  • NIH3T3 cells were transduced with retroviral particles to obtain NIH3T3 feeder cells expressing CD38, CS1 or both targets to generate feeder cell lines.
  • NIH3T3 cells were cultured in DMEM medium: DMEM (manufacturer) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 2 nM glutamine at 37°C + 5% CO2.
  • HEK293-T cells were cultured under the same conditions as NH3T3 cells.
  • Lentiviral production HEK293T cells were co-transfected with lentiviral CAR vector and packaging plasmids (pMD2G and psPAX2, all from Addgene) using calcium phosphate precipitation method (ThermoFisher) following the manufacturer’s protocol. Lentiviral supernatants were collected at 72-hours post-transfection and concentrated using high-speed ultracentrifugation. To generate the lentiviral stocks, the resulting concentrated lentivirus batches were suspended in PBS and stored at -80°C. CAR-T cell production and Genome editing Primary peripheral blood cells (PBMCs) were obtained from the “Etableau für du Sang”.
  • PBMCs Genome editing Primary peripheral blood cells
  • PBMCs were isolated from cytapheretic residues by centrifugation on a Pancoll (Human; PANTM Biotech) gradient (2:1 ratio of diluted blood to Pancoll), using Leucosep tubes (ThermoFisher) according to the manufacturers protocol, and frozen at -80°C in RPMI 20% SVF medium containing10% DMSO.
  • ribo-nucleoprotein (RNP) complexes were generated by incubating single guide RNA (sgRNA) targeting CD38 (100 ⁇ M, Sigma-Aldrich) was complexed with the Cas9nuclease (ThermoFisher) at a 1:2 molecular ratios, respectively, for 20minutes at room- temperature.
  • the RNP complex was then electroporated with 5x106 activated T cells in 100 ⁇ L of 2M electroporation buffer (Chicaybam et al., 2013) in a 2B-Nucleofactor Unit (Lonza).
  • Transfected T cells were maintained in culture, using CD3/CD28 dynabeads in OpTmizer (containing 5ng/ml of IL-2 instead of 10 ng/ml; indicated form here as normal conditions ) at 37°C + 5% CO2, for another 24hours prior to transduction with lentiviral particles.
  • Lentiviral transduction was performed in culture plates coated with retronectin (15mg/ml, Takara Bio), T cells were combined with lentiviral particles at MOI10 or higher at 37°C + 5% CO2. After 24hours the medium was changed and cells were kept in culture under standard conditions.
  • mice were fixed in a 5% formalin solution (Sigma-Aldrich) in PBS prior to analysis. To determine reconstitution in the humanized mice cells were treated with Fc block (BD, #564220) for 10min at RT, followed by staining with Zombie UVTM (Biolegend, # 423107, 1/1000) according to the manufacturers protocol.
  • Fc block BD, #564220
  • Zombie UVTM Biolegend, # 423107, 1/1000
  • Cytotoxicity assay The cytotoxicity of CAR-T cells was determined by assessing tumour cell viability following a co-culture with CAR-T cells. Sixteen hours prior to the cytotoxicity assay, anti-CD3/CD28 beads were magnetically removed from the cultures, and CAR-T cells were mixed with luciferase expressing target cells at different effector to target (E/T) ratios.
  • CAR-T cells were co- cultured in feeder cells at an initial ration of 1:4 (25000:100000) CAR-T to feeder ratio. Every 2-3 days the CAR-T were transferred to fresh feeder cells.
  • Cell proliferation was determined by counting the viable cells, death cells were excluded by trypan Blue (Invitrogen) staining, using the Countess II (Invitrogen). Cell numbers were quantified using ImageJ (ImageJ) software.
  • Cytokine production Cytokine secretion was determined by collecting the supernatant of the long-term proliferation assay. The supernatant was collected at day 4 and stored at -20°C.
  • MM1.S.luc xenograft model Six to 12-week-old NOD/SCID/IL-2Rg null mice were inoculated with 3x106 MM1.SLuc cells by tail vein injection (i.v) at day 0, followed, 14 days later, by infusion of 106 CAR-T. Bioluminescence was measured with the IVIS Imaging System (PerkinElmer) every 7 days after tumour injection.
  • the study contained 6 mice in each group which were injected with CAR-T cells generated from 3 different healthy donors. Data was further analysed using Aura imaging software (Spectral Instruments Imaging) Subcutaneous MM1.S.luc xenograft model MM1.Sluc cells (0.5x106 cells per mouse) were injected sub cutaneous on the back of NSG mice. MM1.Swt cells were injected in the left flank and MM1.SCS1ko cells were injected in the right flank of the mice. CAR-T cells (106 cells per mouse) were administered IV in the tail vein 4 days post tumor injection. Tumor progression was followed by luminescent imaging every 7 days using the IVIS Imaging System. The study contained 4 mice in each group which were injected with CAR-T cells originating from 2 different healthy donors.
  • IMDM IMDM (Gibco) completed with 20%BIT (StemcellTM technologies), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 nM glutamine, 50ng/ml SCF (Miltenyi Biotec), 50ng/ml FLT3 (Miltenyi Biotec) 20ng/ml TPO (Miltenyi Biotec) , 10ng/ml IL-3 (Miltenyi Biotec) for 16H at 37°C + 5% CO2.
  • the CD34+ cells (4 different donors) were pooled and cells (0.35x106 per mouse) were administered IV in the tail vein in sub lethally irradiated NSG mice.5-weeks post injection of CD34+ cells mice injected with CAR-T cells (106 cells per mouse) IV in the tail vein. Each CAR-T was produced from 2 different healthy donors. Blood was collected from the mice to monitor the reconstitution prior to CAR-T cell injection. Blood, serum and bone marrow were collected post CAR-T cell treatment for further analysis by flow cytometry. Prior to analysis blood and organ samples were passed on a Pancoll gradient (2:1 ratio of diluted blood to Pancoll).
  • DNA sequences encoding the anti-CD381G CAR and the anti-CS1 CCR were cloned into a P2A-based bicistronic construct, which allows the expression of two separate polypeptides from a single gene ( Figure 1a). All sequences coding for 1G, 2G and DCAR were cloned under the control of the spleen focus-forming virus (SFFV) promoter into a lentiviral vector and were stably expressed on T cells isolated from healthy human donors. FACS analysis of transduced T cells stained with a anti human scFv revealed strong expression of the CARs and/or CCR transgenes in around 90% of infected T cells (data not shown).
  • SFFV spleen focus-forming virus
  • the DCAR construct allows co-expression of the anti-CD38 CAR and the anti-CS1 CCR, while 1G and 2G vectors induced a higher expression level of the anti-CD38 CAR at the surface of transduced T cells.
  • Deletion of CD38 in anti-CD38 DCAR-T enhances their cytotoxic activity against MM cells Since CD38 is upregulated on activated T cells, we investigated whether this process could interfere with the expression or the function of anti-CD38 CAR.
  • CD38 edited T cells from healthy donors were transduced with the different vectors, expanded in vitro for 7-14 days with anti-CD3/CD28 beads plus interleukine- 2 (IL-2) and IL-7, then co-cultured overnight with MM1.Sluc at various E:T ratios and the luciferase levels were measured to determine the cytotoxic activity.
  • IL-2 interleukine- 2
  • 1G anti-CD38 CAR-T and not transduced T cells displayed little or no proliferation over time. Furthermore, we used this NIH cells model to measure the production of IL-2 and tumour necrosis factor-alpha (TNF-a) by T cells transduced with the different constructs. After 96 hours in culture on NIH38CS1 production of IL-2 was much lower in 1G than in 2G anti-CD38 CAR-T cultures (18.73 pg/mL compare with 580 pg/mL) ( Figure 3c). Remarkably, DCAR-T secreted an intermediate level of IL-2 (183 pg/mL), while no production was detected in the culture medium of not transduced control T cells.
  • TNF-a was readily produced by all transduced T cells, however the levels were higher in 1G and 2G anti-CD38 CAR-T compared with DCAR-T (1.2 ⁇ g/mL, 2.7 ⁇ g/mL and 0.45 ⁇ g/mL, respectively as average) (Figure 3c). These results indicate that DCAR-T responded to stimulation by CD38 and CS1 expressing targets by secreting strong levels of IL-2 and lower levels of TNF-a compare to 1G and 2G anti-CD38 CAR-T, in vitro.
  • MM1.S cells inactivated for both CD38 and CS1 appeared significantly resistant to DCAR-T cell lysis as 60% of the cells remained viable at the E:T of 1:2, as average, and 90% at the E:T of 1:10 ( Figure 4a).
  • MM cells inactivated for CD38 MM1.S38ko
  • CS1 MM1.SCS1ko
  • NIH3T3 cells expressing CD38 (NIH38), CS1 (NIHCS1), CD38 and CS1 (NIH38CS1) or none (NIHwt) were used each cell line to support CD38 edited DCAR-T cell expansion in vitro.
  • DCAR-T stimulated with either NIHwt or NIH38 cells expanded very poorly ( Figure 4b).
  • culture on NIH38CS1 feeder cells triggered a robust proliferation of DCAR-T, with cells numbers increasing from 25000 at day 0 to more than 1.5x106 at day 14.
  • DCAR-T co- culture on NIHCS1 cells readily expanded, albeit at a lower level compare to those cultured on NIH38CS1 cells expressing both CD38 and CS1 antigens.
  • NIH3T3 cellular models to investigate in vitro cytokine production by edited DCAR-T in response to engagement of anti-CD38 CAR, anti-CS1 CCR or both.
  • the medium of DCAR-T cultured for 96 hours on NIHwt contained low concentrations of IL-2 and TNF- ⁇ ( Figure 4c).
  • DCAR-T produced weak levels of IL-2 and TNF-a when cultured on NIH38.
  • Dimopoulos M.A., Oriol, A., Nahi, H., San-Miguel, J., Bahlis, N.J., Usmani, S.Z., Rabin, N., Orlowski, R.Z., Komarnicki, M., Suzuki, K., et al. (2016). Daratumumab, Lenalidomide, and Dexamethasone for Multiple Myeloma. N Engl J Med 375, 1319-1331. Dimopoulos, M.A., Richardson, P., and Lonial, S. (2022). Treatment Options for Patients With Heavily Pretreated Relapsed and Refractory Multiple Myeloma.

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Abstract

Le traitement par lymphocytes T du récepteur antigénique chimérique (CAR) pour le myélome multiple (MM) ciblant l'antigène de maturation des lymphocytes B (BCMA) induit des taux de réponse globaux élevés. Cependant, une rechute se produit fréquemment et de nouvelles stratégies pour des cellules CAR-T ciblant des cellules MM s'imposent. Les cellules CAR-T ciblant CD38 représentent une alternative potentielle, mais l'expression de CD38 sur des cellules T activées et d'autres cellules hématopoïétiques augmente les problèmes d'efficacité et de sécurité d'une telle thérapie. Ici, les inventeurs ont développé DCAR, un système CAR double ciblant CD38 et SLAMF7 (CS1) par l'intermédiaire respectivement de récepteurs d'activation et de co-stimulation divisés. En outre, les inventeurs montrent que l'inactivation de CRISPR/Cas9 du gène CD38 améliore l'activité anti-MM de DCAR-T in vitro. Les DCAR-T éditées ont développé de fortes réponses spécifiquement à l'encontre des cellules MM exprimant à la fois les antigènes CD38 et CS1 in vitro et in vivo. Plus important encore, les inventeurs attestent que, contrairement à CAR anti-CD38, qui suscite une réaction immunitaire grave contre des cellules hématopoïétiques dans un modèle de souris humanisé, DCAR-T ne présente aucun signe de toxicité. En conséquence, la présente invention concerne de nouvelles cellules CAR-T à double division et des utilisations de ces dernières pour le traitement de malignités hématologiques CD38-positives.
EP23765243.3A 2022-09-06 2023-09-05 Nouvelles cellules car-t à double division destinées au traitement de malignités hématologiques cd38-positives Pending EP4583905A1 (fr)

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