WO2019129850A1 - Cellules génétiquement modifiées immédiatement disponibles pour une thérapie - Google Patents

Cellules génétiquement modifiées immédiatement disponibles pour une thérapie Download PDF

Info

Publication number
WO2019129850A1
WO2019129850A1 PCT/EP2018/097079 EP2018097079W WO2019129850A1 WO 2019129850 A1 WO2019129850 A1 WO 2019129850A1 EP 2018097079 W EP2018097079 W EP 2018097079W WO 2019129850 A1 WO2019129850 A1 WO 2019129850A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
tcr
antibody
alpha beta
uniprot
Prior art date
Application number
PCT/EP2018/097079
Other languages
English (en)
Inventor
Caleph WILSON
Laurent Poirot
Philippe Duchateau
Original Assignee
Cellectis
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 Cellectis filed Critical Cellectis
Publication of WO2019129850A1 publication Critical patent/WO2019129850A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464413—CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/804—Blood cells [leukemia, lymphoma]
    • 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 A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection
    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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 A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • 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
    • 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

Definitions

  • the step of purification is crucial for depleting the alpha beta TCR-positive T cell fraction as much as possible, as this fraction could be directly responsible for GvHD when the engineered cells are injected into patients. Moreover, because the final product will undergo amplification once in a patient, even a tiny number of TCR-positive cells, when amplified, will result in the occurrence of GvHD. Despite sophisticated and cost-effective techniques of purification, homogenous populations devoid of detrimental activity when transplanted into a patient, are difficult to obtain and remains a challenge.
  • a method for manufacturing non alloreactive cells comprising:
  • alpha beta T Cell Receptor alpha beta TCR
  • a TCR+ alpha beta T Cell Receptor
  • engineered cells comprising at least 99.9% of engineered cells with an inactivated alpha TCR gene
  • engineered cells comprising at least 99.99% of engineered cells with an inactivated alpha TCR gene.
  • the method according to any one of items 1 to 12, which further comprises a differentiation step, resulting in the production of matured cells expressing a recombinant receptor provided that said recombinant receptor is not a recombinant alpha beta TCR that can bind to a reagent binding to alpha beta TCR and maturation comprises acquiring a cytotoxic activity.
  • the fill and finish step comprises thawing a frozen sample collected from a healthy donor, said sample preferably comprising T cells or stem cells and said sample preferably being blood, tissue or a blood derived or tissue derived product.
  • a composition comprising cells comprising engineered cells expressing alpha beta TCR on their surface in less than 80% of the total cells, preferably less than 10% of the total cells, more preferably less than 5% of the total cells, even more preferably in less than 3% of the total cells, bound to an antibody selectively binding to an antigen present at the surface of cells expressing said cell surface alpha beta TCR.
  • alpha beta T Cell Receptor alpha beta TCR
  • an incubation step wherein said cells are incubated with an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells.
  • Non alloreactive means that allogenic engineered cells when administered into a host which is host from which the cells were not isolated originally induce undetectable immune response (GVHD grade 1 , CRS grade 1 , anamnestic etc) as compared to a response observed with non engineered allogenic cells in a histoincompatible host
  • an incubation step wherein said cells are incubated with a reagent selectively binding to an antigen present at the surface of alpha beta TCR cells, preferably said antigen is an antigen of the endogenous alpha beta TCR.
  • alpha beta T Cell Receptor alpha beta TCR
  • an incubation step wherein said cells are incubated with a reagent, preferably an antibody selectively binding to an antigen present at the surface of alpha beta TCR cells, preferably said antigen is an antigen of the endogenous alpha beta TCR, is provided.
  • a reagent preferably an antibody selectively binding to an antigen present at the surface of alpha beta TCR cells, preferably said antigen is an antigen of the endogenous alpha beta TCR, is provided.
  • an incubation step wherein the cells are incubated with a reagent selectively binding to an antigen present at the surface of cells (still) expressing said endogenous TCR component.
  • the invention may be further summarized by the following items:
  • CAR Chimeric Antigen Receptor
  • the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD38, CS1 and/or CD70, together with an inactivation of the genes encoding respectively CD38, CS1 and/or CD70 in the cells expressing said CARs.
  • a cell surface marker such as CD38, CS1 and/or CD70
  • the recombinant receptors are recombinant chimeric antigen receptors (CAR) comprising a scfv and a TAG.
  • CAR chimeric antigen receptors
  • the reagent consists in or comprises an antibody, or an antibody-drug conjugate or a fragment of an antibody, said fragment of antibody comprising a domain that binds to a protein of the complement or to a receptor of a cell mediating antibody dependent cytotoxicity, said receptor being preferably a Fc receptor.
  • immune checkpoint genes which are genes that can be inactivated or over expressed according to the teaching of the present invention in order to improve the efficiency and fitness of the engineered T-cells.
  • the immune checkpoints gene are preferably selected from such genes having identity to those listed in this table involved into co- inhibitory receptor function, cell death, cytokine signaling, arginine tryptophan starvation, TCR signaling, Induced T-reg repression, transcription factors controlling exhaustion or anergy, and hypoxia mediated tolerance.
  • the edited genes are human edited genes.
  • Table 1 Genes that make allogeneic T-cells more active for immunotherapy when engineered (KO, inactivated, overexpressed %) according to the present invention.
  • polynucleotide sequence(s) which expression mediate(s) interaction with HLA-G, such as ILT2 or ILT4;
  • polynucleotide sequence(s), which expression is(are) involved into the down regulation of T-cell proliferation such as SEMA7A, SHARPIN to reduce Treg proliferation, STAT1 to lower apoptosis, PEA15 to increase IL-2 secretion and RICTOR to favor CD8 memory differentiation; and/or
  • an engineered immune cell bound to an antibody specific for an alpha beta TCR is provided said engineered immune cell express a chimeric antigen receptor (CAR) and comprises a genetic modification reducing or inactivating the expression of a microRNA genomic sequence, more particularly said microRNA genomic sequences are selected from miR21 , mir26A and miR101 .
  • CAR chimeric antigen receptor
  • the method of the present invention further comprises a step of incubation, wherein TCR-positive cells are incubated in the presence of an antibody that binds selectively to alpha beta TCR-positive cells, or selectively to alpha betaTCR expressing cells, such as an antibody selective for CD3, an antibody selective for alphaTCR, an antibody selective for betaTCR, or an antibody selective for alphabetaTCR, preferably an antibody selective for CD3.
  • TCR-positive cells preferably between 15% and 0.001 % of TCR positive cells
  • an anti-TCR antibody at a temperature comprised between from 1 °C to 40°C, preferably between from 4°C to 37°C for 5 minutes to 60 minutes, more preferably for 60 minutes at 4°C.
  • TCR-positive cells (preferably between 15% and 0.001 % of TCR positive cells) incubated in the presence of an anti-TCR antibody at a temperature comprised between from 1 °C to 40°C, preferably between from 4°C to 37°C are then rinsed and frozen.
  • the method further comprises a fill and finish step, wherein the cells are packaged and frozen; and, preferably, the incubation step is performed before the fill and finish step, or the incubation step is performed after the fill and finish step and after optionally thawing the composition.
  • the method successively comprises:
  • the engineered cells for therapy are engineered cells for treating inflammation, the recombinant receptor being able to selectively bind to one or more antigens present or presented at the surface of inflammatory cells, cells exposed to inflammation or inflammatory factors.
  • the cells are T cells.
  • composition of the invention induces no TCR-induced Graft versus Host Disease, (GVHD), regardless of the grade analyzed (grade 1 , 2 3 or 4) as compared to a composition comprising alpha beta TCR-positive cells that was not incubated in the present of an anti-alpha beta TCR-antibody.
  • GVHD Graft versus Host Disease
  • It is another object of the invention to provide a method for purifying engineered cells comprising a step of contacting a composition as described above or obtainable by the method described above with a complement protein, or with cytolytic cells, so as to deplete or eliminate cells still expressing the endogenous TCR component.
  • the present invention makes it possible to overcome the drawbacks of the prior art.
  • the invention provides an efficient process of manufacturing gene-modified cells for therapy, better suited for an implementation at the industrial production scale and for the production of high quality living treatment, with reduced occurrence or reduced magnitude of adverse reactions such as GvHD.
  • a reagent selectively binding to an antigen present at the surface of alpha beta+ TCR cells binds to endogenous alpha beta TCR - expressing cells and allows an antibody -mediated destruction of alpha beta expressing cells in the presence of an appropriate reagent.
  • said activation step preferably comprises contacting the cells with an anti-CD3 antibody, an anti-CD28 antibodies, stromal cells, or any combination of anti-CD3 antibody, anti-CD28 antibody, stromal cells.
  • progenitor cells such as stem cells
  • immature pre-T lymphocytes are engineered to develop into mature T cells ie into cells capable of degranulating upon binding of recombinant receptor to its target.
  • the disruption step is performed by introducing a polynucleotide comprising a sequence coding a Chimeric Antigen Receptor (CAR) into the cells and inserting it into an endogenous TCR component gene locus, preferably the constant region of the TCR alpha component.
  • CAR Chimeric Antigen Receptor
  • composition of the invention wherein the cells comprise T cells expressing a chimeric antigen receptor.
  • a protein of the complement is a protein initiating a complement-dependent antibody response that can lead to the destruction of cells binding to said antibody (Janeway, CA Jr; Travers P; Walport M; et al. (2001). "The complement system and innate immunity”. Immunobiology: The Immune System in Health and Disease. New York: Garland Science. Retrieved 1 December 2017
  • T cell receptors are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen.
  • the TCR is generally made from two chains, alpha and beta, which assemble to form a heterodimer and associates with the CD3-transducing subunits to form the T-cell receptor complex present on the cell surface.
  • Each alpha and beta chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region.
  • V immunoglobulin-like N-terminal variable
  • C constant
  • the variable region of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells.
  • T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction.
  • MHC restriction Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of GVHD. It has been shown that normal surface expression of the TCR depends on the coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusner 1990). The inactivation of TCRalpha or TCRbeta can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD.
  • the product thus obtained is a composition, a pharmaceutical composition, comprising engineered T cells as well as an anti-TCR reagent such as an anti- TCR antibody (or fragment or conjugate thereof) bound to any residual TCR- positive cells in the composition, and a pharmaceutically acceptable vehicle.
  • an anti-TCR reagent such as an anti- TCR antibody (or fragment or conjugate thereof) bound to any residual TCR- positive cells in the composition, and a pharmaceutically acceptable vehicle.
  • the transformation step can be performed before the disruption step. Or it can be performed at the same time as the disruption step.
  • An additional disruption step can also optionally be performed before the transformation step. It can even be performed before the (firstly mentioned) disruption step.
  • the optional purification step and the incubation step can be performed after the transformation / disruption step(s).
  • the optional purification step and the incubation step can be performed after the expansion step and just before the fill and finish step.
  • the optional differentiation and/or maturing step is preferably performed after the expansion step. It can be followed by the incubation step, or by the purification step and then the incubation step.
  • washing, centrifugation, culturing and exchange of culture media can be performed in addition to the above steps, and notably between the main steps specified above.
  • they are obtained from a healthy donor or from a pool of healthy donors. They may also be obtained from a blood bank. Pooling cells from different donors may have a number of advantages as disclosed in detail in WO 2015/075175, which is incorporated herein by reference.
  • the T cells used in the present invention can also be obtained from a cell culture, such as a culture of stem cells.
  • the stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.
  • Representative human cells are CD34+ cells.
  • PBMCs peripheral blood mononuclear cells
  • whole blood buffy coat, leukapheresis or any clinical sampling of blood product.
  • Other sources for the cells include bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue and tumors.
  • An accessory molecule on the surface of the T cells can also be stimulated, using a ligand that binds the accessory molecule.
  • the population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used.
  • Cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells.
  • the method of the invention comprises at least one step of disrupting at least one gene encoding an endogenous T Cell Receptor (TCR) component.
  • TCR component is meant any molecule which is part of the TCR complex.
  • the method also comprises a step of additionally disrupting at least one other gene, which can in particular be another gene encoding a TCR component, or a gene expressing a target for an immunosuppressive agent, or a gene encoding an immune checkpoint function.
  • at least one other gene which can in particular be another gene encoding a TCR component, or a gene expressing a target for an immunosuppressive agent, or a gene encoding an immune checkpoint function.
  • the edited gene can also encode beta-2 microglobulin or any one of the following FILA class ll-related gene selected from the group consisting of regulatory factor X-associated ankyrin-containing protein (RFXANK), regulatory factor 5 (RFX5), regulatory factor X-associated protein (RFXAP), class II transactivator ⁇ CUT A), HLA-DPA (a chain), HLA-DPB (b chain), HLA-DQA, HLA- DQB, HLA-DRA, HLA-DRB, HLA-DMA, HLA-DMB, HLA-DOA and HLA-DOB.
  • RFXANK regulatory factor X-associated ankyrin-containing protein
  • RFX5 regulatory factor 5
  • RFXAP regulatory factor X-associated protein
  • class II transactivator ⁇ CUT A HLA-DPA (a chain), HLA-DPB (b chain), HLA-DQA, HLA- DQB, HLA-DRA, HLA-DRB, HLA-DMA,
  • the (first) disruption step is a step of disrupting the TCRa gene and the additional disruption step is a step of disrupting the TCR gene.
  • each disruption step may comprise degrading or inactivating a gene, for instance by using siRNA, miRNA, antisense RNA molecules, antisense DNA molecules, or agents conveying RNA-directed DNA methylation.
  • the DNA digesting agent may be directly introduced into the cells.
  • the disruption step(s) may include transforming the cells with exogenous material, such as a nucleic acid (preferably a RNA) encoding said DNA digesting agent.
  • each disruption step relies on the expression in the cells of two DNA digesting agents such that said each of the two DNA digesting agents specifically and respectively catalyzes a modification, e.g. cleavage, in a pair of genes (e.g.
  • Non-limiting examples of nucleases include DNase I, Benzonase, Exonuclease I, Exonuclease III, Mung Bean Nuclease, Nuclease BAL 31 , RNase I, S1 Nuclease, Lambda Exonuclease, RecJ and T7 exonuclease.
  • Restriction endonucleases are the most preferred class of nucleases that may be used.
  • the DNA digesting agent is a site-specific nuclease, and more particularly a“rare-cutting" endonuclease, the recognition sequence of which rarely occurs in a genome.
  • the recognition sequence of the site- specific nuclease occurs only once in a genome.
  • the DNA digesting agent is a site-specific Cas nuclease.
  • the Cas nuclease is Cas9.
  • the nuclease is Cas9 and the exogenous material further comprises a guide RNA.
  • Another example of a sequence-specific nuclease system that can be used with the methods and compositions described herein includes the Cas9/CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, which exploits RNA-guided DNA binding and sequence-specific cleavage of target DNA.
  • the guide RNA/Cas9 combination confers site specificity to the nuclease.
  • the DNA digesting agent is another site-specific nuclease such as a zinc finger nuclease.
  • Zinc finger nucleases generally comprise a DNA binding domain (i.e. zinc finger) and a cutting domain (i.e. nuclease).
  • Zinc finger binding domains may be engineered to recognize and bind to any nucleic acid sequence of choice.
  • An engineered zinc finger binding domain may have a novel binding specificity compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.
  • Rational design includes, for example, using databases comprising doublet, triplet, and/or quadruplet nucleotide sequences and individual zinc finger amino acid sequences, in which each doublet, triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence.
  • the zinc finger nuclease may further comprise a nuclear localization signal or sequence (NLS).
  • NLS nuclear localization signal or sequence
  • An NLS is an amino acid sequence which facilitates targeting the zinc finger nuclease protein into the nucleus to introduce a double stranded break at the target sequence in the chromosome.
  • a zinc finger nuclease also includes a cleavage domain.
  • the cleavage domain portion of the zinc finger nuclease may be obtained from any endonuclease or exonuclease.
  • Non-limiting examples of endonucleases from which a cleavage domain may be derived include, but are not limited to, restriction endonucleases and homing endonucleases.
  • the near edges of the recognition sites of the zinc finger nucleases may be separated by 6 nucleotides. In general, the site of cleavage lies between the recognition sites.
  • a zinc finger nuclease may comprise the cleavage domain from at least one type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. Additional restriction enzymes also contain separable binding and cleavage domains, and these also are contemplated by the present disclosure.
  • the targeting endonuclease may be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by a large recognition site, i.e. the recognition site generally ranges from about 12 base pairs to about 40 base pairs. As a consequence of this requirement, the recognition site generally occurs only once in any given genome.
  • Naturally- occurring meganucleases recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Meganucleases can be targeted to specific chromosomal sequence by modifying their recognition sequence.
  • the TALE nuclease may be a mega TAL nuclease, i.e. a fusion protein comprising a TALE DNA binding domain and a meganuclease cleavage domain.
  • the meganuclease cleavage domain is active as a monomer and does not require dimerization for activity.
  • the nuclease domain may also exhibit DNA-binding functionality.
  • the nuclease may be a homing nuclease.
  • Homing endonucleases include l-Scel, l-Ceul, l-Pspl, Vl-Sce, l-SceIN, l-Csml, I Panl, I- Scell, l-Ppol, l-Scelll, l-Crel, l-Tevl, l-Tevll and l-7evlll.
  • the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites.
  • the DNA digesting agent is a site-specific nuclease selected from the group consisting of zinc finger, MEGATAL, TALE and CRISPR/Cas9 nucleases, preferably a TALE nuclease, more preferably a TAL nuclease as those described in WO2014184741
  • an additional catalytic domain can be further expressed in the cells together with the DNA digesting agent (e.g. rare-cutting endonuclease) to increase mutagenesis in order to enhance the capacity to inactivate targeted genes.
  • said additional catalytic domain can be a DNA end processing enzyme.
  • DNA end-processing enzymes include 5’-3' exonucleases, 3’-5' exonucleases, 5’-3' alkaline exonucleases, 5' flap endonucleases, helicases, hosphatase, hydrolases and template-independent DNA polymerases.
  • Non limiting examples of such catalytic domain comprise of a protein domain or catalytically active derivate of the protein domain selected from the group consisting of human Exol, yeast Exol, E. coli Exol, human TREX2, mouse TREX1 , human TREX1 , bovine TREX1 , rat TREX1 , TdT (terminal deoxynucleotidyl transferase) human DNA2, yeast DNA2.
  • said additional catalytic domain has a 3'-5'-exonuclease activity and in more preferred embodiments, said additional catalytic domain is a TREX, more preferably TREX2 catalytic domain.
  • said catalytic domain is formed by a single chain TREX polypeptide. Said additional catalytic domain may be fused to a nuclease fusion protein or chimeric protein optionally by a peptide linker.
  • the disruption step(s) of the method further comprise the introduction of an exogenous nucleic acid into the cells which comprises at least a sequence homologous to a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the exogenous nucleic acid.
  • said exogenous nucleic acid comprises first and second portions which are homologous to the 5' and 3' regions of the target nucleic acid sequence, respectively.
  • Said exogenous nucleic acid in these embodiments also comprises a third portion positioned between the first and the second portion which comprises no homology with the 5' and 3' regions of the target nucleic acid sequence.
  • a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid.
  • homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used within said donor matrix. Therefore, the exogenous nucleic acid is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, shared nucleic acid homologies are located in regions flanking upstream and downstream the site of the break and the nucleic acid sequence to be introduced should be located between the two arms.
  • said exogenous nucleic acid may successively comprise a first region of homology to sequences upstream of said cleavage, a sequence to inactivate one targeted gene selected from the group consisting of CD52, GR, dCK, TCRa and TCR and a second region of homology to sequences downstream of the cleavage.
  • Said polynucleotide introduction step can be simultaneous, before or after the introduction or expression of the material encoding the DNA digesting agent (e.g. rare-cutting endonuclease).
  • the DNA digesting agent e.g. rare-cutting endonuclease
  • exogenous nucleic acid can be used to knock-out a gene, e.g.
  • inactivation of genes from the group consisting of CD52, GR, dCK, TCRa and TCR can be performed at a precise genomic location targeted by a specific nuclease such as a TALE-nuclease, wherein said specific nuclease catalyzes a cleavage and wherein said exogenous nucleic acid successively comprising at least a region of homology and a sequence to inactivate one targeted gene selected from the group consisting of CD52, GR, dCK, TCRa and TCR is integrated by homologous recombination.
  • a specific nuclease such as a TALE-nuclease
  • genes can be, successively or at the same time, inactivated by using several nucleases such as TALE-nucleases respectively and specifically targeting one defined gene and several specific polynucleotides for specific gene inactivation.
  • the various enzymes described above such as endonucleases, TALE- nucleases, DNA-end processing enzymes as well as the exogenous nucleic acids can be introduced as transgenes encoded by one or different plasmid vectors or by electroporation under the form of mRNA.
  • Different transgenes can be included in one vector which comprises a nucleic acid sequence encoding a ribosomal skip sequence such as a sequence encoding a 2A peptide.
  • two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame.
  • Such ribosomal skip mechanisms are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.
  • 2A peptides can be used to express in the cells a rare-cutting endonuclease and a DNA end- processing enzyme.
  • the plasmid vector can contain a selection marker which provides for identification and/or selection of cells which received said vector.
  • Polypeptides may be synthesized in situ in the cells as a result of the introduction of polynucleotides encoding said polypeptides into the cells.
  • the inventors have considered means known in the art to allow delivery into cells or into subcellular compartments of said cells the polynucleotide(s) and/or polypeptides of the invention including the polynucleotide expressing an endonuclease(s), their possible co-effectors (e.g. guide RNA or DNA associated with Cas9 nucleases) as well as the chimeric antigen receptors.
  • These means include viral transduction, electroporation and also liposomal delivery means, polymeric carriers, chemical carriers, lipoplexes, polyplexes, dendrimers, nanoparticles, emulsion, natural endocytosis or phagocytose pathway as non- limiting examples.
  • Methods for introducing a polynucleotide construct into animal cells, in particular into animal genome include as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods.
  • the polynucleotide construct is integrated into the genome of the cell at the TCR locus (TRAC locus).
  • Said polynucleotides may be introduced into the cells by for example, liposomes, recombinant viral vectors (e.g.
  • retroviruses adenoviruses, preferably adenoviruses, more preferably adenoviruses type 6 particles with type 2 ITR).
  • transient transformation methods include for example microinjection, electroporation or particle bombardment.
  • Said polynucleotides may be included in vectors, more particularly plasmids or virus, so as to be expressed in the cells.
  • polynucleotides encoding the endonucleases of the present invention are transfected under mRNA form by electroporation in order to obtain transient expression and avoid chromosomal integration of foreign DNA.
  • the inventors have determined different optimal conditions for mRNA electroporation in primary cell.
  • the inventor used the cytoPulse technology which allows, by the use of pulsed electric fields, to transiently permeabilize living cells for delivery of material into the cells (U.S. patent 6,010,613 and WO 2004/083379). Pulse duration, intensity as well as the interval between pulses can be modified in order to reach the best conditions for high transfection efficiency in primary cells with minimal mortality.
  • the first high electric field pulses allow pore formation, while subsequent lower electric field pulses allow to moving the polynucleotide into the cell.
  • the inventor describes the steps that led to achievement of >95% transfection efficiency of mRNA in T cells, and the use of the electroporation protocol to transiently express different kind of proteins in T cells.
  • step (b) one electrical pulse with a voltage range from 500 to 3000 V per centimeter, preferably 800 V, with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c) ;
  • Electroporation medium can be any suitable medium known in the art.
  • the electroporation medium has conductivity in a range spanning 0.01 to 1.0 milliSiemens.
  • the method of the invention preferably comprises a purification step, wherein cells in which the TCR disruption described above has not occurred (TCR-positive cells) are depleted, so that the population of cells is enriched in cells in which the TCR disruption described above has occurred (TCR-negative cells).
  • Purification may e.g. be performed by contacting the cell composition with particles (such as magnetic particles) coated with an anti-TCR antibody or antibody fragment, so that TCR-positive cells are bound to the particles and removed by separating said particles from the composition.
  • the proportion of TCR-negative cells at the end of the purification step is more than 97%, preferably more than 98%, preferably more than 99%, preferably more than 99.1 %, or more than 99.2%, or more than 99.3%, or more than 99.4%, or more than 99.5%, or more than 99.6%, or more than 99.7%, or more than 99.8%, or more than 99.9%.
  • Methods of identification or isolation of TCR-negative cells include FACS, column chromatography, panning with magnetic beads, western blots, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno fluorescent assays and the like.
  • isolation of TCR-negative cells includes using magnetic beads comprising anti TCR antibodies.
  • isolation of TCR-negative cells includes using magnetic beads comprising anti TCR antibodies 4 days after TCR gene disruption.
  • cells in which said gene or genes have not been properly disrupted can also be depleted in the same manner as described above with respect to the TCR-positive cells, using the same range of techniques.
  • Such purification can be performed simultaneously with the purification described above, or separately. In particular, if two disruption steps are used, two respective purification steps can also be carried out.
  • the method of the invention comprises a transformation step, wherein the cells are modified by introducing a polynucleotide into them which encodes a recombinant receptor.
  • said recombinant receptor is a CAR.
  • the CAR is then expressed at on the surface of the cells.
  • said expression can be conditional (expression to depend on the presence or absence of a drug in the milieu.
  • the transformation step can be performed simultaneously with (to) the disruption step.
  • a polynucleotide encoding a recombinant receptor can be introduced into the cells and inserted at a gene locus, such as at the gene locus of an endogenous TCR component, preferably the endogenous TCR alpha component, so that the endogenous TCR component gene is engineered (inactivated, knocked out, and replaced by a polynucleotide sequence encoding the recombinant receptor.
  • the transformation step can be performed simultaneously with the disruption step.
  • the polynucleotide can be introduced into the cells and inserted at any one of following the gene locus (using a TALEN and AAV6/AAV2 particles for a targeted insertion):
  • the inserted exogenous coding sequence(s) can have the effect of reducing or preventing the expression, by the engineered immune cell of at least one protein selected from the TCR alpha subnit (encoded by the TRAC gene), PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG3 (Uniprot P18627), HAVCR2 (Uniprot Q8TDQ0), BTLA (Uniprot Q7Z6A9), CD160 (Uniprot 095971 ), TIGIT (Uniprot Q495A1 ), CD96 (Uniprot P40200), CRTAM (Uniprot 095727), LAIR1 (Uniprot Q6GTX8), SIGLEC7 (Uniprot (
  • the inserted exogenous coding sequence may have the effect of reducing or preventing the expression of genes encoding or positively regulating suppressive cytokines or metabolites or receptors thereof, in particular TGFbeta (Uniprot:P01137), TGFbR (Uniprot:P37173), IL10 (Uniprot:P22301 ), IL10R (Uniprot: Q13651 and/or Q08334), A2aR (Uniprot: P29274), GCN2 (Uniprot: P15442) and PRDM1 (Uniprot: 075626).
  • TGFbeta Uniprot:P01137
  • TGFbR Uniprot:P37173
  • IL10 Uniprot:P22301
  • IL10R Uniprot: Q13651 and/or Q08334
  • A2aR Uniprot: P29274
  • GCN2 Uniprot: P15442
  • PRDM1 Unipro
  • the inserted exogenous coding sequence may have the effect of reducing or preventing the expression of a gene responsible for the sensitivity of the immune cells to compounds used in standard of care treatments for cancer or infection, such as drugs purine nucleotide analogs (PNA) or 6-Mercaptopurine (6MP) and 6 thio-guanine (6TG) commonly used in chemotherapy. Reducing or inactivating the genes involved into the mode of action of such compounds (referred to as“drug sensitizing genes”) improves the resistance of the immune cells to same.
  • PNA drugs purine nucleotide analogs
  • 6MP 6-Mercaptopurine
  • Examples of drug sensitizing gene are those encoding DCK (Uniprot P27707) with respect to the activity of PNA, such a clorofarabine et fludarabine, HPRT (Uniprot P00492) with respect to the activity of purine antimetabolites such as 6MP and 6TG, and GGH (Uniprot Q92820) with respect to the activity of antifolate drugs, in particular methotrexate.
  • DCK Uniprot P27707
  • HPRT Uniprot P00492
  • purine antimetabolites such as 6MP and 6TG
  • GGH Uniprot Q92820
  • the resulting product is more efficient than non engineered immune cells (even with a CAR) and safer because inducing less side effects.
  • the CAR preferably enables the engineered immune cells to trigger the destruction of pathogens or more preferably of pathological cells, in particular malignant cells.
  • the CAR preferably specifically binds to at least one antigen marker which is present on a target, such as pathological cells.
  • the CAR preferably specifically binds to at least one antigen marker which is present on a target, such as pathological cells and said at least one antigen marker is not bound by the anti TCR antibody used in the present invention to eliminate TCR-expressing cells.
  • the CAR can be single-chain or multi-chain.
  • Multi-chain CAR architectures are advantageous in that the T cell activity is modulated in terms of specificity and intensity.
  • Multiple subunits can shelter additional co-stimulation domains or keep such domains at an appropriate distance.
  • Single-chain CARs are synthetic receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule.
  • the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains can also been used.
  • the signaling domains can e.g. be derived from the cytoplasmic region of the and CD3z or the Fe receptor gamma chains.
  • Signaling domains from co- stimulatory molecules including CD28, OX-40 (CD134) and 4-IBB (CD137) can be been added alone (so-called second generation CARs) or in combination (so- called third generation CARs) to enhance survival and increase proliferation of CAR-modified T cells.
  • CARs targeting an antigen marker which is common to pathological cells and T cells such as CD38
  • further CARs may be expressed that are directed towards other antigen markers not necessarily expressed by the T cells, so as to enhance T cell specificity.
  • the antigen targeted by the CAR can be an antigen from any cluster of differentiation molecules (e.g. CD16, CD64, CD78, CD96, CLL1 , CD1 16, CD1 17, CD71 , CD45, CD123 and CD138), a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvlll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, b-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE- 1 , MN-CA IX, human
  • a tumor-associated surface antigen such
  • the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD38, CS1 and/or CD70, together with an inactivation of the genes encoding respectively CD38, CS1 and/or CD70 in the cells expressing said CARs.
  • the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD38, CS1 together with an inactivation of the genes encoding respectively CD38, CS1 in the cells expressing said CARs.
  • the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD38, HSP70, CD30, FAP, HER2 CD79, CD123, CD22, CLL-1 , MUC-1 GD2, O acetyl GD2, CS1 or CD70.
  • a cell surface marker such as CD38, HSP70, CD30, FAP, HER2 CD79, CD123, CD22, CLL-1 , MUC-1 GD2, O acetyl GD2, CS1 or CD70.
  • the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD38, HSP70, CD30, FAP, HER2 CD79, CD123, CD22, CLL-1 , MUC-1 GD2, O acetyl GD2, CS1 or CD70, BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70, MUC16 - PRAME - TSPAN10, CLAUDIN18.2 - DLL3 - LY6G6D, Liv-1 - CHRNA2 - ADAM 10.
  • a cell surface marker such as CD38, HSP70, CD30, FAP, HER2 CD79, CD123, CD22, CLL-1 , MUC-1 GD2, O acetyl GD2, CS1 or CD70
  • BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70 MUC16 - PRAME - TSPAN10
  • the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70, MUC16 - PRAME - TSPAN10, CLAUDIN18.2 - DLL3 - LY6G6D, Liv-1 - CHRNA2 - ADAM 10.
  • a cell surface marker such as BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70, MUC16 - PRAME - TSPAN10, CLAUDIN18.2 - DLL3 - LY6G6D, Liv-1 - CHRNA2 - ADAM 10.
  • CARs that can be expressed to create multi-specific cells are antigen receptors directed against multiple myeloma or lymphoblastic leukemia antigen markers, such as TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1 ), FKBPII (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708) and FCRL5 (UNIPROT Q68SN8).
  • TNFRSF17 UNIPROT Q02223
  • SLAMF7 UNIPROT Q9NQ25
  • GPRC5D UNIPROT Q9NZD1
  • FKBPII UNIPROT Q9NYL4
  • KAMP3, ITGA8 UNIPROT P53708
  • FCRL5 UNIPROT Q68SN8
  • the multi-chain CAR can comprise several extracellular ligand-binding domains, to simultaneously bind different elements in a target thereby augmenting immune cell activation and function.
  • the extracellular ligand-binding domains can be placed in tandem on the same transmembrane polypeptide, and optionally can be separated by a linker.
  • said different extracellular ligand-binding domains can be placed on different transmembrane polypeptides composing the multi-chain CAR.
  • the cells may express multi-chain CARs comprising different extracellular ligand binding domains.
  • they may express at least a part of FcsRI beta and/or gamma chain fused to a signal-transducing domain and several parts of FcERI alpha chains fused to different extracellular ligand binding domains on their surface.
  • they may express a FcsRI beta and/or gamma chain fused to a signal-transducing domain and several FcsRI alpha chains fused to different extracellular ligand binding domains.
  • two, three, four, five, six or more multi-chain CARs may be expressed in the cells, so as to preferably simultaneously bind different elements in a target, thereby augmenting immune cell activation and function.
  • the signal transducing domain or intracellular signaling domain of the multi-chain CAR of the invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response.
  • the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the multi-chain CAR is expressed.
  • the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines.
  • Preferred examples of signal transducing domain for use in single or multi- chain CAR can be the cytoplasmic sequences of the Fe receptor or T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that has the same functional capability.
  • the signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs.
  • ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases.
  • ITAM used in the invention can include as non-limiting examples those derived from TORz, FcRy, FcR , FcRs, CD3y, CD36, CD3s, CDS, CD22, CD79a, CD79b and CD66d.
  • the signaling transducing domain of the multi-chain CAR can comprise the CD3z signaling domain, or the intracytoplasmic domain of the FcsRI beta or gamma chains.
  • the cells may express multi-chain CARs which specifically target a cell surface marker such as BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70, MUC16 - PRAME
  • chimeric scFv is meant a polypeptide corresponding to a single-chain variable fragment composed of heavy and light chains (VFI and VL, respectively) and of at least one epitope, which was not originally included in said VH and VL chains.
  • the latter epitope is referred to as“mAb-specific epitope” when it has the ability to be bound specifically by a monoclonal antibody.
  • the mAb-specific epitope is not an epitope recognized by the ScFv.
  • the mAb-specific epitope is not derived from the extracellular domain of the CAR.
  • the components of this chimeric scFv i.e.
  • the light and heavy variable fragments of the ligand binding domain and at least one, preferably 3 or 3 mAb specific epitopes) may be joined together by at least one linker, usually a flexible linker. These components are generally joined to the transmembrane domain of the CAR by a hinge.
  • Any hinge allowing the CART of the invention to reach a desired target, bind said target, and trigger an intracellular signal via a CD8alphaTM- 41 BB/CD3 zeta intracellular domains is appropriate.
  • a preferred hinge of the invention is selected from lgG1 , lgG4, CD8alpha, and FcyRIIIa.
  • the extracellular domain of the CAR comprises a scFv formed by at least a VH chain and a VL chain specific to an antigen, and at least one mAb-specific epitope located between the VH and the VL and/ or in the hinge.
  • the mAb specific-epitopes may be bound together by at least one linker or two linkers and to the transmembrane domain of said CAR.
  • the mAb-specific epitope is an epitope to be bound by an epitope-specific mAb for in vitro cell sorting and/or in vivo cell depletion of T cells expressing a CAR comprising such epitope.
  • the CAR comprises
  • extracellular binding domain further comprises a hinge, - a transmembrane domain, and,
  • the extracellular binding domain may comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mAb- specific epitopes, preferably 2 or 3 mAb-specific epitopes.
  • the CAR according to the present invention, wherein the extracellular binding domain comprises the following sequence
  • Vi is VL and V2 is VH or Vx is VH and V2 is VL;
  • Li is a linker suitable to link the VH chain to the VL chain
  • L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain can be identical or different to other occurrence of L in the same extracellular binding domain, and, x is 0 or 1 and each occurrence of x is selected independently from the others; and,
  • the transformation step preferably comprises introducing at least one polynucleotide encoding the recombinant receptor such as a CAR (or CARs) into the cells, and expressing the polynucleotide(s).
  • the nucleic acid sequences of the present invention are codon- optimized for expression in mammalian cells, preferably for expression in human cells.
  • Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the amino acids as the codons that are being exchanged.
  • Methods for introducing a polynucleotide construct encoding a CAR into cells include as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods.
  • Said polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like.
  • transient transformation methods include for example microinjection, electroporation or particle bombardment.
  • the required polynucleotide(s) are be included in one or more vectors, more particularly plasmids or viruses, for being expressed in cells.
  • the vectors can also contain a selection marker which provides for identification and/or selection of cells which received said vector.
  • the polynucleotide may consist in an expression cassette or expression vector (e.g. a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus vector for transfection of a mammalian host cell).
  • a lentivirus vector is particularly preferred in the present invention.
  • an adeno associated virus vector is even more particularly preferred in the present invention as disclosed in PCT/EP2017/076798 incorporated herein by reference or in Wang J, DeClercq JJ, Hayward SB, et al. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Research. 2016;44(3):e30. doi:10.1093/nar/gkv1 121 .
  • Exogenous sequence refers to any nucleotide or nucleic acid sequence that was not initially present at the selected locus in non engineered cells. This sequence may be homologous to, or a copy of, a genomic sequence, or be a foreign sequence introduced into the cell with parts (eg 5’ and 3’ parts of the endogenous sequence for homologous recombination. By opposition “endogenous sequence” means a cell genomic sequence initially present at a locus. The exogenous sequence preferably codes for a polypeptide which expression confers a therapeutic advantage over sister cells that have not integrated this exogenous sequence at the locus. An endogenous sequence that is gene edited by the insertion of a nucleotide or polynucleotide as per the method of the present invention, in order to express a different polypeptide is broadly referred to as an exogenous coding sequence.
  • exogenous coding sequence of the invention comprises a CAR and immune cells comprising them can be prepared by the skilled person according to the methodology disclosed in WO 2013/176915.
  • the recombinant receptor when expressed in engineered cells of the invention does not (must not) bind to the reagent specific for alpha beta TCR expressing cells used at the incubation step, (anti-TCR /anti-CD3 antibody).
  • the recombinant receptor may be any recombinant receptor provided that the recombinant receptor does not bind to the reagent selectively binding to an antigen present at the surface of alpha beta TCR cells, preferably said antigen is an antigen of the endogenous alpha beta TCR.
  • the reagent selectively binding to an antigen present at the surface of alpha beta TCR cells preferably said antigen is an antigen of the endogenous alpha beta TCR does not bind to the CAR or recombinant TCR.
  • Differentiation / maturing A differentiation and/or maturing step may be provided in the method of the invention.
  • the cells may express the recombinant receptor (preferably the CAR) described above; and/or, as a result of this step, the cells may acquire cytotoxic activity, and for instance may express granzyme A, granzyme B and perforin.
  • the recombinant receptor preferably the CAR
  • This step may be performed in the presence of stromal cells.
  • an immunosuppressive agent can be a calcineurin inhibitor, a target of rapamycin, an interleukin-2 u-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite.
  • the CD52 gene is disrupted (e.g. in the disruption step or in the additional disruption step) and the immunosuppressive treatment comprises a humanized antibody targeting the CD52 antigen.
  • the GR gene is disrupted (e.g. in the disruption step or in the additional disruption step) and the immunosuppressive treatment comprises a corticosteroid such as dexamethasone.
  • an FKBP family gene member or a variant thereof is disrupted (e.g. in the disruption step or in the additional disruption step) and the immunosuppressive treatment comprises FK506 also known as Tacrolimus or fujimycin.
  • said FKBP family gene member is FKBP12 or a variant thereof.
  • the cells of the invention (comprising a proportion of cells with an inactivated TCR gene and a proportion of cells still expressing an alpha beta TCR) are incubated with an anti-TCR reagent, i.e. a reagent which selectively binds to an antigen present at the surface of cells which still express the endogenous TCR component.
  • an anti-TCR reagent i.e. a reagent which selectively binds to an antigen present at the surface of cells which still express the endogenous TCR component.
  • the reagent is approved by health authorities.
  • the reagent is an antibody, eve more preferably an antibody approved by health authorities.
  • an antibody fragment may be used.
  • the fragment must have the ability to bind to said antigen, and it must also have the ability to bind to a protein of the complement or to a receptor of a cell mediating antibody dependent cytotoxicity.
  • it is able to bind to a Fc receptor.
  • a monoclonal antibody or antibody fragment, or antibody-drug conjugate
  • a monoclonal antibody or antibody fragment, or antibody-drug conjugate
  • a polyclonal antibody or antibody fragments, or antibody- drug conjugates is used.
  • the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the TCRa chain.
  • the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the TCRa chains.
  • the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the TCR chain.
  • the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the CD3y chain.
  • the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the CD3s chain.
  • the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the CD3z chain.
  • the anti-TCR antibody may be for instance morumonab-CD3, marketed by Janssen-Cilag under the trade name Orthoclone OKT3 (OKT3). Fragments of this antibody or antibody-drug conjugates based on this antibody may also be used.
  • the anti-TCR antibody may also be for instance otelixizumab, also known as TRX4, developed by Tolerx, Inc. in collaboration with GlaxoSmithKline and manufactured by Abbott Laboratories. Fragments of this antibody or antibody- drug conjugates based on this antibody may also be used.
  • the anti-TCR antibody may also be for instance teplizumab, also known as MGA031 and hOKT3y1 (Ala-Ala), developed at MacroGenics, Inc. Fragments of this antibody or antibody-drug conjugates based on this antibody may also be used.
  • Visilizumab (tentative trade name Nuvion, PDL BioPharma Inc.) is a humanized monoclonal antibody.
  • the anti-TCR antibody may also be for instance TOL101 , made by Tolera Therapeutics and described in particular in document US 8,524,234. Fragments of this antibody or antibody-drug conjugates based on this antibody may also be used.
  • the anti-TCR antibody may also be for instance any one of the active antibody disclosed in W02010027797A1 .
  • a rinsing step may be performed after the incubation step in order to eliminate non-bound anti-TCR reagent.
  • the non-bound anti- TCR reagent is not eliminated.
  • activation-induced cell death (AICD) in remaining TCR-positive cells may be induced during manufacturing by repeated exposure of the cells to the anti-TCR reagent and/or by crosslinking bound anti-TCR reagent.
  • the amount of anti- TCR antibody used for incubating cells of the invention is efficient for binding the TCR expressed at the cell surface so that once cells are depleted cell surface expression of the TCR is below detection by FACS analysis using an antibody specific for an anti-alpha beta TCR or an anti-CD3.
  • the amount of anti- TCR antibody used to be combined with cells of the invention may a dose already used in human for the treatment of a pathological condition (as defined by health authorities), preferably in excess for binding all cell surface expressed alpha beta TCR.
  • the amount of anti- TCR antibody used may represent between 1 picogramme (pg) to 10 microgrammes ⁇ g) of antibody for 10 000 total cells (providing that a proportion of cells still express an alpha beta TCR, said proportion ranging from 80% to 0.00001 %).
  • this step results in the selective binding of the reagent specific for alpha beta TCR positive cells to alpha beta TCR positive cells and formation of a complex between both.
  • the cells are sampled and frozen.
  • either the cells together with the anti-TCR reagent may be subjected to this step; or only the engineered cells may be subjected to this step, in which case the incubation step occurs after thawing the cells, such as just before administering the composition to a patient.
  • Remaining TCR-positive cells in a composition of the invention may represent 40% of the total cells, preferably less than 40% of the total cells, more preferably less than 10% of the total cells, even more preferably less than 3% of the total cells, even more more preferably less than 1 % of the total cells, even more more and more preferably less than 0.1 % of the total cells, ideally less that 1 % of the total cells.
  • the mechanisms of elimination of remaining alpha betaTCR-positive cells by antibody (Ab)-mediated cytotoxicity involving the proteins of the complement or by antibody-dependent cell mediated cytotoxicity (ADCC) cell are well known.
  • the step of elimination of remaining alpha betaTCR-positive cells may be carried out in vitro and occurs upon contact of the Ab with proteins of the complement such as proteins of the complement of the patient to which cells are intended to.
  • engineered cells wherein alpha betaTCR-positive cells are bound to the reagent specific for alpha betaTCR-positive cells may be used as a medicament in a patient.
  • the engineered cells obtained by the method of the invention may comprise less than 3%, preferably less than 2%, more preferably less than 1 %, or less than 0.5%, or less than 0.1 %, of cells still expressing the endogenous (alpha beta) TCR component, as determined by flow cytometry analysis.
  • composition comprising alpha beta TCR-positive cells may be combined to an alpha beta TCR reagent to make a composition according to the present invention. Accordingly, the present invention provides a composition comprising between 90% to 0.001 % TCR positive cells.
  • composition comprises between 90% to 0.001 % TCR- positive cells bound to a reagent specific for alpha beta TCR cells.
  • That may be administered with a drug.
  • said drug may be an immunosuppressive agent, preferably an inhibitor of the calcineurin pathway of T cell activation such as, but not limited to, cyclosporine A (CsA), FK-506; or other inhibitors of IL-2 production such as, but not limited to, rapamycin, and combinations of the foregoing. More preferably, the immunosuppressive agent is CsA.
  • allogeneic is meant that the cells or population of cells that are used do not originate from the patient but from one or more donors (donor is not the patient and cells preferably match cells of the cells of the recipient (patient).
  • Said treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disorders and/or GvHD.
  • Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. They may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or solid tumors.
  • Types of cancers to be treated with the cells of the invention include, but are not limited to, melanomas, carcinomas, blastomas, and sarcomas, as well as leukemia or lymphoid malignancies, and other benign and malignant tumors.
  • Adult tumors/cancers and pediatric tumors/cancers are also included.
  • 10 2 to 10 1 ° engineered cells e.g. 10 5 to 10 9 cells, or 10 6 to 10 8 cells
  • 10 2 to 10 1 ° engineered cells can be administered to a patient, in one or more administrations.
  • the cells or populations of cells can be administrated in one or more doses.
  • the effective amount of cells are administrated as a single dose.
  • the effective amount of cells are administrated as more than one dose over a period time.
  • An effective amount means an amount which provides a therapeutic or prophylactic benefit. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
  • the treatment of the invention can be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • cells are administered to a patient in conjunction with (e.g. before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti- CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as CAMPATH, anti- CD3 antibodies or other antibody therapies
  • cytoxin fludaribine
  • cyclosporin FK506, rapamycin
  • mycoplienolic acid steroids
  • steroids FR901228
  • cytokines cytokines
  • irradiation irradiation.
  • the cell compositions of the present invention are administered to a patient in conjunction with (e.g. before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external- beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATFI.
  • the cell compositions of the present invention are administered following B-cell ablative therapy with agents that react with CD20, e.g. rituxan. or rituximab.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the cells of the present invention.
  • the cells are administered before or following surgery.
  • a method of manufacturing“off the shelf engineered cells for therapy comprising:
  • TCR preferably alpha beta TCR, more preferably less than 5% TCR+ cells
  • said antigen is an antigen of the endogenous alpha beta TCR and is approved by health authorities,
  • the step of providing cells may comprise
  • said antigen is an antigen of the endogenous alpha beta TCR and is approved by health authorities,
  • step(b) Sampling and freezing.
  • step(b) Sampling and freezing.
  • step(c) concomitant
  • the percentage (%) of PBMC in total cell population is > 85% with a CD4 and CD8 counts in total blood CD4 counts between 1000-10000
  • CD4 / CD8 ratio in total blood CD4/CD8 ratio 0.5-4.0
  • Purified T cells may be used such as cells comprising more than 85% CD4 and/or CD4 expressing cells.
  • Donors are Negative for the following disease markers: HIV1/2, Cytomegalovirus CMV, Babesiosis, Kala Azar (visceral leishmaniasis), Hepatitis B, Hepatitis C, HTLVI/II, West Nile Virus, Syphilis (Treponema pallidum) and Trypanosoma Cruzi.
  • Hepatitis A Hepatitis E
  • Epstein-Barr Virus EBV Epstein-Barr Virus EBV
  • Toxoplasma No evidence of active infection for the following disease markers was measured: Hepatitis A, Hepatitis E, Epstein-Barr Virus EBV and Toxoplasma.
  • Activation step the step involves CD3+/CD28+ cells and reagent (Ab) binding to said CD3+/CD28+ antigens for their use in a method for preparing allogeneic cells, less alloreactive.
  • Transduction step the step involves CD3+/CD28+ cells and reagent (Ab) binding to said CD3+/CD28+ antigens for their use in a method for preparing allogeneic cells, less alloreactive.
  • retroviral vectors and more preferably of lentiviral vectors is particularly suited for expressing the chimeric antigen receptors into the T-cells.
  • Methods for viral transduction are well known in the art (Walther et al. (2000) Viral Vectors for Gene Transfer. Drugs. 60(2):249- 271 ).
  • Integrative viral vectors allow the stable integration of the polynucleotides in the T-cells genome and to expressing the chimeric antigen receptors over a longer period of time.
  • the use of AAV vectors and more preferably of AAV6/AAV2 or AAV9 vectors is even more particularly suited for expressing the chimeric antigen receptors into the T-cells after insertion into a determined region such as un the region encoding the alpha subunit of the alpha beta TCR, in particular the constant region of the TCR alpha gene (TRAC).
  • a determined region such as un the region encoding the alpha subunit of the alpha beta TCR, in particular the constant region of the TCR alpha gene (TRAC).
  • Methods for viral transduction are well known in the art (MacLeod, Daniel T. et al. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells. Molecular Therapy , Volume 25 , Issue 4 , 949 - 961 .
  • Integrative viral vectors allow the stable integration of the polynucleotides of the invention in the genome of primary cells and expressing a chimeric antigen receptor over a stable period of time, even in TCR negative cells.
  • the method of the present invention comprises a viral transduction or transfection using nanoparticles, and also may be combined with other gene inactivation and/or transgene insertions.
  • a sequence specific reagent a nuclease, preferably a TALE protein
  • the targeted gene integration is operated by homologous recombination or by Non-Homologous End-Joining (NHEJ) into said immune cells.
  • NHEJ Non-Homologous End-Joining
  • Viral vector such as lentiviral or AAV vector may be used as previously reported in for example Wang J, DeClercq JJ, Hayward SB, et al. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Research. 2016;44(3):e30. doi:10.1093/nar/gkv1121.
  • rare cutting endonucleases used in the present study are sequence-specific endonuclease reagents of choice, insofar as their recognition sequences generally range from 10 to 50 successive base pairs, preferably from 6 to 40 bp, and more preferably from 14 to 20 bp. See Arnould S., et al. (W02004067736) (TAL-nuclease).
  • TALE-nuclease Due to their higher specificity, TALE-nuclease have proven to be particularly appropriate sequence specific nuclease reagents for therapeutic applications, especially under heterodimeric forms - i.e. working by pairs with a “right” monomer (also referred to as“5”’ or“forward”) and‘left” monomer (also referred to as“3”” or“reverse”) as reported for instance by Mussolino et al. (TALEN ® facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773).
  • the following reagent was used : a zing finger nuclease (ZFN) as described, for instance, by Urnov F., et al. (Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646- 651 ), or a MegaTAL nuclease as described, for instance by Boissel et al. (MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research 42 (4):2591 -2601 ).
  • the rare - cutting endonuclease is used in the present study is a TALE-protein.
  • the endonuclease reagent is a RNA-guide to be used in conjunction with a RNA guided endonuclease, such as Cas9 or Cpf1 , as per, inter alia, the teaching by Doudna, J., and Chapentier, E., (The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346 (6213):1077), which is incorporated herein by reference.
  • a RNA guided endonuclease such as Cas9 or Cpf1
  • 80% of the endonuclease reagent is degraded by 30 hours, preferably by 24 hours, more preferably by 20 hours after transfection.
  • Expression of the endonuclease reagent is sufficient to KO the TCRalpha and results in more than 95% of the cells expressing undetectable level of TCR (alpha beta TCR expression by flow cytometry using an alpha beta TCR Ab).
  • the half-life of said Rare cutting endonuclease used in the present study may be increased or decreased by adding 5’ terminal sequences polyA of different length and/or specific 3’ sequences to the known mRNA.
  • a rare cutting endonuclease under mRNA form is preferably synthetized with a cap to enhance its stability according to techniques well known in the art, as described, for instance, by Kore A.L., et a/. (Locked nucleic acid (LNA)- modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization (2009) J Am Chem Soc. 131 (18):6364-5).
  • LNA locked nucleic acid
  • electroporation steps that are used to transfect cells performed in closed chambers comprising parallel plate electrodes producing a pulse electric field between said parallel plate electrodes greater than 100 volts/cm and less than 5,000 volts/cm, substantially uniform throughout the treatment volume such as described in WO/2004/083379, which is incorporated by reference, especially from page 23, line 25 to page 29, line 1 1 .
  • One such electroporation chamber preferably has a geometric factor (cm -1 ) defined by the quotient of the electrode gap squared (cm2) divided by the chamber volume (cm 3 ), wherein the geometric factor is less than or equal to 0.1 cm -1 , wherein the suspension of the cells and the sequence-specific reagent is in a medium which is adjusted such that the medium has conductivity in a range spanning 0.01 to 1 .0 milliSiemens.
  • the suspension of cells undergoes one or more pulsed electric fields, preferably 6.
  • the treatment volume of the suspension is scalable, and the time of treatment of the cells in the chamber is substantially uniform.
  • the method of the invention may include additional steps of providing the T-cells from a donor and to inactivate genes thereof involved in MHC recognition (MHC class I and/or class II molecules and or being targets of immunosuppressive drugs such as described for instance in WO 2013/176915.
  • MHC recognition MHC class I and/or class II molecules and or being targets of immunosuppressive drugs such as described for instance in WO 2013/176915.
  • the product obtained may be used as a medicament.
  • the composition of the invention ie, ex vivo engineered T-cells incubated in the presence of an excess of anti-alpha betaTCR antibody and then rinsed to eliminate unbound anti-alpha betaTCR antibody, can be used as a medicament and either re-implanted into a patient from where they originate, as part of an autologous treatment, or to be used as part of an allogeneic treatment.
  • the entire process lasts 18 to 20 days, an expansion phase make cells in good shape for freezing and takes place before the step of incubation cells comprising cells with an anti-TCR antibody.
  • T-cells were cultured from PBMC and activated in order to produce [TCR] neg [PD1 ] neg [B2M] neg therapeutic immune cells endowed with a CAR directed against CD22 antigen.
  • TALEN mRNA were generated from linearized plasmid DNA encoding each TALEN arm of interest.
  • An in vitro RNA synthesis kit for RNA generation was used (Invitrogen #AMB1345-5). RNA was purified using the Qiagen RNAeasy Kit (#74106) and eluted into T solution from BTX (47-0002).
  • T cells were electroporated with a dose response of rriRNAs encoding TRAC TALEN (10pg), PD-1 TALEN (from 30pg to 70pg) and B2M TALEN (from 30pg to 70pg) either simultaneously or sequentially with a 48 hour intervals using Cellectis proprietary AgilPulse electroporator and protocols. After each electroporation step cells were incubated for 15minutes at 30°C and then incubated at 37°C. Thirteen days post thawing positive T-cells were analyzed for triple KO efficacy by first re-stimulating a portion of T cells with TransACT to induce PD-1 expression.
  • T cells were analyzed for CD22 CAR cytotoxicity by co-culturing T cells with CD22 expressing Raji-Luciferase+ targets at effector to target ratios of 30:1 , 15:1 , 5:1 , and 1 :1 for 5 hours before luminescence was quantified using the ONE Glo luminescence kit (Promega).
  • Triple KO CD22 CAR T were as active as their wild type counter part (non gene edited T-cells endowed with the same CAR CD22)
  • TRAC TALEN-treated T-cells and mock transfected cells were incubated with OKT3 monoclonal antibodies in the presence or absence of complement (BRC) (see Figure 1 ).
  • PBMC peripheral blood mononuclear cells
  • Allcells healthy volunteer donors (Allcells), thawed and cultured in X-Vivo-15 (Lonza) media supplemented by 20 ng/ml IL-2 (final concentration) (Miltenyi Biotech) and 5% human serum AB (Seralab).
  • T lymphocytes were activated directly from PBMCs using TransAct reagent (Miltenyi) according to the manufacturer’s instructions one day after PBMC thawing and passaged every 2 to 3 days at 106 cells/ml in the same culture media as above.
  • TRAC TALEN was obtained from Cellectis SA.
  • the target sequences for TRAC TALEN is the following:
  • RNAs were synthesized using the mMessage mMachine T7 Ultra Kit (Life Technologies). RNAs were purified with RNeasy columns (Qiagen) and eluted in cytoporation medium T (Harvard Apparatus). Following process optimization, TALEN mRNAs were produced by a commercial manufacturer (Trilink Biotechnologies).
  • 5x106 T-cells were transfected with 10 pg of each mRNA encoding left and right arms of TALEN.
  • the day before electroporation cells were passaged at 10 L 6 cells/ml in Xvivo-15 + 5% AB human serum + IL-2 20ng/ml.
  • the days of electroporation a 12-well plate containing 2ml complete medium Xvivo-15 + 5% AB human serum + IL-2 20ng/ml was stabilized at 37°C / 5% C02 ahead of transfection.
  • Activated cells were harvested and washed in cytoporation media T by centrifugation 10min at 300 x g.
  • T cells were transfected or not by electrotransfer of 1 pg of mRNA encoding TRAC TALEN per million cells as above. 1 .5h later, rAAV6 donor vector comprising a CAR was added or not to the culture at the multiplicity of infection of 3x104 vg/cell. TCR and CAR expressions were assessed by flow cytometry on viable T cells using CD4, CD8, TCRa mAb, recombinant protein linked to a marker (full length target of the CAR) in combination with a live/dead cell marker. The integration of the CAR at the TRAC locus was more than 40%.
  • Total cells or CAR+ T cells cytolytic capacities towards antigen presenting cells were assessed in a flow-based cytotoxicity assay.
  • the cell viability was measured after 4h or after an overnight coculture with CAR T cells at effector/target ratios set at 10:1 , 5:1 , 2:1 and 1 :1 or 1 :1 , 0.5:1 , 0.2:1 and 0.1 :1 respectively.
  • the integration of the CAR at the TRAC locus is highly efficient since the frequency of CAR+ TCR- cells reached more than 47%.
  • no CAR expression was detected at the CD52 locus when T cells were transfected only with 1 pg of mRNA encoding CD52 TALEN. More than 80% of the population of CAR+ T cells is knocked-out for both TCRa and CD52.
  • TCR negative cells Purification of TCR negative cells resulted in mainly TCRa -negative (about 98%) while around 90% of unmodified T-cells were TCRa -positive, as expected.
  • TALEN-treated or control cells were then dispensed in a 96-well plate at 2x105 cells/well in 100 pi culture media, with or without 1 pg/well OKT3 low-endotoxin monoclonal antibody (Biolegend). Baby rabbit complement (BRC, 50 pi per well, Bio-Rad) was added or PBS as control. The assay was performed in triplicate for each condition.
  • the TCR KO cells bound to anti-TCR Ab of the invention induces less side effects (assimilated to GVHD or due to the lysis of target cells).
  • UCART universal CAR T cells for transfer into a host who is not the donor
  • the method comprising an activation step, a transduction step (or transformation) for expressing a CAR, an editing step for alphabeta TCR KO ( eg TRAC gene targeted), purification of TCR negative cells, and incubation of cells with an anti-TCR antibody, file and finish (freezing).
  • composition (a sample of 2.5x10 6 cells bound to anti-TCR Ab) was then inoculated to five patients suffering ALL (treated group). Five patients received 2.5x10 6 cells processed as previously described (control group).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de fabrication de cellules génétiquement modifiées pour une thérapie, comprenant au moins : - une étape de fourniture, dans laquelle des cellules provenant d'un donneur sont fournies; - une étape de perturbation, les cellules étant modifiées en perturbant au moins un gène codant pour un récepteur de lymphocyte T (TCR) endogène; puis, avant, après ou simultanément, - une étape de transformation, dans laquelle les cellules sont modifiées par introduction d'au moins un polynucléotide codant pour un récepteur chimérique recombinant dans lesdites cellules suivie par : - une étape d'incubation, les cellules étant incubées avec un réactif se liant sélectivement à un antigène présent à la surface de cellules exprimant ledit composant TCR endogène, de préférence se liant au TCR alpha bêta. L'invention concerne également une composition pouvant être obtenue par ce procédé ou comprenant des cellules exprimant TCR liées à un réactif se liant sélectivement à un antigène présent à la surface de cellules exprimant ledit composant TCR endogène.
PCT/EP2018/097079 2017-12-29 2018-12-28 Cellules génétiquement modifiées immédiatement disponibles pour une thérapie WO2019129850A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201762611850P 2017-12-29 2017-12-29
US62/611,850 2017-12-29
DKPA201870014 2018-01-10
DKPA201870014 2018-01-10

Publications (1)

Publication Number Publication Date
WO2019129850A1 true WO2019129850A1 (fr) 2019-07-04

Family

ID=65013669

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2018/097079 WO2019129850A1 (fr) 2017-12-29 2018-12-28 Cellules génétiquement modifiées immédiatement disponibles pour une thérapie

Country Status (1)

Country Link
WO (1) WO2019129850A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114269377A (zh) * 2019-07-26 2022-04-01 南克维斯特公司 作为用于肿瘤溶解的有效治疗性产品的抗体预加载cd16+nk-92细胞
CN117343927A (zh) * 2023-12-06 2024-01-05 上海药明巨诺生物医药研发有限公司 一种工程化细胞的核酸提取方法

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR901228A (fr) 1943-01-16 1945-07-20 Deutsche Edelstahlwerke Ag Système d'aimant à entrefer annulaire
US6010613A (en) 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
WO2004067736A2 (fr) 2003-01-28 2004-08-12 Cellectis Meganuclease sur mesure et son utilisation
WO2004083379A2 (fr) 2003-03-14 2004-09-30 Walters Richard E Procédé d'électroporation ex vivo à grand volume
WO2010027797A1 (fr) 2008-08-26 2010-03-11 Macrogenics Inc. Anticorps des récepteurs des lymphocytes t et leurs méthodes d’utilisation
US8524234B2 (en) 2011-11-03 2013-09-03 Tolera Therapeutics, Inc Antibody for selective inhibition of T-cell responses
WO2013158292A1 (fr) 2012-04-17 2013-10-24 University Of Washington Through Its Center For Commercialization Cellules déficientes en hla de classe ii, cellules déficientes en hla de classe i aptes à exprimer des protéines de hla de classe ii, et leurs utilisations
WO2013176915A1 (fr) 2012-05-25 2013-11-28 Roman Galetto Procédés pour modifier des lymphocytes t résistants allogéniques et immunosuppresseurs pour l'immunothérapie
WO2014011988A2 (fr) 2012-07-13 2014-01-16 The Trustees Of The University Of Pennsylvania Renforcement de l'activité des lymphocytes t car grâce à la co-introduction d'un anticorps bispécifique
WO2014039523A1 (fr) 2012-09-04 2014-03-13 Cellectis Récepteur d'antigène chimérique multicaténaire et utilisations de celui-ci
WO2014184741A1 (fr) 2013-05-13 2014-11-20 Cellectis Procédé permettant d'obtenir par génie génétique des lymphocytes t allogéniques et hautement actifs destinés à l'immunothérapie
WO2014184744A1 (fr) 2013-05-13 2014-11-20 Cellectis Procédés de production, par génie génétique, d'un lymphocyte t hautement actif à vocation immunothérapeutique
WO2015075175A1 (fr) 2013-11-22 2015-05-28 Cellectis Procédé pour générer des lots de cellules t allogéniques dont la puissance a été moyennée
WO2015132598A1 (fr) * 2014-03-05 2015-09-11 Ucl Business Plc Récepteur d'antigène chimérique (car) à domaines de liaison à l'antigène pour la région constante de récepteur bêta du lymphocyte t
WO2016120216A1 (fr) 2015-01-26 2016-08-04 Cellectis Systèmes de récepteurs d'antigènes chimériques dirigés par des mab pour trier/appauvrir les cellules immunitaires génétiquement modifiées

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR901228A (fr) 1943-01-16 1945-07-20 Deutsche Edelstahlwerke Ag Système d'aimant à entrefer annulaire
US6010613A (en) 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
WO2004067736A2 (fr) 2003-01-28 2004-08-12 Cellectis Meganuclease sur mesure et son utilisation
WO2004083379A2 (fr) 2003-03-14 2004-09-30 Walters Richard E Procédé d'électroporation ex vivo à grand volume
WO2010027797A1 (fr) 2008-08-26 2010-03-11 Macrogenics Inc. Anticorps des récepteurs des lymphocytes t et leurs méthodes d’utilisation
US8524234B2 (en) 2011-11-03 2013-09-03 Tolera Therapeutics, Inc Antibody for selective inhibition of T-cell responses
WO2013158292A1 (fr) 2012-04-17 2013-10-24 University Of Washington Through Its Center For Commercialization Cellules déficientes en hla de classe ii, cellules déficientes en hla de classe i aptes à exprimer des protéines de hla de classe ii, et leurs utilisations
WO2013176915A1 (fr) 2012-05-25 2013-11-28 Roman Galetto Procédés pour modifier des lymphocytes t résistants allogéniques et immunosuppresseurs pour l'immunothérapie
WO2014011988A2 (fr) 2012-07-13 2014-01-16 The Trustees Of The University Of Pennsylvania Renforcement de l'activité des lymphocytes t car grâce à la co-introduction d'un anticorps bispécifique
WO2014039523A1 (fr) 2012-09-04 2014-03-13 Cellectis Récepteur d'antigène chimérique multicaténaire et utilisations de celui-ci
WO2014184741A1 (fr) 2013-05-13 2014-11-20 Cellectis Procédé permettant d'obtenir par génie génétique des lymphocytes t allogéniques et hautement actifs destinés à l'immunothérapie
WO2014184744A1 (fr) 2013-05-13 2014-11-20 Cellectis Procédés de production, par génie génétique, d'un lymphocyte t hautement actif à vocation immunothérapeutique
WO2015075175A1 (fr) 2013-11-22 2015-05-28 Cellectis Procédé pour générer des lots de cellules t allogéniques dont la puissance a été moyennée
WO2015132598A1 (fr) * 2014-03-05 2015-09-11 Ucl Business Plc Récepteur d'antigène chimérique (car) à domaines de liaison à l'antigène pour la région constante de récepteur bêta du lymphocyte t
WO2016120216A1 (fr) 2015-01-26 2016-08-04 Cellectis Systèmes de récepteurs d'antigènes chimériques dirigés par des mab pour trier/appauvrir les cellules immunitaires génétiquement modifiées

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. AF414120.1
"GenBank", Database accession no. CR541888.1
"GenBank", Database accession no. CR542051.1
"GenBank", Database accession no. JX049979.1
"GeneBank", Database accession no. AY358337.1
A SAAD ET AL: "Ex vivo T-cell depletion in allogeneic hematopoietic stem cell transplant: past, present and future", BONE MARROW TRANSPLANTATION, vol. 52, no. 9, 20 March 2017 (2017-03-20), GB, pages 1241 - 1248, XP055509875, ISSN: 0268-3369, DOI: 10.1038/bmt.2017.22 *
ALI N.; FLUTTER B.; SANCHEZ RODRIGUEZ R. ET AL.: "Xenogeneic Graft-versus Host-Disease in NOD-scid IL-2Rynull Mice Display a T-Effector Memory Phenotype", PLOS ONE, vol. 7, no. 8, 2012, pages e44219
BOIERI M; SHAH P; DRESSEL R; INNGJERDINGEN M: "The Role of Animal Models in the Study of Hematopoietic Stem Cell Transplantation and GvHD: A Historical Overview", FRONTIERS IN IMMUNOLOGY, vol. 7, 2016, pages 333
BOISSEL ET AL.: "MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering", NUCLEIC ACIDS RESEARCH, vol. 42, no. 4, 2013, pages 2591 - 2601, XP055129962, DOI: doi:10.1093/nar/gkt1224
BROCKER; KARJALAINEN, ADV IMMUNOL, vol. 68, 1998, pages 257 - 269
C A KEEVER-TAYLOR ET AL: "Comparison of immune recovery in recipients of unmanipulated vs T-cell-depleted grafts from unrelated donors in a multicenter randomized phase II-III trial (T-cell depletion trial)", BONE MARROW TRANSPLANTATION, vol. 45, no. 3, 20 July 2009 (2009-07-20), GB, pages 587 - 589, XP055510254, ISSN: 0268-3369, DOI: 10.1038/bmt.2009.170 *
CARPENITO ET AL., PROC. NATL. ACAD. SCI. USA, vol. 106, 2009, pages 3360 - 3365
COHEN; REITER: "T-Cell Receptor-Like Antibodies: Targeting the Intracellular Proteome Therapeutic Potential and Clinical Applications", ANTIBODIES, vol. 2, 2013, pages 517 - 534, XP055440103, DOI: doi:10.3390/antib2030517
DOUDNA, J.; CHAPENTIER, E.: "The new frontier of genome engineering with CRISPR-Cas9", SCIENCE, vol. 346, no. 6213, 2014, pages 1077, XP055162699, DOI: doi:10.1126/science.1258096
ESHHAR ET AL., PROC NATL ACAD SCI U S A, vol. 90, no. 2, 1993, pages 720 - 724
ESHHAR, CANCER IMMUNOL IMMUNOTHER, vol. 45, no. 3- 4, 1997, pages 131 - 1
EYQUEM J; MANSILLA-SOTO J; GIAVRIDIS T ET AL.: "Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection", NATURE, vol. 543, no. 7643, 2017, pages 113 - 117, XP055397283, DOI: doi:10.1038/nature21405
HWU P; YANG JC; COWHERD R; TREISMAN J; SHAFER GE; ESHHAR Z; ROSENBERG SA: "In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes", CANCER RES, vol. 55, 1995, pages 3369 - 73
JANEWAY, CA JR; TRAVERS P; WALPORT M ET AL.: "Immunobiology: The Immune System in Health and Disease", 2001, GARLAND SCIENCE, article "The complement system and innate immunity"
KALOS ET AL., SCI. TRANSL. MED., vol. 3, 2011, pages 95ra73
KORE A.L.: "Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization", J AM CHEM SOC, vol. 131, no. 18, 2009, pages 6364 - 5, XP055088810, DOI: doi:10.1021/ja901655p
MACLEOD, DANIEL T. ET AL.: "Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells", MOLECULAR THERAPY, vol. 25, no. 4, pages 949 - 961, XP055397282, DOI: doi:10.1016/j.ymthe.2017.02.005
MUSSOLINO ET AL.: "TALEN® facilitate targeted genome editing in human cells with high specificity and low cytotoxicity", NUCL. ACIDS RES., vol. 42, no. 10, 2014, pages 6762 - 6773, XP055542508, DOI: doi:10.1093/nar/gku305
NADLER ET AL., J IMMUNOL, vol. 131, no. 1, 1983, pages 244 - 250
P J MARTIN ET AL: "Effects of treating marrow with a CD3-specific immunotoxin for prevention of graft-versus-host disease", BONE MARROW TRANSPLANTATION, vol. 3, 1 October 1988 (1988-10-01), pages 437 - 444, XP055510148 *
POIRIER N.; DILEK N.; MARY C.; VANHOVE B.: "Graft versus host disease in humanized mice is differentially controlled by CD28 and CD80/86 antagonists", JOURNAL OF TRANSLATIONAL MEDICINE, vol. 10, no. 3, 2012, pages 02
POIROT: "Multiplex Genome-Edited T-cell Manufacturing Platform for ''Off-the-Shelf' Adoptive T-cell Immunotherapies", CANCER RES, vol. 75, 2015, pages 3853 - 64, XP055568648, DOI: doi:10.1158/0008-5472.CAN-14-3321
PORTER ET AL., N. ENGL. J. MED., vol. 365, 2011, pages 725 - 733
SAVOLDO ET AL., J. CLIN. INVEST., vol. 121, 2011, pages 1822 - 1826
SOCIE G; BLAZAR BR: "Acute graft-versus-host disease: from the bench to the bedside", BLOOD, vol. 114, no. 20, 2009, pages 4327 - 4336
SONG ET AL., BLOOD, vol. 119, 2012, pages 696 - 706
TORIKAI H ET AL: "A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, UNITED STATES, vol. 119, no. 24, 14 June 2012 (2012-06-14), pages 5697 - 5705, XP002752739, ISSN: 1528-0020, [retrieved on 20120424], DOI: 10.1182/BLOOD-2012-01-405365 *
URNOV F. ET AL.: "Highly efficient endogenous human gene correction using designed zinc-finger nucleases", NATURE, vol. 435, 2005, pages 646 - 651, XP002411069, DOI: doi:10.1038/nature03556
WALTHER ET AL.: "Viral Vectors for Gene Transfer", DRUGS, vol. 60, no. 2, 2000, pages 249 - 271, XP000979501, DOI: doi:10.2165/00003495-200060020-00002
WANG J; DECLERCQ JJ; HAYWARD SB ET AL.: "Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery", NUCLEIC ACIDS RESEARCH, vol. 44, no. 3, 2016, pages e30, XP055278154, DOI: doi:10.1093/nar/gkv1121
WASEEM QASIM ET AL: "Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells", SCIENCE TRANSLATIONAL MEDICINE, vol. 9, no. 374, 25 January 2017 (2017-01-25), US, pages eaaj2013, XP055498786, ISSN: 1946-6234, DOI: 10.1126/scitranslmed.aaj2013 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114269377A (zh) * 2019-07-26 2022-04-01 南克维斯特公司 作为用于肿瘤溶解的有效治疗性产品的抗体预加载cd16+nk-92细胞
CN117343927A (zh) * 2023-12-06 2024-01-05 上海药明巨诺生物医药研发有限公司 一种工程化细胞的核酸提取方法
CN117343927B (zh) * 2023-12-06 2024-03-12 上海药明巨诺生物医药研发有限公司 一种工程化细胞的核酸提取方法

Similar Documents

Publication Publication Date Title
JP6998917B2 (ja) 免疫療法のために同種異系かつ高活性のｔ細胞を操作するための方法
US11365430B2 (en) Methods for engineering T cells for immunotherapy by using RNA-guided Cas nuclease system
US20240026376A1 (en) Method for generating t-cells compatible for allogenic transplantation
JP6673838B2 (ja) 免疫細胞と病的細胞の両方に存在する抗原を標的とするように操作された、免疫療法のための細胞
KR102170533B1 (ko) 암 면역요법을 위한 cd33 특이적 키메라 항원 수용체들
ES2714523T3 (es) Receptor quimérico de antígenos multicatenario y usos del mismo
US9890393B2 (en) Methods for engineering T cells for immunotherapy by using RNA-guided CAS nuclease system
EP2997133B1 (fr) Procédés de production, par génie génétique, d'un lymphocyte t hautement actif à vocation immunothérapeutique
JP2019058189A (ja) 免疫療法のための同種および免疫抑制耐性ｔ細胞を操作するための方法
JP2019047801A (ja) Ｒｎａガイドｃａｓヌクレアーゼ系を用いることによって免疫療法のためにｔ細胞を操作するための方法
AU2016212159A1 (en) Anti-HSP70 specific Chimeric Antigen Receptors (CARs) for cancer immunotherapy
WO2019149743A1 (fr) Association comprenant des cellules immunitaires allogéniques déficientes pour un antigène présent sur les cellules t et sur des cellules pathologiques, et anticorps thérapeutique contre ledit antigène
US20230248825A1 (en) T-cells expressing immune cell engagers in allogenic settings
WO2019129850A1 (fr) Cellules génétiquement modifiées immédiatement disponibles pour une thérapie

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18833441

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18833441

Country of ref document: EP

Kind code of ref document: A1