WO2019129850A1 - Off-the-shelf engineered cells for therapy - Google Patents

Off-the-shelf engineered cells for therapy Download PDF

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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
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Prior art keywords
cells
tcr
antibody
alpha beta
uniprot
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PCT/EP2018/097079
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French (fr)
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Caleph WILSON
Laurent Poirot
Philippe Duchateau
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Cellectis
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Publication of WO2019129850A1 publication Critical patent/WO2019129850A1/en

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    • 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).

Abstract

The invention relates to a method for manufacturing engineered cells for therapy, comprising at least: - a supply step, wherein cells from a donor are provided; - a disruption step, wherein the cells are modified by disrupting at least one gene encoding an endogenous T Cell Receptor (TCR) component; followed by, before, after or concomitantly, - a transformation step, wherein the cells are modified by introducing at least one polynucleotide encoding a recombinant chimeric receptor into said cells followed by: - an incubation step, wherein the cells are incubated with a reagent selectively binding to an antigen present at the surface of cells expressing said endogenous TCR component, preferably binding to the alpha beta TCR. The invention also relates to a composition obtainable by this method or comprising TCR expressing cells bound to a reagent selectively binding to an antigen present at the surface of cells expressing said endogenous TCR component.

Description

OFF-THE-SHELF ENGINEERED CELLS FOR THERAPY
TECHNICAL FIELD
The present invention relates to a method for improving the preparation of composition comprising alpha beta TCR negative cells, a composition wherein alpha beta TCR positive cells are combined (bound) to an antibody specific for cells expressing an alpha beta TCR, such as an anti-alpha/beta TCR antibody, which is implemented at the industrial production scale and exhibit improved therapeutic properties compared to previous“allogeneic” cells preparations.
TECHNICAL BACKGROUND
Hematopoietic stem cell transplantation (HSCT), derived from bone marrow, peripheral blood, or umbilical cord blood can be transplanted into patients. It may be autologous (the patient's own stem cells are used),“allogeneic” (the stem cells come from a donor who is not the patient) or syngeneic (from an identical twin). Similarly, peripheral blood mononuclear cells (PBMC) from a patient for autologous transfer (or transplantation) or from a healthy donor for allogenic transfer can be used.
There is increasing evidences demonstrating that multipotent hematopoietic stem cells, as mononuclear cells (PBMCs), can be genetically modified to reconstitute and repair immunity. Transplantation is also performed for patients with cancers of the blood or bone marrow, such as multiple myeloma or leukemia. Thus, primary cells can be engineered so as to express a Chimeric Antigen Receptor (CAR), or a recombinant TCR, to redirect primary immune cells against pathological cells such as cancer cells.
Chimeric antigen receptors (CARs) are artificial antibody-like molecules designed to convey antigen specificity to T cells. T cells expressing CARs have shown long-term efficacy for the treatment of particular types of cancer (Eshhar, 1997, Cancer Immunol Immunother 45(3- 4) 131 -1 )36; Eshhar et al, 1993, Proc Natl Acad Sci U S A 90(2):720-724; Brocker and Karjalainen, 1998, Adv Immunol 68:257-269). The first generation of CARs include an antigen binding domain, a transmembrane domain and an intracellular domain, such as Oϋ3z, selected to activate the T cell and provide specific immunity. However, the expansion and persistence of these CAR-modified T cells in vivo was hampered by the lack of costimulatory signals after engagement with target antigens, as many tumor cells down-regulate their expression of the costimulatory molecules required for optimal and sustained T-cell function, proliferation and persistence. A second and third generation of CAR constructs were created to boost the T cell response, they have included one and two secondary costimulatory signals in tandem with ΰϋ3z. The costimulatory molecule mimics a“second signal” such as CD28, 4-1 BB, OX- 40, and CD27, that amplifies the activation of the CAR T cells to expand to high numbers and maintain long term functional persistence (Carpenito et al., 2009, Proc. Natl. Acad. Sci. USA 106:3360-3365; Song et al., 2012, Blood 1 19:696-706) and in clinical studies (Porter et al., 201 1 , N. Engl. J. Med. 365:725-733; Kalos et al., 201 1 , Sci. Transl. Med. 3:95ra73; Savoldo et al., 201 1 , J. Clin. Invest. 121 :1822-1826); Hwu P, Yang JC, Cowherd R, Treisman J, Shafer GE, Eshhar Z, Rosenberg SA,1995, “In vivo antitumor activity of T cells redirected with chimeric antibody/T-cell receptor genes.” Cancer Res; 55:3369-73). However, these highly activated T cells resulted in enhanced toxicity due to cytokine storm and tumor lysis syndrome.
CD19 (Cluster of Differentiation 19) glycoprotein specific to the B-cell lineage, is one of the first target against which a CAR was prepared and used in cancer immunotherapies (Nadler, et al., 1983 J Immunol 131 (1 ):244-250); using allogenic CART. The vast majority of B-acute lymphoblastic leukemia (B-ALL) uniformly express CD19, whereas expression is absent on non hematopoietic cells, as well as myeloid, erythroid, and T cells, and bone marrow stem cells. Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Accordingly, CD19 represents an attractive target for immune-based therapies.
Allogeneic transplantation with CART or Hematopoietic stem cell remains a dangerous procedure with many possible complications; including Graft-versus- host disease(GvHD).
GvHD is a medical complication following the receipt of transplanted tissue from a genetically different person. Immune cells (white blood cells) in the donated tissue (the graft) recognize the recipient (the host) as foreign (nonself). The transplanted immune cells then attack the host's body cells. GvHD can also occur after a blood transfusion if the blood products used have not been irradiated or treated with an approved pathogen reduction system. Whereas transplant rejection occurs when the host rejects the graft, GvHD occurs when the graft rejects the host.
To remedy this, it is known to engineer the T cells by modifying one or more genes to reduce or abolish graft versus host disease (GvHD), especially genes of the T cell Receptor (TCR) complex. This can be prepared for example, by using a rare-cutting endonuclease transiently expressed from an mRNA electroporated into the cells.
So far, the most accurate and safest technique to delete a TCR gene has been the use of a TALEN® gene editing tool which is highly specific and efficient. More than 90 percent of the cells can be engineered using this technique with undetectable level of off target as determined by guide seq. analysis. Cells are subsequently grown for about 10 to 12 days to obtain enough injectable doses.
The final step of the manufacturing process typically consists in purifying the TCR-negative cell fraction from grown cells before vialing the product. It is conventional to perform such depletion at the very end of the expansion phase to make sure that the final product is as pure as possible. The overall process lasts about 20 days and is very expensive, especially the final step.
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.
Thus, there is important a need to improve the manufacturing of such gene- modified cells for therapy. SUMMARY OF THE INVENTION
The inventors have identified means to improve the compositions comprising allogeneic cells (less or non alloreactive) for immunotherapy in patients and steps to improve methods to prepare such medicaments. They provide the following:
1. A method for manufacturing non alloreactive cells, comprising:
- a supply step, wherein cells or a population of cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided,
- an incubation step, wherein said cells or population of cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are incubated with an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells resulting in antibody bound cells, or in a population of cells comprising antibody bound cells, immediately followed by :
- a rinsing step for eliminating unbound antibody immediately followed by a fill and finish step, or a fill and finish step
In the file and finish step, antibody bound cells or population of cells comprising antibody bound cells are conditioned as a medicament for immediate use in a patient.
2. The method of item 1 , wherein:
the antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells is selected from an antibody specific for the a TCR, for CD3, an antibody specific for the a TCR already approved by health authorities, an antibody T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab, a fragment thereof, and combinations thereof, provided that said fragment thereof comprises a binding domain for the human complement (allowing an antibody dependent cytotoxicity)
3. The method of item 1 or 2 comprising:
a disruption step, wherein alpha beta TCR cells are genetically modified by disrupting at least one gene encoding a component of the alpha beta TCR to inhibit cell surface expression of alpha beta TCR, said disruption step occurring before or after the supply step and resulting in the production of engineered cells comprising alpha beta -TCR-negative engineered cells and cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface, the disruption step occurring before the incubation step. The engineered cells of the invention comprises at least 80% of engineered cells with an inactivated alpha TCR gene, and encompass :
engineered cells comprising at least 85% of engineered cells with an inactivated alpha TCR gene,
engineered cells comprising at least 90% of engineered cells with an inactivated alpha TCR gene,
engineered cells comprising at least 95% of engineered cells with an inactivated alpha TCR gene,
engineered cells comprising at least 97% of engineered cells with an inactivated alpha TCR gene,
engineered cells comprising at least 99% of engineered cells with an inactivated alpha TCR gene,
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.
4. The method according to any one of item 1 to 3 wherein the non alloreactive cells comprise engineered cells, preferably TALEN®- engineered cells.
5. The method of item 4 wherein the TALEN®- engineered cells, were engineered with a TALEN®- having the following sequences:
MGDPKKKRKVI DIADLRTLGYSQQQQEKI KPKVRSTVAQHH EALVGHGFTHAH IVALSQHPAALGTVAVKY
QDMIAALPEATH EAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQWAIASNNGGKQALETVQRLLPVL
CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQWAIASH DGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQWAIASH DGGKQALETVQRLLPVLCQAHGL
TPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ
VVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAI
ASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN I
GGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQWAIASH DGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTN DH LVALACLGGRPALD
AVKKGLGDPISRSQLVKSELEEKKSELRH KLKYVPH EYI ELI EIARNSTQDRI LEMKVMEFFMKVYGYRGKH LG GSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEF
KFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELUGGEMIKAGTLTLEEVRRKFNNGEINFAAD and
MGDPKKKRKVIDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKY
QDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLNLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAH
GLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLT
PQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQ
QVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV
VAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNN
GGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRP
ALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGK
HLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSV
TEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD. or with a TALEN ® which right domain sequence has at least 90% identity with
MGDPKKKRKVIDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKY
QDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL
CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL
TPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ
VVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAI
ASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI
GGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALD
AVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLG
GSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEF
KFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD and the left domain has at least 90 % identity with
MGDPKKKRKVIDIADLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKY
QDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLNLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAH
GLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLT
PQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQ
QVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV
VAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIA SN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN N
GGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDH LVALACLGGRP
ALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYI ELI EIARNSTQDRILEMKVMEFFM KVYGYRGK
H LGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKH IN PN EWWKVYPSSV
TEFKFLFVSGHFKGNYKAQLTRLNH ITNCNGAVLSVEELUGGEMI KAGTLTLEEVRRKFNNGEIN FAAD.
6. The method according to any one of item 1 to 5 comprising a transformation step, wherein the cells are modified by introducing at least one polynucleotide into the cells, said polynucleotide comprising a sequence encoding a recombinant receptor, provided that said recombinant receptor does not bind an endogenous alpha beta TCR.
7. The method according to any one of items 1 to 6, wherein said cells or a population of cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are T cells or a population of cells comprising T cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface.
8. The method according to any one of items 1 to 7, wherein said antibody consists in or comprises a domain allowing an antibody dependent cytotoxicity.
9. The method according to any one of items 1 to 8, wherein the antibody is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab, fragments thereof,
10. The method according to any one of items 1 to 9, further comprising an activation step, said activation step occurring preferably before the transformation and disruption steps.
11 The method according to any one of items 1 to 10, further comprising an expansion step, wherein the cells are expanded, before the incubation step and before the fill and finish step. 12. The method according to any one of items 1 to 11 , which further comprises at least one step of purification of TCR-negative cells from TCR positive and negative cells.
13. 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.
14. The method according to any one of items 1 to 13, wherein the differentiation step is achieved by expressing a factor selected from IL-2, IL-3, IL-5, IL-7 and IL-15, IL-21 , IL-27 a combination thereof, in the cells, or incubating the cells with an effective dose of at least one growth factor selected from of IL- 2, IL-3, IL-5, IL-7 and IL-15, IL-21 , IL-27 a combination thereof, optionally in the presence of stromal cells.
15. The method according to any one of items 1 to 14, wherein the disruption step is performed before the transformation step; or wherein the disruption step is performed after the transformation step; or wherein the disruption step is performed concomitantly with the transformation step.
16. The method for according to any one of items 1 to 14, comprising an additional disruption step, wherein the cells are modified by disrupting at least one gene, said additional disruption step being performed before the incubation step.
17. The method for according to any one of items 1 to 16, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, said expression being optionally a conditional expression.
18. A method according to any one of items 1 to 17, wherein said fill and finish step comprising a step selected from sampling, packaging, freezing cells, and combinations thereof.
19. The method of any one of items 1 to 18, which successively comprises:
- the supply step; optionally, the activation step;
the disruption step;
the transformation step;
the additional disruption step; the expansion step;
the purification step;
the incubation step; and
the fill and finish step;
or which successively comprises:
the supply step;
optionally, the activation step; the disruption step;
the purification step;
the additional disruption step; the transformation step;
the expansion step;
the incubation step; and
the fill and finish step
or which successively comprises:
the supply step;
optionally, the activation step; the disruption step;
the transformation step;
the additional disruption step; the expansion step;
the differentiation and/or maturing step the purification step;
the incubation step; and
the fill and finish step;
or which successively comprises:
the supply step;
optionally, the activation step; the disruption step;
the purification step;
the additional disruption step; the transformation step;
the expansion step;
the differentiation and/or maturing step the incubation step; and
the fill and finish step. The method of any one of items 1 to 19, wherein the supply 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. The method of any one of items 1 to 20, wherein a disruption step is performed by introducing a nucleic acid encoding a rare-cutting endonuclease, preferably a TALE-nuclease, into the cells and expressing it, the introduction of the nucleic acid encoding a rare-cutting endonuclease being preferably performed by electroporation or by means of an agent allowing nucleic acid transport across cell compartments to a cell nucleus. The method of any one of items 1 to 21 , wherein a disruption step includes the inactivation of at least one gene selected from TCRa and/or TCR , preferably the inactivation of at least TCRa, and more preferably the inactivation of TCRa and at least one gene selected from CD52, dCK, GR, PD1 , CTLA4, beta-2 microglobulin, CBLB and CISH. The method of any one of items 1 to 22, wherein the recombinant receptor can selectively bind to one or more antigens present or presented at the surface of cancer cells and ultimately induces the destruction of cancer cells by allogeneic cells expressing it. The method of any one of items 2 to 23, wherein the engineered cells obtained after the disrupting step comprise between from less than 80 % and to less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component at the cell surface. A composition comprising cells comprising engineered cells expressing alpha beta TCR on their surface in less than 80% of the total cells, preferably in less than 10% of the total cells, more preferably in less than 5% of the total cells, even more preferably in as preferably determined by flow cytometry analysis, in combination with and/or bound to a reagent selectively binding to an antigen present at the surface of cells expressing said cell surface alpha beta TCR. 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.
A composition comprising cells comprising engineered cells expressing alpha beta TCR on their surface in less than 3% of the total cells, bound to an antibody selectively binding to cell surface alpha beta TCR.
A composition comprising cells comprising engineered cells expressing alpha beta TCR on their surface in less than 1 %, or in less than 0.1 % of the total cells, bound to an antibody selectively binding cell surface alpha beta TCR.
26. The composition of item 25 comprising between 20% and 0.001 % of cells wherein cell surface of endogenous alpha beta TCR is detectable, as preferably determined by flow cytometry analysis.
The composition of item 23 comprising between less than 0.3 % of total cells to less than 0.001 % of total cells wherein cell surface of endogenous alpha beta TCR is detectable, as preferably determined by flow cytometry analysis, and bound to an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells.
The composition of item 23 comprising between less than 0.3 % to less than 0.001 % of cells wherein cell surface of endogenous alpha beta TCR is detectable, as preferably determined by flow cytometry analysis, and bound to an antibody selectively binding to alpha beta TCR (a TCR+).
27. The composition according to item 25 or 26, wherein the reagent is or comprises an antibody, an antibody-drug conjugate or a fragment of an antibody comprising a domain binding to a protein of the complement or to a receptor of a cell mediating antibody dependent cytotoxicity, said receptor being preferably a Fc receptor.
28. The composition according to item 27, wherein the antibody or fragment of antibody or antibody-drug conjugate is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab as well as fragments thereof, antibody-drug conjugates thereof and combinations thereof.
29. The composition according to any one of items 25 to 28, wherein said engineered cells express at their surface a recombinant receptor.
30. The composition according to item 29, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, the expression of the CAR being optionally a conditional expression.
31. The composition according to any one of items 25 to 29, wherein the engineered cells comprise less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component, as preferably determined by flow cytometry analysis.
32. The composition according to any one of items 25 to 30, wherein the engineered cells are derive from stem cells or from T cells.
33. The composition according to any one of items 25 to 32, for use in therapy or prophylaxis.
34. The composition according to any one of items 25 to 33, for use in therapy or prophylaxis according to item 33 wherein from 102 to 1010 engineered cells per kg or per m2, preferably from 104 to 1010 engineered cells per kg or per m2 are administered to a patient in one or more administrations.
35. The composition of any one of items 25 to 34, for the treatment or prophylaxis of a cancer, a viral infection, an autoimmune disorder or Graft versus Host Disease.
36. The composition according to any one of items 25 to 35, inducing no Graft versus Host Disease, preferably no alpha beta TCR-mediated Graft versus Host Disease.
37. The composition according to any one of items 25 to 36, for use in a method for eliminating engineered cells still expressing endogenous alpha beta TCR component in vivo and/or in vitro. 38. The composition according to any one of items 25 to 37, for use in an in vivo or in vitro process comprising contacting said composition with at least one protein of the complement.
39. A method for purifying alpha beta TCR negative engineered cells comprising a step of contacting a composition according to any one of item 23 to 36, or obtainable by the method of any one of items 1 to 22 with a complement protein, or with cytolytic cells, so as to deplete or eliminate cells still expressing the endogenous alpha beta TCR component of the composition, preferably in vivo.
In other embodiments the invention provides:
A method for manufacturing allogeneic cells, comprising:
a supply step, wherein cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided,
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.
“Allogenic” means of nonself intraspecies origin or less or non alloreactive cells and designates cells, preferably engineered cells intended to be administered to a host from which the cells were not isolated originally.
Less alloreactive means that allogenic engineered cells when administered into a host which is host from which the cells were not isolated originally induce no or reduced immune response (GVHD, CRS, anamnestic etc) than non engineered allogenic 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
In a preferred embodiment 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 GVHD. The method for manufacturing less alloreactive cells of the present invention, comprises:
a supply step, wherein a population of cells comprising less than 3% alpha beta TCR+ cells (cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface) are provided,
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.
a step wherein cells bound to the antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) are purified, resulting in a population of cells wherein the less than 3% alpha beta TCR+ cells are combined to said antibody and the 97% or so of cells in the population do not express said alpha beta TCR,
a step of fill and finish wherein the population of cells comprising less than 3% alpha beta TCR+ cells combined to (bound to) said antibody are frozen.
The method for manufacturing allogeneic cells as above comprising:
- a disruption step, wherein alpha beta TCR cells are genetically modified by disrupting at least one gene encoding a component of the alpha beta TCR to inhibit cell surface expression of alpha beta TCR, said disruption step occurring before or after the supply step.
- The method for manufacturing allogeneic cells according to the above comprising a transformation step, wherein the cells are modified by introducing at least one polynucleotide into the cells, said polynucleotide comprising a sequence encoding a recombinant receptor, provided that said recombinant receptor does not bind to said reagent selectively binding to an antigen present at the surface of cells expressing an endogenous alpha beta TCR.
A method for manufacturing allogeneic cells, comprising:
a supply step, wherein cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided, a disruption step, wherein alpha beta TCR cells are genetically modified by disrupting at least one gene encoding a component of the alpha beta TCR to inhibit cell surface expression of alpha beta TCR,
a transformation step, wherein the cells are modified by introducing at least one polynucleotide into the cells, said polynucleotide comprising a sequence encoding a recombinant receptor, provided that said recombinant receptor does not bind to the reagent selectively binding to an antigen present at the surface of cells expressing an endogenous alpha beta TCR used in 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.
A method for manufacturing allogeneic cells, comprising:
- a supply step, wherein cells comprising cells expressing a detectable level of alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided,
- 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.
A method for manufacturing allogeneic cells, comprising:
- a supply step, wherein cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided,
- 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.
The invention relates to a method for manufacturing engineered cells for therapy, comprising at least:
a supply step, wherein cells from a donor are provided; - a disruption step, wherein the cells are modified by disrupting at least one gene encoding an endogenous T Cell Receptor (TCR) component; followed by, before, after or concomitantly,
- a transformation step, wherein the cells are modified by introducing at least one polynucleotide encoding a recombinant chimeric receptor into said cells followed by:
- 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 also relates to a composition obtainable by this method or comprising TCR expressing cells bound to a reagent selectively binding to an antigen present at the surface of cells expressing said endogenous TCR component.
The invention may be further summarized by the following items:
1. A method for manufacturing allogeneic cells, comprising:
- a supply step, wherein cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided,
- an incubation step, wherein said cells are incubated with a an antibody selectively binding to an antigen present at the surface of alpha beta TCR cells.
2. The method for manufacturing allogeneic cells of item 1 comprising:
-a disruption step, wherein alpha beta TCR cells are genetically modified by disrupting at least one gene encoding a TCR component, said disruption step occurring before or after the supply step. The method for manufacturing allogeneic cells of above comprising: a disruption step, wherein alpha beta TCR cells are genetically modified by disrupting at least one gene encoding a component of the alpha beta TCR to inhibit cell surface expression of alpha beta TCR, said disruption step occurring before or after the supply step. The method for manufacturing allogeneic cells according to item 1 or 2 comprising a transformation step, wherein the cells are modified by introducing at least one polynucleotide into the cells, said polynucleotide comprising a sequence encoding a recombinant receptor, provided that said recombinant receptor does not bind to said reagent selectively binding to an antigen present at the surface of cells expressing an endogenous alpha beta TCR. The method for manufacturing allogeneic cells according to any one of items 1 to 3, wherein said cells are T cells. The method for manufacturing allogeneic cells according to any one of items 1 to 4, wherein said reagent consists in or comprises an antibody, an antibody-drug conjugate or a fragment of an antibody comprising a domain allowing an antibody dependent cytotoxicity. The method for manufacturing allogeneic cells according to any one of items 1 to 5, wherein the antibody or fragment of antibody or antibody-drug conjugate is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab, fragments thereof, antibody-drug conjugates thereof and combinations thereof. The method for manufacturing allogeneic cells according to any one of items 1 to 6, further comprising an activation step, said activation step occurring preferably before the transformation and disruption steps. The method for manufacturing allogeneic cells according to any one of items 1 to 7, further comprising a step of rinsing (to remove the excess of antibody, antibody fragment, or drug conjugated antibody), successive to the incubation step. The method for manufacturing allogeneic cells according to any one of items 1 to 8, further comprising an expansion step, wherein the cells are expanded, before the incubation step and before the fill and finish step. The method for manufacturing allogeneic cells according to any one of items 1 to 9, which further comprises at least one step of purification of TCR- negative cells from TCR positive and negative cells. The method for manufacturing allogeneic cells according to any one of items 1 to 10, 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 method for manufacturing allogeneic cells according to any one of items 1 to 11 , wherein the differentiation step is achieved by expressing a factor selected from IL-2, IL-3, IL-5, IL-7 and IL-15, IL-21 , IL-27 a combination thereof, in the cells, or incubating the cells with an effective dose of at least one growth factor selected from of IL-2, IL-3, IL-5, IL-7 and IL-15, IL-21 , IL- 27 a combination thereof, optionally in the presence of stromal cells. The method for manufacturing allogeneic cells according to any one of items 1 to 12, wherein the disruption step is performed before the transformation step; or wherein the disruption step is performed after the transformation step; or wherein the disruption step is performed concomitantly with the transformation step. The method for manufacturing allogeneic cells according to any one of items 1 to 13, comprising an additional disruption step, wherein the cells are modified by disrupting at least one gene, said additional disruption step being performed before the incubation step. The method for manufacturing allogeneic cells according to any one of items 1 to 14, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, said expression being optionally a conditional expression. The method for manufacturing allogeneic cells according to any one of iteml to 15, further comprising a fill and finish step, said fill and finish step comprising a step selected from sampling, packaging, freezing cells; and combinations thereof, wherein 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 freezing the composition. ethod of any one of items 1 to 16, which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the transformation step;
- the additional disruption step;
- the expansion step;
- the purification step;
- the incubation step; and
- the fill and finish step;
or which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the purification step;
- the additional disruption step;
- the transformation step;
- the expansion step;
- the incubation step; and
- the fill and finish step.
or which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the transformation step;
- the additional disruption step;
- the expansion step;
- the differentiation and/or maturing step
- the purification step;
- the incubation step; and
- the fill and finish step;
or which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the purification step;
- the additional disruption step;
- the transformation step; - the expansion step;
- the differentiation and/or maturing step
- the incubation step; and
- the fill and finish step.
18. The method of any one of items 1 to 17, wherein, the supply 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.
19. The method of any one of items 1 to 18, wherein a disruption step is performed by introducing a nucleic acid encoding a rare-cutting endonuclease, preferably a TALE-nuclease, into the cells and expressing it, the introduction of the nucleic acid encoding a rare-cutting endonuclease being preferably performed by electroporation or by means of an agent allowing nucleic acid transport across cell compartments to a cell nucleus.
20. The method of any one of items 1 to 19, wherein a disruption step includes the inactivation of at least one gene selected from TCRa and/or TCR , preferably the inactivation of at least TCRa, and more preferably the inactivation of TCRa and at least one gene selected from CD52, dCK, GR, PD1 , CTLA4, beta-2 microglobulin, CBLB and CISH.
21. The method of any one of items 1 to 20, wherein the recombinant receptor can selectively bind to one or more antigens present or presented at the surface of cancer cells and ultimately induces the destruction of cancer cells by allogeneic cells expressing it.
22. The method of any one of items 1 to 21 , wherein the engineered cells obtained after the disrupting step comprise between from less than 80 % and to less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component at the cell surface.
23. The method of any one of items 1 to 21 , wherein the engineered cells obtained after the disrupting step comprise between from less than 80 % and to less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component at the cell surface, as determined by flow cytometry analysis. A composition 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, as preferably determined by flow cytometry analysis, in combination with and/or bound to a reagent selectively binding to an antigen present at the surface of cells expressing said cell surface alpha beta TCR. The composition of item 24 comprising between 20% and 0.001 % of engineered cells wherein at least one gene encoding a component of an endogenous alpha beta TCR is disrupted and/or cells wherein cell surface of endogenous alpha beta TCR is detectable, as preferably determined by flow cytometry analysis. The composition according to item 24 or 25, wherein the reagent is or comprises an antibody, an antibody-drug conjugate or a fragment of an antibody comprising a domain binding to a protein of the complement or to a receptor of a cell mediating antibody dependent cytotoxicity, said receptor being preferably a Fc receptor. The composition according to item 26, wherein the antibody or fragment of antibody or antibody-drug conjugate is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab as well as fragments thereof, antibody-drug conjugates thereof and combinations thereof. The composition according to any one of items 24 to 27, wherein said engineered cells express at their surface a recombinant receptor. The composition according to item 28, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, the expression of the CAR being optionally a conditional expression. The composition according to any one of items 24 to 29, wherein the engineered cells comprise less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component, as preferably determined by flow cytometry analysis.
31. The composition according to any one of items 24 to 30, wherein the engineered cells are derive from stem cells or from T cells.
32. The composition according to any one of items 24 to 31 , for use in therapy or prophylaxis.
33. The composition according to any one of items 24 to 32, for use in therapy or prophylaxis wherein from 102 to 1010 engineered cells per kg or per m2, preferably from 104 to 1010 engineered cells per kg or per m2 are administered to a patient in one or more administrations.
34. The composition of any one of items 24 to 33, for the treatment or prophylaxis of a cancer, a viral infection, an autoimmune disorder or Graft versus Host Disease.
35. The composition according to any one of items 24 to 34, inducing no Graft versus Host Disease, preferably no alpha beta TCR-mediated Graft versus Host Disease.
36. The composition according to any one of items 24 to 35, for use in a method for eliminating engineered cells still expressing endogenous alpha beta TCR component in vivo and/or in vitro.
37. The composition according to any one of items 24 to 36, for use in an in vivo or in vitro process comprising contacting said composition with at least one protein of the complement.
38. A method for purifying alpha beta TCR negative engineered cells comprising a step of contacting a composition according to any one of items 24 to 36, or obtainable by the method of any one of items 1 to 23 with a complement protein, or with cytolytic cells, so as to deplete or eliminate cells still expressing the endogenous alpha beta TCR component of the composition.
The invention provides a population of non alloreactive engineered cells comprising alpha beta -TCR-negative cells engineered cells and cells expressing at least one alpha beta T Cell Receptor (alpha beta TCR) and a CAR, at the cell surface in contact with an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells.
In the population of non alloreactive engineered cells of the invention the antibody bound to TCR-positive cells is an antibody used in human to prevent or treat graft versus host disease (GVHD), already approved by health authorities);
In the population of non alloreactive engineered cells of the invention the antibody is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab, fragments thereof.
In the population of non alloreactive engineered cells of the invention the antibody is T10B9,
In the population of non alloreactive engineered cells of the invention the antibody is BMA031 , In the population of non alloreactive engineered cells of the invention the antibody is TOL101 ,
In the population of non alloreactive engineered cells of the invention the antibody is muromonab-CD3,
In the population of non alloreactive engineered cells of the invention the antibody is otelixizumab,
In the population of non alloreactive engineered cells of the invention the antibody is teplizumab,
In the population of non alloreactive engineered cells of the invention the antibody is visilizumab.
In the population of non alloreactive engineered cells of the invention the antibody or antibody fragment comprises further a domain that can bind to protein of the complement.
Preferably the CAR at the cell surface is targeting and binding 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 telomerase reverse transcriptase, RUI, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY- ES0-1 , LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate- carcinoma tumor antigen-1 (PCTA-1 ), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1 )-l, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MFIC) molecule presenting a tumor-specific peptide epitope, 5T4, RORI, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1 ) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MFIC) molecule, BCMA (CD269, TNFRSF 17), or a virus-specific surface antigen such as an FI IV specific antigen (such as FIIV gp120); an EBV-specific antigen, a CMV-specific antigen, a FIPV- specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface markers. Antigens are not necessarily surface marker antigens but can be also endogenous small antigens presented by FILA class I at the surface of the cells.
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, selected from CD38, FISP70, CD30, FAP, FIER2 CD79, CD123, CD19, CD22, CLL-1 , MUC-1 GD2, O acetyl GD2, CS1 , CD70, and combination thereof, preferably CD19 and CD22, CD123 and CLL-1 .
By way of example, the present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD38, FISP70, CD30, FAP, HER2 CD79, CD123, CD19, 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, and combination thereof,
The present invention encompasses single-chain CARs which target specifically a cell surface marker, such as CD19, BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70, MUC16 - PRAME - TSPAN10, CLAUDIN18.2 - DLL3 - LY6G6D, Liv-1 - CFIRNA2 - ADAM10, and combination thereof. In particular embodiments, the population of non alloreactive engineered cells comprising alpha beta -TCR-negative cells engineered cells and cells expressing at least one alpha beta T Cell Receptor (alpha beta TCR) and a CAR, at the cell surface in contact with an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells is used for the treatment of a patent, preferably a patient with a cancer.
Example of cancers that were found to be affected with the population of non alloreactive engineered cells comprising alpha beta -TCR-negative cells engineered cells and cells expressing at least one alpha beta T Cell Receptor (alpha beta TCR) and a CAR, at the cell surface in contact with an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells.
The invention provides a method for manufacturing engineered cells, comprising at least:
- a supply step, wherein cells from a donor are provided;
- a transformation step, wherein the cells are modified by introducing at least one polynucleotide encoding a recombinant chimeric antigen receptor into said cells;
- a disruption step, wherein the cells are modified by disrupting at least one gene encoding an endogenous T Cell Receptor (TCR) component; followed by:
- an incubation step, wherein the cells are incubated with a reagent selectively binding to an antigen of the endogenous TCR component present at the surface of cells (still) expressing said endogenous TCR component.
A recombinant chimeric antigen receptor is a receptor comprising elements originally from at least two different receptors, preferably from at least two different (separated) human receptors; particular elements may be from mouse origin such as parts of antibodies or scfv, or humanized antibodies or fragments. Elements may be fused together
In some variations, the cells are T cells.
In some variations, the recombinant receptors are recombinant chimeric antigen receptors (CAR). In some variations, the recombinant receptors are recombinant chimeric antigen receptors (CAR) comprising a scfv.
In some variations, the recombinant receptors are recombinant chimeric antigen receptors (CAR) comprising a scfv and a TAG.
Endogenous or genomic for a sequence means that the sequence belongs to the non engineered cells.
Exogenous sequence means a sequence introduced into the cells or a transgene, (a coding sequence introduced into the cell, preferably stably integrated into the genome of the cells.
In some variations, the transformation step is carried out after the disruption step, or concomitantly.
In some variations, the reagent selectively binding to an antigen of the endogenous TCR component is an antibody or antibody fragment, specific for an antigen of the endogenous TCR, preferably an antigen of the endogenous TCR alpha beta, binding to the alpha beta TCR at the cell surface and endowed with a domain allowing the selective destruction of cells expressing said endogenous alpha beta TCR component bound to the antibody or antibody fragment, upon interaction between said antibody or antibody fragment and proteins of the complement.
In some variations, 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.
In some variations, the reagent is selected from T 10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, vedolizumab, visilizumab, fragments thereof, antibody-drug conjugates thereof and combinations thereof.
In some variations, the method further comprises an activation step, wherein the cells are activated, preferably before the transformation and disruption steps; said activation step preferably comprising contacting the cells with an antibody or a fragment of antibody selected from anti-CD3 antibodies and fragments thereof, and/or anti-CD28 antibodies and fragments thereof, and/or incubating the cells in the present of stromal cells.
In some variations, the method further comprises a rinsing step after the incubation step, wherein reagent not bound to the cells is eliminated.
In some variations, the method further comprises an expansion step, wherein the cells are expanded, preferably after the incubation step and before the fill and finish step. In some variations, the method further comprises at least one purification step, wherein cells still expressing said endogenous TCR component are depleted, said purification step being preferably performed before the incubation step and after the disruption step, more preferably from 3 to 4 days after the disruption step and/or from 7 to 20 days after the supply step, even more preferably from 15 to 20 days after the supply step.
In some variations, the method further comprises a differentiation and/or maturing step, resulting in the cells expressing the recombinant receptor and/or the cells having cytotoxic activity; said differentiation and/or maturing step being preferably performed before the incubation step.
In some variations, the differentiation and/or maturing step is achieved by expressing a factor selected from IL-2, IL-7 and IL-15 receptors in the cells, and incubating the cells with an effective dose of at least one of IL-2, IL-7 and IL-15, optionally in the presence of stromal cells.
In some variations, the differentiation and/or maturing step is achieved by incubating the cells with an effective dose of at least one of IL-2, IL-3, IL-5, IL-7 and IL-15, IL-21 optionally in the presence of stromal cells.
In some variations, the disruption step is performed before the transformation step; or the disruption step is performed after the transformation step; or the disruption step is performed concomitantly with the transformation step, preferably by introducing a polynucleotide which encodes a Chimeric Antigen Receptor (CAR) into the cells and inserting it at an endogenous TCR component gene locus such as in the TCR alpha encoding gene, preferably the constant region of the gene encoding the TCR alpha (TRAC gene).
In some variations, the method further comprises an additional disruption step, wherein the cells are modified by disrupting at least one additional gene, said additional disruption step being performed before the incubation step.
In some variations, the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, said CAR expression being optionally a conditional CAR expression.
In some variations, cells are modified by disrupting at least one additional gene, said additional disruption step being preferably performed before the incubation step, even more preferably using a TALE-protein.
The at least one additional gene disrupted, may be one of the gene including, but not limited to Programmed Death 1 (PD-1 , also known as PDCD1 or CD279, accession number: NM_005018), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4, also known as CD152, GenBank accession number AF414120.1 ), LAG3 (also known as CD223, accession number: NM_002286.5), Tim3 (also known as HAVCR2, GenBank accession number: JX049979.1 ), BTLA (also known as CD272, accession number: NM_181780.3), BY55 (also known as CD160, GenBank accession number: CR541888.1 ), TIGIT (also known as VSTM3, accession number: NM_173799), B7H5 (also known as C10orf54, homolog of mouse vista gene, accession number: NM_022153.1 ), LAIR1 (also known as CD305, GenBank accession number: CR542051 .1 ), SIGLEC10 (GeneBank accession number: AY358337.1 ), 2B4 (also known as CD244, accession number: NM_001 166664.1 ), which directly inhibit immune cells.
In a particular embodiment, the genetic modification step of the method relies on the inactivation of one additional gene, (in addition to the TRAC), preferably two genes selected from the group consisting of PD1 , CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1 , SIGLEC10, 2B4, and TCR beta 1 and 2. In embodiments, the genetic modification step of the method relies on the inactivation of two genes selected from the group consisting of PD1 and TCR alpha, PD1 and TCR beta, CTLA-4 and TCR alpha, CTLA-4 and TCR beta, LAG3 and TCR alpha, LAG3 and TCR beta, Tim3 and TCR alpha, Tim3 and TCR beta, BTLA and TCR alpha, BTLA and TCR beta, BY55 and TCR alpha, BY55 and TCR beta, TIGIT and TCR alpha, TIGIT and TCR beta, B7H5 and TCR alpha, B7H5 and TCR beta, LAIR1 and TCR alpha, LAIR1 and TCR beta, SIGLEC10 and TCR alpha, SIGLEC10 and TCR beta, 2B4 and TCR alpha, 2B4 and TCR beta. In another embodiment, the genetic modification step of the method relies on the inactivation of more than two genes. The genetic modification is preferably operated ex-vivo.
Table 1 below, without being exhaustive, show 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. In a human system the edited genes are human edited genes.
Figure imgf000030_0001
Figure imgf000031_0001
Table 1 : Genes that make allogeneic T-cells more active for immunotherapy when engineered (KO, inactivated, overexpressed ...) according to the present invention.
In particular embodiments, an engineered immune cell bound to an antibody specific for an alpha beta TCR, preferably a human alpha beta TCR, is provided said engineered immune cells combined to an alpha beta TCR antibody express a chimeric antigen receptor (CAR) and comprises at least one genetic modification to reduce or inactivate the expression of at least one endogenous polynucleotide sequence selected from: a) polynucleotide sequence(s), which transcription and/or translation is(are) involved into reduction of glycolysis and calcium signaling in response to a low glucose condition, such as SERCA3 to increase calcium signaling, miR101 and mir26A to increase glycolysis, BCAT to mobilize glycolytic reserves; and/or
b) polynucleotide sequence(s), which expression up regulate(s) immune checkpoint proteins (e.g.TIM3, CEACAM, LAG3, TIGIT) expression, such as IL27RA , STAT1 , STAT3; and/or
c) polynucleotide sequence(s), which expression mediate(s) interaction with HLA-G, such as ILT2 or ILT4; and/or
d) 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
e) polynucleotide sequence(s), which expression is(are) involved into the down regulation of T-cell activation, such as mir21 ; and/or f) polynucleotide sequence(s), which expression is(are) involved in signaling pathways responding to cytokines, such as JAK2 and AURKA; and/or
g) polynucleotide sequence(s), which expression is(are) involved in T-cell exhaustion, such as DNMT3, miRNA31 , MT1 A, MT2A, PTGER2. In particular embodiments, 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 .
In particular embodiments, 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 rendering cells resistant to a drug and/or hypersensitive to a (another) drug.
The method of the present invention further comprises a step of incubation, wherein the cells comprising between 90% and 0.00001 % of TCR-positive cells (such as alpha beta TCR positive cells) are incubated in the presence of an antibody that binds to alpha beta TCR-positive cells, or selectively to alpha betaTCR expressing cells, such as an antibody selective for CD3, an anti-TCR antibody preferably an anti-alphaTCR, anti-betaTCR, or anti-alphabetaTCR antibody.
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.
The method of the present invention further comprises a step of incubation, wherein TCR-positive cells are incubated in the presence of an anti- alphabetaTCR antibody, preferably an anti-CD3 antibody, at a temperature comprised between from 1 °C to 40°C, preferably between from 10 °C to 37°C.
The present invention encompasses incubating cells with an antibody blocking the non specific binding of an antibody selective for CD3, an antibody selective for alphaTCR, an antibody selective for betaTCR, or an antibody selective for alphabetaTCR . The method of the present invention further comprises a step of incubation, wherein cells comprising between 40% and 0.00001 % of TCR-positive cells (preferably between 15% and 0.001 % of TCR positive cells) are 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.
In embodiments, between 40% and 0.00001 % of TCR-positive cells (preferably between 15% and 0.001 % of TCR positive cells) are 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 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.
In some variations, 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.
In some variations, the method successively comprises:
- the supply step;
- the activation step;
- the disruption step;
- the transformation step;
- the additional disruption step;
- the expansion step;
- the purification step;
- the incubation step in the presence of an anti-TCR reagent; and
- the fill and finish step;
or which successively comprises:
- the supply step;
- the activation step;
- the disruption step;
- the purification step;
- the additional disruption step;
- the transformation step;
- the expansion step;
- the incubation step in the presence of an anti-TCR reagent; and - the fill and finish step.
or which successively comprises:
- the supply step;
- the activation step;
- the disruption step;
- the transformation step;
- the additional disruption step;
- the expansion step;
- the differentiation and/or maturing step
- the purification step;
- the incubation step; and
- the fill and finish step;
or which successively comprises:
- the supply step;
- the activation step;
- the disruption step;
- the purification step;
- the additional disruption step;
- the transformation step;
- the expansion step;
- the differentiation and/or maturing step
- the incubation step; and
- the fill and finish step.
In some variations, the supply 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 a blood derived product.
In some variations, the disruption step and optionally the additional disruption step are performed by introducing a nucleic acid encoding a rare-cutting endonuclease, preferably a TALE-nuclease, into the cells and expressing it, the introduction of the nucleic acid being preferably performed by electroporation or by means of an agent allowing nucleic acid transport across cell compartments to a cell nucleus.
In some variations, the disruption step and optionally the additional disruption step include the inactivation of at least one gene selected from TCRa and/or TCR , preferably the inactivation of at least TCRa, more preferably the inactivation of at least two alleles TCRa, and even more preferably the inactivation of two alleles TCRa and at least one gene selected from CD52, dCK, GR, PD1 , CTLA4, beta-2 microglobulin, CBLB and CISH or a combination of genes selected from CD52, dCK, GR, PD1 , CTLA4, beta-2 microglobulin, CBLB and CISH.
In some variations, the engineered cells for therapy are engineered cells for cancer therapy, the recombinant receptor being able to selectively bind to one or more antigens present or presented at the surface of cancer cells.
In some variations, 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.
In some variations, the final product (engineered cells that are combined to the anti-TCR reagent) comprise less than 5%, less than 4%, less than 3%, preferably less than 2%, more preferably less than 1 %, even more preferably undetectable level of cells still expressing the endogenous TCR component, as determined by flow cytometry analysis.
It is another object of the invention to provide a composition comprising:
- engineered cells, wherein at least one gene encoding an endogenous T Cell Receptor (TCR) component is disrupted (preferably inactivated) and comprising at least one polynucleotide encoding a recombinant receptor; in combination with
- a reagent selectively binding to an antigen present at the surface of cells still expressing said endogenous TCR component.
In some variations, the cells are T cells.
In some variations, the composition comprises:
- engineered cells, wherein at least one gene encoding an endogenous T Cell Receptor (TCR) component is Knocked out (KO) and comprising at least one polynucleotide encoding a recombinant receptor; in combination with
- a reagent selectively binding cell surface endogenous TCR component, cell surface endogenous alpha beta TCR component.
An alpha beta TCR component may be an alpha TCR component, a beta
TCR component, an alpha beta TCR component.
In some variations, the reagent is or comprises an antibody, or a fragment of an antibody comprising a domain binding 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. In some variations, the reagent is or comprises an antibody-drug conjugate.
In some variations, the antibody or fragment of antibody or antibody-drug conjugate is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab as well as fragments thereof, antibody-drug conjugates thereof and combinations thereof.
In some variations, the antibody or fragment of antibody or antibody-drug conjugate is an anti-TCR therapeutic antibody, preferentially an anti-TCR therapeutic antibody approved by health authority, preferentially an anti-TCR therapeutic antibody approved by health authority that binds to the alpha beta TCR, more preferentially an anti-TCR therapeutic antibody approved by health authority that binds to the alpha beta TCR, and selectively deplete alpha -beta TCR -positive cells, even more preferentially an anti-TCR therapeutic antibody approved by health authority that binds to the alpha beta TCR, and selectively deplete alpha -beta TCR -positive cells in vivo selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab as well as fragments thereof, antibody-drug conjugates thereof and combinations thereof.
, Even more more preferentially an anti-TCR therapeutic antibody approved by health authority that binds to the alpha beta TCR, and selectively deplete alpha -beta TCR -positive cells in vivo which is TOL101 , muromonab- CD3, otelixizumab, teplizumab, visilizumab as well as fragments thereof, antibody-drug conjugates thereof and combinations thereof.
In some variations, the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, the expression of the CAR being optionally a conditional expression.
In some variations, the engineered cells comprise less than 3%, preferably less than 2%, more preferably less than 1 %, of cells still expressing the endogenous TCR component, as determined by flow cytometry analysis.
In some variations, the engineered cells derive from stem cells or from T cells.
The present invention provides a pharmaceutical composition comprising engineered cells of the invention (that is to say combined/bound to an antibody specific for an anti-alpha beta TCR expressing cell) and a pharmaceutically acceptable excipient or vehicle.
In some variations, the composition is for use as a medicament for therapy or prophylaxis. In some variations, from 10° to 1010 engineered cells, preferably from 104 to 1010 engineered cells are administered to a patient in one or more administrations, and preferably in one or two administrations.
In some variations, the composition is for the treatment or prophylaxis of cancer, a viral infection, an autoimmune disorder or Graft versus Host Disease. For the treatment or prophylaxis of cancer, the following cancer may be treated using the composition of the present invention: Acute myeloid leukemia (AML), Chronic myeloid leukemia (CML), Acute lymphoblastic leukemia (ALL), Hodgkin lymphoma (HL) (relapsed, refractory), Non-Hodgkin lymphoma (NHL) (relapsed, refractory), Neuroblastoma, Ewing sarcoma, Multiple myeloma, Myelodysplastic syndromes, Gliomas, other solid tumors.
Other condition can benefit from the composition of the invention such as Thalassemia, Sickle cell anemia, Aplastic anemia, Fanconi anemia, Malignant infantile osteopetrosis, Mucopolysaccharidosis, Immune deficiency syndromes, Autoimmune diseases.
The 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. Moreover, it appears that the selective elimination of TCR positive cells in engineered cell preparations using the antibody selected from the group of the invention have other advantages (such as less inflammation) that will be discussed in the present invention.
In some variations, the composition is for use in a method for eliminating engineered cells still expressing the endogenous TCR component in vivo and/or in vitro.
In some variations, the composition is for use in a process comprising contacting said composition with at least one protein of the complement.
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. In particular, 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.
The invention relies on the realization by the present inventors that incubating the cells with reagent binding to TCR -positive cells such as an anti- TCR antibody (or fragment or conjugate thereof) during manufacturing makes it possible to subsequently eliminate, any TCR-positive cells within the cell population, e.g. when the cells are supplied to a patient.
Thus, adverse reactions due to TCR-positive cells in a patient, such as in particular the occurrence of GvFID, can be better prevented and even completely eliminated for the TCR alpha beta inducing GVFID.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 represents the Frequency of viable cells following incubation of TCR- positive cells with an anti-TCR-antibody and complement - (or control) and complement-mediated cytotoxicity of TRAC TALE protein-treated or mock- transfected cells (averages and standard deviations).
DESCRIPTION OF EMBODIMENTS
The invention will now be described in more detail without limitation in the following description.
Preferably, the method for manufacturing allogeneic cells of the invention, comprises incubating alpha beta TCR expressing cells with a reagent selectively binding to said cells, preferably to the alpha beta TCR, even more preferably to an antigen of the alpha beta TCR.
Preferably, the method for manufacturing allogeneic cells of the invention, comprises incubating alpha beta expressing T cells with a reagent selectively binding to the alpha beta TCR. More preferably said cells comprise cells comprising a gene encoding the TCR that is mutated, deleted or comprises an insertion and that not allow an endogenous TCR to be expressed at the cell surface. Allogeneic cells are intended to be transferred into a recipient. Allogeneic cells are meant to be cells from a donor, preferably from a genetically similar to the recipient, but not identical - This is often a sister or brother, but could be an unrelated donor, or a histo-incompatible donor - A method for manufacturing allogeneic cells aims are preparing cells inducing no undesirable effects such as GVHD, in the recipient.
In the present invention, 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.
Graft-versus-host disease (GvHD) is a medical complication following the receipt of transplanted tissue from a genetically different person. The definition of GVHD is the common definition and encompasses acute and chronic GVHD, each of different grades as disclosed in Socie G, Blazar BR. Acute graft-versus-host disease: from the bench to the bedside. Blood. 2009;1 14(20):4327-4336. doi:10.1 182/blood-2009-06-204669.
In general, an alpha beta TCR is an alpha beta TCR encoded by a T cell (endogenous or genomic alpha beta TCR). An alpha beta TCR, may, in particular embodiments, be an exogenous alpha beta TCR encoded by a transgene (a synthetic gene introduced into cells). Preferably, an alpha beta TCR is the alpha beta TCR encoded by a T cell (endogenous or genomic alpha beta TCR, as direct link between alpha beta TCR and GVHD has been reported in prior art.
The TCR targeted by an antibody binding to TCR positive cells may be any TCR inducing GVHD such as particular gamma delta TCR. In that case the reagent binding to said TCR expressing cells may be chosen to bind the particular gamma delta TCR In the present invention, an antigen present at the surface of alpha beta TCR cells, may be any antigen preferentially and selectively expressed by alpha beta TCR expressing cells such as an antigen of CD4, CD8, CD3, CD28, T-bet, GATA3, RoRyT (RAR-related orphan receptor gamma T, which is encoded by RORC), BCL-6 and FOXP3 (Forkhead box P3), interferon gamma (IFN)-y (Th1 ), interleukin 13 (IL)-13 (Th2), ), interleukin 17, IL-17 (Th17), IL-10 and TGF (Treg) TCR alpha, TCRbeta, CD3 zeta. More preferably said antigen is an antigen of the human alpha beta TCR.
The definition of an alpha beta TCR is meant to be that described in any a textbook: a TCR is a disulfide-linked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (b) chains expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as a:b (or alpha beta ab) T cells, though a minority of T cells express an alternate receptor, formed by variable gamma (y) and delta (d) chains, referred as gamma delta (gd) T cells.
Each chain is composed of two extracellular domains: Variable (V) region and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel b-sheets. The Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the Variable region binds to the peptide/MFIC complex.
According to the present invention, cells comprise at least one cell expressing the alpha beta TCR at the cell surface, from 0.0001 to 99,99% of alpha beta TCR expressing cells over total cells, preferably between 40% and 0.1 % of alpha beta TCR expressing cells, even more preferably between 10% and 1 % of alpha beta TCR expressing cells. In particular embodiments, cells of the invention comprise less than (< 3% of TCR positive cells).
In preferred embodiments, the gene disrupted in the disruption step is gene encoding a component of the endogenous alpha beta T Cell Receptor (TCR); preferably the constant region of the alpha TCR (TRAC gene), Disrupting a gene means introducing a mutation, a deletion or an insertion into a gene or a product of a gene (such as a RNA). Such a step encompasses functionally disrupting said gene, that is modifying its functioning by increasing its activity or decreasing it as compare to the same non disrupted gene.
The method for manufacturing allogeneic cells according to the present invention wherein the transformation step and disruption step are performed at the same time, in the same step and wherein the cells are transformed by introducing at least one polynucleotide into the cells, said polynucleotide comprising a sequence encoding a recombinant receptor and 3’ sequences allowing the integration of said recombinant receptor into a genomic sequence encoding a component of the TCR.
A recombinant receptor is meant to be any receptor that may redirect cells expressing it to a target cell expressing the target binding to said recombinant receptor, preferably a chimeric antigen receptor (CAR) and even more preferably a single-chain variable fragment (scFv).
A single-chain variable fragment ( scFv ) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility. In a CAR, a scfv is linked to a transmembrane domain by a hinge.
The intracellular part of the transmembrane domain may or may not comprise a sequence conferring the CAR the capacity to signal. In that later case, the TM domain may comprise a domain that can form a dimer with another transmembrane domain in a multichain CAR and the second TM domain comprises an intracellular signaling domain.
The method as above wherein a polynucleotide sequence is inserted into a genomic sequence coding for the alpha component of the TCR - constant region (TRAC gene) is preferred; this is resulting in an inactivation of said TRAC gene and in undetectable level (or expression in less than 10% of the total cells) of cell surface alpha beta TCR expression.
The method as above wherein said reagent comprises a binding domain for a protein of the complement and/or a binding domain for a receptor of a cell mediating antibody dependent cytotoxicity, such as a Fc receptor.
All the reagents binding to alpha beta expressing T cells of the present invention have a domain allowing complement binding.
In preferred embodiments, 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.
In a preferred embodiment, said purification step is performed before the incubation step and after the disruption step, more preferably from 3 to 4 days after the disruption step and/or from 6 to 20 days after the supply step, even more preferably from 8 to 20 days after the supply step.
In preferred embodiments, 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.
In preferred embodiments, 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.
A conditional expression is an expression that depends on the presence of a factor or a drug in the cell medium.
The 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
The present invention provides a composition comprising purified engineered TCR negative T cells comprising a percentage of engineered TCR positive T cells bound to an anti-TCR antibody, their use for treating a disease, and a method for preparing said combination which is adapted for being implemented at the industrial production scale.
T cell receptors (TCR) 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. As for immunoglobulin molecules, 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. However, in contrast to immunoglobulins that recognize intact antigen, 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. 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. Thus, still according to the invention, engraftment of the T-cells may be improved by inactivating at least one gene encoding a TCR component. TCR is rendered not functional in the cells by inactivating TCR alpha gene and/or TCR beta gene(s), preferably a TCR alpha gene. Thus, still according to the invention, a mutation, preferably by insertion of a polynuleotide into the TRAC gene is performed to obtain alpha beta TCR-deficient cells.
The method of the invention is for manufacturing engineered cells for therapy. Preferably, the engineered cells are T cells. The method of the invention is also applicable to other cells such as stem cells. Accordingly, the term“T cells” can be replaced by“cells” in general in rest of the description, as appropriate.
The method can comprise at least the following steps:
- a supply step;
- an activation step (optional);
- a disruption step;
- a purification step (optional);
- a transformation step;
- an incubation step;
- a rinsing step (optional);
- an expansion step (optional);
- a differentiation and/or maturing step (optional); and
- a fill and finish step.
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.
Most preferably, all steps of the manufacturing method are performed in vitro.
The purification step, wherein TCR-positive cells are depleted, is optional. Indeed, in some embodiments, the use of the anti-TCR reagent may make it possible to do without the purification step, as TCR-positive cells can be eliminated e.g. in vivo when the cell product is administered to a patient.
In other, preferred embodiments, the at least one purification step is present, preferably before the incubation step. Therefore, TCR-positive cells are depleted within the cell composition, and the use of the anti-TCR reagent makes it possible to target and eliminate e.g. in vivo residual TCR-positive cells.
The order of the steps can be as specified just above. Alternatively, 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.
The incubation step may be provided after the fill and finish step. In this case, the composition is thawed and then the incubation step is performed, for example just before administering the composition to a patient.
Preferably, all steps are performed in a closed and sterile environment, according to cGMP.
Any one of 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.
Supply
The cells used in the present invention are most preferably Mononuclear cells (MNC) or human T cells. In some embodiments, they can be inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T- lymphocytes. Cytotoxic T-lymphocytes are preferred. Cytolytic T-lymphocytes are more preferred.
In some embodiments, the cells are obtained from a patient to be treated.
According to other, preferred, embodiments, 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. Alternatively, and preferably, they can be isolated from various samples of hematologic origin, such as peripheral blood mononuclear cells (PBMCs), 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.
The starting material composition (containing T cells) for the method of the invention may be fresh or frozen. Preferably it is frozen, so that the supply test comprises thawing a sample (e.g. of hematologic origin) and culturing the cells on an appropriate medium.
Examples of appropriate media include those known as Minimal Essential Media, RPMI Media 1640, X-vivo 1 or 5 or 20 (Lonza), A1 M-V, DMEM, MEM, a- MEM, F-12 and Optimizer. They may contain factors necessary for proliferation and viability, including serum (e.g. fetal bovine or human serum), interleukin-2 (IL- 2), insulin, IFN-g, IL-4, IL-7, GM-CSF, GM10, GM2, IL-15, TGFbeta, IL-21 and TNF- or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, a surfactant, plasmanate and reducing agents such as N-acetyl-cysteine and 2- mercaptoethanol. Other possible additives are added amino acids, sodium pyruvate and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g. 37° C) and atmosphere (e.g. air plus 5 % CO2).
Activation
The method of the invention can involve an activation step. By“activation" is meant that the cells are stimulated by contact with at least one and preferably at least two activating substances.
In particular, T cells may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody, or by contact with a protein kinase C activator (e.g. bryostatin) in conjunction with a calcium ionophore.
An accessory molecule on the surface of the T cells can also be stimulated, using a ligand that binds the accessory molecule. For example, 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. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used.
The various activating substances which are used may be in solution or alternatively may be immobilized on a surface such as the surface of beads or particles. The appropriate ratio of particles to cells may depend on particle size relative to the target cells.
In some embodiments of the present invention, the T cells are thus combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In alternative embodiments, the agent- coated beads and the cells are not separated prior to culture but are cultured together.
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.
Additional details can be found in WO 2013/176915 which is incorporated herein by reference.
Disruption and optional additional disruption
The method of the invention comprises at least one step of disrupting at least one gene encoding an endogenous T Cell Receptor (TCR) component. By TCR component is meant any molecule which is part of the TCR complex.
Optionally, 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.
The genes encoding a TCR component which can be disrupted include in particular a gene encoding a TCRa, TCR , TCRy, TCR5, CD3y, CD35, CD3s and ΰϋ3z, preferably TCRa, TCR , CD3y, CD35, CD3s and ΰϋ3z, more preferably a gene encoding a TCRa, TCR , even more preferably TRC a, all alleles encoding said gene(s).
In some embodiments, each disruption step is meant to disrupt a single gene. In alternative embodiments, at least one disruption step, possibly both the (first) disruption step and the additional disruption step, are meant to disrupt at least two genes at the same time with no increase in off site events. When the (first) disruption step is meant to disrupt at least two genes, one of them is a gene encoding a TCR component, and the other(s) can be one or more gene(s) encoding a TCR component and/or one or more genes expressing a target for an immunosuppressive agent or encoding an immune checkpoint function. By “ disrupting” is meant any genetic modification leading to reduced, arrested or abnormal expression of the gene. In preferred embodiments, disrupting means inactivating, i.e. the gene is not expressed in a functional protein form. The gene can be altered (such as mutated) or deleted or a sequence inserted into said gene that interrupt the transcription and/or translation of said gene.
An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. In other words, an immunosuppressive agent is a compound which can diminish the extent and/or amplitude of an immune response. As non-limiting examples, an immunosuppressive agent can be a calcineurin inhibitor, a target of rapamycin, an interleukin-2 alpha-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. Classical cytotoxic immune suppressants act by inhibiting DNA synthesis. Others may act through activation of T cells or by inhibiting the activation of helper cells.
By disrupting the target of an immunosuppressive agent in T cells, immunosuppressive resistance is conferred to T cells for immunotherapy. As non- limiting examples, targets for immunosuppressive agent can be receptors for an immunosuppressive agent such as: CD52, deoxycytidine kinase (dCK), glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.
According to some embodiments, at least one gene encoding an immune checkpoint function can be disrupted, as described in detail in WO 2014/184744, which is incorporated herein by reference. These genes are more particularly those involved in the functions of 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.
They include in particular PD1 , CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1 , LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1 , SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1 , IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1 , SIT1 , FOXP3, PRDM1 , BATF, GUCY1A2, GUCY1A3, GUCY1 B2, GUCY1 B3, CISH, CBLB.
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.
The edited gene can also encode CTIIA or any one of the genes disclosed in WO2013158292.
According to some embodiments, 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.
According to other embodiments, the (first) disruption step is a step of disrupting the TCRa gene and the additional disruption step is a step of disrupting the TCR i and b2 genes.
According to other embodiments, the (first) disruption step is a step of disrupting the TCRa gene and the additional disruption step is a step of disrupting a non-TCR gene which is a gene expressing a target for an immunosuppressive agent (such as e.g. CD52 or GR or dCK).
According to other embodiments, the (first) disruption step is a step of disrupting the
Figure imgf000050_0001
gene and the additional disruption step is a step of disrupting a non-TCR gene which is a gene expressing a target for an immunosuppressive agent selected from a gene encoding CD52, GR, dCK, 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 Q9Y286), SIGLEC9 (Uniprot Q9Y336), CD244 (Uniprot Q9BZW8), TNFRSF10B (Uniprot 014763), TNFRSF10A (Uniprot 000220), CASP8 (Uniprot Q14790), CASP10 (Uniprot Q92851 ), CASP3 (Uniprot P42574), CASP6 (Uniprot P55212), CASP7 (Uniprot P55210), FADD (Uniprot Q13158), FAS (Uniprot P25445), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), SMAD2 (Uniprot Q15796), SMAD3 (Uniprot P84022), SMAD4 (Uniprot Q13485), SMAD10 (Uniprot B7ZSB5), SKI (Uniprot P12755), SKIL (Uniprot P12757), TGIF1 (Uniprot Q15583), IL10RA (Uniprot Q13651 ), IL10RB (Uniprot Q08334), HMOX2 (Uniprot P30519), IL6R (Uniprot P08887), IL6ST (Uniprot P40189), EIF2AK4 (Uniprot Q9P2K8), CSK (Uniprot P41240), PAG1 (Uniprot Q9NWQ8), SIT1 (Uniprot Q9Y3P8), FOXP3 (Uniprot Q9BZS1 ), PRDM1 (Uniprot Q60636), BATF (Uniprot Q16520), GUCY1A2 (Uniprot P33402), GUCY1A3 (Uniprot Q02108), GUCY1 B2 (Uniprot Q8BXH3) and GUCY1 B3 (Uniprot Q02153)). In other embodiments, the method includes the disruption of more than two genes, such as TCRa, TCR , and one or more genes expressing a target for an immunosuppressive agent; or such as TCRa and at least two genes expressing targets for immunosuppressive agents; or such as TCR and at least two genes expressing targets for immunosuppressive agents.
According to some embodiments, 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.
According to some preferred embodiments, each disruption step may rely on the expression of at least one DNA digesting (or cutting) agent in the cells, such that said DNA digesting agent specifically catalyzes a modification, e.g. cleavage, in the targeted gene(s) (preferably by a double-strand break) thereby disrupting or inactivating said targeted gene(s).
The DNA digesting agent may be directly introduced into the cells. Alternatively and preferably, 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 DNA digesting agent may in particular target one gene selected from e.g. CD52, GR, dCK, TCRa and TCR , 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 Q9Y286), SIGLEC9 (Uniprot Q9Y336), CD244 (Uniprot Q9BZW8), TNFRSF10B (Uniprot 014763), TNFRSF10A (Uniprot 000220), CASP8 (Uniprot Q14790), CASP10 (Uniprot Q92851 ), CASP3 (Uniprot P42574), CASP6 (Uniprot P55212), CASP7 (Uniprot P55210), FADD (Uniprot Q13158), FAS (Uniprot P25445), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), SMAD2 (Uniprot Q15796), SMAD3 (Uniprot P84022), SMAD4 (Uniprot Q13485), SMAD10 (Uniprot B7ZSB5), SKI (Uniprot P12755), SKIL (Uniprot P12757), TGIF1 (Uniprot Q15583), IL10RA (Uniprot Q13651 ), IL10RB (Uniprot Q08334), HMOX2 (Uniprot P30519), IL6R (Uniprot P08887), IL6ST (Uniprot P40189), EIF2AK4 (Uniprot Q9P2K8), CSK (Uniprot P41240), PAG1 (Uniprot Q9NWQ8), SIT1 (Uniprot Q9Y3P8), FOXP3 (Uniprot Q9BZS1 ), PRDM1 (Uniprot Q60636), BATF (Uniprot Q16520), GUCY1A2 (Uniprot P33402), GUCY1A3 (Uniprot Q02108), GUCY1 B2 (Uniprot Q8BXH3) and GUCY1 B3 (Uniprot Q02153). The present invention encompasses a means for selectively editing the genes above such as a TALE-protein selective for one of the genes above associated to any editing nuclease.
In other embodiments, 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. CD52 and TCRa, CD52 and TCR , GR and TCRa, GR and TCR , TCRa and TCR , PD1 (Uniprot Q15116) and TCRa, CTLA4 (Uniprot P16410) and TCRa, PPP2CA (Uniprot P67775) and TCRa, PPP2CB (Uniprot P62714) and TCRa, PTPN6 (Uniprot P29350) and TCRa, PTPN22 (Uniprot Q9Y2R2) and TCRa, LAG3 (Uniprot P18627) and TCRa, HAVCR2 (Uniprot Q8TDQ0) and TCRa, BTLA (Uniprot Q7Z6A9) and TCRa, CD160 (Uniprot 095971 ) and TCRa, TIGIT (Uniprot Q495A1 ) and TCRa, CD96 (Uniprot P40200) and TCRa, CRTAM (Uniprot 095727) and TCRa, LAIR1 (Uniprot Q6GTX8) and TCRa, SIGLEC7 (Uniprot Q9Y286) and TCRa, SIGLEC9 (Uniprot Q9Y336) and TCRa, CD244 (Uniprot Q9BZW8) and TCRa, TNFRSF10B (Uniprot 014763) and TCRa, TNFRSF10A (Uniprot 000220) and TCRa, CASP8 (Uniprot Q14790) and TCRa, CASP10 (Uniprot Q92851 ) and TCRa, CASP3 (Uniprot P42574) and TCRa, CASP6 (Uniprot P55212) and TCRa, CASP7 (Uniprot P55210) and TCRa, FADD (Uniprot Q13158) and TCRa, FAS (Uniprot P25445) and TCRa, TGFBRII (Uniprot P37173) and TCRa, TGFRBRI (Uniprot Q15582) and TCRa, SMAD2 (Uniprot Q15796) and TCRa, SMAD3 (Uniprot P84022) and TCRa, SMAD4 (Uniprot Q13485) and TCRa, SMAD10 (Uniprot B7ZSB5) and TCRa, SKI (Uniprot P12755) and TCRa, SKIL (Uniprot P12757) and TCRa, TGIF1 (Uniprot Q15583) and TCRa, IL10RA (Uniprot Q13651 ) and TCRa, IL10RB (Uniprot Q08334) and TCRa, FI M OX2 (Uniprot P30519) and TCRa, IL6R (Uniprot P08887) and TCRa, IL6ST (Uniprot P40189) and TCRa, EIF2AK4 (Uniprot Q9P2K8) and TCRa, CSK (Uniprot P41240) and TCRa, PAG1 (Uniprot Q9NWQ8) and TCRa, SIT1 (Uniprot Q9Y3P8) and TCRa, FOXP3 (Uniprot Q9BZS1 ) and TCRa, PRDM1 (Uniprot Q60636) and TCRa, BATF (Uniprot Q16520) and TCRa, GUCY1A2 (Uniprot P33402) and TCRa, GUCY1A3 (Uniprot Q02108) and TCRa, GUCY1 B2 (Uniprot Q8BXH3) and TCRa and GUCY1 B3 (Uniprot Q02153) and TCRa), thereby inactivating said genes. In other embodiments, more than two DNA digesting agents can be expressed in the cells in order to target and/or inactivate more than two genes.
Alternatively, use may be made of a disrupting agent such as a transposase, for example, a synthetic DNA transposon (e.g. "Sleeping Beauty' transposon system), designed to introduce precisely defined DNA sequences into the genome of the cells.
In further embodiments, the DNA digesting agent is an integrase.
In further and preferred embodiments, the DNA digesting agent is a nuclease. The term“nuclease" is used herein to generally refer to any enzyme that hydrolyzes nucleic acid sequences.
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. Most preferably, 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. Preferably, the recognition sequence of the site- specific nuclease occurs only once in a genome.
In some embodiments, the DNA digesting agent is a site-specific Cas nuclease. In related embodiments, the Cas nuclease is Cas9. In further embodiments, 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. A guide RNA (gRNA) contains about 20 nucleotides that are complementary to a target genomic DNA sequence upstream of a genomic PAM (protospacer adjacent motifs) site (NNG) and a constant RNA scaffold region. The Cas (CRISPR-associated)9 protein binds to the gRNA and the target DNA to which the gRNA binds and introduces a double-strand break in a defined location upstream of the PAM site.
Cas9 harbors two independent nuclease domains homologous to HNH and RuvC endonucleases, and by mutating either of the two domains, the Cas9 protein can be converted to a nickase that introduces single-strand breaks. The method of the invention can be implemented with the single- or double-strand- inducing version of Cas9, as well as with other RNA-guided DNA nucleases, such as other bacterial Cas9-like systems. The sequence-specific nuclease can be engineered, chimeric, or isolated from an organism. The sequence-specific nuclease is advantageously introduced into the cells in the form of an RNA encoding the sequence-specific nuclease, such as an mRNA.
In other embodiments, use can be made of a CRISPR/Cpfl system. In other embodiments, 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.
Alternative methods, such as rational design using a nondegenerate recognition code table may also be used to design a zinc finger binding domain to target a specific sequence. Publicly available web-based tools for identifying potential target sites in DNA sequences and designing zinc finger binding domains may be found at http://www.zincfingertools.org and http://bindr.gdcb.iastate.edu/ZiFiT/, respectively.
A zinc finger binding domain may be designed to recognize and bind a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or preferably from about 9 to about 18 nucleotides in length. In general, the zinc finger binding domains comprise at least three zinc finger recognition regions (i.e. zinc fingers). In some embodiments, the zinc finger binding domain may comprise four zinc finger recognition regions. In other embodiments, the zinc finger binding domain may comprise five zinc finger recognition regions. In still other embodiments, the zinc finger binding domain may comprise six zinc finger recognition regions.
Zinc finger recognition regions and/or multi-fingered zinc finger proteins may be linked together using suitable linker sequences, including for example, linkers of five or more amino acids in length. The zinc finger binding domain described herein may include a combination of suitable linkers between the individual zinc fingers of the protein.
In some embodiments, the zinc finger nuclease may further comprise a nuclear localization signal or sequence (NLS). 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.
A cleavage domain also may be derived from an enzyme or portion thereof, as described above, that requires dimerization for cleavage activity. Two zinc finger nucleases may be required for cleavage, as each nuclease comprises a monomer of the active enzyme dimer. Alternatively, a single zinc finger nuclease may comprise both monomers to create an active enzyme dimer (i.e. an enzyme dimer capable of cleaving a nucleic acid molecule). The two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof).
When two cleavage monomers are used to form an active enzyme dimer, the recognition sites for the two zinc finger nucleases are preferably disposed such that binding of the two zinc finger nucleases to their respective recognition sites places the cleavage monomers in a spatial orientation to each other that allows the cleavage monomers to form an active enzyme dimer, e.g. by dimerizing. As a result, the near edges of the recognition sites may be separated by about 5 to about 18 nucleotides. For instance, the near edges may be separated by about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or 18 nucleotides. It will however be understood that any integral number of nucleotides or nucleotide pairs may intervene between two recognition sites (e.g. from about 2 to about 50 nucleotide pairs or more). The near edges of the recognition sites of the zinc finger nucleases, such as for example those described in detail herein, may be separated by 6 nucleotides. In general, the site of cleavage lies between the recognition sites.
Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at a recognition site), and cleaving DNA at or near the site of binding. Certain restriction enzymes (e.g. type IIS) cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. For example, the type IIS enzyme Fokl catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. Thus, 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. In other embodiments, 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.
In further embodiments, the targeting endonuclease may be a transcription activator-like effector (TALE) nuclease. TALEs are transcription factors from the plant pathogen Xanthomonas that can be readily engineered to bind new DNA targets. TALEs or truncated versions thereof may be linked to the catalytic domain of endonucleases such as Fokl to create targeting endonuclease called TALE nucleases or TALENs. Examples of TALENs useful in the present invention include those disclosed in document WO 2013/176915, which is incorporated herein by reference.
In other embodiments, 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. In addition, the nuclease domain may also exhibit DNA-binding functionality.
In yet other embodiments, the targeting endonuclease may be an artificial targeted DNA double strand break inducing agent (also called an artificial restriction DNA cutter). For example, the artificial targeted DNA double strand break inducing agent may comprise a metal/chelator complex that cleaves DNA and at least one oligonucleotide that is complementary to the targeted cleavage site. The artificial targeted DNA double strand break inducing agent, therefore, does not contain any protein. The metal of the metal/chelator complex may be cerium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, zinc, and the like. The chelator of the metal/chelator complex may be EDTA, EGTA, BAPTA, and so forth. In preferred embodiments, the metal/chelator complex may be Ce(IV)/EGTA. In other preferred embodiments, the artificial targeted DNA double strand break inducing agent may comprise a complex of Ce(IV)/EGTA and two strands of pseudo-complementary peptide nucleic acids (PNAs).
In further embodiments, 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. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. The DNA- binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e. such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous cleavage domain.
In some embodiments, 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
In some embodiments, 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. In particular, said additional catalytic domain can be a DNA end processing enzyme. Non-limiting examples of 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. In preferred embodiments, 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. In other preferred embodiments, 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.
Endonucleolytic breaks are known to stimulate the rate of homologous recombination. Thus, in other embodiments, 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. In particular embodiments, 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. Following cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. Preferably, 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.
In particular, 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). Depending on the location of the target nucleic acid sequence wherein a break event has occurred, such exogenous nucleic acid can be used to knock-out a gene, e.g. when exogenous nucleic acid is located within the open reading frame of said gene, or to introduce new sequences or genes of interest. Sequence insertions by using such exogenous nucleic acid can be used to modify a targeted existing gene, by correction or replacement of said gene (allele swap as a non-limiting example), or to up- or down-regulate the expression of the targeted gene (promoter swap as non-limiting example), said targeted gene correction or replacement.
In preferred embodiments, 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. In other embodiments, several 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. 2A peptides, which were identified in the aphthovirus subgroup of picornaviruses, cause a ribosomal“skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons.
By“codon" is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue.
Thus, 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. As a non-limiting example, 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.
Alternatively, said polypeptides can be produced outside the cell and then introduced thereto.
Delivery methods
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. Preferably, 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). For example, 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.
Electroporation is a preferred method for introducing a polynucleotide construct into animal cells.
As a preferred embodiment of the invention, 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. Generally, the first high electric field pulses allow pore formation, while subsequent lower electric field pulses allow to moving the polynucleotide into the cell. In one aspect of the present invention, 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. In particular the invention relates to a method of transforming T cell comprising contacting said T cell with RNA and applying to T cell an agile pulse sequence consisting of: (a) one electrical pulse with a voltage range from 500 to 3000 V per centimeter, preferably 800 V, a pulse width of 0.1 ms and a pulse interval of 0.2 to 10 ms between the electrical pulses of step (a) and (b);
(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) ; and
(c) 4 electrical pulses with a voltage of 150-325V, preferably 200 V with a pulse width of 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses.
In a preferred embodiment, primary cells are transfected 4 to 5 days after activation, for example 5x106 cells are transfected with 10 pg of each mRNA encoding left and right arms of TALEN.
The day before electroporation, cells were passaged at 106 cells/ml in Xvivo-15 + 5% AB human serum, + IL-2 20ng/ml.
The days of electroporation, T at 25x106 cells/ml. 200 pi (5x106 cells) were mixed with mRNA encoding TRAC TALEN (10 pg for each arm of TALEN), transferred to a 0.4 cm electroporation cuvette (Bio-Rad) and electroporated using PulseAgile system using the following pulse series:
Figure imgf000061_0001
Immediately following electroporation, 200 pi of pre-warmed media was transferred from the above described 12-well plate in to the electroporation cuvette and the whole content of the cuvette was transferred back to the 12-well plate (2.2 ml total) for incubation at 37°C / 5 %C02. 24h post electroporation, cells were centrifuged and passaged at 10L6 cells/ml in Xvivo-15 + 5% AB human serum + IL-2 20ng/ml. 7 days later, efficiency of TRAC gene inactivation was estimated by flow cytometry analysis using anti-TCRab-specific antibodies (Miltenyi).
In particular embodiments, the method of transforming T cell comprising contacting said T cell with RNA and applying to T cell an agile pulse sequence consisting of:
(a) one electrical pulse with a voltage of 500, 600, 700, 800, 900, 1000, 1200, 1400, 1800, 2000, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per centimeter, a pulse width of 0.1 ms and a pulse interval of 0.2, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between the electrical pulses of step (a) and (b);
(b) one electrical pulse with a voltage range from of 500, 600, 700, 800, 900, 1000, 1200, 1400, 1800, 2000, 2250, of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V 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); and
(c) 4 electrical pulses with a voltage of 50, 100, 110 ,120 ,130 ,140 , 150 , 160, 170, 180, 190, 200, 220, 250, 300, 325 V with a pulse width of 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses.
Any values included in the value range described above are disclosed in the present application. Electroporation medium can be any suitable medium known in the art. Preferably, the electroporation medium has conductivity in a range spanning 0.01 to 1.0 milliSiemens.
Purification / depletion
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. Preferably, 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.
In a preferred embodiment, isolation of TCR-negative cells includes using magnetic beads comprising anti TCR antibodies.
In a more preferred embodiment, isolation of TCR-negative cells includes using magnetic beads comprising anti TCR antibodies 4 days after TCR gene disruption.
If more than one gene is disrupted in the disruption step, and/or if one or more genes are disrupted in an additional disruption step, 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.
Transformation
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.
Preferably, said recombinant receptor is a CAR.
More preferably, said recombinant receptor is a CAR comprising a structure scfv-CD8alpha hinge-CD8alpha transmembrane domain-4-1 BB- CD3 zeta or scfv-lgG1 hinge-CD8alpha transmembrane domain -4-1 BB-CD3 zeta or scfv- FcyRIIIa hinge-CD8alpha transmembrane domain -4-1 BB-CD3 zeta.
Examples of Sequences of the different CAR components
Figure imgf000064_0001
The CAR is then expressed at on the surface of the cells. In some embodiments, said expression can be conditional (expression to depend on the presence or absence of a drug in the milieu.
The transformation step can be distinct from the disruption step described above and be performed before or after the disruption step.
Alternatively, the transformation step can be performed simultaneously with (to) the disruption step. In particular, 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.
In individual embodiment, the transformation step can be performed simultaneously with the disruption step. In particular, 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):
For instance, 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 Q9Y286), SIGLEC9 (Uniprot Q9Y336), CD244 (Uniprot Q9BZW8), TNFRSF10B (Uniprot 014763), TNFRSF10A (Uniprot 000220), CASP8 (Uniprot Q14790), CASP10 (Uniprot Q92851 ), CASP3 (Uniprot P42574), CASP6 (Uniprot P55212), CASP7 (Uniprot P55210), FADD (Uniprot Q13158), FAS (Uniprot P25445), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), SMAD2 (Uniprot Q15796), SMAD3 (Uniprot P84022), SMAD4 (Uniprot Q13485), SMAD10 (Uniprot B7ZSB5), SKI (Uniprot P12755), SKIL (Uniprot P12757), TGIF1 (Uniprot Q15583), IL10RA (Uniprot Q13651 ), IL10RB (Uniprot Q08334), HMOX2 (Uniprot P30519), IL6R (Uniprot P08887), IL6ST (Uniprot P40189), EIF2AK4 (Uniprot Q9P2K8), CSK (Uniprot P41240), PAG1 (Uniprot Q9NWQ8), SIT1 (Uniprot Q9Y3P8), FOXP3 (Uniprot Q9BZS1 ), PRDM1 (Uniprot Q60636), BATF (Uniprot Q16520), GUCY1A2 (Uniprot P33402), GUCY1A3 (Uniprot Q02108), GUCY1 B2 (Uniprot Q8BXH3) and GUCY1 B3 (Uniprot Q02153). The gene editing introduced in the genes encoding the above proteins is preferably combined with an inactivation of TCR in CAR T cells.
Preference is given to inactivation of PD1 , TRAC, B2M and/or CTLA4, in combination with the expression of non-endogenous immunosuppressive polypeptide, such as a PD-L1 ligand and/or CTLA-4 Ig.
List of genes that may constitute immune check points and improve the activity and/or survival of engineered immune cells
Figure imgf000066_0001
Figure imgf000067_0001
Inhibiting suppressive cytokines/metabolites
According to another aspect of the invention, 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).
Preference is given to engineered immune cells in which a sequence encoding IL-2, IL-12 or IL-15 replaces the sequence of at least one of the above endogenous genes.
Preference is given to engineered immune cells in which a sequence encoding IL-2, IL-12, IL-7, IL-5, or IL-21 replaces the sequence of at least one of the above endogenous genes.
Preference is given to engineered immune cells in which a sequence encoding IL-2, IL-12, IL-7, IL-5, or IL-21 replaces the sequence of at least one of the above endogenous genes and a sequence encoding a CAR is inserted into the TRAC gene.
Inducing resistance to chemotherapy drugs
According to another aspect of the present method, 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.
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.
This enables the combination of cells with anti-TCR antibodies of the invention to be used after or in combination with conventional anti-cancer chemotherapies.
The resulting product is more efficient than non engineered immune cells (even with a CAR) and safer because inducing less side effects.
In particular embodiments, 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. In general, 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. In so-called first generation CARs, 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.
In addition to 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.
As examples, the antigen targeted by the CAR (at least one antigen marker) 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 telomerase reverse transcriptase, RUI, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ES0-1 , LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1 ), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1 )-l, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MFIC) molecule presenting a tumor-specific peptide epitope, 5T4, RORI, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1 ) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MFIC) molecule, BCMA (CD269, TNFRSF 17), or a virus-specific surface antigen such as an FI IV specific antigen (such as FIIV gp120); an EBV-specific antigen, a CMV-specific antigen, a FlPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface markers. Antigens are not necessarily surface marker antigens but can be also endogenous small antigens presented by FILA class I at the surface of the cells.
By way of example, 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. By way of example, 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.
By way of example, 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.
By way of example, 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.
By way of example, 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.
Examples of 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).
Multi-chain CARs may in particular be derived from FcsRI. In this architecture, the high affinity IgE binding domain of FcsRI alpha chain is replaced by an extracellular ligand-binding domain such as scFv to redirect T cell specificity against cell targets and the N and/or C-termini tails of FcsRI beta chain are used to place costimulatory signals in normal juxtamembrane positions.
Multi-chain CARs may in particular comprise at least two of the following components:
a) one polypeptide comprising the transmembrembrane domain of FcsRI alpha chain and an extracellular ligand-binding domain,
b) one polypeptide comprising a part of N- and C- terminal cytoplasmic tail and the transmembrane domain of FcsRI beta chain and/or
c) at least two polypeptides comprising each a part of intracytoplasmic tail and the transmembrane domain of FcsRI gamma chain, whereby different polypeptides multimerize together spontaneously to form a dimeric, trimeric or tetrameric CAR. According to such architectures, ligand binding domains and signaling domains are borne on separate polypeptides. The different polypeptides are anchored into the membrane in a close proximity allowing interactions with each other. In such architectures, the signaling and costimulatory domains can be in juxtamembrane positions (i.e. adjacent to the cell membrane on its internal side), which is deemed to allow improved function of co-stimulatory domains. The multi- subunit architecture also offers more flexibility and possibilities of designing CARs with more control on T cell activation. For instance, it is possible to include several extracellular antigen recognition domains having different specificity to obtain a multi-specific CAR architecture.
It is also possible to control the relative ratio between the different subunits in a multi-chain CAR. This type of architecture is described in document WO 2014/039523, which is incorporated herein by reference.
The assembly of the different chains as part of a single multi-chain CAR is made possible, for instance, by using the different alpha, beta and gamma chains of the high affinity receptor for IgE (FcsRI) to which the signaling and co- stimulatory domains are fused. The gamma chain comprises a transmembrane region and cytoplasmic tail containing one immunoreceptor tyrosine-based activation motif (ITAM).
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. In some embodiments, the extracellular ligand-binding domains can be placed in tandem on the same transmembrane polypeptide, and optionally can be separated by a linker. In other embodiments, 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. In particular embodiments, 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. In other embodiments, 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.
Thus, two, three, four, five, six or more multi-chain CARs, each one comprising different extracellular ligand binding domains, 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. In other words, 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.
For example, 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. Examples of 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. In preferred embodiments, 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.
In particular embodiments, the signal transduction domain of a multi-chain CAR comprises a co-stimulatory signal molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response. Ligand binding-domains can e.g. be an scFv from a monoclonal antibody. Bispecific or multi-specific CARs can be as described in WO 2014/01 1988 which is incorporated by reference.
Similarly, to what was described above with respect to single-chain CARs, the cells may express multi-chain CARs which specifically target 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.
Similarly, to what was described above with respect to single-chain CARs, the cells may express multi-chain CARs which specifically target a cell surface marker such as BCMA - CD33 - EGFRVIII - Flt3 - WT1 - CD70, MUC16 - PRAME
- TSPAN10, CLAUDIN18.2 - DLL3 - LY6G6D, Liv-1 - CHRNA2 - ADAM 10.
In some embodiments, the CAR used in the present invention has an extracellular binding domain that comprises a scFv formed by at least a VFI chain and a VL chain specific to an antigen, preferably a cell surface marker antigen, which also comprises a tag such as a monoclonal antibody (mAb)-specific epitope, as disclosed in WO 2016120216.
The tag can be optionally used in the method to perform enriching, sorting and/or depleting of the cells comprising the CAR. The scFc is preferably a chimeric scFv.
By“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. In some embodiments, the mAb-specific epitope is not an epitope recognized by the ScFv. In some embodiments, 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.
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.
In some embodiments 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
- an extracellular binding domain, wherein said extracellular binding domain further comprises a hinge, - a transmembrane domain, and,
- an intracellular domain.
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-Li-VHL -Epitopel-iL -;
V1 -L1 -V2-(L)x-Epitopel-(L)x-Epitope2-(L)x-;
V1 -L1 -V2-(L)x-Epitopel-(L))<-Epitope2-(L))<-Epitope3-(L))<-;
(L -Epitopel-a -VHi-Vj;
(L)x-Epitopel-(L)x-Epitope2-(L)x-V1 -L1 -V2;
Epitopel-(L)x-Epitope2-(L)x-Epitope3-(L)x-V1 -L1 -V2;
(L)x-Epitopel-(L)x-V1 -L1 -V2-(L)x-Epitope2-(L)x;
(L)x-Epitopel-(L)x-V1 -L1 -V2-(L)x-Epitope2-(L)x-Epitope3-(L)x-;
(Ljx-Epitopel-ILjx-Vi-U-Vz-iLjx-EpitopeZ-iLjx-EpitopeS-iLjx-EpitopeAiL),,-;
(Ljx-Epitopel-ILjx-EpitopeZ-ILjx-Vi-U-Vz-iLjx-Epitopea-iLjx-;
(Ljx-Epitopel-ILjx-EpitopeZ-iLjx-Vi-U-Vz-iLjx-EpitopeS-iLjx-EpitopeAiLjx-;
Vi-i Ux-Epitopel-i Ux-Vj;
V1 -(L)x-Epitopel-(L)x-V2-(L)x-Epitope2-(L)x;
V!-ILjx-Epitopel-iLjx-Vz-iLjx-EpitopeZ-iLjx-EpitopeS-iLjx;
V!-ILjx-Epitopel-iLjx-Vz-iLjx-EpitopeZ-iLjx-EpitopeS-iLjx-EpitopeAiLjx;
(L)x-Epitopel-(L)x-V1 -(L)x-Epitope2-(L)x-V2; or,
(Ljx-Epitopel-ILjx-V!-ILjx-EpitopeZ-iLjx-Vz-iLjx-Epitopea-iLjx;
wherein, 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,
Epitope 1 , Epitope 2 and Epitope 3 are mAb-specific epitopes and can be identical or different.
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).
Use can be made of a recombinant DNA construct comprising one or more sequences encoding a CAR as defined above, and wherein the sequence encoding the extracellular domain is contiguous with and in the same reading frame as a sequence encoding a transmembrane domain and an intracellular domain. An exemplary CAR construct may comprise an optional leader sequence, an extracellular cell target antigen binding domain, a hinge, a transmembrane domain, and an intracellular inhibitory signaling domain
Preferably, 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 are known in the art and 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. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment.
Most preferably, in the transformation step 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). The use of a lentivirus vector is particularly preferred in the present invention. The use of 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” or transgene 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.
The 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).
In other word, 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.
Alternatively, 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. As a result of this step, 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.
In this step, the cells may be incubated with an effective dose of IL-2, IL-7 and/or IL-15. This may trigger the desired response, as the cells express the IL-2 receptor, the IL-7 receptor and/or the IL-15 receptor.
This step may be performed in the presence of stromal cells.
Expansion
The method of the invention may also comprise a step of expanding the cells, optionally in the presence of an immunosuppressive agent as disclosed above (immunosuppressive treatment). Preferably, the immunosuppressive agent is the target of one of the genes which are disrupted as described above.
As a non-limiting example, 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.
In some embodiments, 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.
In other embodiments, 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.
In other embodiments, 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. In another embodiment, said FKBP family gene member is FKBP12 or a variant thereof.
In other embodiments, a cyclophilin 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 cyclosporine.
Incubation with the anti-TCR reagent
According to the invention, 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.
Preferably, the reagent is approved by health authorities.
Preferably, the reagent is an antibody, eve more preferably an antibody approved by health authorities.
Alternatively, an antibody fragment may be used. In this case, 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. Preferably, it is able to bind to a Fc receptor.
Alternatively, an antibody-drug conjugate may be used, wherein a drug, preferably a cytotoxic drug, is coupled to an anti-TCR antibody. This makes it possible for instance to further deplete remaining TCR-positive cells during manufacturing.
Preferably, a monoclonal antibody (or antibody fragment, or antibody-drug conjugate) may be used.
Alternatively, a polyclonal antibody (or antibody fragments, or antibody- drug conjugates) is used.
In some embodiments, the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the TCRa chain.
In other embodiments, the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the TCRa chains.
In other embodiments, the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the TCR chain.
In other embodiments, the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the CD3y chain.
In other embodiments, the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the CD3s chain.
In other embodiments, the anti-TCR antibody (or antibody fragment, or antibody-drug conjugate) targets the CD3z chain.
Anti-TCR antibodies are in particular mentioned in the review article“T-Cell Receptor-Like Antibodies: Targeting the Intracellular Proteome Therapeutic Potential and Clinical Applications" by Cohen and Reiter in Antibodies 2013, 2, 517-534; doi:10.3390/antib2030517.
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.
The anti-TCR antibody may also be for instance visilizumab, developed by PDL BioPharma 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 T10B9, for instance the MEDI-500 clone provided by Medlmmune Corp. 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 .
The anti-TCR antibody may also be for instance BMA031 , which can be supplied by Creative Biolabs. Fragments of this antibody or antibody-drug conjugates based on this antibody may also be used.
Mixtures of several antibodies or antibody fragments may alternatively be used.
Optionally, a rinsing step may be performed after the incubation step in order to eliminate non-bound anti-TCR reagent. Alternatively, the non-bound anti- TCR reagent is not eliminated.
In some embodiments, 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.
In particular embodiments, 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 %).
According to the present invention, 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.
Excess of reagent may be eliminated by rinsing the preparation. In one particular embodiment, engineered cells may be incubated in the present of human immunoglobulin before incubation with said reagent to block any eventual non specific binding site.
Fill and finish
During the final step of fill and finish, the cells are sampled and frozen. As mentioned above, 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.
Elimination of alpha betaTCR-positive cells
According to the present invention, remaining TCR-positive cells in a composition of the invention - can be depleted and preferably substantially eliminated in vitro and/or in vivo, in particular after administration to a patient. The anti-TCR reagent bound to said remaining TCR-positive cells triggers an elimination response (destruction of TCR expressing cells) when put in contact with a protein of the complement or with cytolytic cells.
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.
Accordingly, 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.
Additionally, or alternatively, the use of antibody-drug conjugates, or repeated stimulation by an anti-TCR reagent, or crosslinking of bound reagent, as described above, may also lead to a depletion or substantial elimination of remaining TCR-positive cells.
As a result, 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.
Any cells 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.
Preferably said composition comprises between 90% to 0.001 % TCR- positive cells bound to a reagent specific for alpha beta TCR cells.
Therapeutic applications
The T cell product prepared by the method of the present invention, and therefore including or in combination with the anti-TCR reagent, can be used as a medicament. It constitutes first a pharmaceutical composition
That may be administered with a drug.
In particular embodiments, 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.
In some embodiments, said medicament can be used for treating pathologies such as cancer in a patient, more particularly a human patient, in need thereof.
In another aspect, the invention provides a medicament for its use in a method for treating patients, said method comprising at least one of the following steps: providing a composition obtained or obtainable by the method of the present invention; and administrating said composition to the patients.
Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that the cells, cell lines or population of cells that are used originate from the patient (donor is the recipient).
By 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.
The administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the cell compositions of the present invention are preferably administered by intravenous injection.
The administration of the cells or population of cells can consist of the administration of 104-109 cells per kg body weight, preferably 105 to 106 cells/kg body weight including all integer values of cell numbers within those ranges.
Alternatively, 102 to 101° engineered cells (e.g. 105 to 109 cells, or 106 to 108 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. In other embodiments, the effective amount of cells are administrated as a single dose. In other embodiments, 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.
In other embodiments, an effective amount, or number of cells or a composition comprising those cells are administrated parenterally. Said administration can be an intravenous administration. Said administration can be direct injection into a tumor.
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. In certain embodiments of the present invention, 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. In further embodiments, 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. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). In further embodiments, 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. In other embodiments, 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. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the cells of the present invention. In some embodiments, the cells are administered before or following surgery.
Examples Preparation of allogeneic (off the shelf) cells.
GENERAL METHOD OF MANUFACTURING.
The following general method is part of the present invention
A method of manufacturing“off the shelf engineered cells for therapy comprising:
- 1 providing engineered allogeneic cells comprising cells with surface expression of TCR, preferably alpha beta TCR, more preferably less than 5% TCR+ cells,
- 2 combining said cells with a reagent selectively binding to an antigen present at the surface of said TCR+ -expressing cells, preferably said antigen is an antigen of the endogenous alpha beta TCR and is approved by health authorities,
3 Optionally eliminating the excess of unbound reagent, by eliminating unbound antibody.
4 Sampling and freezing.
Other steps may be part of the method according to the present invention
The step of providing cells may comprise
(a) providing cells,
(a’) activating cells
- (b) optionally engineering cells by expressing a recombinant receptor, preferably a chimeric antigen receptor, wherein the chimeric antigen receptor comprises at least one extracellular binding domain which is specific for an antigen and which additionally comprises a tag or an antibody specific binding site
- (c) engineering said cells by inactivating at least one TCR gene and/or at least one TCR gene and at least one more gene -to provide cells comprising cells with surface expression of TCR, preferably alpha beta TCR,
- (d) a step of purification of TCR cells and
- combining said cells with a reagent selectively binding to an antigen present at the surface of said TCR -expressing cells, preferably said antigen is an antigen of the endogenous alpha beta TCR and is approved by health authorities,
Optionally eliminating the excess of unbound reagent,
Sampling and freezing. The method may be carried out with step(b) and (c) concomitant, step
(b) before (c)or step (c) before step (b).
Starting materials:
CELLS
Samples of cells from 106 to >/7E9 or more total MNCs collected by leukapheresis from between 18 to 30 years old healthy volunteers are used.
Preferably, the percentage (%) of PBMC in total cell population is > 85% with a CD4 and CD8 counts in total blood CD4 counts between 1000-10000
CD8 counts: 1000-6500
CD4 / CD8 ratio in total blood CD4/CD8 ratio: 0.5-4.0
% CD3 (CD3 over CD45) >45%
25% > % CD4 (CD4 over CD3)>70%
10%> % CD8 (CD8over CD3)>60%
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.
No evidence of active infection for the following disease markers was measured: Hepatitis A, Hepatitis E, Epstein-Barr Virus EBV and Toxoplasma.
Compliance with EU (Tissues and Cells Directive 2004/23/EC and sister directives 2006/17/EC and 2006/86/EC, and ’’blood” directives 2002/98/EC and 2005/62/EC, 2004/33/EC, 2005/61 /EC). Directives and US requirements signed by responsible person, including but not limited to deferral, exclusion, testing unless excluded at mentioned in the table below, including the new FDA Guidance for Industry, Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells, Tissues, and Cellular and Tissue- Based Products. Should the requirements evolve with a donor screening test for Zika virus, or product test for Zika virus before the time of initiation of the procurement campaigns, they will be implemented.
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
According to the present invention, the use of 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.
According to particular embodiments, 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). 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 . Alternatively the method described in Eyquem J, Mansilla- Soto J, Giavridis T, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543(7643):1 13-1 17. doi:10.1038/nature21405, may be used.
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.
According to the method of the present invention, a sequence specific reagent (a nuclease, preferably a TALE protein) and the targeted gene integration is operated by homologous recombination or by Non-Homologous End-Joining (NHEJ) into said immune cells.
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.
Double/Triple KO- KO and Kl
The Method for gene engineering with optimized yield is described in PCT/EP2017/066355 and in PCT/EP2017/076798.
Briefly, 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).
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).
Alternatively, 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 ). Preferably the rare - cutting endonuclease is used in the present study is a TALE-protein.
In another example, 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.
These nucleases are used in the present invention for gene editing ie for introducing a mutation, deletion or insertion into an endogenous (genomic) sequence. The Rare cutting endonucleases used in the present study may be introduced preferably under mRNA form for TAL-protein, into cells by electroporation, or lipofection.
Under these conditions, 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).
ELECTROPORATION
In general, 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 (cm3), 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. With the method, 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.
The product obtained may be used as a medicament. Thus, 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.
Expansion
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.
Cell Purification
Optimization of purification of TCR-cells and TCR+ cells depletion is the object of the present invention.
In particular embodiments, it is an object of the present invention to provide a method that exclude using conventional coated beads that are very expensive for purifying TCR negative-cells.
Steps of fill and finish
- Sampling
- - Use of Cryo media for Freezing. All compositions are tested before freezing and after freezing and thawing.
Example of non alloreactive T cells MHC-deficient
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.
Furthermore, sequential TALEN electroporation was performed to generate genes knock-out and triple KO CAR T cells, the cells were edited sequentially with TALEN specific for TRAC gene; PD-1 and B2M. In all cases, T- cells were transduced with viral particles (AAV6) for the expression of CAR targeting the antigen CD22.
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).
Frozen human PBMCs from two different donors are thawed at 2 x106 cells per ml on day prior activation and transduction step, in complete X-Vivo media (X- VIVO 15, Lonza#04-418Q; 5% Human serum AB, Gemini #100-318; 20ng/mL IL- 2, Miltenyi#130-097-743;). One day post thawing, cells are transduced as described in (Poirot et a/. (2015) Multiplex Genome-Edited T-cell Manufacturing Platform for“Off-the-Shelf Adoptive T-cell Immunotherapies Cancer Res. 75: 3853-64) with lentiviral particles allowing the expression of a Chimeric Antigen Receptor targeting CD22 antigen containing a mimotope sequence. Cells are further activated the same day using anti CD3 and anti CD28 antibod ies-coated beads (TransAct, Miltenyi) according to manufacturer’s protocol for 4 days.
At day 5 post thawing, 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. Two days later, re-stimulated cells were labeled with antibodies at a 1 :50 dilution of each antibody for 15 minutes at 4°C (Miltenyi; TCR#130-091 -236, HLA-ABC#130-101-467, PD-1 #130-099-878). For all the different donors tested sequential editing provide the best triple KO efficacy ranging from 20 up to 40%.
Triple KO T-cells were then enriched using a biotin and column based negative purification system for TOR and B2M dKO cells (Miltenyi; biotin-TCR #130-098-219, bitoin-HLA-ABC#130-101 -463, Biotin beads #130-090-485, MS columns#130-042-201 ). Under this purification scheme, only TOR and B2M positive cells bind the MS column, and the TCR/B2M dKO cells of interest are enriched in the flowthrough fraction with 97% or greater purity. Triple KO CAR-T cells enriched for TCR/B2M dKO were further incubated for an additional two days before assessing CAR-T cells activity. On day 15, 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)
Sequence of TALEN ® used in experiments.
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
In vitro demonstration of the efficiency of the composition: Depletion of OKT3-coated TCR+ cells
To model what is expected in patients infused with engineered cells coated with anti-CD3 or anti-TCR antibodies, 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 ).
Methods
Cell culture Frozen peripheral blood mononuclear cells (PBMC) were obtained from 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.
TALEN
TRAC TALEN was obtained from Cellectis SA. The target sequences for TRAC TALEN is the following:
TTGTCCCACAGATATCCagaaccctgaccctgCCGTGTACCAGCTGAGA Two 17-bp recognition sites (upper case letters) are separated by a 15-bp spacer. mRNA
mRNAs 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).
Cell transfection
4 to 5 days after activation, 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 10L6 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. Cells were resuspended in Cytoporation media T at 25x106 cells/ml. 200 pi (5x106 cells) were mixed with mRNA encoding TRAC TALEN (10 pg for each arm of TALEN), transferred to a 0.4 cm electroporation cuvette (Bio-Rad) and electroporated using PulseAgile system using the following pulse series:
Figure imgf000096_0001
Figure imgf000097_0001
Immediately following electroporation, 200 pi of pre-warmed media was transferred from the above described 12-well plate in to the electroporation cuvette and the whole content of the cuvette was transferred back to the 12-well plate (2.2 ml total) for incubation at 37°C / 5 %C02. 24h post electroporation, cells were centrifuged and passaged at 10L6 cells/ml in Xvivo-15 + 5% AB human serum + IL-2 20ng/ml. 7 days later, efficiency of TRAC gene inactivation was estimated by flow cytometry analysis using TCRa -specific antibodies (Miltenyi).
TALEN -targeted CAR gene integration into the TRAC locus.
3 days after activation, 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 results show that the cytolytic activity of these cells was comparable to that of CAR expressing cells obtained by other method (classical transduction) 3 days after activation, T cells were transfected or not by electrotransfer of 1 pg of each mRNA encoding TRAC and CD52 TALEN per million cells. The results show that this 2-in-1 strategy of TCR KO and CAR Kl can be extended to the use of more than one TALEN. The integration of the CAR at the TRAC locus is highly efficient since the frequency of CAR+ TCR- cells reached more than 47%. Importantly, 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.
Purification of TCR negative cells resulted in mainly TCRa -negative (about 98%) while around 90% of unmodified T-cells were TCRa -positive, as expected. Complement-dependent cytotoxicity assay
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. After 12 to 15 h incubation at 37°C / 5% C02, cells were recovered, washed in PBS, labeled with eFIuor 780 viability marker (eBioscience) and cell viability was assessed by flow cytometry, for which data were acquired on a Macsquant X analyzer (Miltenyi) and analyzed using FlowJo software (FlowJo LLC).
The results show that mock transfected cells are all eliminated by complement- mediated cytotoxicity whereas TCR-negative cells in TRAC TALEN-treated sample are resistant to complement. These data demonstrate that OKT3-coated TCRa + cells are efficiently eliminated by complement mediated cytotoxicity.
To unsure that CAR -positive cells do not express endogenous TCR that may be expressed later during the process, cells obtained after TCR depletion were cultured for a week in the presence of anti-CD3/anti-CD28 antibodies and then analyzed by flow cytometry for TCR expression.
Under these conditions, cell surface expression of TCR was undetectable demonstrating that the lack of expression of alpha beta TCR is stable in time.
Models of GVHD used to analyzed the composition engineered cells+anti- TCR antibody.
Based on the models described in: Poirier N., Dilek N., Mary C. and Vanhove B. (2012). Graft versus host disease in humanized mice is differentially controlled by CD28 and CD80/86 antagonists. Journal of Translational Medicine, 10(Suppl. 3):02, and AN N., Flutter B., Sanchez Rodriguez R., et al. (2012). Xenogeneic Graft-versus Host-Disease in NOD-scid IL-2RYnull Mice Display a T-Effector Memory Phenotype. PLoS ONE, 7(8):e44219, the capacity of cells and composition according to the present invention to induce GVHD was tested. 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. 2016;7:333. doi:10.3389/fimmu.2016.00333.
Cells obtained according to the process below and incubated in the presence of the absence of human complement or control (serum) (for mediating in vitro cytotoxicity) were inoculated into individual mouse for GVHD analysis. As a control, anti-TCR antibody, complement and combinations of cells plus complement (without Ab) were inoculated to individuals.
None of the individuals treated with the composition (from 104 to 108 cells) per mice of 30-40g of the invention develop one sign of GVHD whereas inviduals treated with TCR positive cells (expressing 90% alpha beta TCR) induced signs of GVHD about 30 to 60 days after inoculation (weight loss 1/3 of body mass was lost) and eventually died.
Clinical DATA
Under autologous transfer, 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) were prepared according to the method of the invention and following the GMP practices, 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).
The composition (a sample of 2.5x106 cells bound to anti-TCR Ab) was then inoculated to five patients suffering ALL (treated group). Five patients received 2.5x106 cells processed as previously described (control group).
Under these conditions, one out of 5 patients had grade 1 GVHD in the control group none in the treated group.
In arm corresponding to patients treated with the composition of the invention no GVHD is detected, regardless of the dose of cells administered (104 to 108 cells /kg). Thus, GVHD frequency is reduced as compared to prior art treatment and no grade 3, grade 3 grade 2 or grade 1 GVHD was measured.
Thus, when the composition of the invention comprising cells bound to anti-alpha beta TCR antibody were administered into patients in need thereof, no GVHD was detected in 11/11 patients.

Claims

1. A method for manufacturing non alloreactive cells, comprising:
- a supply step, wherein cells or a population of cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are provided,
- an incubation step, wherein said cells or population of cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are incubated with an antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells resulting in antibody bound cells, or in a population of cells comprising antibody bound cells, immediately followed by :
- a rinsing step for eliminating unbound antibody immediately followed by a fill and finish step, or a fill and finish step.
2. The method of claim 1 , wherein:
the antibody selectively binding to an antigen present at the surface of alpha beta TCR positive (a TCR+) cells is selected from an antibody specific for the a TCR, for CD3, an antibody specific for the a TCR already approved by health authorities, an antibody T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab, a fragment thereof, and combinations thereof, provided that said fragment thereof comprises a binding domain for the human complement (allowing an antibody dependent cytotoxicity).
3. The method of claim 1 or 2 comprising:
a disruption step, wherein alpha beta TCR cells are genetically modified by disrupting at least one gene encoding a component of the alpha beta TCR to inhibit cell surface expression of alpha beta TCR, said disruption step occurring before or after the supply step and resulting in the production of engineered cells comprising alpha beta -TCR-negative engineered cells and cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface, the disruption step occurring before the incubation step.
4. The method according to any one of claim 1 to 3 wherein the non alloreactive cells comprise engineered cells, preferably TALEN®- engineered cells.
5. The method of claim 4 wherein the TALEN®- engineered cells, were engineered with a TALEN®- having the following sequences:
MGDPKKKRKVI DIADLRTLGYSQQQQEKI KPKVRSTVAQHH EALVGHGFTHAH IVALSQHPAALGTVAVKY
QDMIAALPEATH EAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL
CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGL
TPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ
VVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPEQVVAI
ASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN I
GGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGK
QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDH LVALACLGGRPALD
AVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPH EYIELIEIARNSTQDRI LEM KVM EFFMKVYGYRGKH LG
GSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYN LPIGQADEMQRYVEENQTRN KH I NPNEWWKVYPSSVTEF
KFLFVSGH FKGNYKAQLTRLNH ITNCNGAVLSVEELLIGGEM IKAGTLTLEEVRRKFN NGEIN FAAD and
MGDPKKKRKVIDIADLRTLGYSQQQQEKIKPKVRSTVAQH HEALVGHGFTHAH IVALSQHPAALGTVAVKY
QDMIAALPEATH EAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLN LTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAH
GLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLT
PQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN NGGKQALETVQRLLPVLCQAHGLTPQ
QVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV
VAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN N
GGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDH LVALACLGGRP
ALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYI ELI EIARNSTQDRILEMKVMEFFM KVYGYRGK
H LGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKH IN PN EWWKVYPSSV
TEFKFLFVSGHFKGNYKAQLTRLN HITNCNGAVLSVEELUGGEMIKAGTLTLEEVRRKFN NGEIN FAAD. or with a TALEN ® which right domain sequence has at least 90% identity with
MGDPKKKRKVI DIADLRTLGYSQQQQEKI KPKVRSTVAQHH EALVGHGFTHAH IVALSQHPAALGTVAVKY
QDMIAALPEATH EAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVL
CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQA
HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGL
TPEQVVAIASN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ
VVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAI
ASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN I
GGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGK QALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTN DH LVALACLGGRPALD AVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPH EYIELIEIARNSTQDRI LEM KVM EFFMKVYGYRGKH LG GSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRN KHI N PNEWWKVYPSSVTEF KFLFVSGH FKGNYKAQLTRLNH ITNCNGAVLSVEELLIGGEM IKAGTLTLEEVRRKFN NGEIN FAAD and the left domain has at least 90 % identity with
MGDPKKKRKVI DIADLRTLGYSQQQQEKI KPKVRSTVAQHH EALVGHGFTHAH IVALSQHPAALGTVAVKY
QDMIAALPEATH EAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN
ALTGAPLN LTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC
QAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN IGGKQALETVQALLPVLCQAH
GLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLT
PQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQ
QVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV
VAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIA
SN IGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN N
GGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDH LVALACLGGRP
ALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYI ELI EIARNSTQDRILEMKVMEFFM KVYGYRGK
H LGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKH IN PN EWWKVYPSSV
TEFKFLFVSGH FKGNYKAQLTRLNH ITNCNGAVLSVEELLIGGEMI KAGTLTLEEVRRKFN NGEIN FAAD.
6. The method according to any one of claim 1 to 5 comprising a transformation step, wherein the cells are modified by introducing at least one polynucleotide into the cells, said polynucleotide comprising a sequence encoding a recombinant receptor, provided that said recombinant receptor does not bind an endogenous alpha beta TCR.
7. The method according to any one of claim 1 to 6, wherein said cells or a population of cells comprising cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface are T cells or a population of cells comprising T cells expressing alpha beta T Cell Receptor (alpha beta TCR) at the cell surface.
8. The method according to any one of claim 1 to 7, wherein said antibody consists in or comprises a domain allowing an antibody dependent cytotoxicity.
9. The method according to any one of claim 1 to 8, wherein the antibody is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab, fragments thereof,
10. The method according to any one of claims 1 to 9, further comprising an activation step, said activation step occurring preferably before the transformation and disruption steps.
11 The method according to any one of claims 1 to 10, further comprising an expansion step, wherein the cells are expanded, before the incubation step and before the fill and finish step.
12. The method according to any one of claims 1 to 11 , which further comprises at least one step of purification of TCR-negative cells from TCR positive and negative cells.
13. The method according to any one of claims 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.
14. The method according to any one of claims 1 to 13, wherein the differentiation step is achieved by expressing a factor selected from IL-2, IL- 3, IL-5, IL-7 and IL-15, IL-21 , IL-27 a combination thereof, in the cells, or incubating the cells with an effective dose of at least one growth factor selected from of IL-2, IL-3, IL-5, IL-7 and IL-15, IL-21 , IL-27 a combination thereof, optionally in the presence of stromal cells.
15. The method according to any one of claims 1 to 14, wherein the disruption step is performed before the transformation step; or wherein the disruption step is performed after the transformation step; or wherein the disruption step is performed concomitantly with the transformation step.
16. The method for according to any one of claims 1 to 14, comprising an additional disruption step, wherein the cells are modified by disrupting at least one gene, said additional disruption step being performed before the incubation step.
17. The method for according to any one of claims 1 to 16, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, said expression being optionally a conditional expression.
18. A method according to any one of claims 1 to 17, wherein said fill and finish step comprising a step selected from sampling, packaging, freezing cells, and combinations thereof.
19. The method of any one of claims 1 to 18, which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the transformation step;
- the additional disruption step;
- the expansion step;
- the purification step;
- the incubation step; and
- the fill and finish step;
or which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the purification step;
- the additional disruption step;
- the transformation step;
- the expansion step;
- the incubation step; and
- the fill and finish step
or which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the transformation step;
- the additional disruption step;
- the expansion step;
- the differentiation and/or maturing step
- the purification step;
- the incubation step; and
- the fill and finish step; or which successively comprises:
- the supply step;
- optionally, the activation step;
- the disruption step;
- the purification step;
- the additional disruption step;
- the transformation step;
- the expansion step;
- the differentiation and/or maturing step
- the incubation step; and
- the fill and finish step.
20. The method of any one of claims 1 to 19, wherein the supply 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.
21. The method of any one of claims 1 to 20, wherein a disruption step is performed by introducing a nucleic acid encoding a rare-cutting endonuclease, preferably a TALE-nuclease, into the cells and expressing it, the introduction of the nucleic acid encoding a rare-cutting endonuclease being preferably performed by electroporation or by means of an agent allowing nucleic acid transport across cell compartments to a cell nucleus.
22. The method of any one of claims 1 to 21 , wherein a disruption step includes the inactivation of at least one gene selected from TCRa and/or TCR , preferably the inactivation of at least TCRa, and more preferably the inactivation of TCRa and at least one gene selected from CD52, dCK, GR, PD1 , CTLA4, beta-2 microglobulin, CBLB and CISH.
23. The method of any one of claims 1 to 22, wherein the recombinant receptor can selectively bind to one or more antigens present or presented at the surface of cancer cells and ultimately induces the destruction of cancer cells by allogeneic cells expressing it.
24. The method of any one of claims 2 to 23, wherein the engineered cells obtained after the disrupting step comprise between from less than 80 % and to less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component at the cell surface.
25. A composition comprising cells comprising engineered cells expressing alpha beta TCR on their surface in less than 80% of the total cells, preferably in less than 10% of the total cells, more preferably in less than 5% of the total cells, even more preferably in as preferably determined by flow cytometry analysis, in combination with and/or bound to a reagent selectively binding to an antigen present at the surface of cells expressing said cell surface alpha beta TCR.
26. The composition of claim 25 comprising between 20% and 0.001 % of cells wherein cell surface of endogenous alpha beta TCR is detectable, as preferably determined by flow cytometry analysis.
27. The composition according to claim 25 or 26, wherein the reagent is or comprises an antibody, an antibody-drug conjugate or a fragment of an antibody comprising a domain binding to a protein of the complement or to a receptor of a cell mediating antibody dependent cytotoxicity, said receptor being preferably a Fc receptor.
28. The composition according to claim 27, wherein the antibody or fragment of antibody or antibody-drug conjugate is selected from T10B9, BMA031 , TOL101 , muromonab-CD3, otelixizumab, teplizumab, visilizumab as well as fragments thereof, antibody-drug conjugates thereof and combinations thereof.
29. The composition according to any one of claims 25 to 28, wherein said engineered cells express at their surface a recombinant receptor.
30. The composition according to claim 29, wherein the recombinant receptor is a Chimeric Antigen Receptor (CAR), and said CAR is expressed at the surface of the cells, the expression of the CAR being optionally a conditional expression.
31. The composition according to any one of claims 25 to 29, wherein the engineered cells comprise less than 3%, preferably less than 2%, more preferably less than 1 %, of cells expressing the endogenous alpha beta TCR component, as preferably determined by flow cytometry analysis.
32. The composition according to any one of claims 25 to 30, wherein the engineered cells are derived from stem cells or from T cells.
33. The composition according to any one of claims 25 to 32, for use in therapy or prophylaxis.
34. The composition according to any one of claims 25 to 33, for use in therapy or prophylaxis according to claim 33 wherein from 102 to 1010 engineered cells per kg or per m2, preferably from 104 to 1010 engineered cells per kg or per m2 are administered to a patient in one or more administrations.
35. The composition of any one of claims 25 to 34, for the treatment or prophylaxis of a cancer, a viral infection, an autoimmune disorder or Graft versus Host Disease.
36. The composition according to any one of claims 25 to 35, inducing no Graft versus Host Disease, preferably no alpha beta TCR-mediated Graft versus Host Disease.
37. The composition according to any one of claims 25 to 36, for use in a method for eliminating engineered cells still expressing endogenous alpha beta TCR component in vivo and/or in vitro.
38. The composition according to any one of claims 25 to 37, for use in an in vivo or in vitro process comprising contacting said composition with at least one protein of the complement.
39. A method for purifying alpha beta TCR negative engineered cells comprising a step of contacting a composition according to any one of claim 23 to 36, or obtainable by the method of any one of claims 1 to 22 with a complement protein, or with cytolytic cells, so as to deplete or eliminate cells still expressing the endogenous alpha beta TCR component of the composition, preferably in vivo.
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN114269377A (en) * 2019-07-26 2022-04-01 南克维斯特公司 Antibody pre-loaded CD16+ NK-92 cells as an effective therapeutic product for tumor lysis
CN117343927A (en) * 2023-12-06 2024-01-05 上海药明巨诺生物医药研发有限公司 Nucleic acid extraction method of engineering cells

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR901228A (en) 1943-01-16 1945-07-20 Deutsche Edelstahlwerke Ag Ring gap magnet system
US6010613A (en) 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
WO2004067736A2 (en) 2003-01-28 2004-08-12 Cellectis Custom-made meganuclease and use thereof
WO2004083379A2 (en) 2003-03-14 2004-09-30 Walters Richard E Large volume ex vivo electroporation method
WO2010027797A1 (en) 2008-08-26 2010-03-11 Macrogenics Inc. T-cell receptor antibodies and methods of use thereof
US8524234B2 (en) 2011-11-03 2013-09-03 Tolera Therapeutics, Inc Antibody for selective inhibition of T-cell responses
WO2013158292A1 (en) 2012-04-17 2013-10-24 University Of Washington Through Its Center For Commercialization Hla class ii deficient cells, hla class i deficient cells capable of expressing hla class ii proteins, and uses thereof
WO2013176915A1 (en) 2012-05-25 2013-11-28 Roman Galetto Methods for engineering allogeneic and immunosuppressive resistant t cell for immunotherapy
WO2014011988A2 (en) 2012-07-13 2014-01-16 The Trustees Of The University Of Pennsylvania Enhancing activity of car t cells by co-introducing a bispecific antibody
WO2014039523A1 (en) 2012-09-04 2014-03-13 Cellectis Multi-chain chimeric antigen receptor and uses thereof
WO2014184741A1 (en) 2013-05-13 2014-11-20 Cellectis Methods for engineering allogeneic and highly active t cell for immunotheraphy
WO2014184744A1 (en) 2013-05-13 2014-11-20 Cellectis Methods for engineering highly active t cell for immunotherapy
WO2015075175A1 (en) 2013-11-22 2015-05-28 Cellectis Method for generating batches of allogeneic t-cells with averaged potency
WO2015132598A1 (en) * 2014-03-05 2015-09-11 Ucl Business Plc Chimeric antigen receptor (car) with antigen binding domains to the t cell receptor beta constant region
WO2016120216A1 (en) 2015-01-26 2016-08-04 Cellectis mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR901228A (en) 1943-01-16 1945-07-20 Deutsche Edelstahlwerke Ag Ring gap magnet system
US6010613A (en) 1995-12-08 2000-01-04 Cyto Pulse Sciences, Inc. Method of treating materials with pulsed electrical fields
WO2004067736A2 (en) 2003-01-28 2004-08-12 Cellectis Custom-made meganuclease and use thereof
WO2004083379A2 (en) 2003-03-14 2004-09-30 Walters Richard E Large volume ex vivo electroporation method
WO2010027797A1 (en) 2008-08-26 2010-03-11 Macrogenics Inc. T-cell receptor antibodies and methods of use thereof
US8524234B2 (en) 2011-11-03 2013-09-03 Tolera Therapeutics, Inc Antibody for selective inhibition of T-cell responses
WO2013158292A1 (en) 2012-04-17 2013-10-24 University Of Washington Through Its Center For Commercialization Hla class ii deficient cells, hla class i deficient cells capable of expressing hla class ii proteins, and uses thereof
WO2013176915A1 (en) 2012-05-25 2013-11-28 Roman Galetto Methods for engineering allogeneic and immunosuppressive resistant t cell for immunotherapy
WO2014011988A2 (en) 2012-07-13 2014-01-16 The Trustees Of The University Of Pennsylvania Enhancing activity of car t cells by co-introducing a bispecific antibody
WO2014039523A1 (en) 2012-09-04 2014-03-13 Cellectis Multi-chain chimeric antigen receptor and uses thereof
WO2014184741A1 (en) 2013-05-13 2014-11-20 Cellectis Methods for engineering allogeneic and highly active t cell for immunotheraphy
WO2014184744A1 (en) 2013-05-13 2014-11-20 Cellectis Methods for engineering highly active t cell for immunotherapy
WO2015075175A1 (en) 2013-11-22 2015-05-28 Cellectis Method for generating batches of allogeneic t-cells with averaged potency
WO2015132598A1 (en) * 2014-03-05 2015-09-11 Ucl Business Plc Chimeric antigen receptor (car) with antigen binding domains to the t cell receptor beta constant region
WO2016120216A1 (en) 2015-01-26 2016-08-04 Cellectis mAb-DRIVEN CHIMERIC ANTIGEN RECEPTOR SYSTEMS FOR SORTING/DEPLETING ENGINEERED IMMUNE CELLS

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 (en) * 2019-07-26 2022-04-01 南克维斯特公司 Antibody pre-loaded CD16+ NK-92 cells as an effective therapeutic product for tumor lysis
CN117343927A (en) * 2023-12-06 2024-01-05 上海药明巨诺生物医药研发有限公司 Nucleic acid extraction method of engineering cells
CN117343927B (en) * 2023-12-06 2024-03-12 上海药明巨诺生物医药研发有限公司 Nucleic acid extraction method of engineering cells

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