WO2019106163A1 - Reprogramming of genetically engineered primary immune cells - Google Patents

Reprogramming of genetically engineered primary immune cells Download PDF

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WO2019106163A1
WO2019106163A1 PCT/EP2018/083180 EP2018083180W WO2019106163A1 WO 2019106163 A1 WO2019106163 A1 WO 2019106163A1 EP 2018083180 W EP2018083180 W EP 2018083180W WO 2019106163 A1 WO2019106163 A1 WO 2019106163A1
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cells
uniprot
cell
engineered
immune
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David Sourdive
Stephan REYNIER
Laurent Poirot
Philippe Duchateau
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Cellectis
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • 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
    • 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
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    • C07K—PEPTIDES
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention pertains to the field of adaptive cell immunotherapy and provides with methods for producing hematopoietic engineered cells lines by reprogramming primary cells that have been genetically engineered. By engineering primary cells -before their reprogramming into multipotent cells - the cells can be tested or even screen for their sought therapeutic properties.
  • the multipotent cells obtainable by these methods offer a renewable source for the production of“off-the-shelf” therapeutic cells that can be differentiated in-vitro or in-vivo into immune effector cells. of the invention
  • Effector immune cells are essential mediators of immune defense against infectious pathogens and cancer. Their insufficiency, which occurs in hereditary or acquired immune deficiencies, results in life threatening infections, increased cancer incidence, and disrupted immunoregulation. Conversely, under impaired regulation T-cells can be harmful and cause normal tissue destruction, as seen in autoimmune disorders, graft rejection, and graft- versus-host disease (GVHD).
  • GVHD graft- versus-host disease
  • T-cells develop from precursors that rearrange germline antigen receptor VDJ genes in the thymus, thereby generating clonotypic T cell receptors (TCRs) that undergo positive and negative thymic selection.
  • TCRs clonotypic T cell receptors
  • the resulting T cells are self-restricted and tolerant of self- tissues.
  • the newly generated T cells known as naive T cells, initially circulate throughout the body with a low frequency of precursors for any given antigen specificity. Upon encountering antigen, T cells expand and acquire effector and/or memory functions to amount an immune response.
  • Adoptive cell therapies utilizing“donor-derived T cells,” harvested from a healthy donor can expose the recipient to the risk of normal tissue destruction by graft versus host (GVH) responses.
  • GSH graft versus host
  • Autologous T-cells are devoid of such toxic potential. However, they may be lacking therapeutic potential or be functionally impaired in patients with refractory infections or progressing cancer. Allogeneic and autologous effector immune cells thus have their respective advantages and disadvantages.
  • T cells may be isolated from surgically removed tumors, which are enriched in tumor-reactive T cells relative to peripheral blood.
  • T umor infiltrating lymphocytes TILs
  • TILs can be isolated at quite a high frequency from melanoma specimens, but this technique is not feasible or effective in many other tumor types [Wu,R. et al. (2012) Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J. 18:160-175].
  • Immune cell therapy is thus currently limited by the availability of effector immune cells to be safely engrafted and prompt to fight the disease specifically addressed by the patient.
  • Peripheral blood T cells are the most easily accessible and perfectly suitable cell source for this purpose. Most current therapies utilizing engineered T cells process autologous peripheral blood T cells that are targeted to tumor antigens following retroviral transduction of a TCR or a chimeric antigen receptor (CAR). In recent years, a few clinical trials have resulted in encouraging and sometimes dramatic clinical responses [Couzin-Frankel, J. (2013) Breakthrough of the year 2013. Cancer immunotherapy. Science. 342:1432-1433].
  • TCRs are typically cloned from patients’ tumor-reactive T cells from humanized murine models, or through the use of phage display technology.
  • tumor recognition is typically mediated by a single chain variable fragment (scFv) derived from a monoclonal antibody or an antigen-binding region isolated from an immunoglobulin (Ig) heavy and light chain library.
  • scFv single chain variable fragment
  • Ig immunoglobulin
  • CARs function independently of HLA and can therefore be used in any genetic background.
  • the genetic modification of autologous peripheral blood T lymphocytes to generate tumor-targeted T cells is now a well- established approach that was developed in a handful of academic centers.
  • T cells engineered to express specific CARs or TCRs may be initiated from Ficoll-purified PBMCs, which are next activated with anti-CD3 monoclonal antibody (mAb) in the presence of irradiated allogeneic feeder cells and transduced with a vector encoding either the CAR or TCR a and b chains
  • mAb monoclonal antibody
  • the functional, proliferative, and persistence potential of adoptively transferred T lymphocytes is determined by multiple factors. These include the TCR or CAR design, the manufacturing platform, the selected T cell subsets, and the differentiation stage of the harvested T cells.
  • Peripheral blood T cells comprise naive (TN), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and terminal effector (TE) cells.
  • TN naive
  • TCM stem cell memory
  • TCM central memory
  • TEM effector memory
  • TE terminal effector
  • T cells can be easily harvested from donors, their use is compromised by the high alloreactive potential.
  • TCRs are naturally prone to react against non-autologous tissues, recognizing either allogeneic HLA molecules or other polymorphic gene products, referred to as minor antigens [Afzali, B. et al. (2007) Allorecognition and the alloresponse: clinical implications. Tissue Antigens 69:545-556]. This propensity underlies the high risk of graft rejection in transplant recipients and of GVHD in recipients of donor-derived T cells.
  • the endogenous TCR cannot be tamed, one may abrogate its expression, making the engineered TCR or CAR the sole driver of T cell activation and clonal expansion.
  • TCR T-cell receptor
  • CAR chimeric antigen receptor
  • a cassette encoding the chimeric antigen receptor (CAR CD19) was introduced randomly into the genome using a retroviral vector, and second, the primary cells were electroporated with capped mRNA encoding TALE-nuclease heterodimers, which were transiently expressed to induce double strand break disruption in the TCRalpha alleles (WO2013176915).
  • T cells can cause GVHD, their precursors do not, as they undergo positive and negative selection in the recipient’s thymus. Taking advantage of this requires the ability to expand T cell precursors in culture, which is now possible due to advances in understanding T cell development [D.K. Shah, J.C. Ziiniga-Pflucker (2014) An overview of the intrathymic intricacies of T cell development. J. Immunol. 192:4017-4023] T cell precursors lack the ability to initiate GVH reactions because they complete their differentiation in the recipient’s thymus wherein they become restricted to host MHC and yield T lymphocytes that are host tolerant.
  • T cell-derived iPSCs T cell-derived iPSCs (TiPSCs) expressing a CAR (CAR-TiPSC) provide an effective means to concomitantly exploit the unlimited proliferative potential of iPSCs and direct the antigen specificity of iPSC-derived T cells.
  • CAR-TiPSC T cells generated in culture expanded robustly upon CD19 engagement by the CAR (up to 1 ,000-fold over 3 weeks) and showed anti-tumor efficacy against a CD19+ lymphoma in a xenogeneic murine model, comparable to their natural counterparts harvested from peripheral blood (from the same donor) and transduced with the same CAR [Themeli et al.
  • the present invention provides with methods wherein, instead of engineering iPS cells as suggested above, primary immune cells from donors or patient are first gene-edited prior to being reprogrammed, and then turned into precursor cells, especially CD34+ cells, to either form cells available for engraftment into a patient host or to establish cell lines available for a continuous production of useful engineered hematopoietic cells.
  • Performing genetic modifications in multipotent or pluripotent cells requires cumbersome and complicated handling. Maintaining, culturing, passaging multiple undifferentiated multipotent or pluripotent clones requires significant resources and is labor intensive.
  • Performing genetic modifications in multipotent or pluripotent cells requires handling that may affect their potency. Transduction, exposure to penetration facilitating agents, electroporation, single cell isolation or other methods used to introduce genetic changes in cells may impair their potency (e.g. single cell culture of pluripotent stem cells has an adverse effect on preserving their undifferentiated state).
  • Desired genetic modifications may not necessarily lead to identifiable or selectable phenotypic changes in pluripotent or multipotent cells. It is therefore more complicated to identify or isolate cells that carry the desired modifications (e.g. inactivation of a gene expressed only in a more differentiated lineage).
  • the genetic background of the cells used to make the bank may not be tested for its suitability for or performance in cells that have a chosen more differentiated type and may also carry desired genetic modifications. Any contribution from the genetic background to the final performance of the desired differentiated cells that may also carry desired genetic modifications can only be tested once the entire engineering, banking, differentiation process is done, which may be resource consuming to do on multiple candidate genetic background when trying to identify a better one.
  • the method of the present invention broadly relies on:
  • the method can be implemented by: performing genetic modifications (either or both addition of genetic material and/or genetic deletions or gene inactivations through gene editing and/or transgenesis and/or transduction) in primary nucleated blood cells, such as PBMCs or T-cells ; such cell type allows for efficient genetic modifications that can be combined or done sequentially while culturing said cells over multiple days or weeks; selecting, isolating or enriching for the cells that have undergone the desired modification or combination of modifications.
  • the method can be implemented by: testing the behavior of primary nucleated blood cells, such as PBMCs or T-cells, coming from different individuals, and selecting those that have a more desirable phenotype, selecting, isolating or enriching for the cells that have the desired genetic background (e.g., T-cells that have a receptor with a desirable specificity/affinity or structure that lead to a desirable T-cell phenotype when confronted with an antigen in the organism, for example:
  • - T-cells carrying a receptor that allows them to have a long term memory phenotype in organisms that have cleared an acute antigen); reprogramming such cells (which genetic makeup may, in addition, have been functionally validated in said cell type) into pluripotent stem cells.
  • the invention may further comprise a step of conditioning, by which the cells previously obtained are differentiated into hematopoietic cells, especially CD34 + hematopoietic stem cells.
  • This“conditioning” step may include a characterization step to ensure that they are suitable for administration into a patient for permanent or transient engraftment.
  • Figure 1 Schematic representation of the hematopoiesis lineage.
  • Figure 2 Schematic representation of examples of genetic engineering that can be performed to confer additional therapeutic properties to the immune cells prior to their reprogramming as per the present invention.
  • the panels correspond to the main functions, which can be boosted or reduced by genetic alteration.
  • the invention encompasses any alteration of the expression of one or several genes from the different panels, alone or in combination, to be stably maintained in multipotent cells in view of their redifferentiation into immune therapeutic cells. Legends can be read as follows: A - Alloreactivity/ Engraftment: weakening self and non-self-recognition mechanisms in order to produce engineered allogeneic cells that are less alloreactive.
  • B - Inhibiting suppressive cytokines/metabolites reducing the production by the immune cells of cytokines and metabolites, the secretion of which have a negative influence on immune cells activation.
  • C - T-cell proliferation inhibiting genetic pathways that down regulate T-cell proliferation.
  • D - T-cell activation inhibiting genetic pathways that down regulate T-cell activation.
  • E - T-cell exhaustion inhibiting genetic pathways that trigger T-cell exhaustion.
  • F - Resistance to tumor-induced glucose deprivation inhibiting genetic pathways that down regulate T-cells proliferation and activation in response to glucose deprived microenvironment.
  • G - Immune checkpoint receptors reducing control of immune checkpoints on the activation of the engineered immune cells by inhibiting their receptors and pathways controlled by these receptors.
  • CAR chimeric antigen receptor
  • Figure 3 Schematic representation of a method to engineer hematopoietic cells lines according to the invention, comprising the following steps described in Example 1 :
  • NK or T-cells 5- Differentiating the cells from said cell line(s) to produce therapeutic effector cells, such as NK or T-cells. This step can take place in vivo if the cell lines are engrafted into patients as hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • Figure 4 Schematic representation of the donor sequences used in the experimental section to insert IL-15 exogenous coding sequence at the CD25 and PD1 loci and also the anti-CD22 CAR exogenous coding sequence at the TRAC locus.
  • A donor template (designated IL-15m-CD25) designed for site directed insertion of IL-15 at the CD25 locus for obtaining co-transcription of CD25 and IL-15 polypeptides by the immune cell. Sequences are detailed in the examples.
  • B donor template (designated IL-15m-PD1 ) designed for site directed insertion of IL-15 at the PD1 locus for obtaining transcription of IL-15 under the transcriptional activity of the promoter of PD1 endogenous gene.
  • the PD1 right and Left border sequences can be selected so as to keep the PD1 endogenous coding sequence intact or disrupted. In this later case, PD1 is knocked-out while IL-15 is Knocked- in and transcribed.
  • C donor template designed for site directed insertion of a chimeric antigen receptor (ex: anti-CD22 CAR) into the TCR locus (ex: TRAC).
  • the left and right borders are chosen so as to disrupt the TCR in order to obtain [TCR] neg [CAR] pos engineered immune cells suitable for allogeneic transplant into patients.
  • Figure 5 Flow cytometry measures of the frequency of targeted integration of IL-15m at either the PD1 or CD25 locus by using respectively PD1 or CD25 TALEN ® , in a context where an anti-CD22 CAR is also integrated at the TRAC locus using TRAC TALEN ® .
  • A mock transfected primary T-cells.
  • B primary T-cells transfected with the donor sequences described in figure 4 (B and C) and specific TALEN ® for the double integration at the TCR and PDI loci.
  • C primary T-cells transfected with the donor sequences described in figure 4 (A and C) and specific TALEN ® for the double integration at the TCR and CD25 loci.
  • FIG. 6 Schematic representation of the exogenous sequences used in the experimental section to transfect the primary immune cells to obtain the results shown in figures 7 and 8.
  • FIG. 7 and 8 Flow cytometry measures for LNGFR expression among viable T-cells transfected with donor templates of figure 6 and specific TALEN ® (TCR and CD25), upon antiCD3/CD28 non-specific activation (Dynabeads ® ) and upon CAR dependent tumor cell activation (raji tumor cells).
  • LNGFR expression was specifically induced in [CAR anti-CD22] positive cells upon CAR/tumor engagement.
  • Figure 9 and 10 Flow cytometry measures for CD25 expression among viable T-cells transfected with donor templates of figure 6 and specific TALEN ® (TCR and CD25) upon antiCD3/CD28 non-specific activation (Dynabeads ® ) and Tumor cell activation (raji tumor cells).
  • CD25 expression was specifically induced in [CAR anti-CD22] pos i t i ve ce
  • FIG 11 Schematic representation of the exogenous sequences used in the experimental section to transfect the primary immune cells to obtain the results shown in figures 13 and 14.
  • Figure 12 and 13 Flow cytometry measures for LNGFR expression among viable T-cells transfected with donor templates of figure 1 1 and specific TALEN ® (TCR and PD1 ) upon antiCD3/CD28 non-specific activation (Dynabeads ® ) and Tumor cell activation (raji tumor cells). As shown in figure 13, LNGFR expression was specifically induced in [CAR anti-CD22] positive cells upon CAR/tumor engagement.
  • Figure 14 Flow cytometry measures for endogenous PD1 expression among viable T-cells transfected with donor templates of figure 1 1 upon antiCD3/CD28 non-specific activation (Dynabeads ® ) and Tumor cell activation (raji tumor cells) with and without using TALEN ® (TCR and PD1 ).
  • PD1 was efficiently Knocked-out by TALEN treatment (8% remaining expression of PD1 out of 54 %).
  • Figure 15 Diagram showing IL-15 production in [CAR] positive (CARm) and [CAR] negative engineered immune cells according to the invention transfected with the donor template described in Figure 4 (B) and TALEN ® for insertion of IL-15 exogenous coding sequences into the PD1 locus.
  • IL15 which transcription was under control of endogenous PD1 promoter, was efficiently induced upon antiCD3/CD28 non-specific activation (Dynabeads ® ) and Tumor cell activation (raji tumor cells) and secreted in the culture media.
  • Figure 16 Graph showing the amount of IL-15 secreted over time (days) post activation by the immune cells engineered according to the invention.
  • A Cells engineered by integration of the IL-15 coding sequence at the CD25 locus using the DNA donor templates described in Figures 4A (IL-15m_CD25) and/or 2C (CARm).
  • B Cells engineered by integration of the IL-15 coding sequence at the PD1 locus using the DNA donor templates described in Figures 4B (IL-15m_PD1 ) and/or 2C (CARm). Integrations at both loci show similar IL-15 secretion profiles. Secretion of IL-15 is significant increased by tumor specific activation of CAR.
  • Figure 17 Graph reporting number of Raji-Luc tumor cells expressing CD22 antigen (luciferase signal) over time in a survival assay (serial killing assay) as described in the examples.
  • the immune cells have been engineered to integrate IL-15 coding sequences at the PD1 (A) or CD25 locus (B) and to express anti-CD22-CAR at the TCR locus (thereby disrupting TCR expression).
  • PBMCs peripheral blood cells
  • IL-15 coding sequences at the PD1 (A) or CD25 locus (B)
  • anti-CD22-CAR at the TCR locus (thereby disrupting TCR expression).
  • tumor cells are regularly added to the culture medium, while being partially or totally eliminated by the CAR positive cells.
  • the re-expression of IL-15 at either PD1 or CD25 cells dramatically helps the elimination of the tumor cells by the CAR positive cells.
  • the present invention refers to a method for producing engineered progenitor hematopoietic stem cells (HSC) or generating an engineered cell line to produce effector cells for immune therapy, wherein said method comprises the steps of:
  • step b) Genetically engineering said cell to modify its therapeutic properties; and c) Processing the immune cell engineered in step b) into at least one multipotent cell line.
  • Effector cells are the relatively short-lived activated cells that defend the body in an immune response.
  • Activated T cells which include cytotoxic T cells and helper T cells are preferred effector cells to carry out cell-mediated responses.
  • the category of effector T cell is a broad one that includes various T cell types that actively respond to a stimulus, such as co-stimulation. This includes helper, killer, regulatory, and potentially other T cell types.
  • immune cell is meant a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response, such as typically CD3 or CD4 positive cells.
  • the immune cell according to the present invention may be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes.
  • primary cell or“primary cells” are intended cells taken directly from living tissue (e.g. biopsy material) and established for growth in vitro for a limited amount of time, meaning that they can undergo a limited number of population doublings. Primary cells are opposed to continuous tumorigenic or artificially immortalized cell lines.
  • Non- limiting examples of such cell lines are CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT- 1 16 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
  • Primary immune cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and from tumors, such as tumor infiltrating lymphocytes.
  • PBMC peripheral blood mononuclear cells
  • said immune cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection.
  • said cell is part of a mixed population of immune cells which present different phenotypic
  • CD4, CD8 and CD56 positive cells are provided from donors or patients through a variety of methods known in the art, as for instance by leukapheresis techniques as reviewed by Schwartz J.et a/. (Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013) J Clin Apher. 28(3): 145-284).
  • a cell line is obtained from a cell that has acquired capacity of self-renewal and which can produce cells over more than 10 4 generations, more generally over more than 10 5 generations, mostly over than 10 6 generations, if not being an immortal cell line.
  • Multipotent cells describe at least progenitor cells which have the potential to produce cells that can differentiate into discrete cell types.
  • Multipotent cells may be pluripotent cells according to the invention, in particular induced pluripotent stem cells (iPS).
  • iPS induced pluripotent stem cells
  • Induced multipotent stem cells are typically derived from a non-multipotent cell, such as a primary immune cell, by "forcing” the expression of so-called“reprogramming factors” into such cells.
  • Oct-3/4 and certain products of the Sox gene family have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible.
  • Klf1 , Klf2, Klf4, and Klf5 have been identified to increase the induction efficiency.
  • The“Yamanaka factors”, OCT4, SOX2, KLF4, and C-MYC have been found to be pivotal, in particular when expressed using retrovirus vectors [Yamanaka, K. et al. (2008). "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors”. Science. 322 (5903): 949-53].
  • Lentiviral expression of reprogramming factors has been used to induce multipotent cells from human peripheral blood cells [Staerk, J. et al. (2010). "Reprogramming of human peripheral blood cells to induced pluripotent stem cells”. Cell stem cell. 7 (1 ): 20-4] [Loh, YH. et al. (2010). "Reprogramming of T cells from human peripheral blood”. Cell stem cell. 7 (1 ): 15-9]. Although non-retroviral approaches have demonstrated lower efficiency levels so far, adenovirus can also be useful to transiently express reprogramming factors to avoid adverse insertional tumorigenic mutagenesis.
  • One preferred approach according to the present invention is to transfect primary cells with mRNA that encode the reprogramming factors to obtain a transient expression thereof.
  • This transfection is preferably performed by electroporation.
  • electroporation of MicroRNAs short RNA molecules that bind to complementary sequences on messenger RNA
  • miR-291 , miR-294 and miR-295 can be performed to block the expression of genes committed in maintaining cell differentiation [Bao X. et al. (2013) “MicroRNAs in somatic cell reprogramming”. Current Opinion in Cell Biology. 25 (2): 208- 214]
  • HDAC histone deacetylase
  • valproic acid which act the same signaling pathway as transcription factors c-Myc and Sox2
  • HMT histone methyl transferase
  • BIX-01294 Desponts et al. (2008) "Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 3 (5): 568-74]
  • ALK5 inhibitor SB431412, MEK (mitogen-activated protein kinase) inhibitor PD0325901 and Thiazovivin can also be used to increase the efficiency of reprogramming factors.
  • a cocktail of seven small-molecule compounds including DZNep is also available to induce multipotency [Bo, F. et al. (2009) Molecules that Promote or Enhance Reprogramming of Somatic Cells to Induced Pluripotent Stem Cells. Cell Stem Cell. 4 (4):301 -312].
  • Generation of iPS cells is also possible without any genetic alteration of the adult cell by repeated treatment of cells with certain proteins channeled into the cells via poly-arginine anchors as described by [Zhou H. et al. (2009) "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins". Cell Stem Cell.
  • iPSCs protein- induced pluripotent stem cells. All the above methods are envisioned as part of the method of the present invention to induce the engineered primary immune cells to lower differentiation stages to create hematopoietic cell lines, especially hematopoietic stem cells (HSC), useful for sourcing cells for immune therapy.
  • HSC hematopoietic stem cells
  • Hematopoietic lineage is meant the differentiation stages by which blood-cell development progresses from a hematopoietic stem cell (HSC), which can undergo either self-renewal or differentiation into a multilineage committed progenitor cell: a common lymphoid progenitor (CLP) or a common myeloid progenitor (CMP).
  • HSC are typically CD34 positive cells, and more typically CD150+ and CD48- when using SLAM markers as described by Oguro, H., et al. (2013)“SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors” Cell stem cell, 13(1 ), 102-16.
  • a CLP gives rise to the lymphoid lineage of white blood cells or leukocytes-the natural killer (NK) cells and the T and B lymphocytes.
  • a CMP gives rise to the myeloid lineage, which comprises the rest of the leukocytes, the erythrocytes (red blood cells), and the megakaryocytes that produce platelets important in blood clotting.
  • Cells undergoing these differentiation process express a stage- and lineage-specific set of surface markers. Therefore cellular stages are identified by the specific expression patterns of these genes.
  • a simplified representation of the hematopoietic lineage is presented in Figure 1.
  • the cells of the present invention comprise genetic modifications for the purpose of producing effector immune cells with an improved suitability for cell therapy, in either autologous or allogeneic cell therapy schemes.
  • therapeutic properties encompasses the different ways such cells can be improved in the perspective of their use in therapeutic treatments.
  • the cells are genetically engineered to confer them a therapeutic advantage benefit (i.e. therapeutic potency) or to facilitate their use or their production.
  • the genetic engineering can concur to the effector cells having better survival, faster growth, shorter cell cycles, improved immune activity, be more functional, more differentiated, more specific with respect to their target cells, more sensitive or resistant to drugs, less sensitive to glucose deprivation, oxygen or amino acid depletion (i.e. resilient to tumor microenvironment).
  • Progenitor cells may be more productive, better tolerated by the recipient patient, more likely to produce cells that will differentiate in the desired effector cells.
  • Gene editing is meant any methods aiming to introduce or withdraw genetic material from a cell.
  • Gene editing is meant a genetic engineering allowing genetic material to be added, removed, or altered at particular locations (loci) in the genome, including punctual mutations. Gene editing generally involves sequence specific reagents.
  • “seguence-specific reagent” is meant any active molecule that has the ability to specifically recognize a selected polynucleotide sequence at a genomic locus, referred to as“target sequence”, which is generally of at least 9 bp, more preferably of at least 10 bp and even more preferably of at least 12 pb in length, in view of modifying the expression of said genomic locus.
  • Said expression can be modified by mutation, deletion or insertion into coding or regulatory polynucleotide sequences, by epigenetic change, such as by methylation or histone modification, or by interfering at the transcriptional level by interacting with transcription factors or polymerases.
  • sequence-specific reagents are endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, endonucleases, end-processing enzymes such as exonucleases, and more particularly cytidine deaminases such as those coupled with the CRISPR/cas9 system to perform base editing (i.e. nucleotide substitution) without necessarily resorting to cleavage by nucleases as described for instance by Hess, G.T. et al. [Methods and applications of CRISPR-mediated base editing in eukaryotic genomes (2017) Mol Cell. 68(1 ): 26-43.
  • said sequence-specific reagent is preferably a sequence-specific nuclease reagent, such as a RNA guide coupled with a guided endonuclease.
  • the present invention aims to improve the therapeutic potential of immune cells through gene editing techniques, especially by gene targeted integration.
  • gene targeting integration is meant any known site-specific methods allowing to insert, replace or correct a genomic coding sequence into a living cell.
  • said gene targeted integration involves homologous gene recombination at the locus of the targeted gene to result the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of several nucleotides (i.e. polynucleotide), and more preferably a coding sequence.
  • exogenous nucleotide preferably a sequence of several nucleotides (i.e. polynucleotide), and more preferably a coding sequence.
  • “Rare-cutting endonucleases” are sequence-specific endonuclease reagents of choice, insofar as their recognition sequences generally range from 10 to 50 successive base pairs, preferably from 12 to 30 bp, and more preferably from 14 to 20 bp.
  • said endonuclease reagent is a nucleic acid encoding an“engineered” or“programmable” rare-cutting endonuclease, such as a homing endonuclease as described for instance by Arnould S., et a/. [W02004067736], a zinc finger nuclease (ZFN) as described, for instance, by Urnov F., et a/. [Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651 ], a TALE-Nuclease as described, for instance, by Mussolino et a/.
  • an“engineered” or“programmable” rare-cutting endonuclease such as a homing endonuclease as described for instance by Arnould S., et a/.
  • ZFN zinc finger nuclease
  • Urnov F. et a
  • a novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (201 1 ) Nucl. Acids Res. 39(21 ):9283-9293], 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 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
  • the endonuclease reagent is transiently expressed into the cells, meaning that said reagent is not supposed to integrate into the genome or persist over a long period of time, such as be the case of RNA, more particularly mRNA, proteins or complexes mixing proteins and nucleic acids (eg: Ribonucleoproteins).
  • the endonuclease reagent is degraded by 30 hours, preferably by 24, more preferably by 20 hours after transfection.
  • An endonuclease under mRNAform 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 steps that are used to transfect primary immune cells, such as PBMCs are typically 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 W02004083379, 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.
  • the treatment volume of the suspension is scalable, and the time of treatment of the cells in the chamber is substantially uniform.
  • 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 a/. [TALEN ® facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773].
  • sequence specific reagent is preferably under the form of nucleic acids, such as under DNA or RNA form encoding a rare cutting endonuclease a subunit thereof, but they can also be part of conjugates involving polynucleotide(s) and polypeptide(s) such as so-called“ribonucleoproteins”.
  • conjugates can be formed with reagents as Cas9 or Cpf1 (RNA-guided endonucleases) or Argonaute (DNA-guided endonucleases) as recently respectively described by Zetsche, B. et al.
  • Exogenous sequence refers to any nucleotide or nucleic acid sequence that was not initially present at the selected locus. This sequence may be homologous to, or a copy of, a genomic sequence, or be a foreign sequence introduced into the cell. 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.
  • a 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
  • drug is meant any substance (other than food that provides nutritional support) that, when inhaled, injected, smoked, consumed, absorbed or dissolved under the tongue causes a temporary physiological change in the body.
  • the effector cells resulting from the methods herein described are made resistant to certain immunosuppressive drugs, meaning that they can better resist to molecules used in standard cancer care treatments for lymphodepleting patients. This resistance to the drug is assessed by comparing the LD 5 o of the gene edited cells with respect to this drug in comparison with non gene edited cells in experimental conditions. The LD 5 O has to be statistically higher in such experimental conditions using a t-test to conclude that the cells are“resistant”.
  • CAR Chimeric Antigen Receptor
  • scFv single-chain antibody
  • Binding moieties based on receptor or ligand domains have also been used successfully.
  • the signaling domains of CARs are generally derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains, which are generally combined with signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS and 4-1 BB (CD137) to enhance survival and increase proliferation of the cells.
  • CARs are generally expressed in effector immune cells to redirect their immune activity against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors.
  • a component of a CAR is any functional subunit of a CAR that is encoded by an exogenous polynucleotide sequence introduced into the cell. For instance, this component can help to interact with the target antigen, the stability or the localization of the CAR into the cell.
  • the cell generated by the present invention can express a variety of CARs or recombinant (modified) TCRs, such as those commonly described in the literature, under single-chain or multiple subunits (multi-chain) as described in WO2014039523.
  • Preferred recombinant TCR to be used in the present invention are those directed against antigen specific of cancer cells, such as MART-1 , MAGE-1 , MAGE-2, MAGE-3 MAGE-12, BAGE, GAGE, NY-ESO-1 , or overexpressed in cancer cells, such as a- Fetoprotein, Telomerase catalytic protein, G-250, MUC-1 , CarcinoEmbryonic antigen (CEA), p53, Her-2/Neu and WT1 [Rosenberg S.A., (2001 ) Progress in human tumour immunology and immunotherapy Nature. 41 1 (6835):380-4]
  • CARs can bind tumor antigen as diverse as one selected from: CD19 molecule (CD19); membrane spanning 4-domains A1 (MS4A1 also known as CD20); CD22 molecule (CD22); CD24 molecule (CD24); CD248 molecule (CD248); CD276 molecule (CD276 or B7H3); CD33 molecule (CD33); CD38 molecule (CD38); CD44v6; CD70 molecule (CD70); CD72; CD79a; CD79b; interleukin 3 receptor subunit alpha (IL3RA also known as CD123); TNF receptor superfamily member 8 (TNFRSF8 also known as CD30); KIT proto-oncogene receptor tyrosine kinase (CD1 17); V-set pre-B cell surrogate light chain 1 (VPREB1 or CD179a); adhesion G protein-coupled receptor E5 (ADGRE5 or CD97); TNF receptor superfamily member 17 (TNFRSF17 also known as BCMA); SLAM family member 7 (
  • the invention preferably involves CARs directed against cancer marker antigens, such as CD19, CD22, CD33, 5T4, ROR1 , CD38, CD52, CD123, CS1 , BCMA, Flt3, CD70, EGFRvlll, WT1 , HSP-70 and CCL1.
  • cancer marker antigens such as CD19, CD22, CD33, 5T4, ROR1 , CD38, CD52, CD123, CS1 , BCMA, Flt3, CD70, EGFRvlll, WT1 , HSP-70 and CCL1.
  • Such CARs having preferably one structure as described in W02016120216.
  • the immune cells are endowed with a CAR directed against the CD19 antigen in view of treating leukemia, more especially acute lymphoblastic leukemia (ALL), such as the CAR described in example 1 of WO2014184143, or one integrated at the TCR locus such as described in example 5 of WO2017062451 , which are both incorporated herein by reference.
  • ALL acute lymphoblastic leukemia
  • the CAR polynucleotide sequences are not introduced or expressed into the cell lines until the step of cell differentiation (i.e. not during the reprogramming step).
  • the multipotent cells once obtained, can be transduced with retroviral vectors comprising the CAR polynucleotide to initiate differentiation.
  • Expression of said CAR in said cells generally helps and can even trigger cell differentiation into effector cells, in particular into T-cells or NK-cells.
  • signalization sequences from specific receptors or effectors involved into cell differentiation, especially into differentiation from HSC -> CLP (common Lymphoid Progenitor cells) can be introduced into the cell or included into the CAR structure to stimulate said differentiation.
  • the CAR polynucleotide sequences can be introduced into the genome of the primary cell during the initial genetic engineering step, for instance by site directed integration (e.g. integration of an exogenous sequence encoding the CAR by homologous recombination) and its expression being repressed or being inactive when the cells get reprogrammed.
  • site directed integration e.g. integration of an exogenous sequence encoding the CAR by homologous recombination
  • the exogenous sequence encoding the CAR is introduced into an endogenous locus, such as one indicated in figure 2 under A, B, C, D, E, F and G, more preferably at a locus selected from those encoding TCRalpha, TCRbeta, PD1 , CTLA4, DCK and B2M.
  • This introduction is generally performed with the effect of inactivating at least one endogenous coding sequence present at such locus.
  • the genetic engineering of the present invention can have the effect of reducing or preventing the expression of at least one protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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
  • A“recombinant or engineered TCR” is protein expressed into an immune cell to redirect its immune activity against a desired type of cells, especially cancer and infected cells having specific markers at their surface. It can replace or be-co-expressed with the endogenous TCR.
  • recombinant TCR are single-chain TCRs comprising an open reading frame where the variable (V) a and nb domains are paired with a protein linker. This involves the molecular cloning of the TCR genes known to be specific for an antigen of choice. These chains are then introduced into T cells usually by means of a retroviral vector.
  • a component of a recombinant or engineered TCR is any functional subunit of a TCR, such as a recombined TCRa or TCR3, which is encoded by an exogenous polynucleotide sequence introduced into the cell.
  • pre-T By“pre-T”, is meant“pre-T-cell receptor”, which comprises a pre-Talpha (pTalpha) chain, which has the ability to transduce signals during the early stages of T-cell development as described by Harald von Boehmer [Unique features of the pre-T-cell receptor a-chain: not just a surrogate (2005) Nature Reviews Immunology. 5, 571-577]
  • the present invention is drawn to the multipotent cells obtainable by the present invention whether or not they have resulted into an established cell line.
  • the invention is drawn to multipotent or iPS cells, and more especially genetically engineered iPS cells, which have been gene edited into at least one gene encoding a protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (Uniprot P18627), HAVCR2 (Uniprot Q8TDQ0), BTLA (Uniprot Q7Z6A9), CD160 (Uniprot 095971 ), TIGIT (Uniprot Q495A1 ), CD96 (Uniprot P40200), CRTAM (Uniprot 095727), LAIR1
  • Multipotent or iPS cells according to the invention can be gene edited to be differentiated into more potent immune therapeutic cells by inactivation or repression of at least one gene encoding a protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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), SIGLEC
  • Multipotent or iPS cells according to the invention can be gene edited to be differentiated into immune therapeutic cells resistant to drugs by inactivation or repression of at least one gene encoding a protein selected from CD52 (Uniprot P31358), GR (Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150), DCK (Uniprot P27707), HPRT (Uniprot P00492), GGH (Uniprot Q92820).
  • CD52 Uniprot P31358
  • GR Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150
  • DCK Uniprot P27707
  • HPRT Uniprot P00492
  • GGH Uniprot Q92820
  • Multipotent or iPS cells according to the invention can be gene edited to be differentiated into less alloreactive immune therapeutic cells, by inactivation or repression of at least one gene encoding a protein selected from TCRalpha and/or TCRbeta, 32m and HLA.
  • multipotent or iPS cells according to the invention can be gene edited in at least two genes, such as for instance:
  • the present invention comes along with a general method for generating an engineered cell line to produce effector cells for immune therapy, said method comprising the step of differentiating cells from an induced multipotent cell line, which originates from a cell that has been genetically engineered, preferably as a primary cell, prior to being induced to a multipotent stage.
  • said cell has been engineered when it was a primary immune cell to modify its immune properties before being reprogrammed as a pluripotent cell and possibly further, as an effector immune cell, such as a CAR positive T- cell or NK cell.
  • an effector immune cell such as a CAR positive T- cell or NK cell.
  • Such multipotent or iPS cells are preferably engineered before having been reprogrammed.
  • Such genetic engineering generally aim at:
  • suppressive cytokines and metabolites such as an inactivation of TGFb, TGFbR, IL-10, IL-10R, GCN2 or PRDM1.
  • T-cells proliferation and activation in Tumor induced inhibitory environment in particular glucose deprived microenvironment, such as miR101 , miR26a, SERCA or BCAT.
  • the engineered cells which have been processed into iPS cells by expression of at least one reprogramming factor selected from OCT4, SOX2, KLF4, C-MYC, NANOG, LIN28, being preferably transiently expressed are generally subjected to an additional step where they are differentiated into hematopoietic stem cells, in particular CD34 + cells.
  • Such cells are useful for administration into patients, especially in view of achieving bone marrow transplantation.
  • Said method may thus comprise an additional step comprising assaying, sorting or screening the engineered cells, which are CD34 +.
  • the engineered hematopoietic CD34 + stem cells of the present invention get engrafted into their patient host, they can produce hematopoietic cells having the same genetic modifications conferring improved therapeutic properties such as previously proposed, in particular displaying:
  • Therapeutic properties may also be obtained or combined with the previous ones by integration of exogenous nucleic acids (eg: transgene) coding for various polypeptides such as:
  • CAR Chimeric Antigen Receptor
  • cytokine such as an interleukin or/and their receptors
  • T-cells activity such as IL-2, IL-12, IL- 15 or IL-18, which expression are particularly beneficial to improve their cytotoxicity against infected or malignant cells.
  • other interleukins are known to stimulate Treg (Regulatory T-Cells), such as IL-10 and TGFbeta, which secretion in the culture medium in-vitro or in the Treg environment in-vivo is particularly useful to mitigate certain self-immune reactions or syndromes cause by T-cell hyperactivation.
  • Treg Regulatory T-Cells
  • HLA component for instance to improve cells engraftment
  • DHFR dihydrofolate reductase
  • IMPDH2 inosine monophosphate dehydrogenase 2
  • MGMT calcineurin or methylguanine transferase
  • TAM Tumor Associated Macrophages
  • the cells are engineered to introduce an exogenous nucleic acid sequence encoding a CAR or a sequence coding for an interleukin gene at a precise locus, such as the TCR locus.
  • Said CAR or a sequence coding for an interleukin gene may also be favorably introduced at the PD1 , PDL1 , CTLA4, TIM3 or LAG3 locus, where such sequences may or may not inactivate the expressions of the genes originally present at said loci.
  • CAR architectures may be designed to be controllable by molecules that can be administered to patients.
  • CARs or recombinant TCRs may comprise dimerization or cleavable domains to make them inducible or inactivated by a soluble compound, such as streptavidin, or rapamycin CID.
  • Exogenous coding sequence can be introduced by using viral vectors, such as a lentiviral vector or by using rare-cutting endonucleases combined with an AAV vector comprising the nucleic acid sequence to be integrated.
  • viral vectors such as a lentiviral vector
  • rare-cutting endonucleases combined with an AAV vector comprising the nucleic acid sequence to be integrated.
  • a method of the present invention as previously described may further comprise one step of transient expression of reprogramming factors such as TERT.
  • reprogramming factors such as TERT.
  • Such reprogramming factors can be introduced into the cells under protein or RNA forms, for instance by electroporation and/or using nanoparticles.
  • Viral vectors, especially non-integrative viral vectors, such as for instance episomal vectors, may also be used to reprogram the cells.
  • the engineered hematopoietic cells or cell lines originate from primary cells preferably obtained from a healthy donor.
  • Said primary cell are generally mononuclear cell, such as peripheral blood cells (PBMC) isolated by leukapheresis, or may also originate from cord blood cells.
  • PBMC peripheral blood cells
  • Such primary cells are preferably T-cells or NK cells.
  • the primary cells may originate from patients, in particular as Tumor Infiltrating Lymphocytes (TIL).
  • TIL Tumor Infiltrating Lymphocytes
  • the primary cells may also originate from lymphoid progenitor cells, such as hematopoietic stem cells.
  • step(s) of redifferentiation can take place.
  • the step(s) is (are) preferably performed by contacting the cells with notch ligand positive cells, such as OP9-DL1 cells.
  • Said notch ligand positive cells can be engineered stromal cells that can form three dimension structures such as in artificial thymic organoids such as described by Seet, C. et a/. [Generation of mature T cells from human hematopoietic step and progenitor cells in artificial thymic organoids (2017) Nat Methods. 14(5): 521-530] .
  • Said notch ligand positive cells can be engineered to express specific antigen, such as to drive the differentiation of the cells towards a desired hematopoietic lineage.
  • the cells are differentiated in until obtaining functional progenitor, such as CD34 + cells, or more mature immune cells, such as CD3 +, CD4 + or CD8+.
  • the cells are differentiated until obtaining functional effector T-cells.
  • the cell products resulting from the present invention include the genetically engineered induced multipotent cell obtainable at the different steps of the re-programming process illustrated in Figure 3, especially an induced multipotent cell, wherein said cell has been genetically modified at the TCR locus.
  • Said induced multipotent cell, as well as all the cells deriving thereof by differentiation through the hematopoietic lineages can comprise two, preferably three, more preferably four genetic modifications as previously described, in particular at least one introduced at one locus encoding a protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (Uniprot P18627), HAVCR2 (Uniprot Q8TDQ0), BTLA (Uniprot Q7Z6A9), CD160 (Uniprot 095971 ), TIGIT (Uniprot Q495A1 ), CD96 (Uniprot P40200), CRTAM (Uniprot 095727), LAIR1 (Uniprot Q6GTX
  • the present invention extends to the induced engineered multipotent cell line(s) stably established for producing multipotent cells, which can be further differentiated according to the invention, as well as the intermediate cells produced by such cell line(s).
  • the invention encompasses primary immune cells, which has been genetically engineered and further transfected with, proteins or nucleic acids, such as mRNA, encoding at least one reprogramming factor(s) to induce pluripotency, such as OCT4, SOX2, KLF4, C-MYC, TERT, NANOG and/or LIN28, and thereby the engineered cells comprising same.
  • proteins or nucleic acids such as mRNA
  • encoding at least one reprogramming factor(s) to induce pluripotency such as OCT4, SOX2, KLF4, C-MYC, TERT, NANOG and/or LIN28
  • the present inventions extends to libraries or bank of cells or cell lines useful to produce different types of engineered hematopoietic cells, especially effector immune cells, which may originate from various donors and target different disease or groups of patients.
  • compositions comprising a therapeutically effective amount of effector immune cells produced or differentiated from the induced multipotent cell lines established by the methods of the present invention described in this specification, and more specifically by the methods comprising at least one of the following steps of:
  • RNA editing step into primary T-cells by electroporation of mRNA encoding rare-cutting endonuclease, preferably TALE-nucleases, as illustrated in the experimental section.
  • Such step can be performed to inactivate CCR5 and provide with hematopoietic cells, which are subsequently resistant to HIV.
  • Such step can also be performed to obtain the insertion of a CAR at the TCR locus, for instance [CAR] pos [TCR] neg [dcK] neg effector immune cells resistant to purine nucleotide analogs (PNA), such as fludarabine and chlorofarabine.
  • PNA purine nucleotide analogs
  • Such cells can be reprogrammed into iPS cells and further differentiation into CD34 + cells, HSC or cells lines, for their use in allogeneic cell immunotherapy treatments.
  • TCR-deficient T-cells can be isolated and/or purified in order to be subsequently reprogrammed into iPS cells. Such cells may be assayed by using transient expression of a CAR or any attributes for their suitability in therapy before being actually transformed with reprogramming factors as illustrated herein in the experimental section.
  • the induced multipotent cells are picked, isolated and colonies are checked for the genetic modifications initially performed on the primary cells.
  • cell lines are established for their continuous production of cells which may be stored or differentiated into hematopoietic cells as illustrated in the experimental section.
  • the cells can be co-cultured with notch ligand positive stromal cells, such as in Artificial Thymic Organoid (ATO) cultures, until obtaining CD34 +, CD4+ and/or CD8+ immune cells.
  • ATO Artificial Thymic Organoid
  • the engineered Pluripotent cells obtainable by the present invention which may be TCR negative, can be transduced with lentiviral vectors for re- expression of CAR or a recombinant TCR to obtain differentiation into adoptive T-cells useful for cell immunotherapy.
  • the CAR may have been inserted into the genome of the cells by homologous recombination during the initial gene editing step.
  • TCAR anti-CD22l pos effector immune cells by gene editing of primary PBMC cells, reprogramming and differentiation by co-culture in artificial thymic organoids.
  • TALEN ® designate TALE-nucleases developed by Cellectis (8, rue de la Croix Jarry, 75013 PARIS), which are fusions of bespoke TALE binding domains with the nuclease domain of Fok1 as originally described by Voytas et al. (WO2011072246).
  • Cell culture reagents, X-vivo-15 are obtained from Lonza (Basel, Switzerland cat#BE04-418Q), IL-2 from Miltenyi Biotech (Bergisch Gladbach, Germany, cat#130-097- 748), human serum AB from Seralab (West Wales, UK cat#GEM-100-318), human T activator CD3/CD28 from Life Technologies (Beverly, MA, cat#11132D), MACS-LD column from Miltenyi Biotech (cat#130-042-901 ), fixable viability dye eFluor780 from eBioscience (San Diego, CA, cat#65-0865-14).
  • CFSE dye are obtained from Life Technologies (cat#C34554) and anti-IFNy ELISA kit are obtained from R&D systems (Minneapolis, MN, cat#DIF50).
  • PBMCs Mononuclear cells
  • the PBMCs are activated and transfected according to the procedure described in Galetto, R. et al. [Pre-TCRa supports CD3- dependent reactivation and expansion of TCRodeficient primary human T-cells (2014) Mol Ther Methods Clin Dev.14021f] Briefly regarding transfection, 4 days after their activation by Dynabeads human T activator CD3/CD28, 5.10 6 T cells are simultaneously transfected with 5 pg of each mRNA encoding left and right arms of TALEN ® targeting the TCRa constant chain and dCK exon 2 (Uniprot P27707).
  • Transfection is performed using Agilpulse technology, by applying two 0.1 ms pulses at 3,000 V/cm followed by four 0.2 ms pulses at 325 V/cm in 0.4 cm gap cuvettes and a final volume of 200 pi of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA). Cells were then immediately diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 (final concentration) and 5% human serum AB. Transfected T cells are eventually diluted at 1 x 10 6 /ml and kept in culture at 37 °C in the presence of 5% CO2 and 20 ng/ml IL-2 (final concentration) and 5% human AB serum for further characterization.
  • X-vivo-15 was obtained for Lonza (cat#BE04-418Q), IL-2 from Miltenyi Biotech (cat#130-097-748), human serum AB from Seralab (cat#GEM-100-318), human T activator CD3/CD28 from Life Technology (cat#1 1 132D), QBEND10-APC from R&D Systems (cat#FAB7227A), vioblue-labeled anti-CD3, PE-labeled anti-LNGFR, APC-labeled anti- CD25 and PE-labeled anti-PD1 from Miltenyi (cat# 130-094-363, 130-1 12-790, 130-109-021 and 130-104-892 respectively) 48 wells treated plates (CytoOne, cat#CC7682-7548), human IL-15 Quantikine ELISA kit from R&D systems (cat#S1500), ONE-Glo from Promega (cat#E61 10).
  • AAV6 batches containing the different matrices were obtained from Virovek, PBMC cells were obtained from Allcells, (cat#PB004F) and Raji-Luciferase cells were obtained after Firefly Luciferase-encoding lentiviral particles transduction of Raji cells from ATCC (cat#CCL-86).
  • PBMC cells were first thawed, washed, resuspended and cultivated in X-vivo-15 complete media (X-vivo-15, 5% AB serum, 20 ng/ml_ IL-2). One day later, cells were activated by Dynabeads human T activator CD3/CD28 (25 uL of beads/1 E 6 CD3 positive cells) and cultivated at a density of 1 E 6 cells/mL for 3 days in X-vivo complete media at 37°C in the presence of 5% CO2. Cells were then split in fresh complete media and transduced/transfected the next day according to the following procedure.
  • TALEN ® is a standard format of TALE-nucleases resulting from a fusion of TALE with Fok-1 Transfection was performed using Pulse Agile technology, by applying two 0.1 mS pulses at 3,000 V/cm followed by four 0.2 mS pulses at 325 V/cm in 0.4 cm gap cuvettes and in a final volume of 200 pi of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts). Electroporated cells were then immediately transferred to a 12-well plate containing 1 mL of prewarm X-vivo-15 serum-free media and incubated for 37°C for 15 min.
  • a serial killing assay was performed.
  • the principle of this assay is to challenge CAR T-cell antitumor activity everyday by a daily addition of a constant amount of tumor cells.
  • T umor cell proliferation, control and relapse could be monitored via luminescence read out thanks to a Luciferase marker stably integrated in Tumor cell lines.
  • the mixture is incubated 24 hours before determining the luminescence of 25 uL of cell suspension using ONE-Glo reagent.
  • Cells mixture are then spun down, the old media is discarded and substituted with 1 ml. of fresh complete X-vivo-15 media containing 2.5x10 5 Raji-Luc cells and the resulting cell mixture is incubated for 24 hours. This protocol is repeated 4 days.
  • This example describes methods to improve the therapeutic outcome of CAR T-cell therapies by integrating an IL-15/soluble IL-15 receptor alpha heterodimer (I L15/sl L15ra) expression cassette under the control of the endogenous T-cell promoters regulating PD1 and CD25 genes. Because both genes are known to be upregulated upon tumor engagement by CAR T-cells, they could be hijacked to re-express IL- 1 L15/sl L15ra only in vicinity of a tumor.
  • I L15/sl L15ra IL-15/soluble IL-15 receptor alpha heterodimer
  • This method aims to reduce the potential side effects of I L15/sl L15ra systemic secretion while maintaining its capacity to reduced activation induced T-cell death (AICD), promote T-cell survival, enhance T-cell antitumor activity and to reverse T-cell anergy.
  • AICD activation induced T-cell death
  • the method developed to integrate I L15/sl L15ra at PD1 and CD25 loci consisted in generating a double-strand break at both loci using TALEN in the presence of a DNA repair matrix vectorized by AAV6.
  • This matrix consists of two homology arms embedding IL15/slL15ra coding regions separated by a 2A cis acting elements and regulatory elements (stop codon and polyA sequences).
  • the targeted endogenous gene could be inactivated or not via specific matrix design.
  • the insertion matrix was designed to knock-in (Kl) I L15/sl L15ra without inactivating CD25 because the protein product of this gene is regarded as essential for T-cell function.
  • Kl knock-in
  • the insertion matrix was designed to prevent its expression while enabling the expression and secretion of I L15/sl L15ra.
  • FIG. 4A To illustrate this approach and demonstrate the feasibility of double targeted insertion in primary T-cells, three different matrices were designed (figure 4A, 4B and 4C).
  • the first one named CARm represented by SEQ ID NO:13 was designed to insert an anti- CD22 CAR cDNA at the TRAC locus in the presence of TRAC TALEN ® (SEQ ID NO:3 and 4).
  • the second one, IL-15_CD25m (SEQ ID NO:14) was designed to integrate IL15, slL15ra and the surface marker named ALNGFR cDNAs separated by 2A cis-acting elements just before the stop codon of CD25 endogenous coding sequence using CD25 TALEN ® (SEQ ID NO:5 and 6).
  • the third one, IL-15_PD1 m contained the same expression cassette and was designed to integrate in the middle of the PD1 open reading frame using PD1 TALEN ® (SEQ ID NO:7 and 8).
  • the three matrices contained an additional 2A cis-acting element located upstream expression cassettes to enable co- expression of I L15/sl L15ra and CAR with the endogenous gene targeted.
  • TCRa3-deficient cells are purified according to the protocol described by Valton, J. et a/. [Efficient strategies for TALEN-mediated genome editing in mammalian cell lines (2014) Methods, 69:151 -170]. Briefly, about 10 7 T cells recovered 6 days after TALEN ® treatment are labeled with biotin conjugated anti-TCRa3 antibody MicroBeads before being loaded onto a MACS LD-Column placed in the magnetic field of a MACS Separator. Using this procedure, the magnetically labeled CD3-positive cells are retained in the column while the unlabeled TCRa3-deficient cells can be recovered in the flow through. One round of purification is usually necessary to obtain a homogeneous population of TCRa3-deficient cells (purity > 99%).
  • Clofarabine, fludarabine (fludarabine-phosphate), and cytarabine are obtained from Sigma (St Louis, MO, cat #C7495,# F9813, and #C3350000 respectively), diluted according to the manufacturer protocol. The concentrations of diluted PNA solutions were accurately determined by spectrophotometry. IC50 of a given drug is defined as the concentration of drug need to decrease the cellular viability by 50%.
  • T-cells are incubated in the presence of increasing concentration of drugs (from 0 to 100 pmol/l typicially) for 48 hours and in a total volume of 100 pi X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum.
  • Drugs from 0 to 100 pmol/l typicially
  • a total volume of 100 pi X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum.
  • Cells are washed with 100 mI of phosphate buffer saline (PBS) and then labeled by eFIuor 780 for 15 minutes at 4 °C according to the manufacturer protocol.
  • PBS phosphate buffer saline
  • neg Cells are cultured in the presence of clinically relevant doses of PNA for 10 days in the presence of a combination of clofarabine and cytarabine (1 and 10 pmol/l respectively) reported as being consistent with their respective average Cmax after their uptake in human patients [Valton, J. et a/. A Multidrug-resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy Molecular Therapy 23(9): 1507-1518].
  • CAR anti-CD22 has been constructed by assembling sequences encoding 41 BB costimulatory domain, the CD3z activation domain, the CD8a transmembrane domain, a CD8a hinge and ScFv of the antibody anti-CD22 m971 formerly used by Haso W. et al. [Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia (2013) Blood 14;121 (7):1 165-74] . The resulting polynucleotide sequence has been cloned into an expression vector and transcribed into mRNA.
  • RNA Transfection is performed using Agilpulse technology, by applying two 0.1 ms pulses at 3,000 V/cm followed by four 0.2 ms pulses at 325 V/cm in 0.4 cm gap cuvettes and a final volume of 200 mI of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA). Cells are then immediately diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 (final concentration) and 5% human serum AB. Transfected T cells are eventually diluted at 1 c 10 6 /ml and kept in culture at 37 °C in the presence of 5% CO2 and 20 ng/ml IL-2 (final concentration) and 5% human AB serum.
  • the cytolytic activity and specificity of the engineered CAR T cells are assessed according to the flow cytometry-based cytotoxicity assay described in Zhao, B. et al.
  • This assay consists of labeling 10 4 NALM-16 (CD22 + ) and 10 4 SUP-T1 (CD22 negative control) cells with 1 pmol/l CellTrace CFSE and 1 pmol/l CellTrace violet respectively (Life Technology) and coincubate them with 10 5 effector CAR T cells (E/T ratio of 10:1 ) in a final volume of 100 mI X-Vivo-15 media, for 5 hours at 37 °C.
  • Cells are then recovered and labeled with eFluor780 viability marker before being fixed by 4% PFA as described above. Fixed cells are then analyzed by flow cytometry to determine their viability. The frequency of specific cell lysis is calculated using the following formula:
  • Frequency of specific cell lysis (Via NALM-16/Via SUP-T1 )/(Via NALM-16/Via SUP-T1 ) where Via NALM-16 and Via SUP-T1 correspond respectively to the % of viable NALM-16 and SUP-T1 cells obtained after 5h in the presence of CAR T cells and where Via NALM- 16 and Via SUP-T1 correspond respectively to the % of NALM-16 and SUP-T1 cells obtained after 5h in the absence of CAR T cells.
  • PBMC B 20 x 10 6 PBMC freshly purified from a donor B (PBMC B) are labeled by a 10-minute incubation with 2 nmol/l CFSE in the dark, at 37 °C in a final volume of 5 ml.
  • CFSE- labeled PBMC B were then diluted two times in fetal bovine serum, and washed with X- Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum. Cell number and viability is determined and their concentration adjusted to 1 c 10 6 viable cells/ml.
  • CFSE-labeled PBMC B are incubated with the engineered CAR T cells that have originated from a different blood donor (CAR T cell A) in X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum.
  • CAR T cell A a different blood donor
  • CFSE labeled PBMC viability, activation and proliferation are determined after 6 days of incubation, by flow cytometry and anti IFNy ELISA.
  • AAV6-assisted vectorization of matrices in the presence of mRNA encoding TRAC TALEN (SEQ ID NO:3 and 4) and PD1 TALEN ® (SEQ ID NO:7 and 8) or CD25 TALEN ® (SEQ ID NO:5 and 6) enabled expression of the anti CD22 CAR in up to 46% of engineered T-cells (figure 5).
  • IL15 was secreted in the media only by T-cells that were co-treated by both CARm and IL15m matrices along with their corresponding TALEN ® (figure 15).
  • T- cell treated with either one of these matrices were unable to secrete any significant level of IL15 with respect to resting T-cells.
  • IL-15 secretion level was found transitory, with a maximum peak centered at day 4 (Figure 16).
  • CAR T-cell were co-cultured in the presence of tumor cells at E:T ratio of 5:1 for 4 days. Their antitumor activity was challenged everyday by pelleting and resuspended them in a culture media lacking IL-2 and containing fresh tumor cells. Antitumor activity of CAR T-cell was monitored everyday by measuring the luminescence of the remaining Raji tumor cells expressing luciferase. Our results showed that CAR T-cells co-expressing IL-15 had a higher antitumor activity than those lacking IL15 at all time points considered (figure 17).
  • Table 2 polynucleotide sequences referred to in example 2.
  • RNA have been synthesized with the MEGAscript T7 kit (Ambion, Austin, TX), with 1 .6 pg of purified tail PCR product to template each 40 pl_ reaction.
  • a custom ribonucleoside blend was used comprising 3'-0-Me-m7G(5')ppp(5')G ARCA cap analog (New England Biolabs), adenosine triphosphate and guanosine triphosphate (USB, Cleveland, OH), 5- methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies, San Diego, CA).
  • RNA reprogramming cocktails are prepared by pooling individual 100 ng/pL RNA stocks to produce a 100 ng/pL (total) blend. Volumetric ratios used for pooling are as follows: 170:160:420:130:90 (KLF4:c-MYC:OCT4:SOX2:LIN28). RNA Transfection of reprogramming factors was performed every two days using Agilpulse technology, by applying two 0.1 ms pulses at 3,000 V/cm followed by four 0.2 ms pulses at 325 V/cm in 0.4 cm gap cuvettes and a final volume of 200 pi of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA).
  • Transfected T cells are eventually diluted at 1 c 10 6 /ml and kept in culture at 37 °C in the presence of 5% CO2 and 20 ng/ml IL-2 (final concentration) and 5% human AB serum
  • Gamma-irradiated human neonatal fibroblast feeders (GlobalStem, Rockville, MD) are seeded at 33,000 cells/cm2. Nutristem media was replaced daily, 4 hr after transfection, and supplemented with 100 ng/mL bFGF and 200 ng/mL B18R (eBioscience, San Diego, CA). Where applied, VPA is added to media at 1 mM final concentration on days 8-15 of reprogramming. Low-oxygen experiments were carried out in a NAPCO 8000 WJ incubator (Thermo Scientific). Media are equilibrated at 5% 02 for approximately 4 hr before use. Cultures are passaged with TrypLE Select recombinant protease (Invitrogen). Y27632 ROCK inhibitor (Watanabe et al., 2007) is used at 10 mM in recipient plates until the next media change.
  • iPS colonies are mechanically picked and transferred to MEF-coated 24-well plates with standard hESC medium containing 5 mM Y27632 (BioMol, Plymouth Meeting, PA).
  • the hESC media comprises DMEM/F12 supplemented with 20% Knockout Serum Replacement (Invitrogen), 10 ng/mL of bFGF (Gembio, West Sacramento, CA), 1 x nonessential amino acids (Invitrogen), 0.1 mM b-ME (Sigma), 1 mM L-glutamine (Invitrogen), plus antibiotics.
  • Clones are mechanically passaged once more to MEF-coated 6-well plates, and then expanded via enzymatic passaging with collagenase IV (Invitrogen).
  • Colonies with well-defined hESC-like morphology are first observed from 20 days after first transfection colonies with distinct flat and compact morphology with clear-cut round edges reminiscent of hES cells after a slightly longer latency of around 35 days as described by Warren, L. et al. [Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA (2010) Cell Stem Cell. 7 - 5 (5): 618-630] are isolated.
  • the iPS cell lines are analyzed in Immunohistochemistry for expression of multipotent cells markers. Colonies that are stained positive for Tra-1 -81 , NANOG, OCT4, Tra-1 -60, SSEA4 markers by multicolor flow cytometry analysis are selected and passaged to establish homogenous cell cultures (cell lines) by using Inside Stain Kit (Miltenyi Biotec GmbH ref: #130-090-477). Different cell lines are obtained in this way that are frozen and banked, to be later processed for differentiation, for instance by co-culture with notch ligand positive stromal cells, such as in Artificial Thymic Organoid (ATO) cultures.
  • ATO Artificial Thymic Organoid
  • Lentiviral vectors comprising the polynucleotide sequence encoding the CAR anti-CD22 transiently expressed in above step 2 are used to transduce the multipotent cells previously obtained for obtaining stable expression of the CAR in these cells, and further in the cells that will be differentiated into effector cells for their antigen dependent activation.
  • 10 6 of the above cells are plated in 6-well non-treated plates coated with 20 pg/ml Retronectin (Clontech, Mountain View, CA) in 1 ml X-VIVO-15 (Lonza, Basel, Switzerland) supplemented with 50 ng/ml of recombinant human SCF, FLT3L, and TPO, and 10 ng/ml IL-3 (Peprotech, Rocky Hill, NJ) for 12— 18 h , after which concentrated lentiviral supernatant was to a final concentration of 1-2x10 7 TU/ml. Mock-transduced cells are cultured in identical conditions without addition of lentiviral vector.
  • OP9-DL1 cells are prepared as described by Seet, C. et al. [Generation of mature T cells from human hematopoietic step and progenitor cells in artificial thymic organoids (2017) Nat Methods. 14(5): 521-530].
  • MS5-hDLL1 can be used, which are murine stromal cell line transduced with a lentiviral vector encoding human DLL1 and eGFP.
  • the cells are set up in monolayer cultures by seeding cells into 0.1 % gelatin-coated 12 well plates 1-2 days prior to use to achieve 70-80% confluence. Medium is aspirated from monolayers and 1 .5x10 4 FACS purified to select CD34+CD3- cells.
  • the cells transduced with the CAR in step 5 are plated on stromal monolayers in 2 ml of medium composed of MEMa, 20% FBS, 30 mM L-Ascorbic acid, 5 ng/ml rhFLT3L, and 5 ng/ml rhlL-7.
  • Cells are transferred to new stromal cell monolayers every 4-5 days by harvesting cells, filtering through a 50 pm nylon strainer, and replating in fresh medium. When confluent, cells are split into multiple wells containing fresh stromal layers.
  • CD8SP or CD4SP cells from ATOs are isolated by magnetic negative selection using the CD8+ or CD4+ Isolation Kits (Miltenyi) and sorted by FACS to further deplete CD45RO+ cells (containing immature naive T cells and CD4ISP precursors).
  • Purified T cell populations are plated in 96-well U-bottom plates in 200 pi AIM V (ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio- Products, West Sacramento, CA). PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego, CA) are added to each well and incubated for 6h.
  • Cells are stained for CD3, CD4, and CD8 (Biolegend, San Diego, CA) and UV455 fixable viability dye (eBioscience, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNy, TNFa, IL-2, IL-4, or IL-17A (Biolegend, San Diego, CA).
  • ATO-derived CD8SP or CD4SP T cells are isolated by negative selection MACS as above (with further FACS purification of CD4SP T cells as described above) and labeled with 5 mM CFSE (Biolegend, San Diego, CA). Labeled cells are incubated with anti- CD3/CD28 beads (ThermoFisher Scientific, Grand Island, NY) in AIM V/5% human AB serum with 20 ng/ml rhlL-2 (Peprotech, Rocky Hill, NJ).
  • 5x103-1 x104 ATO-derived CD8SP or CD4SP T cells isolated as above are plated in 96- well U-bottom plates in 200 mI, and activated/expanded with anti-CD3/28 beads and either 20 ng/mL IL-2 or 5 ng/mL IL-7 and 5 ng/mL IL-15 (Peprotech). Beads are removed on day 4, and fresh medium and cytokines are added every 2-3 days with replating into larger wells as needed. Cells are counted weekly with a hemacytometer.
  • ATO derived CAR positive cells are diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% CTSTM Immune Cell SR and diluted at 1x10 6 cells/ml and kept in culture at 37°C in the presence of 5% C02.
  • day 3 7 and 1 1 , cell viability, CD4 and CD8 phenotypes, CAR positive cells frequency are assessed by flow cytometry by using a recombinant CD22 protein corresponding to the membrane proximal domain of CD22 or a recombinant CD22 protein corresponding to the whole extracellular domain of CD22.
  • Cytolytic activity of anti-CD22 CAR + ATO derived cells is assessed in a flow-based cytotoxicity assay after an overnight coculture with antigen presenting cells (NALM-16) at 37°C in the presence of 5% C02.
  • CAR positive cells and target cells are cocultured in X-Vivo-15 medium at effector (CAR + ) : target ratios of 0.1 :1 , 0.2:1 , 0.5:1 and 1 :1.
  • Culture medium was supplemented with 5% CTSTM Immune Cell SR.
  • NALM-16 target cells were stained with CFSE while SUP-T1 are stained with the CellT race violet proliferation marker.
  • cell viability is measured and the percentage of specific lysis is calculated after normalization to non- specific target cell lysis.
  • the degranulation activity of CART cells is measured using a flow-based degranulation assay.
  • CART and target cells are cocultured in X-Vivo-15 medium at effector (CAR + ):target ratios of 2:1.
  • CAR + effector
  • target ratios 2:1.
  • cell viability is measured and the degranulation activity as represented by CD107a expression is determined by flow cytometry on CD8 CART cells.
  • CART + T cells IFN-g secretion capacity towards antigen presenting cells is assessed in an ELISA immunoassay Quantikine® measuring IFN-g secretion (ELISA Human IFN-y Immunoassay KIT, R&D Systems).
  • NSG NOD scid gamma
  • mice First group of mice is treated with mock cells (not transduced with CAR anti CD22), a second group is treated with non-genetically engineered CAR positive ATO-derived [CAR] pos[TCR]pos [DCKjpos + combination of clofarabine and cytarabine (1 and 10 pmol/l respectively), a third group is treated with engineered ATO-derived [CARjpos [TCRjneg [DCKjneg cells without PNA treatment, a fourth group is treated with engineered ATO-derived [CARjpos [TCRjneg [DCKjneg cells + combination of clofarabine and cytarabine (1 and 10 pmol/l respectively).
  • ATO-derived [CAR] pos [TCR] neg [DCK] neg cells are expanded in-vivo for 14 days. Injection of PBS into control mice is also performed. Tumor bioluminescence is repeated every 3-4 days for at least 21 days, after which mice are sacrificed based on disease burden criteria. Monitoring show a reduction of the tumor burden in mice injected with the ATO- derived [CAR] pos [TCR] neg [DCK] neg cells in third and fourth group, while tumor progression is observed in the first and second groups of mock cells.

Abstract

The invention pertains to the field of adaptive cell immunotherapy and provides with methods for producing hematoipoietic engineered cells lines by reprogramming primary cells that have been genetically engineered. By engineering primary cells before their reprogramming into multipotent cells, the cells can be tested or even screen for their sought therapeutical properties. The multipotent cells obtainable by these methods offer a renewable source for the production of "off the shelf" therapeutic cells that can be differentiated in vitro or in vivo into immune effector cells.

Description

Reprogramming of genetically engineered primary immune cells
Field of the invention
The invention pertains to the field of adaptive cell immunotherapy and provides with methods for producing hematopoietic engineered cells lines by reprogramming primary cells that have been genetically engineered. By engineering primary cells -before their reprogramming into multipotent cells - the cells can be tested or even screen for their sought therapeutic properties. The multipotent cells obtainable by these methods offer a renewable source for the production of“off-the-shelf” therapeutic cells that can be differentiated in-vitro or in-vivo into immune effector cells.
Figure imgf000002_0001
of the invention
Effector immune cells are essential mediators of immune defense against infectious pathogens and cancer. Their insufficiency, which occurs in hereditary or acquired immune deficiencies, results in life threatening infections, increased cancer incidence, and disrupted immunoregulation. Conversely, under impaired regulation T-cells can be harmful and cause normal tissue destruction, as seen in autoimmune disorders, graft rejection, and graft- versus-host disease (GVHD).
T-cells develop from precursors that rearrange germline antigen receptor VDJ genes in the thymus, thereby generating clonotypic T cell receptors (TCRs) that undergo positive and negative thymic selection. The resulting T cells are self-restricted and tolerant of self- tissues. The newly generated T cells, known as naive T cells, initially circulate throughout the body with a low frequency of precursors for any given antigen specificity. Upon encountering antigen, T cells expand and acquire effector and/or memory functions to amount an immune response.
The infusion of effector immune cells, or adoptive transfer, has proven to overcome the limitations of active immunization in some pathologies. The therapeutic use of isolated T cells began with allogeneic bone marrow transplantation (BMT). The use of whole marrow grafts containing donor T cells revealed the beneficial (graft-versus-tumor responses), but at the same time the deleterious (GVHD) effects of adoptive T cell transfer [Ferrara, J.L. and Deeg, H.J. (1991 ) Graft-versus-host disease. N. Engl. J. Med., 324: 667-674] Adoptive cell therapies utilizing“donor-derived T cells,” harvested from a healthy donor can expose the recipient to the risk of normal tissue destruction by graft versus host (GVH) responses. Autologous T-cells are devoid of such toxic potential. However, they may be lacking therapeutic potential or be functionally impaired in patients with refractory infections or progressing cancer. Allogeneic and autologous effector immune cells thus have their respective advantages and disadvantages.
For some cancers, T cells may be isolated from surgically removed tumors, which are enriched in tumor-reactive T cells relative to peripheral blood. T umor infiltrating lymphocytes (TILs) can be isolated at quite a high frequency from melanoma specimens, but this technique is not feasible or effective in many other tumor types [Wu,R. et al. (2012) Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes for metastatic melanoma: current status and future outlook. Cancer J. 18:160-175].
Immune cell therapy is thus currently limited by the availability of effector immune cells to be safely engrafted and prompt to fight the disease specifically addressed by the patient.
The goal pursued by immune cell engineering over the last years is to rapidly generate populations of immune cells specific for any antigen and, furthermore, to enhance their therapeutic (e.g., anti-tumor) functions. Peripheral blood T cells are the most easily accessible and perfectly suitable cell source for this purpose. Most current therapies utilizing engineered T cells process autologous peripheral blood T cells that are targeted to tumor antigens following retroviral transduction of a TCR or a chimeric antigen receptor (CAR). In recent years, a few clinical trials have resulted in encouraging and sometimes dramatic clinical responses [Couzin-Frankel, J. (2013) Breakthrough of the year 2013. Cancer immunotherapy. Science. 342:1432-1433].
TCRs are typically cloned from patients’ tumor-reactive T cells from humanized murine models, or through the use of phage display technology. In CARs, tumor recognition is typically mediated by a single chain variable fragment (scFv) derived from a monoclonal antibody or an antigen-binding region isolated from an immunoglobulin (Ig) heavy and light chain library. Unlike TCR-mediated antigen recognition, CARs function independently of HLA and can therefore be used in any genetic background. The genetic modification of autologous peripheral blood T lymphocytes to generate tumor-targeted T cells is now a well- established approach that was developed in a handful of academic centers. The power and promise of TCR and CAR therapies utilizing these manufacturing processes are best illustrated by the very significant clinical results obtained with NY-ESO-1 TCR [Robbins, P.F et al. (201 1 ) Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J. Clin. Oncol. 29:917-924] and CD19 CAR T cells [Brentjens, R.J. et al. (2013), CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia Sci. Transl. Med., 5 p. 177ra138].
These cell manufacturing processes combine T cell activation and transduction steps to generate expanded, genetically targeted T cell products. For example, T cells engineered to express specific CARs or TCRs may be initiated from Ficoll-purified PBMCs, which are next activated with anti-CD3 monoclonal antibody (mAb) in the presence of irradiated allogeneic feeder cells and transduced with a vector encoding either the CAR or TCR a and b chains [ Till, B.G. M.C. Jensen, J. Wang, X. Qian, A.K. Gopal, D.G. Maloney, C.G. Lindgren, Y. Lin, J.M. Paet al. (2012) CD20-specific adoptive immunotherapy for lymphoma using a chimeric antigen receptor with both CD28 and 4-1 BB domains: pilot clinical trial results. Blood 1 19:3940-3950].
The functional, proliferative, and persistence potential of adoptively transferred T lymphocytes is determined by multiple factors. These include the TCR or CAR design, the manufacturing platform, the selected T cell subsets, and the differentiation stage of the harvested T cells. Peripheral blood T cells comprise naive (TN), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and terminal effector (TE) cells. Several groups have investigated which of these T cell subsets are best suited for use in different adoptive therapy settings. Polyclonal CD8+ TCM isolation from leukopheresis products, followed by CD3/CD28 activation without exogenous feeder cells and cell expansion in IL- 2/IL-15, has thus been developed on a clinical scale and is currently in use for the generation of autologous CAR-redirected CD19-specific CD8+ TE/CM for adoptive transfer after autologous hematopoietic stem cell transplantation (HSCT) for high-risk CD19+ non- Hodgkin lymphomas [Wang, X. et al. (2012) Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. J. Immunother., 35:689-701].
The promising clinical results of engineered T cell therapy could be further amplified and broadened if potent and histocompatible T cells were readily available. Autologous approaches have a proven track record, but personalized manufacture may be challenging in some instances, for example in patients with chemotherapy or HIV-induced immune deficiency or in small infants. While T cells can be easily harvested from donors, their use is compromised by the high alloreactive potential. Owing to their ontogeny, TCRs are naturally prone to react against non-autologous tissues, recognizing either allogeneic HLA molecules or other polymorphic gene products, referred to as minor antigens [Afzali, B. et al. (2007) Allorecognition and the alloresponse: clinical implications. Tissue Antigens 69:545-556]. This propensity underlies the high risk of graft rejection in transplant recipients and of GVHD in recipients of donor-derived T cells.
If the endogenous TCR cannot be tamed, one may abrogate its expression, making the engineered TCR or CAR the sole driver of T cell activation and clonal expansion.
Recently engineered T-cells disrupted in their T-cell receptor (TCR) using TALE- nucleases, endowed with chimeric antigen receptor (CAR) targeting CD19 malignant antigen, referred to as“UCART19” product, have shown therapeutic efficacy and moderate alloreactivity in at least two infants who had refractory leukemia [Leukaemia success heralds wave of gene-editing therapies (2015) Nature 527:146-147] To obtain such UCART19 cells, two steps of genetic engineering were performed. First, a cassette encoding the chimeric antigen receptor (CAR CD19) was introduced randomly into the genome using a retroviral vector, and second, the primary cells were electroporated with capped mRNA encoding TALE-nuclease heterodimers, which were transiently expressed to induce double strand break disruption in the TCRalpha alleles (WO2013176915).
Despite this success, allogeneic immune cell approaches are still labor intensive and constrained by the limited replicative potential of mature T cells [Gattinoni, L. et al. (2012) Paths to sternness: building the ultimate antitumour T cell. Nat. Rev. Cancer. 12:671 -684]
While T cells can cause GVHD, their precursors do not, as they undergo positive and negative selection in the recipient’s thymus. Taking advantage of this requires the ability to expand T cell precursors in culture, which is now possible due to advances in understanding T cell development [D.K. Shah, J.C. Ziiniga-Pflucker (2014) An overview of the intrathymic intricacies of T cell development. J. Immunol. 192:4017-4023] T cell precursors lack the ability to initiate GVH reactions because they complete their differentiation in the recipient’s thymus wherein they become restricted to host MHC and yield T lymphocytes that are host tolerant. When transduced with a CAR, allogeneic lymphoid progenitors yield tumor- targeted T cells without causing GVHD [Zakrzewski, J.L. et al. (2008) Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors. Nat. Biotechnol. 26:453-461 ] The main advantage of using immune cell precursors for immunotherapy is that this approach does not require strict histocompatibility between donors and recipients. In mice, this therapy works with unrelated fully mismatched cells just as well as with autologous cells. T cell precursor immunotherapy may therefore allow for a true“off-the- shelf” therapy, if lymphoid progenitor cell manufacturing can be scaled up.
The development of cellular therapeutics relying on functionally validated, banked, broadly histocompatible cell types would have a major impact on the applicability and cost of adoptive immune cell therapies. This prospect raises the challenge of artificially generating ideal T cells rather than modifying those naturally formed.
It has been previously demonstrated that T cell-derived iPSCs (TiPSCs) expressing a CAR (CAR-TiPSC) provide an effective means to concomitantly exploit the unlimited proliferative potential of iPSCs and direct the antigen specificity of iPSC-derived T cells. CAR-TiPSC T cells generated in culture expanded robustly upon CD19 engagement by the CAR (up to 1 ,000-fold over 3 weeks) and showed anti-tumor efficacy against a CD19+ lymphoma in a xenogeneic murine model, comparable to their natural counterparts harvested from peripheral blood (from the same donor) and transduced with the same CAR [Themeli et al. (2013) Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat. Biotechnol. 31 :928-933]. The latter study provided the proof of principle that human iPSC-derived T lymphocytes generated in vitro possessed anti-tumor function in vivo.
While natural, autologous T cells represent nowadays the best-defined cell source for adoptive cell therapy, and they are the cornerstone of present cell-based cancer immunotherapy. Induced, engineered T cells derived from allogeneic pluripotent stem cell sources may play an important role in the future.
To achieve this goal, the present invention provides with methods wherein, instead of engineering iPS cells as suggested above, primary immune cells from donors or patient are first gene-edited prior to being reprogrammed, and then turned into precursor cells, especially CD34+ cells, to either form cells available for engraftment into a patient host or to establish cell lines available for a continuous production of useful engineered hematopoietic cells.
Summary of the invention
Performing genetic modifications can have limited efficiency in multipotent or pluripotent cells due partly to the fact that:
(i) the frequency of cells that undergo the exact desired modification or combination of modifications (or moreover without additional unwanted modifications) can be low, implying complex sorting and purification steps;
(ii) Performing genetic modifications in multipotent or pluripotent cells requires cumbersome and complicated handling. Maintaining, culturing, passaging multiple undifferentiated multipotent or pluripotent clones requires significant resources and is labor intensive. (iii) Performing genetic modifications in multipotent or pluripotent cells requires handling that may affect their potency. Transduction, exposure to penetration facilitating agents, electroporation, single cell isolation or other methods used to introduce genetic changes in cells may impair their potency (e.g. single cell culture of pluripotent stem cells has an adverse effect on preserving their undifferentiated state).
(iv) Desired genetic modifications may not necessarily lead to identifiable or selectable phenotypic changes in pluripotent or multipotent cells. It is therefore more complicated to identify or isolate cells that carry the desired modifications (e.g. inactivation of a gene expressed only in a more differentiated lineage).
(v) Desired genetic modifications or combinations of modifications that are intended to procure a desired phenotype in a more differentiated lineage cannot be validated before the extensive work required to engineer pluripotent or multipotent cells and to differentiate them into the desired cell type.
(vi) When engineering pluripotent or multipotent cells, the genetic background of the cells used to make the bank may not be tested for its suitability for or performance in cells that have a chosen more differentiated type and may also carry desired genetic modifications. Any contribution from the genetic background to the final performance of the desired differentiated cells that may also carry desired genetic modifications can only be tested once the entire engineering, banking, differentiation process is done, which may be resource consuming to do on multiple candidate genetic background when trying to identify a better one.
In order to circumvent the above limitations, the method of the present invention broadly relies on:
(a) designing or selecting a genetic makeup of cells (e.g. by performing genetic engineering in a genetic background that may be or not be selected) of a cell type allowing efficient genetic modifications and/or efficient isolation of cells with desired genetic modifications and/or efficient testing or validation of desired genetic makeup in said cells, and
(b) reprogramming the cell type or phenotype of said engineered cells into another cell type or phenotype, such as obtaining a cell line that can be banked and from which a desired cell type with desired genetic modifications can further be obtained. As an example, the method can be implemented by: performing genetic modifications (either or both addition of genetic material and/or genetic deletions or gene inactivations through gene editing and/or transgenesis and/or transduction) in primary nucleated blood cells, such as PBMCs or T-cells ; such cell type allows for efficient genetic modifications that can be combined or done sequentially while culturing said cells over multiple days or weeks; selecting, isolating or enriching for the cells that have undergone the desired modification or combination of modifications. This can allow both (a) easy selection of genetic modifications that lead to phenotypic differences in such cell types and (b) providing cells of said type with the desired genetic modifications to be used to validate such combinations of cell type with genetic modifications through testing performed on such cells. reprogramming such engineered cells with the desired genetic modifications (which consequences may, in addition, have been functionally validated in said cell type) into pluripotent stem cells. banking said iPS cells or a derivative thereof that can be further differentiated into cells that express the phenotypic traits which were engineered in the original primary blood cells before reprogrammation into IPS cells.
As a second example, the method can be implemented by: testing the behavior of primary nucleated blood cells, such as PBMCs or T-cells, coming from different individuals, and selecting those that have a more desirable phenotype, selecting, isolating or enriching for the cells that have the desired genetic background (e.g., T-cells that have a receptor with a desirable specificity/affinity or structure that lead to a desirable T-cell phenotype when confronted with an antigen in the organism, for example:
- T-cells carrying a receptor that allows them to remain functional and/or clear a chronic antigen in the organism, or
- T-cells carrying a receptor that allows them to have a long term memory phenotype in organisms that have cleared an acute antigen); reprogramming such cells (which genetic makeup may, in addition, have been functionally validated in said cell type) into pluripotent stem cells. banking said iPS cells or a derivative thereof that can be further differentiated into cells, such as CD34 + cells, which express the phenotypic traits that were engineered in the original primary blood cells before their reprogrammation into IPS cells.
The invention may further comprise a step of conditioning, by which the cells previously obtained are differentiated into hematopoietic cells, especially CD34 + hematopoietic stem cells. This“conditioning” step may include a characterization step to ensure that they are suitable for administration into a patient for permanent or transient engraftment.
Among others, the methods of the present invention can present several benefits:
To perform genetic modifications on differentiated cells prior to their
reprogrammation, avoids the problem of having to maintain the genomic integrity of the cells under prolonged undifferentiated state.
To perform genetic modifications on cells that have been chosen to express a phenotypic difference resulting from said genetic modifications allows for easier identification or selection of the desired modified cells.
To perform genetic modifications on cells that have been chosen to express a phenotypic difference resulting from said genetic modifications allows for procuring a (differentiated) cell preparation including cells expressing phenotypic differences resulting from such desired genetic modifications. It allows performing tests and validating that said designed genetic makeup.
Brief description of the Figures and Tables:
Figure 1 : Schematic representation of the hematopoiesis lineage.
Figure 2: Schematic representation of examples of genetic engineering that can be performed to confer additional therapeutic properties to the immune cells prior to their reprogramming as per the present invention. The panels correspond to the main functions, which can be boosted or reduced by genetic alteration. The invention encompasses any alteration of the expression of one or several genes from the different panels, alone or in combination, to be stably maintained in multipotent cells in view of their redifferentiation into immune therapeutic cells. Legends can be read as follows: A - Alloreactivity/ Engraftment: weakening self and non-self-recognition mechanisms in order to produce engineered allogeneic cells that are less alloreactive.
B - Inhibiting suppressive cytokines/metabolites: reducing the production by the immune cells of cytokines and metabolites, the secretion of which have a negative influence on immune cells activation.
C - T-cell proliferation: inhibiting genetic pathways that down regulate T-cell proliferation.
D - T-cell activation: inhibiting genetic pathways that down regulate T-cell activation.
E - T-cell exhaustion: inhibiting genetic pathways that trigger T-cell exhaustion.
F - Resistance to tumor-induced glucose deprivation: inhibiting genetic pathways that down regulate T-cells proliferation and activation in response to glucose deprived microenvironment.
G - Immune checkpoint receptors: reducing control of immune checkpoints on the activation of the engineered immune cells by inhibiting their receptors and pathways controlled by these receptors.
Optionally, the engineered immune cell may be endowed with a chimeric antigen receptor (CAR), which is preferably integrated at a locus X (X = any of the gene expressing the proteins referred to in panels A to G) and/or further transgenes such as those listed in the dotted line panel.
Figure 3: Schematic representation of a method to engineer hematopoietic cells lines according to the invention, comprising the following steps described in Example 1 :
1- Gene edited step into primary immune cells
2- Selection step to keep the functional engineered cells
3- Expression of reprogramming factors into said selected cells
4- Isolating multipotent resulting cells, preferably iPS cells, to generate cell lines
5- Differentiating the cells from said cell line(s) to produce therapeutic effector cells, such as NK or T-cells. This step can take place in vivo if the cell lines are engrafted into patients as hematopoietic stem cells (HSC).
Figure 4: Schematic representation of the donor sequences used in the experimental section to insert IL-15 exogenous coding sequence at the CD25 and PD1 loci and also the anti-CD22 CAR exogenous coding sequence at the TRAC locus. A: donor template (designated IL-15m-CD25) designed for site directed insertion of IL-15 at the CD25 locus for obtaining co-transcription of CD25 and IL-15 polypeptides by the immune cell. Sequences are detailed in the examples. B: donor template (designated IL-15m-PD1 ) designed for site directed insertion of IL-15 at the PD1 locus for obtaining transcription of IL-15 under the transcriptional activity of the promoter of PD1 endogenous gene. The PD1 right and Left border sequences can be selected so as to keep the PD1 endogenous coding sequence intact or disrupted. In this later case, PD1 is knocked-out while IL-15 is Knocked- in and transcribed. C: donor template designed for site directed insertion of a chimeric antigen receptor (ex: anti-CD22 CAR) into the TCR locus (ex: TRAC). In general, the left and right borders are chosen so as to disrupt the TCR in order to obtain [TCR]neg[CAR]pos engineered immune cells suitable for allogeneic transplant into patients.
Figure 5: Flow cytometry measures of the frequency of targeted integration of IL-15m at either the PD1 or CD25 locus by using respectively PD1 or CD25 TALEN®, in a context where an anti-CD22 CAR is also integrated at the TRAC locus using TRAC TALEN®. These results show efficient targeted integration of both the CAR anti-CD22 at the TRAC locus together and the IL-15 coding sequence at the PD1 or CD25 loci. A: mock transfected primary T-cells. B: primary T-cells transfected with the donor sequences described in figure 4 (B and C) and specific TALEN® for the double integration at the TCR and PDI loci. C: primary T-cells transfected with the donor sequences described in figure 4 (A and C) and specific TALEN® for the double integration at the TCR and CD25 loci.
Figure 6: Schematic representation of the exogenous sequences used in the experimental section to transfect the primary immune cells to obtain the results shown in figures 7 and 8.
Figure 7 and 8: Flow cytometry measures for LNGFR expression among viable T-cells transfected with donor templates of figure 6 and specific TALEN® (TCR and CD25), upon antiCD3/CD28 non-specific activation (Dynabeads®) and upon CAR dependent tumor cell activation (raji tumor cells). As shown in figure 8, LNGFR expression was specifically induced in [CAR anti-CD22] positive cells upon CAR/tumor engagement.
Figure 9 and 10: Flow cytometry measures for CD25 expression among viable T-cells transfected with donor templates of figure 6 and specific TALEN® (TCR and CD25) upon antiCD3/CD28 non-specific activation (Dynabeads®) and Tumor cell activation (raji tumor cells). As shown in figure 10, CD25 expression was specifically induced in [CAR anti-CD22] positive ce||s Up0n CAR/tumor engagement.
Figure 11 : Schematic representation of the exogenous sequences used in the experimental section to transfect the primary immune cells to obtain the results shown in figures 13 and 14. Figure 12 and 13: Flow cytometry measures for LNGFR expression among viable T-cells transfected with donor templates of figure 1 1 and specific TALEN® (TCR and PD1 ) upon antiCD3/CD28 non-specific activation (Dynabeads®) and Tumor cell activation (raji tumor cells). As shown in figure 13, LNGFR expression was specifically induced in [CAR anti-CD22] positive cells upon CAR/tumor engagement.
Figure 14: Flow cytometry measures for endogenous PD1 expression among viable T-cells transfected with donor templates of figure 1 1 upon antiCD3/CD28 non-specific activation (Dynabeads®) and Tumor cell activation (raji tumor cells) with and without using TALEN® (TCR and PD1 ). PD1 was efficiently Knocked-out by TALEN treatment (8% remaining expression of PD1 out of 54 %).
Figure 15: Diagram showing IL-15 production in [CAR]positive (CARm) and [CAR]negative engineered immune cells according to the invention transfected with the donor template described in Figure 4 (B) and TALEN® for insertion of IL-15 exogenous coding sequences into the PD1 locus. IL15, which transcription was under control of endogenous PD1 promoter, was efficiently induced upon antiCD3/CD28 non-specific activation (Dynabeads®) and Tumor cell activation (raji tumor cells) and secreted in the culture media.
Figure 16: Graph showing the amount of IL-15 secreted over time (days) post activation by the immune cells engineered according to the invention. A: Cells engineered by integration of the IL-15 coding sequence at the CD25 locus using the DNA donor templates described in Figures 4A (IL-15m_CD25) and/or 2C (CARm). B: Cells engineered by integration of the IL-15 coding sequence at the PD1 locus using the DNA donor templates described in Figures 4B (IL-15m_PD1 ) and/or 2C (CARm). Integrations at both loci show similar IL-15 secretion profiles. Secretion of IL-15 is significant increased by tumor specific activation of CAR.
Figure 17: Graph reporting number of Raji-Luc tumor cells expressing CD22 antigen (luciferase signal) over time in a survival assay (serial killing assay) as described in the examples. The immune cells (PBMCs) have been engineered to integrate IL-15 coding sequences at the PD1 (A) or CD25 locus (B) and to express anti-CD22-CAR at the TCR locus (thereby disrupting TCR expression). In this assay, tumor cells are regularly added to the culture medium, while being partially or totally eliminated by the CAR positive cells. The re-expression of IL-15 at either PD1 or CD25 cells dramatically helps the elimination of the tumor cells by the CAR positive cells. Detailed description of the invention
Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology.
All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes l-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
The present invention refers to a method for producing engineered progenitor hematopoietic stem cells (HSC) or generating an engineered cell line to produce effector cells for immune therapy, wherein said method comprises the steps of:
a) Providing primary immune cells from a donor or a patient;
b) Genetically engineering said cell to modify its therapeutic properties; and c) Processing the immune cell engineered in step b) into at least one multipotent cell line.
Various embodiments of the invention are provided through the features given in the claims in view of the common practice and knowledge of one skilled in the art. The invention more particularly involves cells and reagents with the following characteristics:
Effector cells: Effector cells are the relatively short-lived activated cells that defend the body in an immune response. Activated T cells, which include cytotoxic T cells and helper T cells are preferred effector cells to carry out cell-mediated responses. The category of effector T cell is a broad one that includes various T cell types that actively respond to a stimulus, such as co-stimulation. This includes helper, killer, regulatory, and potentially other T cell types.
By“immune cell” is meant a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response, such as typically CD3 or CD4 positive cells. The immune cell according to the present invention may be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes or helper T-lymphocytes.
By“primary cell” or“primary cells” are intended cells taken directly from living tissue (e.g. biopsy material) and established for growth in vitro for a limited amount of time, meaning that they can undergo a limited number of population doublings. Primary cells are opposed to continuous tumorigenic or artificially immortalized cell lines. Non- limiting examples of such cell lines are CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT- 1 16 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.
Primary immune cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and from tumors, such as tumor infiltrating lymphocytes. In some embodiments, said immune cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, said cell is part of a mixed population of immune cells which present different phenotypic
characteristics, such as comprising CD4, CD8 and CD56 positive cells. Primary immune cells are provided from donors or patients through a variety of methods known in the art, as for instance by leukapheresis techniques as reviewed by Schwartz J.et a/. (Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue (2013) J Clin Apher. 28(3): 145-284).
A cell line is obtained from a cell that has acquired capacity of self-renewal and which can produce cells over more than 104 generations, more generally over more than 105 generations, mostly over than 106 generations, if not being an immortal cell line.
Multipotent cells describe at least progenitor cells which have the potential to produce cells that can differentiate into discrete cell types. Multipotent cells may be pluripotent cells according to the invention, in particular induced pluripotent stem cells (iPS). Induced multipotent stem cells, are typically derived from a non-multipotent cell, such as a primary immune cell, by "forcing" the expression of so-called“reprogramming factors” into such cells. Oct-3/4 and certain products of the Sox gene family (Sox1 , Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klf1 , Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency. The“Yamanaka factors”, OCT4, SOX2, KLF4, and C-MYC, have been found to be pivotal, in particular when expressed using retrovirus vectors [Yamanaka, K. et al. (2008). "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors". Science. 322 (5903): 949-53]. Lentiviral expression of reprogramming factors has been used to induce multipotent cells from human peripheral blood cells [Staerk, J. et al. (2010). "Reprogramming of human peripheral blood cells to induced pluripotent stem cells". Cell stem cell. 7 (1 ): 20-4] [Loh, YH. et al. (2010). "Reprogramming of T cells from human peripheral blood". Cell stem cell. 7 (1 ): 15-9]. Although non-retroviral approaches have demonstrated lower efficiency levels so far, adenovirus can also be useful to transiently express reprogramming factors to avoid adverse insertional tumorigenic mutagenesis.
One preferred approach according to the present invention is to transfect primary cells with mRNA that encode the reprogramming factors to obtain a transient expression thereof. This transfection is preferably performed by electroporation. Alternatively, electroporation of MicroRNAs (short RNA molecules that bind to complementary sequences on messenger RNA), such as miR-291 , miR-294 and miR-295, can be performed to block the expression of genes committed in maintaining cell differentiation [Bao X. et al. (2013) “MicroRNAs in somatic cell reprogramming". Current Opinion in Cell Biology. 25 (2): 208- 214]
Alternative techniques have also been developed, such as chemical compouds mimicking transcription factors, such as histone deacetylase (HDAC) inhibitor valproic acid, which act the same signaling pathway as transcription factors c-Myc and Sox2, and also inhibitors of histone methyl transferase (HMT), such as BIX-01294 [Desponts et al. (2008) "Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds". Cell Stem Cell. 3 (5): 568-74] ALK5 inhibitor SB431412, MEK (mitogen-activated protein kinase) inhibitor PD0325901 and Thiazovivin can also be used to increase the efficiency of reprogramming factors. A cocktail of seven small-molecule compounds including DZNep is also available to induce multipotency [Bo, F. et al. (2009) Molecules that Promote or Enhance Reprogramming of Somatic Cells to Induced Pluripotent Stem Cells. Cell Stem Cell. 4 (4):301 -312]. Generation of iPS cells is also possible without any genetic alteration of the adult cell by repeated treatment of cells with certain proteins channeled into the cells via poly-arginine anchors as described by [Zhou H. et al. (2009) "Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins". Cell Stem Cell. 4 (5): 381-4] The acronym given for those iPSCs are piPSCs (protein- induced pluripotent stem cells). All the above methods are envisioned as part of the method of the present invention to induce the engineered primary immune cells to lower differentiation stages to create hematopoietic cell lines, especially hematopoietic stem cells (HSC), useful for sourcing cells for immune therapy.
By“Hematopoietic lineage” is meant the differentiation stages by which blood-cell development progresses from a hematopoietic stem cell (HSC), which can undergo either self-renewal or differentiation into a multilineage committed progenitor cell: a common lymphoid progenitor (CLP) or a common myeloid progenitor (CMP). HSC are typically CD34 positive cells, and more typically CD150+ and CD48- when using SLAM markers as described by Oguro, H., et al. (2013)“SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors” Cell stem cell, 13(1 ), 102-16.
A CLP gives rise to the lymphoid lineage of white blood cells or leukocytes-the natural killer (NK) cells and the T and B lymphocytes. A CMP gives rise to the myeloid lineage, which comprises the rest of the leukocytes, the erythrocytes (red blood cells), and the megakaryocytes that produce platelets important in blood clotting. Cells undergoing these differentiation process express a stage- and lineage-specific set of surface markers. Therefore cellular stages are identified by the specific expression patterns of these genes. A simplified representation of the hematopoietic lineage is presented in Figure 1.
The cells of the present invention comprise genetic modifications for the purpose of producing effector immune cells with an improved suitability for cell therapy, in either autologous or allogeneic cell therapy schemes.
The term“therapeutic properties” encompasses the different ways such cells can be improved in the perspective of their use in therapeutic treatments. This means that the cells are genetically engineered to confer them a therapeutic advantage benefit (i.e. therapeutic potency) or to facilitate their use or their production. For instance, the genetic engineering can concur to the effector cells having better survival, faster growth, shorter cell cycles, improved immune activity, be more functional, more differentiated, more specific with respect to their target cells, more sensitive or resistant to drugs, less sensitive to glucose deprivation, oxygen or amino acid depletion (i.e. resilient to tumor microenvironment). Progenitor cells may be more productive, better tolerated by the recipient patient, more likely to produce cells that will differentiate in the desired effector cells. These examples of “therapeutic properties” are given as examples without limitation.
By“Genetic engineering” is meant any methods aiming to introduce or withdraw genetic material from a cell. By“gene editing” is meant a genetic engineering allowing genetic material to be added, removed, or altered at particular locations (loci) in the genome, including punctual mutations. Gene editing generally involves sequence specific reagents.
By“seguence-specific reagent” is meant any active molecule that has the ability to specifically recognize a selected polynucleotide sequence at a genomic locus, referred to as“target sequence”, which is generally of at least 9 bp, more preferably of at least 10 bp and even more preferably of at least 12 pb in length, in view of modifying the expression of said genomic locus. Said expression can be modified by mutation, deletion or insertion into coding or regulatory polynucleotide sequences, by epigenetic change, such as by methylation or histone modification, or by interfering at the transcriptional level by interacting with transcription factors or polymerases.
Examples of sequence-specific reagents are endonucleases, RNA guides, RNAi, methylases, exonucleases, histone deacetylases, endonucleases, end-processing enzymes such as exonucleases, and more particularly cytidine deaminases such as those coupled with the CRISPR/cas9 system to perform base editing (i.e. nucleotide substitution) without necessarily resorting to cleavage by nucleases as described for instance by Hess, G.T. et al. [Methods and applications of CRISPR-mediated base editing in eukaryotic genomes (2017) Mol Cell. 68(1 ): 26-43. According to a preferred aspect of the invention, said sequence-specific reagent is preferably a sequence-specific nuclease reagent, such as a RNA guide coupled with a guided endonuclease.
The present invention aims to improve the therapeutic potential of immune cells through gene editing techniques, especially by gene targeted integration.
By“gene targeting integration” is meant any known site-specific methods allowing to insert, replace or correct a genomic coding sequence into a living cell.
According to a preferred aspect of the present invention, said gene targeted integration involves homologous gene recombination at the locus of the targeted gene to result the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of several nucleotides (i.e. polynucleotide), and more preferably a coding sequence.
“Rare-cutting endonucleases” are sequence-specific endonuclease reagents of choice, insofar as their recognition sequences generally range from 10 to 50 successive base pairs, preferably from 12 to 30 bp, and more preferably from 14 to 20 bp.
According to a preferred aspect of the invention, said endonuclease reagent is a nucleic acid encoding an“engineered” or“programmable” rare-cutting endonuclease, such as a homing endonuclease as described for instance by Arnould S., et a/. [W02004067736], a zinc finger nuclease (ZFN) as described, for instance, by Urnov F., et a/. [Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651 ], a TALE-Nuclease as described, for instance, by Mussolino et a/. [A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (201 1 ) Nucl. Acids Res. 39(21 ):9283-9293], 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 ].
According to another embodiment, 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.
According to a preferred aspect of the invention, the endonuclease reagent is transiently expressed into the cells, meaning that said reagent is not supposed to integrate into the genome or persist over a long period of time, such as be the case of RNA, more particularly mRNA, proteins or complexes mixing proteins and nucleic acids (eg: Ribonucleoproteins).
In general, 80% the endonuclease reagent is degraded by 30 hours, preferably by 24, more preferably by 20 hours after transfection. An endonuclease under mRNAform 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]
In general, electroporation steps that are used to transfect primary immune cells, such as PBMCs are typically 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 W02004083379, 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. In general, the suspension of cells undergoes one or more pulsed electric fields. 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.
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 a/. [TALEN®facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773].
As previously stated, the sequence specific reagent is preferably under the form of nucleic acids, such as under DNA or RNA form encoding a rare cutting endonuclease a subunit thereof, but they can also be part of conjugates involving polynucleotide(s) and polypeptide(s) such as so-called“ribonucleoproteins”. Such conjugates can be formed with reagents as Cas9 or Cpf1 (RNA-guided endonucleases) or Argonaute (DNA-guided endonucleases) as recently respectively described by Zetsche, B. et al. [Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System (2015) Cell 163(3): 759-771 ] and by Gao F. et al. [DNA-guided genome editing using the Natronobacterium gregoryi Argonaute (2016) Nature Biotech ], which involve RNA or DNA guides that can be complexed with their respective nucleases.
“Exogenous sequence” refers to any nucleotide or nucleic acid sequence that was not initially present at the selected locus. This sequence may be homologous to, or a copy of, a genomic sequence, or be a foreign sequence introduced into the cell. 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. A 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
By“drug” is meant any substance (other than food that provides nutritional support) that, when inhaled, injected, smoked, consumed, absorbed or dissolved under the tongue causes a temporary physiological change in the body. According to a preferred aspect of the present invention the effector cells resulting from the methods herein described are made resistant to certain immunosuppressive drugs, meaning that they can better resist to molecules used in standard cancer care treatments for lymphodepleting patients. This resistance to the drug is assessed by comparing the LD5o of the gene edited cells with respect to this drug in comparison with non gene edited cells in experimental conditions. The LD5O has to be statistically higher in such experimental conditions using a t-test to conclude that the cells are“resistant”.
By“Chimeric Antigen Receptor (CAR)” is meant recombinant receptors comprising a targeting moiety that is 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 heavy variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains of CARs are generally derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains, which are generally combined with signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS and 4-1 BB (CD137) to enhance survival and increase proliferation of the cells. CARs are generally expressed in effector immune cells to redirect their immune activity against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors. A component of a CAR is any functional subunit of a CAR that is encoded by an exogenous polynucleotide sequence introduced into the cell. For instance, this component can help to interact with the target antigen, the stability or the localization of the CAR into the cell.
The cell generated by the present invention can express a variety of CARs or recombinant (modified) TCRs, such as those commonly described in the literature, under single-chain or multiple subunits (multi-chain) as described in WO2014039523.
Preferred recombinant TCR to be used in the present invention are those directed against antigen specific of cancer cells, such as MART-1 , MAGE-1 , MAGE-2, MAGE-3 MAGE-12, BAGE, GAGE, NY-ESO-1 , or overexpressed in cancer cells, such as a- Fetoprotein, Telomerase catalytic protein, G-250, MUC-1 , CarcinoEmbryonic antigen (CEA), p53, Her-2/Neu and WT1 [Rosenberg S.A., (2001 ) Progress in human tumour immunology and immunotherapy Nature. 41 1 (6835):380-4]
CARs can bind tumor antigen as diverse as one selected from: CD19 molecule (CD19); membrane spanning 4-domains A1 (MS4A1 also known as CD20); CD22 molecule (CD22); CD24 molecule (CD24); CD248 molecule (CD248); CD276 molecule (CD276 or B7H3); CD33 molecule (CD33); CD38 molecule (CD38); CD44v6; CD70 molecule (CD70); CD72; CD79a; CD79b; interleukin 3 receptor subunit alpha (IL3RA also known as CD123); TNF receptor superfamily member 8 (TNFRSF8 also known as CD30); KIT proto-oncogene receptor tyrosine kinase (CD1 17); V-set pre-B cell surrogate light chain 1 (VPREB1 or CD179a); adhesion G protein-coupled receptor E5 (ADGRE5 or CD97); TNF receptor superfamily member 17 (TNFRSF17 also known as BCMA); SLAM family member 7 (SLAMF7 also known as CS1 ); L1 cell adhesion molecule (L1 CAM); C-type lectin domain family 12 member A (CLEC12A also known as CLL-1 ); tumor-specific variant of the epidermal growth factor receptor (EGFRvlll); thyroid stimulating hormone receptor (TSHR); Fms related tyrosine kinase 3 (FLT3); ganglioside GD3 (GD3); Tn antigen (Tn Ag); lymphocyte antigen 6 family member G6D (LY6G6D); Delta like canonical Notch ligand 3 (DLL3); Interleukin- 13 receptor subunit alpha-2 (IL-13RA2); Interleukin 1 1 receptor subunit alpha (IL1 1 RA); mesothelin (MSLN); Receptor tyrosine kinase like orphan receptor 1 (ROR1 ); Prostate stem cell antigen (PSCA); erb-b2 receptor tyrosine kinase 2 (ERBB2 or Her2/neu); Protease Serine 21 (PRSS21 ); Kinase insert domain receptor (KDR also known as VEGFR2); Lewis y antigen (LewisY); Solute carrier family 39 member 6 (SLC39A6); Fibroblast activation protein alpha (FAP); Hsp70 family chaperone (HSP70); Platelet- derived growth factor receptor beta (PDGFR-beta); Cholinergic receptor nicotinic alpha 2 subunit (CHRNA2); Stage-Specific Embryonic Antigen-4 (SSEA-4); Mucin 1 , cell surface associated (MUC1 ); mucin 16, cell surface associated (MUC16); claudin 18 (CLDN18); claudin 6 (CLDN6); Epidermal Growth Factor Receptor (EGFR); Preferentially expressed antigen in melanoma (PRAME); Neural Cell Adhesion Molecule (NCAM); ADAM metallopeptidase domain 10 (ADAM10); Folate receptor 1 (FOLR1 ); Folate receptor beta (FOLR2); Carbonic Anhydrase IX (CA9); Proteasome subunit beta 9 (PSMB9 or LMP2); Ephrin receptor A2 (EphA2); Tetraspanin 10 (TSPAN10); Fucosyl GM1 (Fuc-GM1 ); sialyl Lewis adhesion molecule (sLe); TGS5 ; high molecular weight- melanoma-associated antigen (HMWMAA); o-acetyl- GD2 ganglioside (OAcGD2); tumor endothelial marker 7- related (TEM7R); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61 ); ALK receptor tyrosine kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1 ); hexasaccharide portion of globoH glycoceramide (GloboH); NY-BR-1 antigen; uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1 ); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein- coupled receptor 20 (GPR20); lymphocyte antigen 6 family member K (LY6K); olfactory receptor family 51 subfamily E member 2 (OR51 E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1 ); ETV6-AML1 fusion protein due to 12;21 chromosomal translocation (ETV6-AML1 ); sperm autoantigenic protein 17 (SPA17); X Antigen Family, Member 1 E (XAGE1 E); TEK receptor tyrosine kinase (Tie2); melanoma cancer testis antigen- 1 (MAD-CT-1 ); melanoma cancer testis antigen-2 (MAD-CT-2); Fos- related antigen 1 ; p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N- Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B 1 ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Cytochrome P450 1 B 1 (CYP1 B 1 ); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES 1 ); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1 ); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF);; bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor- like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda- like polypeptide 1 (IGLL1 ).
The invention preferably involves CARs directed against cancer marker antigens, such as CD19, CD22, CD33, 5T4, ROR1 , CD38, CD52, CD123, CS1 , BCMA, Flt3, CD70, EGFRvlll, WT1 , HSP-70 and CCL1.
Even more preferred are CARs directed against CD22, CD38, 5T4, CD123, CS1 , HSP-70 and CCL1. Such CARs having preferably one structure as described in W02016120216. As a preferred embodiment of the invention, the immune cells are endowed with a CAR directed against the CD19 antigen in view of treating leukemia, more especially acute lymphoblastic leukemia (ALL), such as the CAR described in example 1 of WO2014184143, or one integrated at the TCR locus such as described in example 5 of WO2017062451 , which are both incorporated herein by reference.
According to one aspect of the present invention, the CAR polynucleotide sequences are not introduced or expressed into the cell lines until the step of cell differentiation (i.e. not during the reprogramming step). Typically, the multipotent cells, once obtained, can be transduced with retroviral vectors comprising the CAR polynucleotide to initiate differentiation. Expression of said CAR in said cells generally helps and can even trigger cell differentiation into effector cells, in particular into T-cells or NK-cells. According to one aspect, signalization sequences from specific receptors or effectors involved into cell differentiation, especially into differentiation from HSC -> CLP (common Lymphoid Progenitor cells) can be introduced into the cell or included into the CAR structure to stimulate said differentiation.
According to another aspect, the CAR polynucleotide sequences can be introduced into the genome of the primary cell during the initial genetic engineering step, for instance by site directed integration (e.g. integration of an exogenous sequence encoding the CAR by homologous recombination) and its expression being repressed or being inactive when the cells get reprogrammed.
According to a preferred aspect of the invention, the exogenous sequence encoding the CAR is introduced into an endogenous locus, such as one indicated in figure 2 under A, B, C, D, E, F and G, more preferably at a locus selected from those encoding TCRalpha, TCRbeta, PD1 , CTLA4, DCK and B2M. This introduction is generally performed with the effect of inactivating at least one endogenous coding sequence present at such locus.
For instance, the genetic engineering of the present invention can have the effect of reducing or preventing the expression of at least one protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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. A“recombinant or engineered TCR” is protein expressed into an immune cell to redirect its immune activity against a desired type of cells, especially cancer and infected cells having specific markers at their surface. It can replace or be-co-expressed with the endogenous TCR. In general, such recombinant TCR are single-chain TCRs comprising an open reading frame where the variable (V) a and nb domains are paired with a protein linker. This involves the molecular cloning of the TCR genes known to be specific for an antigen of choice. These chains are then introduced into T cells usually by means of a retroviral vector. Consequently, expression of the cloned TCRa and TCR3 genes endows the transduced T cell with a functional specificity determined by the pairing of these new genes [Ping, Y. et a/. (2017) T-cell receptor-engineered T cells for cancer treatment: current status and future directions. Protein Cell DOI 10.1007/s13238-016-0367] A component of a recombinant or engineered TCR is any functional subunit of a TCR, such as a recombined TCRa or TCR3, which is encoded by an exogenous polynucleotide sequence introduced into the cell.
By“pre-T”, is meant“pre-T-cell receptor”, which comprises a pre-Talpha (pTalpha) chain, which has the ability to transduce signals during the early stages of T-cell development as described by Harald von Boehmer [Unique features of the pre-T-cell receptor a-chain: not just a surrogate (2005) Nature Reviews Immunology. 5, 571-577]
The present invention is drawn to the multipotent cells obtainable by the present invention whether or not they have resulted into an established cell line. In particular, the invention is drawn to multipotent or iPS cells, and more especially genetically engineered iPS cells, which have been gene edited into at least one gene encoding a protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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), CD52 (Uniprot P31358), GR (Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150), DCK (Uniprot P27707), HPRT (Uniprot P00492), GGH (Uniprot Q92820), 32m (P61769) and HLA.
Multipotent or iPS cells according to the invention can be gene edited to be differentiated into more potent immune therapeutic cells by inactivation or repression of at least one gene encoding a protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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) and SIT1 (Uniprot Q9Y3P8).
Multipotent or iPS cells according to the invention can be gene edited to be differentiated into immune therapeutic cells resistant to drugs by inactivation or repression of at least one gene encoding a protein selected from CD52 (Uniprot P31358), GR (Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150), DCK (Uniprot P27707), HPRT (Uniprot P00492), GGH (Uniprot Q92820).
Multipotent or iPS cells according to the invention can be gene edited to be differentiated into less alloreactive immune therapeutic cells, by inactivation or repression of at least one gene encoding a protein selected from TCRalpha and/or TCRbeta, 32m and HLA.
Advantageously, multipotent or iPS cells according to the invention can be gene edited in at least two genes, such as for instance:
- TCR and PD1 ,
- TCR and CTLA4,
- TCR and DCK,
- TCR and GR,
- TCR and GGH,
- TCR and HPRT, - TCR and CD52,
- TCR and HLA,
- TCR and b2pΊ,
- PD1 and CTLA4,
- PD1 and DCK,
- PD1 and GR,
- PD1 and CD52,
- PD1 and b2pΊ,
- PD1 and CD52,
- CTLA4 and DCK,
- CTLA4 and GR,
- CTLA4 and GGH,
- CTLA4 and HPRT,
- CTLA4 and CD52,
- CTLA4 and HLA,
- CTLA4 and b2pΊ,
- DCK and GR,
- DCK and GGH,
- DCK and HPRT,
- DCK and CD52,
- DCK and HLA,
- DCK and b2pΊ,
- GR and GGH,
- GR and HPRT,
- GR and CD52,
- GR and HLA,
- GR and b2hi,
The present invention comes along with a general method for generating an engineered cell line to produce effector cells for immune therapy, said method comprising the step of differentiating cells from an induced multipotent cell line, which originates from a cell that has been genetically engineered, preferably as a primary cell, prior to being induced to a multipotent stage. Typically, said cell has been engineered when it was a primary immune cell to modify its immune properties before being reprogrammed as a pluripotent cell and possibly further, as an effector immune cell, such as a CAR positive T- cell or NK cell. As per the present invention, such multipotent or iPS cells are preferably engineered before having been reprogrammed. Such genetic engineering generally aim at:
- reducing alloreactivity, for instance by gene editing of TCR or 32m;
- conferring drug resistance, for instance by gene editing of DCK, GR or CD52,
- Improving homing, for instance by expression of lymphoid homing receptors,
- Improving cell survival, for instance by gene editing PD1 or CTLA4;
- Improving safety, for instance by expressing suicide genes such as rituximab epitopes,
- conferring hypersensitivity to a drug or pro-drug, by expression of a drug activator,
- redirecting antigen/ tissu specificity, for instance by expression of a CAR or a recombinant TCR;
- reducing control of immune checkpoints on the activation of the engineered immune cells by inhibiting their receptors and pathways controlled by these receptors,
- modifying HLA profile,
- inhibiting production of suppressive cytokines and metabolites, such as an inactivation of TGFb, TGFbR, IL-10, IL-10R, GCN2 or PRDM1.
- inhibiting genetic pathways that down regulate effector cell’s activation, inhibiting genetic pathways that trigger effector cell’s exhaustion, such as those controlled by PD1 , PDL1 , CTLA4, TIM3 or LAG 3; and/or
- inhibiting genetic pathways that down regulate T-cells proliferation and activation in Tumor induced inhibitory environment, in particular glucose deprived microenvironment, such as miR101 , miR26a, SERCA or BCAT.
According to one aspect of the invention, the engineered cells which have been processed into iPS cells by expression of at least one reprogramming factor selected from OCT4, SOX2, KLF4, C-MYC, NANOG, LIN28, being preferably transiently expressed, are generally subjected to an additional step where they are differentiated into hematopoietic stem cells, in particular CD34 + cells. Such cells are useful for administration into patients, especially in view of achieving bone marrow transplantation. Said method may thus comprise an additional step comprising assaying, sorting or screening the engineered cells, which are CD34 +.
Once the engineered hematopoietic CD34 + stem cells of the present invention get engrafted into their patient host, they can produce hematopoietic cells having the same genetic modifications conferring improved therapeutic properties such as previously proposed, in particular displaying:
- reduced alloreactivity,
- drug resistant,
- Improved homing, - Improved in-vivo survival,
- Improved safety
- hypersensitive to a drug or pro-drug,
- specific for an antigen,
- lower sensitivity to immune checkpoints such as PD1 , PDL1 , CTLA4, TIM3 or
LAG 3;
- HLA profile more compatible with that of the recipient host;
- lower sensitivity to suppressive cytokines or metabolites, through inactivation of TGFb, TGFbR, IL-10, IL-10R, GCN2 or PRDM1 ;
- resistant to viruses, such as HIV;
- lower exhaustion, and/or
- improved fitness in glucose deprived microenvironment.
These are examples of the advantages of using engineered CD34+ cells of the present invention into bone marrow cells engraftment protocols.
Therapeutic properties may also be obtained or combined with the previous ones by integration of exogenous nucleic acids (eg: transgene) coding for various polypeptides such as:
- a component of Chimeric Antigen Receptor (CAR);
- a component of a recombinant TCR;
- pre-T ;
- a cytokine, such as an interleukin or/and their receptors;
Different interleukins are known to stimulate T-cells activity, such as IL-2, IL-12, IL- 15 or IL-18, which expression are particularly beneficial to improve their cytotoxicity against infected or malignant cells. On another hand, other interleukins are known to stimulate Treg (Regulatory T-Cells), such as IL-10 and TGFbeta, which secretion in the culture medium in-vitro or in the Treg environment in-vivo is particularly useful to mitigate certain self-immune reactions or syndromes cause by T-cell hyperactivation.
- a HLA component, for instance to improve cells engraftment ;
- a product conferring resistance to a drug, such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut and Lckmut;
- a product conferring sensitivity to a drug;
- a peptide inhibitor of FOXP3; and/or
- a secreted inhibitor of Tumor Associated Macrophages (TAM), such as a
CCR2/CCL2 neutralization agent. According to a preferred embodiment of the invention, the cells are engineered to introduce an exogenous nucleic acid sequence encoding a CAR or a sequence coding for an interleukin gene at a precise locus, such as the TCR locus. Said CAR or a sequence coding for an interleukin gene may also be favorably introduced at the PD1 , PDL1 , CTLA4, TIM3 or LAG3 locus, where such sequences may or may not inactivate the expressions of the genes originally present at said loci. CAR architectures may be designed to be controllable by molecules that can be administered to patients. In particular, CARs or recombinant TCRs may comprise dimerization or cleavable domains to make them inducible or inactivated by a soluble compound, such as streptavidin, or rapamycin CID.
Exogenous coding sequence can be introduced by using viral vectors, such as a lentiviral vector or by using rare-cutting endonucleases combined with an AAV vector comprising the nucleic acid sequence to be integrated.
A method of the present invention as previously described may further comprise one step of transient expression of reprogramming factors such as TERT. Such reprogramming factors can be introduced into the cells under protein or RNA forms, for instance by electroporation and/or using nanoparticles. Viral vectors, especially non-integrative viral vectors, such as for instance episomal vectors, may also be used to reprogram the cells.
A method according to any one of claims 1 to 17, wherein said reprogramming factors under protein or RNA forms are introduced into the primary cells using nanoparticles.
According to the present invention, the engineered hematopoietic cells or cell lines originate from primary cells preferably obtained from a healthy donor. Said primary cell are generally mononuclear cell, such as peripheral blood cells (PBMC) isolated by leukapheresis, or may also originate from cord blood cells. Such primary cells are preferably T-cells or NK cells. Alternatively the primary cells may originate from patients, in particular as Tumor Infiltrating Lymphocytes (TIL). The primary cells may also originate from lymphoid progenitor cells, such as hematopoietic stem cells.
Once the primary cells have been genetically engineered and reprogrammed as per the present invention, one or several step(s) of redifferentiation can take place. The step(s) is (are) preferably performed by contacting the cells with notch ligand positive cells, such as OP9-DL1 cells. Said notch ligand positive cells can be engineered stromal cells that can form three dimension structures such as in artificial thymic organoids such as described by Seet, C. et a/. [Generation of mature T cells from human hematopoietic step and progenitor cells in artificial thymic organoids (2017) Nat Methods. 14(5): 521-530] . Said notch ligand positive cells can be engineered to express specific antigen, such as to drive the differentiation of the cells towards a desired hematopoietic lineage. The cells are differentiated in until obtaining functional progenitor, such as CD34 + cells, or more mature immune cells, such as CD3 +, CD4 + or CD8+. According to a preferred embodiment, the cells are differentiated until obtaining functional effector T-cells.
The cell products resulting from the present invention include the genetically engineered induced multipotent cell obtainable at the different steps of the re-programming process illustrated in Figure 3, especially an induced multipotent cell, wherein said cell has been genetically modified at the TCR locus.
Said induced multipotent cell, as well as all the cells deriving thereof by differentiation through the hematopoietic lineages, can comprise two, preferably three, more preferably four genetic modifications as previously described, in particular at least one introduced at one locus encoding a protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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), CD52 (Uniprot P31358), GR (Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150), DCK (Uniprot P27707), HPRT (Uniprot P00492), GGH (Uniprot Q92820), 32m (P61769) and HLA. The selected loci can encode polypeptides sharing identity with the previous proteins, preferably at least 75 % identity, preferably more than 80 %, more preferably at least 95 % identity.
The present invention extends to the induced engineered multipotent cell line(s) stably established for producing multipotent cells, which can be further differentiated according to the invention, as well as the intermediate cells produced by such cell line(s).
As an intermediary stage, the invention encompasses primary immune cells, which has been genetically engineered and further transfected with, proteins or nucleic acids, such as mRNA, encoding at least one reprogramming factor(s) to induce pluripotency, such as OCT4, SOX2, KLF4, C-MYC, TERT, NANOG and/or LIN28, and thereby the engineered cells comprising same.
The present inventions extends to libraries or bank of cells or cell lines useful to produce different types of engineered hematopoietic cells, especially effector immune cells, which may originate from various donors and target different disease or groups of patients.
The inventions also encompasses compositions comprising a therapeutically effective amount of effector immune cells produced or differentiated from the induced multipotent cell lines established by the methods of the present invention described in this specification, and more specifically by the methods comprising at least one of the following steps of:
1 - Gene editing step into primary T-cells by electroporation of mRNA encoding rare-cutting endonuclease, preferably TALE-nucleases, as illustrated in the experimental section. Such step can be performed to inactivate CCR5 and provide with hematopoietic cells, which are subsequently resistant to HIV. Such step can also be performed to obtain the insertion of a CAR at the TCR locus, for instance [CAR]pos [TCR]neg [dcK]neg effector immune cells resistant to purine nucleotide analogs (PNA), such as fludarabine and chlorofarabine. Such cells can be reprogrammed into iPS cells and further differentiation into CD34 + cells, HSC or cells lines, for their use in allogeneic cell immunotherapy treatments.
2- Prior to cell reprogramming, selecting primary cells which have been genetically engineered and/or screened in view of improving their therapeutic properties. For instance, TCR-deficient T-cells can be isolated and/or purified in order to be subsequently reprogrammed into iPS cells. Such cells may be assayed by using transient expression of a CAR or any attributes for their suitability in therapy before being actually transformed with reprogramming factors as illustrated herein in the experimental section.
3- Reprogramming of the selected primary cells into pluripotent iPS cells. Several techniques have been described to reprogram cells into multipotent cells, such as the transient expression of so-called Yamanaka factors as illustrated in the experimental section.
4- Isolating multipotent cells originating from genetically engineered primary immune cells to generate cell lines. In general, the induced multipotent cells are picked, isolated and colonies are checked for the genetic modifications initially performed on the primary cells. Then, cell lines are established for their continuous production of cells which may be stored or differentiated into hematopoietic cells as illustrated in the experimental section. For instance, the cells can be co-cultured with notch ligand positive stromal cells, such as in Artificial Thymic Organoid (ATO) cultures, until obtaining CD34 +, CD4+ and/or CD8+ immune cells.
5- Optionally transducing the hematopoietic generated cells with retroviral vectors for CAR (re)expression. The engineered Pluripotent cells obtainable by the present invention, which may be TCR negative, can be transduced with lentiviral vectors for re- expression of CAR or a recombinant TCR to obtain differentiation into adoptive T-cells useful for cell immunotherapy. In some instances, as illustrated in the experimental section, the CAR may have been inserted into the genome of the cells by homologous recombination during the initial gene editing step.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to limit the scope of the claimed invention
EXAMPLES
Generation of TCAR anti-CD22lpos effector immune cells by gene editing of primary PBMC cells, reprogramming and differentiation by co-culture in artificial thymic organoids.
1 - Gene edited step into primary T-cells
1 -1 Generation of |TCR1neg rdcK1neg effector immune cells resistant to purine nucleotide analogs (PNA), such as fludarabine and chlorofarabine.
Electroporation of T-cells from PBMCs with mRNA encoding TALEN®
TALEN® designate TALE-nucleases developed by Cellectis (8, rue de la Croix Jarry, 75013 PARIS), which are fusions of bespoke TALE binding domains with the nuclease domain of Fok1 as originally described by Voytas et al. (WO2011072246).
Cell culture reagents, X-vivo-15 are obtained from Lonza (Basel, Switzerland cat#BE04-418Q), IL-2 from Miltenyi Biotech (Bergisch Gladbach, Germany, cat#130-097- 748), human serum AB from Seralab (West Sussex, UK cat#GEM-100-318), human T activator CD3/CD28 from Life Technologies (Beverly, MA, cat#11132D), MACS-LD column from Miltenyi Biotech (cat#130-042-901 ), fixable viability dye eFluor780 from eBioscience (San Diego, CA, cat#65-0865-14). CFSE dye are obtained from Life Technologies (cat#C34554) and anti-IFNy ELISA kit are obtained from R&D systems (Minneapolis, MN, cat#DIF50).
Mononuclear cells (PBMCs) are obtained from the peripheral blood samples collected by venipuncture of healthy donors using Ficoll density centrifugation.
To generate TCR and dCK-deficient T cells, the PBMCs are activated and transfected according to the procedure described in Galetto, R. et al. [Pre-TCRa supports CD3- dependent reactivation and expansion of TCRodeficient primary human T-cells (2014) Mol Ther Methods Clin Dev.14021f] Briefly regarding transfection, 4 days after their activation by Dynabeads human T activator CD3/CD28, 5.106 T cells are simultaneously transfected with 5 pg of each mRNA encoding left and right arms of TALEN® targeting the TCRa constant chain and dCK exon 2 (Uniprot P27707).
Transfection is performed using Agilpulse technology, by applying two 0.1 ms pulses at 3,000 V/cm followed by four 0.2 ms pulses at 325 V/cm in 0.4 cm gap cuvettes and a final volume of 200 pi of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA). Cells were then immediately diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 (final concentration) and 5% human serum AB. Transfected T cells are eventually diluted at 1 x 106/ml and kept in culture at 37 °C in the presence of 5% CO2 and 20 ng/ml IL-2 (final concentration) and 5% human AB serum for further characterization.
This first set of experiments were performed to inactivate TCRalpha and the DCK genes by NHEJ through expression of mRNAs encoding the TALEN heterodimers corresponding to SEQ ID NO:1 and 2 (DCK2-R1 and DCK-R2) and SEQ ID NO:3 and 4 (TRAC-L and TRAC-R) listed in Table 1. These endonucleases were directed to the respective polynucleotide target sequences SEQ ID NO:9 and SEQ ID NO:10.
1 -2 TALEN®-mediated double targeted integration of IL-15 and CAR encoding matrices in T-cells at TCR, CD25 or PD1 endogenous loci by homologous recombination prior to reprogramming.
Materials
X-vivo-15 was obtained for Lonza (cat#BE04-418Q), IL-2 from Miltenyi Biotech (cat#130-097-748), human serum AB from Seralab (cat#GEM-100-318), human T activator CD3/CD28 from Life Technology (cat#1 1 132D), QBEND10-APC from R&D Systems (cat#FAB7227A), vioblue-labeled anti-CD3, PE-labeled anti-LNGFR, APC-labeled anti- CD25 and PE-labeled anti-PD1 from Miltenyi (cat# 130-094-363, 130-1 12-790, 130-109-021 and 130-104-892 respectively) 48 wells treated plates (CytoOne, cat#CC7682-7548), human IL-15 Quantikine ELISA kit from R&D systems (cat#S1500), ONE-Glo from Promega (cat#E61 10). AAV6 batches containing the different matrices were obtained from Virovek, PBMC cells were obtained from Allcells, (cat#PB004F) and Raji-Luciferase cells were obtained after Firefly Luciferase-encoding lentiviral particles transduction of Raji cells from ATCC (cat#CCL-86).
Methods a) Transfection-transduction
The double targeted integration at TRAC and PD1 or CD25 loci were performed as follows. PBMC cells were first thawed, washed, resuspended and cultivated in X-vivo-15 complete media (X-vivo-15, 5% AB serum, 20 ng/ml_ IL-2). One day later, cells were activated by Dynabeads human T activator CD3/CD28 (25 uL of beads/1 E6 CD3 positive cells) and cultivated at a density of 1 E6 cells/mL for 3 days in X-vivo complete media at 37°C in the presence of 5% CO2. Cells were then split in fresh complete media and transduced/transfected the next day according to the following procedure. On the day of transduction-transfection, cells were first de-beaded by magnetic separation (EasySep), washed twice in Cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts) and resuspended at a final concentration of 28E6 cells/mL in the same solution. Cellular suspension was mixed with 5 pg mRNA encoding TRAC TALEN® arms (SEQ ID NO:3 and 4) in the presence or in the absence of 15 pg of mRNA encoding arms of either CD25 or PD1 TALEN® (SEQ ID NO:5 and 6 and SEQ ID NO:7 and 8 respectively) in a final volume of 200 pi. TALEN® is a standard format of TALE-nucleases resulting from a fusion of TALE with Fok-1 Transfection was performed using Pulse Agile technology, by applying two 0.1 mS pulses at 3,000 V/cm followed by four 0.2 mS pulses at 325 V/cm in 0.4 cm gap cuvettes and in a final volume of 200 pi of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts). Electroporated cells were then immediately transferred to a 12-well plate containing 1 mL of prewarm X-vivo-15 serum-free media and incubated for 37°C for 15 min. Cells were then concentrated to 8E6 cells/mL in 250 pL of the same media in the presence of AAV6 particles (MOI=3E5 vg/cells) comprising the donor matrices in 48 wells regular treated plates. After 2 hours of culture at 30°C, 250 pL of Xvivo-15 media supplemented by 10% AB serum and 40 ng/ml IL-2 was added to the cell suspension and the mix was incubated 24 hours in the same culture conditions. One day later, cells were seeded at 1 E6 cells/mL in complete X-vivo-15 media and cultivated at 37°C in the presence of 5% CO2. b) Activation-dependent expression of ALNGFR and secretion of IL15
Engineered T-cells were recovered from the transfection-transduction process described earlier and seeded at 1 E6 cells/mL alone or in the presence of Raji cells (E:T=1 :1 ) or Dynabeads (12.5 uL/1 E6 cells) in 100 pL final volume of complete X-vivo-15 media. Cells were cultivated for 48 hours before being recovered, labeled and analyzed by flow cytometry. Cells were labeled with two independent sets of antibodies. The first sets of antibodies, aiming at detecting the presence of ALNGFR, CAR and CD3 cells, consisted in QBEND10-APC (diluted 1/10), vioblue-labeled anti CD3 (diluted 1/25) and PE-labeled anti- ALNGFR (diluted 1/25). The second sets of antibodies, aiming at detecting expression of endogenous CD25 and PD1 , consisted in APC-labeled anti-CD25 (diluted 1/25) and vioblue-labeled anti PD1 (diluted 1/25).
The same experimental set up was used to study IL-15 secretion in the media. Cells mixture were kept in co-culture for 2, 4, 7 and 10 days before collecting and analyzing supernatant using an IL-15 specific ELISA kit. c) Serial killing assay
To assess the antitumor activity of engineered CAR T-cells, a serial killing assay was performed. The principle of this assay is to challenge CAR T-cell antitumor activity everyday by a daily addition of a constant amount of tumor cells. T umor cell proliferation, control and relapse could be monitored via luminescence read out thanks to a Luciferase marker stably integrated in Tumor cell lines.
Typically, CAR T-cells are mixed to a suspension of 2.5x105 Raji-luc tumor cells at variable E:T ratio (E:T=5:1 or 1 :1 ) in a total volume of 1 ml. of Xvivo 5% AB, 20 ng/uL IL-2. The mixture is incubated 24 hours before determining the luminescence of 25 uL of cell suspension using ONE-Glo reagent. Cells mixture are then spun down, the old media is discarded and substituted with 1 ml. of fresh complete X-vivo-15 media containing 2.5x105 Raji-Luc cells and the resulting cell mixture is incubated for 24 hours. This protocol is repeated 4 days.
Experiments and results
This example describes methods to improve the therapeutic outcome of CAR T-cell therapies by integrating an IL-15/soluble IL-15 receptor alpha heterodimer (I L15/sl L15ra) expression cassette under the control of the endogenous T-cell promoters regulating PD1 and CD25 genes. Because both genes are known to be upregulated upon tumor engagement by CAR T-cells, they could be hijacked to re-express IL- 1 L15/sl L15ra only in vicinity of a tumor. This method aims to reduce the potential side effects of I L15/sl L15ra systemic secretion while maintaining its capacity to reduced activation induced T-cell death (AICD), promote T-cell survival, enhance T-cell antitumor activity and to reverse T-cell anergy.
The method developed to integrate I L15/sl L15ra at PD1 and CD25 loci consisted in generating a double-strand break at both loci using TALEN in the presence of a DNA repair matrix vectorized by AAV6. This matrix consists of two homology arms embedding IL15/slL15ra coding regions separated by a 2A cis acting elements and regulatory elements (stop codon and polyA sequences). Depending on the locus targeted and its involvement in T-cell activity, the targeted endogenous gene could be inactivated or not via specific matrix design. When CD25 gene was considered as targeted locus, the insertion matrix was designed to knock-in (Kl) I L15/sl L15ra without inactivating CD25 because the protein product of this gene is regarded as essential for T-cell function. By constrast, because PD1 is involved in T-cell inhibition/exhaustion of T-cells, the insertion matrix was designed to prevent its expression while enabling the expression and secretion of I L15/sl L15ra.
To illustrate this approach and demonstrate the feasibility of double targeted insertion in primary T-cells, three different matrices were designed (figure 4A, 4B and 4C). The first one named CARm represented by SEQ ID NO:13 was designed to insert an anti- CD22 CAR cDNA at the TRAC locus in the presence of TRAC TALEN® (SEQ ID NO:3 and 4). The second one, IL-15_CD25m (SEQ ID NO:14) was designed to integrate IL15, slL15ra and the surface marker named ALNGFR cDNAs separated by 2A cis-acting elements just before the stop codon of CD25 endogenous coding sequence using CD25 TALEN® (SEQ ID NO:5 and 6). The third one, IL-15_PD1 m (SEQ ID NO:15), contained the same expression cassette and was designed to integrate in the middle of the PD1 open reading frame using PD1 TALEN® (SEQ ID NO:7 and 8). The three matrices contained an additional 2A cis-acting element located upstream expression cassettes to enable co- expression of I L15/sl L15ra and CAR with the endogenous gene targeted.
2 -Selection step of efficient engineered cells
2.1 - Selection of the rdcK1neg effector primary immune cells resistant to
Figure imgf000037_0001
fludarabine and chlorofarabine
Isolation of T CRo -deficient T cells using magnetic separation.
TCRa3-deficient cells are purified according to the protocol described by Valton, J. et a/. [Efficient strategies for TALEN-mediated genome editing in mammalian cell lines (2014) Methods, 69:151 -170]. Briefly, about 107 T cells recovered 6 days after TALEN® treatment are labeled with biotin conjugated anti-TCRa3 antibody MicroBeads before being loaded onto a MACS LD-Column placed in the magnetic field of a MACS Separator. Using this procedure, the magnetically labeled CD3-positive cells are retained in the column while the unlabeled TCRa3-deficient cells can be recovered in the flow through. One round of purification is usually necessary to obtain a homogeneous population of TCRa3-deficient cells (purity > 99%).
Clofarabine, fludarabine (fludarabine-phosphate), and cytarabine are obtained from Sigma (St Louis, MO, cat #C7495,# F9813, and #C3350000 respectively), diluted according to the manufacturer protocol. The concentrations of diluted PNA solutions were accurately determined by spectrophotometry. IC50 of a given drug is defined as the concentration of drug need to decrease the cellular viability by 50%. To determine IC50 of a clofarabine, fludarabine and cytarabine, 105 T-cells are incubated in the presence of increasing concentration of drugs (from 0 to 100 pmol/l typicially) for 48 hours and in a total volume of 100 pi X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum. Cells are washed with 100 mI of phosphate buffer saline (PBS) and then labeled by eFIuor 780 for 15 minutes at 4 °C according to the manufacturer protocol. Labeling is stopped by addition of PBS 2% fetal veal serum (SVF) and cells are eventually fixed by 4% paraformaldehyde (PFA) before being analyzed by flow cytometry to determine their viability. Evolution of cell viability as a function of PNA concentration is fitted with the drift R package software using the formula: y = 1/(1 + EXP((LOG10(x) - LOG10(IC50))/z))*100 with y, x, and z corresponding to viability frequency, the concentration of drug, and the scale parameter.
After the purification step mentioned above [TCR]neg Cells are cultured in the presence of clinically relevant doses of PNA for 10 days in the presence of a combination of clofarabine and cytarabine (1 and 10 pmol/l respectively) reported as being consistent with their respective average Cmax after their uptake in human patients [Valton, J. et a/. A Multidrug-resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy Molecular Therapy 23(9): 1507-1518].
Evaluation of Cytotoxic functionality of PNA resistant engineered cells by transient expression of mRNA encoding CAR anti-CD22
CAR anti-CD22 has been constructed by assembling sequences encoding 41 BB costimulatory domain, the CD3z activation domain, the CD8a transmembrane domain, a CD8a hinge and ScFv of the antibody anti-CD22 m971 formerly used by Haso W. et al. [Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia (2013) Blood 14;121 (7):1 165-74] . The resulting polynucleotide sequence has been cloned into an expression vector and transcribed into mRNA. RNA Transfection is performed using Agilpulse technology, by applying two 0.1 ms pulses at 3,000 V/cm followed by four 0.2 ms pulses at 325 V/cm in 0.4 cm gap cuvettes and a final volume of 200 mI of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA). Cells are then immediately diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 (final concentration) and 5% human serum AB. Transfected T cells are eventually diluted at 1 c 106/ml and kept in culture at 37 °C in the presence of 5% CO2 and 20 ng/ml IL-2 (final concentration) and 5% human AB serum.
Flow-based cytotoxicity assay.
The cytolytic activity and specificity of the engineered CAR T cells are assessed according to the flow cytometry-based cytotoxicity assay described in Zhao, B. et al.
[Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res. (2010) 70:9053- 9061]. This assay consists of labeling 104 NALM-16 (CD22+) and 104 SUP-T1 (CD22 negative control) cells with 1 pmol/l CellTrace CFSE and 1 pmol/l CellTrace violet respectively (Life Technology) and coincubate them with 105 effector CAR T cells (E/T ratio of 10:1 ) in a final volume of 100 mI X-Vivo-15 media, for 5 hours at 37 °C. Cells are then recovered and labeled with eFluor780 viability marker before being fixed by 4% PFA as described above. Fixed cells are then analyzed by flow cytometry to determine their viability. The frequency of specific cell lysis is calculated using the following formula:
Frequency of specific cell lysis = (Via NALM-16/Via SUP-T1 )/(Via NALM-16/Via SUP-T1 ) where Via NALM-16 and Via SUP-T1 correspond respectively to the % of viable NALM-16 and SUP-T1 cells obtained after 5h in the presence of CAR T cells and where Via NALM- 16 and Via SUP-T1 correspond respectively to the % of NALM-16 and SUP-T1 cells obtained after 5h in the absence of CAR T cells.
Allogeneic mixed lymphocyte reaction.
20 x 106 PBMC freshly purified from a donor B (PBMC B) are labeled by a 10-minute incubation with 2 nmol/l CFSE in the dark, at 37 °C in a final volume of 5 ml. CFSE- labeled PBMC B were then diluted two times in fetal bovine serum, and washed with X- Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum. Cell number and viability is determined and their concentration adjusted to 1 c 106 viable cells/ml. To perform mixed lymphocyte reaction, CFSE-labeled PBMC B are incubated with the engineered CAR T cells that have originated from a different blood donor (CAR T cell A) in X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% human AB serum. CFSE labeled PBMC viability, activation and proliferation are determined after 6 days of incubation, by flow cytometry and anti IFNy ELISA.
2.2 - Selection of primary effector cells TCAR anti-CD221pos by
Figure imgf000039_0001
integration of the CAR polynucleotide sequence at the TCR locus.
We first assessed the efficiency of double targeted insertion in T-cells by transducing them with one of the AAV6 encoding IL15/slL15ra matrices (SEQ ID NO:41 ; pCLS30519) along with the one encoding the CAR and subsequently transfected the corresponding TALEN®. AAV6-assisted vectorization of matrices in the presence of mRNA encoding TRAC TALEN (SEQ ID NO:3 and 4) and PD1 TALEN® (SEQ ID NO:7 and 8) or CD25 TALEN® (SEQ ID NO:5 and 6) enabled expression of the anti CD22 CAR in up to 46% of engineered T-cells (figure 5).
To determine the extent of IL15m integration at CD25 and PD1 locus, engineered T-cells were activated with either antiCD3/CD28 coated beads or with CD22 expressing Raji tumor cells. 2 days post activation, cells were recovered and analyzed by FACS using LNGFR expression as I L15/sl L15ra secretion surrogate (figure 6 and 7). Our results showed that antiCD3/CD28 coated beads induced expression of ALNGFR by T-cells containing IL- 15m_CD25 or IL-15m_PD1 , independently of the presence of the anti CD22 CAR (figure 6A-B). Tumor cells however, only induced expression of ALNGFR by T-cell treated by both CARm and IL-15m. This indicated that expression of ALNGFR could be specifically induced through tumor cell engagement by the CAR (figures 7 and 8).
As expected the endogenous CD25 gene was still expressed in activated treated T- cells (figures 9 and 10) while PD1 expression was strongly impaired (figure 14).
To verify that expression of ALNGFR correlated with secretion of IL15 in the media, T-cells expressing the anti-CD22 CAR and ALNGFR were incubated in the presence of CD22 expressing Raji tumor cells (E:T ratio = 1 :1 ) for a total of 10 days. Supernatant were recovered at day 2, 4, 7 and 10 and the presence of I L 15 was quantified by ELISA assay. Our results showed that IL15 was secreted in the media only by T-cells that were co-treated by both CARm and IL15m matrices along with their corresponding TALEN® (figure 15). T- cell treated with either one of these matrices were unable to secrete any significant level of IL15 with respect to resting T-cells. Interestingly, IL-15 secretion level was found transitory, with a maximum peak centered at day 4 (Figure 16).
To assess whether the level of secreted IL-15 could impact CAR T-cell activity, CAR T-cell were co-cultured in the presence of tumor cells at E:T ratio of 5:1 for 4 days. Their antitumor activity was challenged everyday by pelleting and resuspended them in a culture media lacking IL-2 and containing fresh tumor cells. Antitumor activity of CAR T-cell was monitored everyday by measuring the luminescence of the remaining Raji tumor cells expressing luciferase. Our results showed that CAR T-cells co-expressing IL-15 had a higher antitumor activity than those lacking IL15 at all time points considered (figure 17).
Thus, together our results showed that we have developed a method allowing simultaneous targeted insertions of CAR and IL15 cDNA at TRAC and CD25 or PD1 loci. This double targeted insertion led to robust expression of an antiCD22 CAR and to the secretion of I L 15 in the media. Levels of secreted I L15 were sufficient to enhance the activity of CAR T-cells. Table 1 : Polypeptide sequences referred to in the examples.
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Table 2: polynucleotide sequences referred to in example 2.
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0002
Once the previous types of cells have been tested for (1 ) their reduced allogenicity due to their inactivation of TCR and/or (2) their resistance to PNA due to the inactivation of DCK and (3) their functionality with respect to their constant or transient expression of CAR, they can be processed for dedifferentiation (i.e. reprogramming step). 3 - Reprogramming of the selected rdcK1neg peripheral blood T-cells into
Figure imgf000046_0001
pluripotent iPS cells
Transient repeated expression of Yamanaka factors
RNA have been synthesized with the MEGAscript T7 kit (Ambion, Austin, TX), with 1 .6 pg of purified tail PCR product to template each 40 pl_ reaction. A custom ribonucleoside blend was used comprising 3'-0-Me-m7G(5')ppp(5')G ARCA cap analog (New England Biolabs), adenosine triphosphate and guanosine triphosphate (USB, Cleveland, OH), 5- methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies, San Diego, CA). Final nucleotide reaction concentrations were 6 mM for the cap analog, 1 .5 mM for guanosine triphosphate, and 7.5 mM for the other nucleotides. Reactions are incubated 3-6 hr at 37°C and DNase treated as directed by the manufacturer. RNA was purified with Ambion MEGAclear spin columns, then treated with Antarctic Phosphatase (New England Biolabs) for 30 min at 37°C to remove residual 5'- triphosphates. Treated RNA was repurified, quantitated by Nanodrop (Thermo Scientific, Waltham, MA), and adjusted to 100 ng/pL working concentration by addition of Tris-EDTA (pH 7.0). RNA reprogramming cocktails are prepared by pooling individual 100 ng/pL RNA stocks to produce a 100 ng/pL (total) blend. Volumetric ratios used for pooling are as follows: 170:160:420:130:90 (KLF4:c-MYC:OCT4:SOX2:LIN28). RNA Transfection of reprogramming factors was performed every two days using Agilpulse technology, by applying two 0.1 ms pulses at 3,000 V/cm followed by four 0.2 ms pulses at 325 V/cm in 0.4 cm gap cuvettes and a final volume of 200 pi of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA). Cells are then immediately diluted in X- Vivo-15 media supplemented by 20 ng/ml IL-2 (final concentration) and 5% human serum AB. Transfected T cells are eventually diluted at 1 c 106/ml and kept in culture at 37 °C in the presence of 5% CO2 and 20 ng/ml IL-2 (final concentration) and 5% human AB serum
Gamma-irradiated human neonatal fibroblast feeders (GlobalStem, Rockville, MD) are seeded at 33,000 cells/cm2. Nutristem media was replaced daily, 4 hr after transfection, and supplemented with 100 ng/mL bFGF and 200 ng/mL B18R (eBioscience, San Diego, CA). Where applied, VPA is added to media at 1 mM final concentration on days 8-15 of reprogramming. Low-oxygen experiments were carried out in a NAPCO 8000 WJ incubator (Thermo Scientific). Media are equilibrated at 5% 02 for approximately 4 hr before use. Cultures are passaged with TrypLE Select recombinant protease (Invitrogen). Y27632 ROCK inhibitor (Watanabe et al., 2007) is used at 10 mM in recipient plates until the next media change.
iPS colonies are mechanically picked and transferred to MEF-coated 24-well plates with standard hESC medium containing 5 mM Y27632 (BioMol, Plymouth Meeting, PA). The hESC media comprises DMEM/F12 supplemented with 20% Knockout Serum Replacement (Invitrogen), 10 ng/mL of bFGF (Gembio, West Sacramento, CA), 1 x nonessential amino acids (Invitrogen), 0.1 mM b-ME (Sigma), 1 mM L-glutamine (Invitrogen), plus antibiotics. Clones are mechanically passaged once more to MEF-coated 6-well plates, and then expanded via enzymatic passaging with collagenase IV (Invitrogen).
Colonies with well-defined hESC-like morphology are first observed from 20 days after first transfection colonies with distinct flat and compact morphology with clear-cut round edges reminiscent of hES cells after a slightly longer latency of around 35 days as described by Warren, L. et al. [Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA (2010) Cell Stem Cell. 7 - 5 (5): 618-630] are isolated.
4 - Isolating multipotent resulting iPS cells to generate cell lines
The iPS cell lines are analyzed in Immunohistochemistry for expression of multipotent cells markers. Colonies that are stained positive for Tra-1 -81 , NANOG, OCT4, Tra-1 -60, SSEA4 markers by multicolor flow cytometry analysis are selected and passaged to establish homogenous cell cultures (cell lines) by using Inside Stain Kit (Miltenyi Biotec GmbH ref: #130-090-477). Different cell lines are obtained in this way that are frozen and banked, to be later processed for differentiation, for instance by co-culture with notch ligand positive stromal cells, such as in Artificial Thymic Organoid (ATO) cultures.
5- Transduction with lentiviral vectors for CAR anti-CD22 expression.
Figure imgf000048_0001
Transduction of iPS derived cells with CAR anti-CD22 lentiviral vectors
Lentiviral vectors comprising the polynucleotide sequence encoding the CAR anti-CD22 transiently expressed in above step 2 are used to transduce the multipotent cells previously obtained for obtaining stable expression of the CAR in these cells, and further in the cells that will be differentiated into effector cells for their antigen dependent activation.
106 of the above cells are plated in 6-well non-treated plates coated with 20 pg/ml Retronectin (Clontech, Mountain View, CA) in 1 ml X-VIVO-15 (Lonza, Basel, Switzerland) supplemented with 50 ng/ml of recombinant human SCF, FLT3L, and TPO, and 10 ng/ml IL-3 (Peprotech, Rocky Hill, NJ) for 12— 18 h , after which concentrated lentiviral supernatant was to a final concentration of 1-2x107 TU/ml. Mock-transduced cells are cultured in identical conditions without addition of lentiviral vector.
6- Differentiating the engineered multipotent cells from said cell linefs) into T-cells
Artificial Thymic Organoid (ATO) cultures
OP9-DL1 cells are prepared as described by Seet, C. et al. [Generation of mature T cells from human hematopoietic step and progenitor cells in artificial thymic organoids (2017) Nat Methods. 14(5): 521-530]. Alternatively MS5-hDLL1 can be used, which are murine stromal cell line transduced with a lentiviral vector encoding human DLL1 and eGFP. The cells are set up in monolayer cultures by seeding cells into 0.1 % gelatin-coated 12 well plates 1-2 days prior to use to achieve 70-80% confluence. Medium is aspirated from monolayers and 1 .5x104 FACS purified to select CD34+CD3- cells. The cells transduced with the CAR in step 5, are plated on stromal monolayers in 2 ml of medium composed of MEMa, 20% FBS, 30 mM L-Ascorbic acid, 5 ng/ml rhFLT3L, and 5 ng/ml rhlL-7. Cells are transferred to new stromal cell monolayers every 4-5 days by harvesting cells, filtering through a 50 pm nylon strainer, and replating in fresh medium. When confluent, cells are split into multiple wells containing fresh stromal layers.
T cell cytokine assays
Mature CD8SP or CD4SP cells from ATOs are isolated by magnetic negative selection using the CD8+ or CD4+ Isolation Kits (Miltenyi) and sorted by FACS to further deplete CD45RO+ cells (containing immature naive T cells and CD4ISP precursors). Purified T cell populations are plated in 96-well U-bottom plates in 200 pi AIM V (ThermoFisher Scientific, Grand Island, NY) with 5% human AB serum (Gemini Bio- Products, West Sacramento, CA). PMA/ionomycin/protein transport inhibitor cocktail or control protein transport inhibitor cocktail (eBioscience, San Diego, CA) are added to each well and incubated for 6h. Cells are stained for CD3, CD4, and CD8 (Biolegend, San Diego, CA) and UV455 fixable viability dye (eBioscience, San Diego, CA) prior to fixation and permeabilization with an intracellular staining buffer kit (eBioscience, San Diego, CA) and intracellular staining with antibodies against IFNy, TNFa, IL-2, IL-4, or IL-17A (Biolegend, San Diego, CA).
T cell activation and proliferation assays
ATO-derived CD8SP or CD4SP T cells are isolated by negative selection MACS as above (with further FACS purification of CD4SP T cells as described above) and labeled with 5 mM CFSE (Biolegend, San Diego, CA). Labeled cells are incubated with anti- CD3/CD28 beads (ThermoFisher Scientific, Grand Island, NY) in AIM V/5% human AB serum with 20 ng/ml rhlL-2 (Peprotech, Rocky Hill, NJ). For in vitro cell expansion assays, 5x103-1 x104 ATO-derived CD8SP or CD4SP T cells isolated as above are plated in 96- well U-bottom plates in 200 mI, and activated/expanded with anti-CD3/28 beads and either 20 ng/mL IL-2 or 5 ng/mL IL-7 and 5 ng/mL IL-15 (Peprotech). Beads are removed on day 4, and fresh medium and cytokines are added every 2-3 days with replating into larger wells as needed. Cells are counted weekly with a hemacytometer.
7 - T cell activation and proliferation assays
In vitro cytotoxicity assays
ATO derived CAR positive cells are diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% CTS™ Immune Cell SR and diluted at 1x106 cells/ml and kept in culture at 37°C in the presence of 5% C02. At day 3, 7 and 1 1 , cell viability, CD4 and CD8 phenotypes, CAR positive cells frequency are assessed by flow cytometry by using a recombinant CD22 protein corresponding to the membrane proximal domain of CD22 or a recombinant CD22 protein corresponding to the whole extracellular domain of CD22.
Cytolytic activity of anti-CD22 CAR+ ATO derived cells is assessed in a flow-based cytotoxicity assay after an overnight coculture with antigen presenting cells (NALM-16) at 37°C in the presence of 5% C02. CAR positive cells and target cells are cocultured in X-Vivo-15 medium at effector (CAR+) : target ratios of 0.1 :1 , 0.2:1 , 0.5:1 and 1 :1. Culture medium was supplemented with 5% CTS™ Immune Cell SR. To distinguish positive CD22 positive (NALM-16) and negative (SUP-T1 ) tumor cell lines, NALM-16 target cells were stained with CFSE while SUP-T1 are stained with the CellT race violet proliferation marker. At the end of the coculture, cell viability is measured and the percentage of specific lysis is calculated after normalization to non- specific target cell lysis.
At the same time point, the degranulation activity of CART cells is measured using a flow-based degranulation assay.
CART and target cells are cocultured in X-Vivo-15 medium at effector (CAR+):target ratios of 2:1. At the end of the coculture, cell viability is measured and the degranulation activity as represented by CD107a expression is determined by flow cytometry on CD8 CART cells.
As a last test of CART cells activity, the ability of CART cells to secrete IFN-y following coculture with irradiated antigen presenting cells for 24h is evaluated. CAR+ T cells IFN-g secretion capacity towards antigen presenting cells (NALM-16) is assessed in an ELISA immunoassay Quantikine® measuring IFN-g secretion (ELISA Human IFN-y Immunoassay KIT, R&D Systems).
In vivo tumor assays
4-6 week old male NOD scid gamma (NSG) (Jackson Laboratory, Bar Harbor, Maine) are subcutaneously implanted with 2x105 NALM-16 cells, which can be imaged for tumor bioluminescence on day 3 by intraperitoneal injection of luciferin. First group of mice is treated with mock cells (not transduced with CAR anti CD22), a second group is treated with non-genetically engineered CAR positive ATO-derived [CAR] pos[TCR]pos [DCKjpos + combination of clofarabine and cytarabine (1 and 10 pmol/l respectively), a third group is treated with engineered ATO-derived [CARjpos [TCRjneg [DCKjneg cells without PNA treatment, a fourth group is treated with engineered ATO-derived [CARjpos [TCRjneg [DCKjneg cells + combination of clofarabine and cytarabine (1 and 10 pmol/l respectively).
ATO-derived [CAR]pos [TCR]neg [DCK]neg cells are expanded in-vivo for 14 days. Injection of PBS into control mice is also performed. Tumor bioluminescence is repeated every 3-4 days for at least 21 days, after which mice are sacrificed based on disease burden criteria. Monitoring show a reduction of the tumor burden in mice injected with the ATO- derived [CAR]pos [TCR]neg [DCK]neg cells in third and fourth group, while tumor progression is observed in the first and second groups of mock cells.

Claims

1. A method for producing engineered progenitor hematopoietic stem cells (HSC) or generating an engineered cell line to produce therapeutic cells for immune therapy, wherein said method comprises the steps of:
a) Providing primary immune cells from a donor or a patient;
b) Genetically engineering said cell to modify its therapeutic properties; and c) Processing the immune cell engineered in step b) into at least one multipotent cell line.
2. A method according to claim 1 , wherein said method comprises an additional step of:
d) Culturing or banking the cells from the previous step(s) for subsequent differentiation through the hematopoiesis lineage.
3. A method according to claim 2, wherein said method comprises an additional step of:
e) Conditioning the hematopoietic cells obtained in step d) as hematopoietic stem cells CD34 + for administration into a patient.
4. A method according to any one of claims 1 to 3, wherein said method comprises an additional step, prior to step c), comprising assaying, sorting or screening the engineered primary cells for their immune properties, prior to inducing the multipotent cells.
5. A method according to any one of claims 1 to 4, wherein said method comprises an additional step, comprising assaying, sorting or screening different multipotent cell lines induced in step c).
6. A method according to any one of claims 1 to 5, wherein said genetic engineering of step b) aims at:
- reducing alloreactivity,
- conferring drug resistance,
- Improving homing,
- Improving cell survival,
- Improving safety - conferring hypersensitivity to a drug or pro-drug,
- redirecting antigen/ tissu specificity,
- reducing control of immune checkpoints on the activation of the engineered immune cells by inhibiting their receptors and pathways controlled by these receptors,
- modifying HLA profile,
- inhibiting production of suppressive cytokines and metabolites, such as an inactivation of TGFb, TGFbR, IL-10, IL-10R, GCN2 or PRDM1.
- inhibiting genetic pathways that down regulate effector cell’s activation, inhibiting genetic pathways that trigger effector cell’s exhaustion, such as those controlled by PD1 , PDL1 , CTLA4, TIM3 or LAG 3 and/or
- inhibiting genetic pathways that down regulate T-cells proliferation and activation in Tumor induced inhibitory environment, such as glucose deprived microenvironment.
7. A method according to any one of claims 1 to 6, wherein said genetic engineering is performed by using sequence-specific endonuclease reagent(s).
8. A method according to claim 7, wherein said sequence-specific endonuclease reagents are selected from TALE-nucleases, ZFN, RNA-guided endonucleases, such as Cas9 or Cpf1 , MegaTAL or engineered meganucleases.
9. A method according to any one of claims 1 to 8, wherein said genetic engineering in step b) comprises the step of introducing an exogenous nucleic acid sequence into the primary cell’s genome.
10. A method according to claim 9, wherein said exogenous nucleic acid codes for:
- a component of Chimeric Antigen Receptor (CAR);
- a component of a recombinant TCR;
- pre-T ;
- a cytokine, such as an interleukin (eg. IL-2, IL-12, IL-15 and IL-18), or/and their receptors;
- a HLA component; - a product conferring resistance to a drug, such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT), mTORmut and Lckmut;
- a product conferring sensitivity to a drug;
- a peptide inhibitor of FOXP3; and/or
- a secreted inhibitor of Tumor Associated Macrophages (TAM), such as a CCR2/CCL2 neutralization agent.
1 1. A method according to claim 10, wherein a CAR is introduced at the TCR locus.
12. A method according to claim 10, wherein a CAR or a sequence coding for an
interleukin gene is introduced at the PD1 , PDL1 , CTLA4, TIM3 or LAG3 locus.
13. A method according to any one of claims 10 to 12, wherein said CAR is inducible, preferably inducible by a soluble compound, such as streptavidin, or rapamycin CID.
14. A method according to any one of claims 10 to 13, wherein the exogenous coding sequence is introduced by using viral vectors, such as a lentiviral vector or an AAV vector.
15. A method according to any one of claims 1 to 14, wherein step b) further comprises the step of transient expression of reprogramming factors such as TERT.
16. A method according to any one of claims 1 to 15, wherein said reprogramming factors are introduced into the cells under protein or RNA forms.
17. A method according to any one of claims 1 to 16, wherein said reprogramming factors under protein or RNA forms are introduced into the primary cells by electroporation.
18. A method according to any one of claims 1 to 17, wherein said reprogramming factors under protein or RNA forms are introduced into the primary cells using nanoparticles.
19. A method according to any one of claims 1 to 18, wherein the primary cell in step a) is obtained from a healthy donor.
20. A method according to any one of claims 1 to 19, wherein said primary cell is a
mononuclear cell, such as peripheral blood cells (PBMC).
21. A method according to any one of claims 1 to 19, wherein said primary cell is a T-cells or a NK cell.
22. A method according to any one of claims 1 to 20, wherein said primary cells are Tumor Infiltrating Lymphocytes (TIL).
23. A method according to any one of claims 1 to 20, wherein said primary cells are cord blood T-cells.
24. A method according to any one of claims 1 to 19, wherein said primary cells are
lymphoid progenitor cells, such as hematopoietic stem cells.
25. A method according to any one of claims 1 to 24, wherein the cells are frozen after step c).
26. A method according to any one of claims 1 to 25, wherein step e) of redifferentiation is performed by contacting the cells with notch ligand positive cells, such as OP9-DL1 cells.
27. A method according to claim 26, wherein said notch ligand positive cells form three dimension structures such as in artificial thymic organoids.
28. A method according to claim 26, wherein said notch ligand positive cells are
engineered stromal cells.
29. A method according to claim 28, wherein said notch ligand positive cells are
engineered to express specific antigen, such as to activate the immune cells.
30. A method according to any one of claims 1 to 29, wherein the cells are differentiated in step e) until obtaining functional progenitor or mature immune cells.
31. A method according to claim 30, wherein the cells are differentiated until obtaining functional effector T-cells or NK cells.
32. A method for generating an engineered cell line to produce effector cells for immune therapy, said method comprising the step of differentiating cells from an induced multipotent cell line, wherein said cell line comes from a cell that has been genetically engineered prior to being induced to a multipotent stage.
33. A method according to claim 32, wherein said cell line has been engineered when it was a primary immune cell.
34. A method according to claim 33, wherein said primary immune cell was genetically engineered to modify its immune properties.
35. A method according to any one of claims 32 to 34, wherein said induced multipotent cell line has been obtained according to the method of any one of claims 1 to 31.
36. A genetically engineered induced multipotent cell, wherein said cell derives from a
formerly genetically engineered primary cell, in particular a genetically modified lymphocyte.
37. An induced multipotent cell, wherein said cell has been genetically modified at the TCR locus.
38. An induced multipotent cell according to claim 37, wherein said modification at the TCR locus comprises the insertion of an exogenous coding sequence such as one encoding a CAR or a recombinant TCR.
39. An induced multipotent cell, wherein said cell comprises at least two, preferably at least three, more preferably at least four genetic modifications combining a CAR with at least one modification into the TCR locus.
40. An induced multipotent cell, wherein said cell comprises at least one genetic modification into at least one locus selected from one encoding PD1 (Uniprot Q15116), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (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), CD52 (Uniprot P31358), GR (Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150), DCK (Uniprot P27707), HPRT (Uniprot P00492), GGH (Uniprot Q92820), 32m (P61769) and HLA.
41. An induced multipotent cell according to claim 40, wherein said cell further comprises one genetic modification at the TCR locus.
42. An induced multipotent cell according to claim 40 or 41 , wherein said cell further
comprises an exogenous sequence encoding a CAR.
43. An induced multipotent cell line stably established from an induced multipotent cell according to any one of claims 36 to 42.
44. A primary immune, which has been genetically engineered, said cell being transfected with proteins or nucleic acids, such as mRNA, encoding at least one reprogramming factor(s) to induce pluripotency.
45. An engineered primary immune cell according to claim 44, wherein said at least one reprogramming factor is selected from OCT4, SOX2, KLF4, C-MYC, NANOG, LIN28.
46. A primary immune, which has been genetically engineered, said cell being transfected with proteins or nucleic acids, such as mRNA, encoding TERT.
47. A bank comprising different multipotent cells according to claim 36 to 43.
48. Composition comprising a therapeutically effective amount of immune cells
differentiated from an induced multipotent cell line according to any one of claims 36 to 43.
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