JP2019047801A - Methods for engineering T cells for immunotherapy by using RNA-guided CAS nuclease system - Google Patents

Abstract

To develop genetically engineered, preferably non-alloreactive T-cells for immunotherapy.SOLUTION: Genetically engineered T-cells for immunotherapy are developed to specifically target a selection of key genes in T-cells by using RNA-guided endonucleases, in particular Cas9/CRISPR system. The engineered T-cells are also intended to express chimeric antigen receptors (CAR) to redirect their immune activity towards malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer and viral infections.SELECTED DRAWING: None

Description

FIELD OF THE INVENTION The present invention relates to methods of developing genetically engineered, preferably non-alloreactive T cells for immunotherapy. The method involves using RNA-guided endonucleases, in particular the Cas9 / Crisper system, to specifically target various key loci present in T cells. Engineered T cells are also intended to express a chimeric antigen receptor (CAR) to reinduce its immune activity to malignant or infected cells. The present invention paves the way to standard and affordable adoptive immunotherapeutic strategies using T cells to treat cancer, viral infections and autoimmune diseases.

BACKGROUND OF THE INVENTION Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy for treating viral infections and cancer. T cells used for adoptive immunotherapy can be generated either by expansion of antigen specific T cells or redirecting T cells through genetic modification (Park, Rosenberg et al. 2011). Transfer of viral antigen-specific T cells is a well-established procedure used for the treatment of viral infections associated with transplantation and rare virus-related malignancies. Similarly, isolation and transfer of tumor specific T cells has shown success in the treatment of melanoma.

  Novel specificity in T cells was successfully generated through gene transfer of transgenic T cell receptor or chimeric antigen receptor (CAR) (Jena, Dotti et al. 2010). CAR is a synthetic receptor consisting of targeting moieties associated with one or more signaling domains as a single fusion molecule. Generally, the binding moiety of CAR consists of the antigen binding domain of a single chain antibody (scFv), which comprises the light chain variable fragment of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domain for the first generation CAR is derived from the cytoplasmic region of the CD3ζ or Fc receptor γ chain. Although first generation CAR has been shown to successfully redirect T cell cytotoxicity, it has not been able to provide longer term expansion and antitumor activity in vivo. To enhance the survival of CAR-modified T cells and increase proliferation, signaling domains derived from costimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) can be Second generation) or combined (third generation) added. CAR has successfully enabled T cells to be redirected to antigens expressed on the surface of tumor cells derived from various malignancies, including lymphomas and solid tumors (Jena, Dotti et al. al. 2010).

  Current protocols for treating patients using adoptive immunotherapy are based on autologous cell transfer. In this approach, T lymphocytes are harvested from the patient, genetically modified or selected ex vivo, optionally cultured in vitro to amplify cell numbers, and finally infused into the patient. In addition to lymphocyte infusion, the host may also engraft T cells or other ways to support T cell involvement in the immune response, such as preconditioning (using radiation or chemotherapy) and lymphocytes. The administration of growth factors (eg, IL-2) may be manipulated. Each patient is given an individually generated treatment using their own lymphocytes (i.e., autologous therapy). Self-medication is facing major technical and logistical obstacles for practical applications. Production requires expensive dedicated facilities and specialists, and must be produced in a short time after diagnosing the patient. In many cases, pretreatment of the patient results in diminished immune function and the patient's lymphocytes do not function well and may be present in very small numbers. Because of these disorders, each patient's autologous cell preparation is virtually a new product, resulting in significant variability in efficacy and safety.

  Ideally, one would like to use standardized therapies that can be prefabricated, characterized in detail, and immediately administered to patients, allogeneic therapeutic cells. Allogeneic means that the cells are obtained from individuals belonging to the same species but are not genetically similar. However, at present, the use of allogeneic cells has many drawbacks. In an immunocompetent host, allogeneic cells are rapidly rejected. This is a process called host versus graft rejection (HvG). Because of this, the potency of the introduced cells is greatly limited. In non-immune hosts, allogeneic cells can engraft, but their endogenous T cell receptor (TCR) specificity recognizes the host tissue as foreign, resulting in graft versus host disease ( GvHD) happens. Graft versus host disease (GvHD) can lead to significant tissue damage and death.

  In order to effectively obtain allogeneic cells, we have previously disclosed a method of genetically engineering T cells, in which the method TALEN.RTM. (Cellectis, 8, rue de la By using a specific TAL-nuclease, which is better known by Croix Jarry (75013 PARIS), various effector genes, in particular genes encoding T cell receptors, are inactivated. This method uses RNA transfection as part of the platform and has been found to be highly efficient in primary cells, so it is possible to mass produce allogeneic T cells (WO 2013/176915).

  Recently, new genome manipulation tools have been developed based on components of the type II Prokaryotic Crisper (Clustered Regularly Interspaced Short palindromic Repeat) adaptive immune system of the bacterium S. pyogenes. This multicomponent system is called the RNA-guided Cas nuclease system (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) or more simply Crisper, and the Cas endonuclease and this nuclease have some specific genomes It involves guide RNA molecules that have the ability to move into sequence. The endonuclease can cleave DNA when the RNA guide hybridizes to the genomic sequence. The Crisper / Crisper-related (Cas) system is: (1) that the foreign genetic material called "spacer" is retained in a clustered array in the host genome; (2) the spacer expresses short guide RNA (crRNA) (3) crRNA is required to bind to a specific part of foreign DNA called protospacer, and (4) crisper related nuclease (Cas) is required to degrade protospacer. The specificity of binding to foreign DNA is controlled by unique spacer elements within the pre-crRNA. The pre-crRNA, when transcribed with tracrRNA, directs the Cas9 nuclease to the protospacer: crRNA heteroduplex, inducing double strand break (DSB) formation. In addition, Cas9 nuclease cleaves DNA only when a specific sequence known as the protospacer flanking motif (PAM) is immediately downstream of the protospacer sequence. The classical PAM sequence in S. pyogenes is 5'-NGG-3 ', N refers to any nucleotide. Later, it was proved that expression of only one type of chimeric crRNA: tracrRNA transcript is sufficient for Cas9 nuclease to sequence specifically cleave the target DNA sequence. This chimeric crRNA: tracrRNA transcript is usually expressed as two different RNAs in the natural type II crisper system. By adapting the S. pyogenes-derived endogenous type II Crisper / Cas system for use in mammalian cells, several groups independently show that the RNA guide Cas9 has high efficiency and minimal off-target effects We have shown that we can efficiently introduce the correct double-strand break into the endogenous genomic locus of mammalian cells (Cong et al. 2013, Mali et al. 2013, Cho et al. 2013). In addition, several Cas9 nuclease variants have been developed to exploit the features of Cas9 nuclease for further applications in genome engineering and transcriptional regulation. For example, one of the Cas9 nuclease variants functions as a nickase (Jinek et al. 2012) and cleaves the complementary strand of DNA rather than both strands as wild-type Cas9. This allows the DNA template to be repaired using a high fidelity pathway rather than NHEJ, thereby preventing indel formation at the targeted locus and possibly elsewhere in the genome. The potential off target / toxic effects are reduced while maintaining the ability to undergo homologous recombination (Cong et al., 2013). Most recently, paired nicking has been shown to reduce off-target activity in cell lines to 1 / 1,500 to 1/50 and to facilitate gene knockout in mouse zygotes without losing on-target cleavage efficiency (Ran et al. al., 2013).

While RNA-guided endonucleases such as the Cas9 / Crisper system appear to be an attractive approach to genetically engineering mammalian cells, the use of RNA-guided endonucleases in primary cells, especially T cells, is less :
-The adverse effect of T cells upon introduction of DNA into the T cell cytoplasm, ie a high apoptosis rate is observed when the cells are transformed with a DNA vector,
-The Crisper system requires that Cas9 be stably expressed in cells, but the long-term expression of Cas9 in living cells may result in chromosomal defects,
-The specificity of the current RNA guide endonuclease is determined solely by the sequence containing 11 nucleotides (N12-20 NGG, where NGG stands for PAM), thus identifying the target sequence of the desired genetic locus, which is unique to the genome Being very difficult to do is a major obstacle.

  The present application aims to provide a solution to these limitations in order to efficiently implement RNA-guided endonucleases in T cells.

  The present invention discloses a method of engineering T cells, in particular allogeneic T cells obtainable from donors, by using an RNA guide endonuclease such as Cas9 / Crisper, in a manner suitable for immunotherapeutic purposes.

  The method of the present invention more specifically modifies the genome of T cells involved in immunotherapy by inactivating or replacing genes involved in MHC recognition and / or immune checkpoint proteins. Make it possible. In particular, the methods of the present invention provide specific relevant target sequences within the genome in order for the guide RNA to inactivate the TCR component without inducing cell death.

  According to some preferred embodiments, the modified cells involved in immunotherapy comprise an exogenous set encoding single chain and multi-subunit chimeric antigen receptor (CAR) for specific cell recognition It further comprises a replacement polynucleotide. More specifically, modified T cells for treating lymphoma become available that have a CAR directed against the CD19 antigen.

  The present invention relates to isolated cells or cell lines comprising the genetic recombination shown in the detailed description, examples and figures, as well as proteins, polypeptides useful for implementing RNA-guided endonucleases in T cells. Or all of the vectors are included.

As a result of the present invention, the modified T cells are ideally "off the shelf" as a therapeutic product in a method for treating or preventing cancer, infection or autoimmune disease. It can be used as a product.
[Invention 1001]
(A) At least
-RNA guide endonucleases, and
-A specific guide RNA which directs the endonuclease to at least one target locus within the T cell genome
Recombining T cells by introduction and / or expression of
(B) A method of preparing T cells for immunotherapy, which comprises the steps of growing the resulting cells.
[Invention 1002]
An amino acid sequence showing at least 80% amino acid sequence identity with the RuvC domain or HNH domain (SEQ ID NO: 1 or SEQ ID NO: 2) derived from Cas9 of S. pyogenes (S. Pyogenes) The method of the present invention 1001, comprising
[Invention 1003]
The method of the invention 1001 or 1002, wherein the RNA guide endonuclease is Cas9.
[Invention 1004]
The method of the invention 1002, wherein the RNA guide endonuclease is split into at least two polypeptides, one polypeptide comprising RuvC and another polypeptide comprising HNH.
[Invention 1005]
RNA-guided endonuclease is expressed in stabilized or inactive form,
The method of the invention 1004, wherein the endonuclease is activated when it is activated by an enzyme produced by T cell or its polypeptide structure is destabilized in T cell.
[Invention 1006]
The method of any of the inventions 1001-1005, wherein the guide RNA is carried by a peptide nucleic acid (PNA) or a locked nucleic acid (LNA).
[Invention 1007]
The method of any of the inventions 1001-1006, wherein the RNA guide endonuclease is expressed from the transfected mRNA.
[Invention 1008]
The method of the invention 1007, wherein the RNA guide endonuclease is expressed from the transfected mRNA and the guide RNA is expressed from plasmid DNA.
[Invention 1009]
The method of the invention 1007 or 1008, wherein the transfected mRNA is stabilized by including a modified nucleobase or a polyadenylation sequence.
[Invention 1010]
The method of the invention 1007, wherein the transfected mRNA is stabilized by the addition of ARCA (anti-reverse Cap analog).
[Invention 1011]
The method of the invention 1007, wherein the transfected mRNA is stabilized by the addition of a 5 'UTR or a 3' UTR.
[Invention 1012]
The method of the invention 1007, wherein the transfected mRNA is conjugated to at least one cell penetrating peptide.
[Invention 1013]
The method of the invention 1012 wherein the cell penetrating peptide is selected from penetratin, TAT, polyarginine peptide, pVEC, MPG, transportan, guanidinium rich molecule transporter.
[Invention 1014]
The method of the invention 1013 wherein the penetrating peptide comprises an amino acid sequence selected from RQIRIWFQNRRMRWRR, RQIRIWFQNRRMRWRRC, and / or RQIKIWFQNRRMKWKKC.
[Invention 1015]
The method of any of the inventions 1001 to 1014, wherein the guide RNA and / or the mRNA is introduced into the cells by electroporation.
[Invention 1016]
The method of any of the inventions 1001-1006, wherein the endonuclease is expressed from a DNA vector.
[Invention 1017]
The method of the invention 1016, wherein expression of the endonuclease is induced when the guide RNA is produced in the cell.
[Invention 1018]
The method of the invention 1016, wherein the DNA vector is not integrated into the genome.
[Invention 1019]
The method of the invention 1016, wherein the DNA vector is an episomal vector.
[Invention 1020]
The method of the invention 1016, wherein the DNA vector is a transposon.
[Invention 1021]
The method of any of the inventions 1001-100, wherein the guide RNA is a transcript from a DNA vector.
[Invention 1022]
The method of the invention 1021, wherein the DNA vector comprises a U6 promoter in order to cause guide RNA to be transcribed more efficiently in T cells.
[Invention 1023]
The method of the invention 1001, wherein the endonuclease is expressed from a non-integrating viral vector.
[Invention 1024]
The method of the invention 1001, wherein the endonuclease is expressed when the retroviral vector or lentiviral vector is integrated into the chromosome.
[Invention 1025]
The method of the present invention 1001, wherein the nuclease and / or the guide RNA is introduced into T cells upon formation of a viral capsid by mock transduction.
[Invention 1026]
The method of the invention 1001, wherein the endonuclease and / or the guide RNA is transfected using a liposomal vector.
[Invention 1027]
The method of any of the inventions 1001-1002 wherein at least one target locus encodes a component of a T cell receptor (TCR).
[Invention 1028]
The method of the invention 1027 wherein the component of the T cell receptor is TCRα.
[Invention 1029]
The method of the invention 1028, wherein the guide RNA specifically hybridizes to a target sequence of TCRα selected from SEQ ID NO: 6 to SEQ ID NO: 52 listed in Table 2.
[Invention 1030]
The method of any of the inventions 1001-1029, wherein the at least one target locus encodes an HLA gene.
[Invention 1031]
The method of any of the claims 1001-1026, wherein at least one target locus is involved in the regulation of expression of a component of the T cell receptor (TCR).
[Invention 1032]
At least one type of target locus is CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8 , CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIL, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, FOXP3, SIF1 The method according to any of the present inventions 1001 to 1026, which is involved in the expression of an immune checkpoint protein selected from PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.
[Invention 1033]
The method of the invention 1032 wherein the locus is involved in expression of PD1 gene or CTLA-4 gene.
[Invention 1034]
At least two inactivated genes are PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ2B4 and TCRα, The method of the invention 1032 selected from the group consisting of 2B4 and TCRβ.
[Invention 1035]
The method of any of the claims 1001-1026, wherein the at least one target locus confers T cell sensitivity to a chemotherapeutic or immunosuppressive drug.
[Invention 1036]
The method of the invention 1035, wherein the targeted locus is CD52.
[Invention 1037]
The method of the invention 1035, wherein the targeted locus is hypoxanthine-guanine phosphoribosyltransferase (HPRT).
[Invention 1038]
The method of the invention 1035, wherein the targeted locus encodes a glucocorticoid receptor (GR).
[Invention 1039]
The method of any of the inventions 1001 to 1038, wherein homologous recombination is induced at the targeted locus so as to introduce exogenous DNA or a mutation into the T cell genome.
[Invention 1040]
The method of any of the inventions 1001 to 1038, further comprising the step of introducing a chimeric antigen receptor (CAR) into T cells.
[Invention 1041]
The method of any of the present invention 1001 to 1040, wherein the T cells of step (a) are derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.
[Invention 1042]
The method of the invention 1041 wherein the T cells of step (a) are derived from CD4 + T lymphocytes and / or CD8 + T lymphocytes.
[Invention 1043]
The method of any of the inventions 1001 to 1042, wherein the transformed T cells grow in vitro.
[Invention 1044]
The method of any of the inventions 1001 to 1042, wherein the transformed T cells proliferate in vivo.
[Invention 1045]
The method of any of the present invention 1001 to 1044 for preparing T cells to be used as a medicine.
[Invention 1046]
The method of the invention 1014 for preparing T cells for treating cancer or viral infection in a patient in need.
[Invention 1047]
The method of the invention 1046 for treating a lymphoma.
[Invention 1048]
An engineered T cell obtainable by the method of any of the present invention 1001 to 1047.
[Invention 1049]
Treating the patient, comprising the steps of: (a) preparing a population of modified T cells according to any of the methods of the invention 1001 to 1047; and (b) administering the transformed T cells to the patient. How to do it.
[Invention 1050]
The method of the invention 1049 wherein the patient is diagnosed with cancer, viral infection, autoimmune disorder, or graft versus host disease (GvHD).
[Invention 1051]
The method of the invention 1050 wherein the T cells are from a patient to be treated.
[Invention 1052]
The method of the invention 1051 wherein the T cells are derived from a donor.

Schematic representation of the normal relationship between T cells and antigen presenting cells. Fig. 5 is a schematic view of a recombinant therapeutic T cell according to the present invention for a patient's tumor cells. Schematic representation of multi-subunit CAR that can be introduced into RNA-guided endonuclease engineered T cells according to the present invention. FIG. 1 is a schematic diagram of an example of a method of engineering human allogeneic cells for immunotherapy. Flow cytometric analysis of crisper activity on TCR genes in T cells (experimental results from example 2). 5 × 10 ⁶ T cells were transfected with 10 μg of Cas9 mRNA plus 10 μg of plasmid DNA expressing TRAC gene-specific sgRNA under the control of the U6 promoter in the presence of caspase inhibitor. Reading: Flow cytometry analysis with TCRαβ antibody 3 days after transfection. Control: non-transfected cells. Flow cytometric analysis of crisper activity on CD52 gene in T cells (experimental results from example 2). 5 × 10 ⁶ T cells were transfected with 10 μg of Cas9 mRNA plus 10 μg of plasmid DNA expressing CD52 gene specific sgRNA under the control of the U6 promoter in the presence of caspase inhibitor. Reading: flow cytometry analysis with CD52 antibody 3 days after transfection. Control: non-transfected cells.

Table 1: Various cytopulse programs used to determine the lowest voltage required for electroporation in PBMC-derived T cells. Table 2: Various voltages used to determine the lowest voltage required for electroporation in PBMC-derived T cells. Cytopulse program Table 3: Target sequences for non-genotoxic Cas9 / Crisper disruption of TCR alpha

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise herein, all technical and scientific terms used are commonly understood by one of ordinary skill in the areas of gene therapy, biochemistry, genetics, and molecular biology Have the same meaning as

  Although suitable methods and materials are described herein, all methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are specifically incorporated herein by reference to the subject matter relating to the subject matter in the adjacent sentence, paragraph or section in which the reference is cited. It is incorporated by reference in its entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise specified.

  The practice of the present invention, unless otherwise indicated, is within the skill of the art, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and conventional methods of immunology. Will use the technology of Such techniques are explained fully in the literature. 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). Oligosaccharide Synthesis (MJ Gait ed., 1984); Mullis et al., US Patent 4,683, 195; Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ). Higgins eds. 1984); Culture Of Animal Cells (RI Freshshney, 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 EN ZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), in particular, volumes 154 and 155 (Wu et al. eds.) and Volume 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MPCalos 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 I-IV (DM Weir and CCBlackwell, eds., 1986); See Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

  In a general aspect, the invention relates to methods for new adoptive immunotherapeutic strategies in the treatment of cancer and infections.

The main object of the present invention is to genetically recombine T cells with an RNA guide endonuclease such as Cas9 in order to generate suitable cells in cell therapy. In a first aspect, the method of the invention comprises
(A) At least
-RNA guide endonucleases, and
-A specific guide RNA that directs the endonuclease to at least one target locus within the T cell genome
Recombining T cells by introduction and / or expression of
(B) relates to a method of preparing T cells for immunotherapy, comprising the steps of growing the resulting cells.

  By "RNA-guide endonuclease" is meant a polypeptide whose activity and specificity depend on the binding of the endonuclease and an RNA molecule. The entire sequence of this RNA molecule, or more generally, a fragment of this RNA molecule, can identify the target sequence within the genome. The fragment of this RNA molecule is preferably a sequence longer than 8 nucleobases, more preferably a sequence longer than 10 nucleobases, and even more preferably a sequence longer than 12 nucleobases. The RNA molecule is generally capable of hybridizing to the target sequence and mediating the endonuclease activity of the endonuclease. An example of an RNA guide endonuclease is cas9 which is part of the Cas9 / Crisper system.

Cas9
Cas9, also called Csn1 (COG 3513), is a large protein involved in both the biosynthesis of crRNA and the destruction of invading DNA. Cas 9 includes S. thermophilus (Sapranauskas, Gasiunas et al. 2011), Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012), and S. pijogenes. It has been described in various bacterial species such as (S. Pyogenes) (Deltcheva, Chylinski et al. 2011). This large Cas9 protein (> 1200 amino acids) contains two putative nuclease domains in the middle of the protein, namely the HNH (McrA-like) nuclease domain and a split RuvC-like nuclease domain (RNase H fold) (Haft, Selengut et al. 2005; Makarova, Grisin et al. 2006).

  "Cas9" also refers to engineered endonucleases or Cas9 homologs capable of processing target nucleic acid sequences. In certain embodiments, Cas9 can induce cleavage of a nucleic acid target sequence that can correspond to either double stranded or single stranded cleavage. The Cas9 variant may be a naturally occurring, non-naturally occurring Cas9 endonuclease, and a Cas9 endonuclease obtained by protein engineering or random mutagenesis. The Cas9 variant according to the invention is, for example, a mutation, ie a deletion from at least one residue or an insertion of at least one residue within the amino acid sequence of the S. pyogenes Cas9 endonuclease (COG 3513-SEQ ID NO: 3) Or it can be obtained by substitution. In the frame aspect of the invention, such Cas9 variants remain functional, ie retain the ability to process the target nucleic acid sequence. The Cas9 variant may also be a S. pyogenes Cas9 homolog, which may comprise a deletion from at least one residue or an insertion or substitution of at least one residue within the amino acid sequence of S. pyogenes Cas9. If the final construct has the desired activity, in particular the ability to bind to the guide RNA or nucleic acid target sequence, any combination of deletion, insertion and substitution may be made to reach the final construct.

  The RuvC / RNase H motif includes proteins (RNase H, RuvC, DNA transposase, and retrovirus integrase, and PIWI domain of Argonaute protein) that exhibit a wide range of nucleolytic functions that act on both RNA and DNA. In the present invention, the RuvC catalytic domain of the Cas9 (SEQ ID NO: 4) protein can be characterized by the sequence motif: D- [I / L] -GXXSXGWA, where X is any of the 20 naturally occurring amino acids And [I / L] is isoleucine or leucine (SEQ ID NO: 1). In other respects, the invention includes at least a D- [I / L] -GXXSXGWA sequence, wherein X is any one of the twenty naturally occurring amino acids and [I / L] is isoleucine or leucine. For the Cas9 variant which is (SEQ ID NO: 1).

  The HNH motif is unique to many nucleases acting on double stranded DNA, including colicin, restriction enzymes, and homing endonucleases. The domain HNH (SMART ID: SM00507, SCOP name: HNH family) binds to a wide range of DNA binding proteins and performs various binding and cleavage functions (Gorbalenya 1994; Shub, Goodrich-Blair et al. 1994). Some of these proteins are hypothetical or putative proteins whose function has not been elucidated in detail. Proteins of known function include a wide range of cellular processes including bacterial toxicity, homing function in group I and group II introns and inteins, recombination, developmentally controlled DNA rearrangement, phage packaging, and restriction endonuclease activity. (Dalgaard, Klar et al. 1997). These proteins were found in viruses, archaebacteria, eubacteria and eukaryotes. Interestingly, like the LAGLI-DADG and GIY-YIG motifs, the HNH motif is often associated with the endonuclease domain of self-growing elements such as inteins, group I and group II introns (Gorbalenya 1994) Dalgaard, Klar et al. 1997). The HNH domain can be characterized by the presence of conserved Asp / His residues flanked by conserved His (amino terminus) and His / Asp / Glu (carboxy terminus) residues at some distance. A significant number of these proteins may also have a CX2C motif on either side of the central Asp / His residue. Structurally, the HNH motif appears as a central hairpin consisting of a twisted β-strand, flanked on both sides by an α-helix (Kleanthous, Kuhlmann et al. 1999). The large HNH domain of Cas9 is represented by SEQ ID NO: 5. In the present invention, the HNH motif can be characterized by the sequence motif: Y-X-D-H-X-P-X-S-X-X-X-D-X-S, where X represents any one of the twenty naturally occurring amino acids (SEQ ID NO: 2). The present invention relates to a Cas9 variant comprising at least a YXX-D-H-XX-P-X-S-XX-XX-D-X-S sequence, wherein X represents any one of the twenty naturally occurring amino acids (SEQ ID NO: 2).

  The present invention may be of particular interest to facilitate targeted multigenic recombination and to create an inducible nuclease system by introducing a guide RNA into Cas9 cells. For the purpose of the present invention, the present inventors are able to process target nucleic acid sequences together with guide RNA or separately with two separate split Cas9 RuvC domain and HNH domain, Cas9 protein Proved to be able to separate.

  RuvC domains and HNH domains from different RNA guide endonucleases or Cas homologs can also be assembled to improve nuclease efficiency or specificity. Domains from different species may be split into two proteins and may be fused together to form a variant Cas protein. The Cas9 split system is particularly suitable for inducible genomic targeting methods and is believed to avoid the potential toxic effects of Cas9 overexpression in cells. In fact, the first split Cas9 domain may be introduced into a cell, preferably by stably transforming said cell with a transgene encoding said split domain. The two split parts may then reassemble into the cell at the desired time to introduce a complementary split part of Cas9 into the cell such that the functional Cas9 protein is reconstituted.

  Reducing the size of split Cas9 as compared to wild-type Cas9 facilitates vectorization and delivery to cells, for example, by using a cell permeable peptide. With re-arranging domains derived from different Cas proteins, it is possible to modulate specificity and nuclease activity, for example, by targeting PAM motifs slightly different from S. pyogenes Cas9 Become.

Split Cas 9 system
Inspired by the previous characterization of the RuvC domain and the HNH domain, we engineered the Cas9 protein to create a split Cas9 protein. Surprisingly, the inventors have shown that these two split Cass 9 can treat the nucleic acid target together or separately. This observation makes it possible to develop a new Cas9 system using split Cas9 protein. Each split Cas9 domain can be prepared and used separately. Thus, this split system offers several advantages for directing and delivering RNA-guided endonucleases to T cells, allowing the delivery of even shorter and / or inactive proteins, and at a desired time It is particularly suitable for inducing genomic manipulation in T cells, thus limiting the potential toxicity of the integrated Cas9 nuclease.

  "Split Cas9" as used herein means a truncated or truncated form of Cas9 protein or Cas9 variant containing either RuvC domain or HNH domain, and a truncated or truncated form of Cas9 protein or Cas9 variant containing both of these domains Does not mean cut off. Such "split Cas9" may be used independently with the guide RNA, eg, one split Cas9 provides RuvC domain and the other split Cas9 complementarily used to provide HNH domain May be Different split RNA guide endonucleases with either RuvC domain and / or NHN domain may be used together.

  Each Cas9 split domain may be derived from the same Cas9 homolog or from different Cas9 homologs. Many Cas9 homologs have been identified in genomic databases.

As a genome manipulation method, the present invention
(A) selecting a target nucleic acid sequence, optionally comprising a PAM motif in the cell;
(B) preparing a guide RNA comprising a sequence complementary to the target nucleic acid sequence;
(C) preparing at least one split Cas9 domain;
(D) introducing a guide RNA and the split Cas9 domain into cells such that the split Cas9 domain processes the target nucleic acid sequence in the cell.

  The Cas9 split domain (RuvC domain and HNH domain) may be introduced simultaneously into the cell to process target nucleic acid sequences in the cell or may be introduced sequentially. The Cas9 split domain and guide RNA may be introduced into cells by using cell penetrating peptides or other transfection methods as described below.

  In another aspect of the invention, only one split Cas9 domain, called compact Cas9, is introduced into the cells. In fact, surprisingly, the inventors have shown that the split Cas9 domain containing the RuvC motif described above can cleave the target nucleic acid sequence independently of the split domain containing the HNH motif. Thus, we were able to prove that the RuvC split domain was sufficiently stable for binding, without requiring the presence of the HNH domain for the guide RNA to bind to the target nucleic acid sequence. In a preferred embodiment, the split Cas9 domain alone can nick the target nucleic acid sequence.

  Each split domain may be fused at least one active domain at the N-terminus and / or C-terminus. The active domain may be nuclease (eg, endonuclease or exonuclease), polymerase, kinase, phosphatase, methylase, demethylase, acetylase, deacetylase, topoisomerase, integrase, transposase, ligase, helicase, recombinase, transcription activator (eg, It may be selected from the group consisting of VP64, VP16), transcription inhibitors (eg, KRAB), DNA end treatment enzymes (eg, Trex2, Tdt), reporter molecules (eg, fluorescent protein, lacZ, luciferase).

  The HNH domain is responsible for the nicking of one strand of the target double stranded DNA, and the RuvC-like RNase H fold domain is responsible for the nicking of the other strand (including PAM motif) of the double stranded nucleic acid target (Jinek, Chylinski et al. 2012). However, in wild-type Cas9, these two domains result in blunt-end cleavage within the same target sequence (protospacer) of the entry DNA, which is in close proximity to PAM (Jinek, Chylinski et al. 2012). Cas9 may be nickase and induces nick events within various target sequences.

  By way of non-limiting example, Cas9 or Split Cas9 may contain mutations in the catalytic residues of either the HNH domain or the RuvC-like domain to induce nick events in various target sequences. As a non-limiting example, the catalytic residues of Cas9 protein correspond to amino acids D10, D31, H840, H868, N882, and N891, or a catalyst corresponding to a position aligned using the CLUSTALW method to a homolog of Cas family members It is a residue. All these residues can be replaced with any other amino acid, preferably an alanine residue. Mutation of the catalytic residue means substitution with another amino acid or deletion or addition of an amino acid that induces inactivation of at least one of the catalytic domains of cas9. (reference). In particular embodiments, Cas9 or split Cas9 may comprise one or more of the mutations. In another particular embodiment, split Cas9 comprises only one of two RuvC and HNH catalytic domains. In the present invention, Cas9, Cas9 homologs, engineered Cas9, and functional variants thereof derived from different species can be used. The present invention contemplates the use of any RNA guide endonuclease or split RNA guide endonuclease variant to effect nucleic acid cleavage at the gene sequence of interest.

  Preferably, said Cas9 variant has an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably 95% identity with Cas9 of S. pyogenes (COG 3513-SEQ ID NO : 3).

  In another aspect of the present invention, Cas9 or split Cas9 has no nucleotide strand cleavage activity. The resulting Cas9 or split Cas9 is coexpressed with a guide RNA designed to contain the complement of the target nucleic acid sequence. The expression of Cas9 without nucleotide strand cleavage activity specifically silences the gene of interest. This system is called CRISPR interference (Crisp CRISPR i) (Qi, Larson et al. 2013). Silencing means that the gene of interest is not expressed in the form of a functional protein. Silencing may occur at the transcriptional stage or at the translation stage. According to the invention, silencing may occur by directly blocking transcription, more particularly by blocking transcription elongation, targeting an important cis-acting motif within any promoter, It may also occur by sterically blocking the binding of cis-acting motifs to cognate trans-acting transcription factors. Cas9 without nucleotide strand cleavage activity contains both nonfunctional HNH and RuvC domains. In particular, Cas9 or split Cas9 polypeptides comprise inactivating mutations of the catalytic residues of the RuvC-like domain and the HNH domain. For example, the catalytic residue required for cleaved Cas9 activity may be D10, D31, H840, H865, H868, N882, and N891 of S. pyogenes Cas9 (COG 3513-SEQ ID NO: 3), homologs of Cas family members It may be a position aligned using the CLUSTALW method described in. The residues contained in the HNH motif or RuvC motif may be the residues described in the preceding paragraph. All these residues can be replaced with any one of the other amino acids, preferably an alanine residue. Mutation of the catalytic residue means substitution with another amino acid or deletion or addition of an amino acid that induces inactivation of at least one of the catalytic domains of cas9.

  In another specific embodiment, Cas9 or each split domain may be fused at least one active domain at the N-terminus and / or C-terminus. The active domain may be nuclease (eg, endonuclease or exonuclease), polymerase, kinase, phosphatase, methylase, demethylase, acetylase, deacetylase, topoisomerase, integrase, transposase, ligase, helicase, recombinase, transcription activator (eg, It may be selected from the group consisting of VP64, VP16), transcription inhibitors (eg, KRAB), DNA end treatment enzymes (eg, Trex2, Tdt), reporter molecules (eg, fluorescent protein, lacZ, luciferase).

RNA-guided endonuclease / inducible nuclease activity of guide RNA system
Given the potential non-specific interactions with various RNAs in T cells and the potential toxicity of RNA guide endonucleases in T cells by predictable off-site targeting, we: We sought a solution that induced the nuclease activity of the RNA guide endonuclease transiently, ideally during the lifetime of the guide RNA, in cells.

  As a first solution, RNA guide endonuclease can be expressed in stabilized or inactive form. When activated by an enzyme produced by T cells or when the polypeptide structure is destabilized in T cells, this stabilized or inactive form becomes an activated form. Conditional protein stability can be achieved, for example, by fusing the endonuclease with a stabilizing / destabilizing protein based on the FKBP / rapamycin system, in which protein conformational changes are induced by small molecules, as a non-limiting example. You can get it.

  Chemical or light induced dimerization of protein partners fused to the endonuclease protein can also be used to lock or unlock the endonuclease.

  When the RNA guide endonuclease is split into two polypeptides as suggested above, each split may be fused to a partner protein. Both partner proteins dimerize when small molecules are added and reconstitute the active endonuclease in living cells. Such systems may be based, as non-limiting examples, on the use of FKBB / FRB as dimerization partner and rapamycin as small molecule. As another example, a protein that can undergo large conformational changes when bound to small molecules or metabolites inserted into endonuclease proteins (a single polypeptide chain consists of two "splits"). Binding (or non-binding) of small molecules switches the active and inactive conformations of Cas9. Such systems may be based on the use of calmodulin and Ca 2+ as non-limiting examples.

  Each half of the split endonuclease may also be fused with a photosensitive partner protein. Such systems may rely on blue light using, as non-limiting examples, cryptochrome 2 (CRY2) and CIB1 as a fusion partner, and ultraviolet light using UV-B photoreceptors UVR8 and COP1 fusion partners You may rely on For example, both light and chemicals may be combined using phytochrome B and PIF 6 partners (red light coupled, far red light dissociated) and exogenous PCB chromophores.

Guide RNA
The method of the present invention comprises the steps of providing an engineered guide RNA. The guide RNA corresponds to the nucleic acid sequence comprising the complementary sequence. Preferably, the guide RNA corresponds to crRNA and tracrRNA which may be used separately and may be fused together.

  In the natural type II crisper system, the crisper targeting RNA (crRNA) targeting sequence is transcribed from a DNA sequence known as a protospacer. The protospacers are clustered in the bacterial genome in groups called crisper arrays. The protospacer is a short sequence (about 20 bp) of known foreign DNA, separated by short palindromic repeats and kept as recorded for future encounters. To generate crRNAs, the crisper array is transcribed and the RNA is processed to separate individual recognition sequences between repeats. The crisper locus containing the spacer is transcribed into long pre-crRNA. The processing of crisper array transcripts (pre-crRNAs) into individual crRNAs depends on the presence of transactivated crRNAs (tracrRNAs) which have sequences complementary to palindromic repeats. The tracr RNA hybridizes to the repeat region separating the pre-crRNA spacer and initiates dsRNA cleavage by endogenous RNase III, after which another cleavage event occurs within each spacer by Cas9, thereby causing A mature crRNA in which tracrRNA and Cas9 remain bound is generated to form a Cas9-tracrRNA: crRNA complex. Engineered crRNAs and tracrRNAs can target selected nucleic acid sequences, thereby eliminating the need for total RNase III and crRNA processing (Jinek, Chylinski et al. 2012).

  In the present invention, crRNA is engineered to include a sequence that is complementary to a portion of a target nucleic acid, such that the target nucleic acid sequence can be targeted, preferably so that the target nucleic acid sequence can be cleaved. In a particular embodiment, the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 12 nucleotides, which is complementary to the target nucleic acid sequence. In further detailed embodiments, the crRNA is a sequence of at least 30 nucleotides, including at least 10 nucleotides, preferably 12 nucleotides, complementary to the target nucleic acid sequence.

  In another aspect, the crRNA may be engineered to include longer sequences that are complementary to the target nucleic acid. In fact, the inventors have shown that the RuvC split Cas9 domain can cleave the target nucleic acid sequence using only guide RNA. Thus, the guide RNA can bind to the target nucleic acid sequence in the absence of the HNH split Cas9 domain. The crRNAs need not produce the stabilizing effect of HNH split domain binding and may be designed to include longer complementary sequences, preferably more than 20 bp, to enhance the annealing between DNA-RNA duplexes. Thus, the crRNA may comprise more than 20 bp complementary sequences to the target nucleic acid sequence. Such crRNAs make it possible to increase the specificity of Cas9 activity.

  In order to improve targeting rates, the crRNA may also contain a complementary sequence at the 5 'end followed by 4 to 10 nucleotides (Cong, Ran et al. 2013; Mali, Yang et al. 2013). . In a preferred embodiment, the complementary sequence of crRNA is followed at the 3 'end by a nucleic acid sequence called a repeat sequence or a 3' extension sequence.

  Several co-expressions of several crRNAs can be used simultaneously, with separate complementary regions for two different genes and targeting both genes. Thus, in certain aspects, the crRNA may be engineered to simultaneously recognize different target nucleic acid sequences. In this case, the same crRNA contains at least two separate sequences complementary to a portion of different target nucleic acid sequences. In a preferred embodiment, the complementary sequences are spaced by repetitive sequences.

  In a further embodiment of the invention, the guide RNA is generated by a peptide nucleic acid (PNA) or a locked nucleic acid (LNA), in particular in order to improve the stability of the guide RNA in T cells.

PAM Motifs All potential selected target nucleic acid sequences in the present invention may have at their 3 'end a specific sequence called a protospacer flanking motif or a protospacer related motif (PAM). PAM is present in the target nucleic acid sequence but not in the crRNA generated to target it. Preferably, the protospacer flanking motif (PAM) may correspond to 2 to 5 nucleotides beginning immediately adjacent to the protospacer at the leader distal end or near the protospacer. The arrangement and location of PAM differ by different systems. The PAM motif may be, for example, non-limiting examples, NNAGAA, NAG, NGG, NGGNG, AWG, CC, CC, CCN, TCN, TTC (Shah SA, RNA biology 2013). Different type II systems have different PAM requirements. For example, the S. pyogenes system requires an NGG sequence, where N can be any nucleotide. The S. thermophilus type II system requires NGGNG (Horvath and Barrangou 2010) and NNAGAAW (Deveau, Barrangou et al. 2008), whereas different S. variant systems require either NGG or NAAR (Van der Ploeg 2009). Tolerate. PAM is not limited to the region adjacent to the proto-spacer, but may be part of the proto-spacer (Mojica, Diez-Villasenor et al. 2009). In certain embodiments, the Cas9 protein may be engineered to not recognize any PAM motifs, or may be engineered to recognize non-natural PAM motifs. In this case, the selected target sequence may comprise smaller PAM motifs or larger PAM motifs with any combination of amino acids. In a preferred embodiment, the selected target sequence comprises a PAM motif comprising at least three, preferably four, more preferably five nucleotides which the Cas9 variant according to the invention recognizes.

  The co-expression of several crRNAs with separate complementary regions to two different genes and targeting both genes can be used simultaneously. Thus, in certain embodiments, crRNAs may be engineered to simultaneously recognize different target nucleic acid sequences. In this case, the same crRNA contains at least two separate sequences complementary to a portion of different target nucleic acid sequences. In a preferred embodiment, the complementary sequences are spaced by repetitive sequences.

  The crRNA according to the present invention may also be modified to enhance the stability of secondary structure and / or the binding affinity of crRNA for Cas9. In certain embodiments, the crRNA may comprise a 2 ', 3'-cyclic phosphate. The 2 ', 3'-cyclic phosphate terminus has a number of cellular processes: tRNA splicing, nucleotide strand cleavage by several ribonucleases, autocleavage by RNA ribozymes, and unfolded proteins in the endoplasmic reticulum. It appears to be involved in the response to various cellular stresses including accumulation and oxidative stress (Schutz, Hesselberth et al. 2010). We speculated that the 2 ', 3'-cyclic phosphate enhanced the stability of crRNA or the affinity / specificity of crRNA for Cas9. Thus, the present invention provides a modified crRNA containing 2 ', 3'-cyclic phosphate, and a crasper / cas system (Jinek, Chylinski et al. 2012; Cong, Ran et al.) Using the modified crRNA. 2013; Mali, Yang et al. 2013) on methods for genome manipulation.

  The guide RNA may also include transactivated crisper RNA (TracrRNA). The transactivated crisper RNA according to the invention is characterized by an anti-repeat sequence which can base pair with at least part of the 3 'extended sequence of crRNA to form tracrRNA: crRNA, also called guide RNA (gRNA) . The tracr RNA contains a sequence that is complementary to a region of crRNA. Using a guide RNA that contains a fusion of crRNA and tracrRNA and forms a hairpin that mimics the tracrRNA-crRNA complex (Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) , Can induce target nucleic acid cleavage via Cas9 endonuclease. The guide RNA may comprise two separate sequences that are complementary to a portion of the two target nucleic acid sequences, preferably spaced apart by repetitive sequences.

Homologous recombination Nucleotide cleavage is known to stimulate the rate of homologous recombination. Thus, in another preferred embodiment, the invention relates to a method for inducing homologous gene targeting within a nucleic acid target sequence by using an RNA guide endonuclease. The method provides the cell (eg, donor DNA) with an exogenous nucleic acid that includes a sequence homologous to at least a portion of the target nucleic acid sequence such that homologous recombination occurs between the target nucleic acid sequence and the exogenous nucleic acid. The method may further include the step of

  In certain embodiments, the exogenous nucleic acid comprises a first portion homologous to the 5 'region of the target nucleic acid sequence and a second portion homologous to the 3' region. The exogenous nucleic acid in these embodiments also comprises a third portion disposed between the first portion and the second portion which is not homologous to the 5 'and 3' regions of the target nucleic acid sequence. . After the target nucleic acid sequence is cleaved, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp, more preferably more than 200 bp are used in said donor matrix. Therefore, the homologous sequence is preferably 200 bp to 6000 bp, more preferably 1000 bp to 2000 bp. In practice, shared nucleic acid homology is placed in the region upstream and downstream of the cleavage site and adjacent to the cleavage site, the nucleic acid sequence to be introduced has to be placed between these two arms.

  Depending on the location of the target nucleic acid sequence where the cleavage event occurred, such exogenous nucleic acid is used, for example, to knock out the gene when the exogenous nucleic acid is placed within the open reading frame of said gene It may also be used to introduce new sequences or genes of interest. Sequence insertion by use of such exogenous nucleic acid may be used to modify an existing target gene by correction or exchange of said gene (as a non-limiting example allelic replacement) The target gene may be used to upregulate or downregulate the expression of the target gene by correcting or replacing the target gene (as a non-limiting example, promoter replacement).

Methods of Delivery The methods of the invention involve introducing into a cell a molecule of interest, such as a guide RNA (crRNA, tracrRNa, or fusion guide RNA), split Cas9, Cas9, exogenous nucleic acid, DNA end processing enzyme. As a result of introducing into a cell a polynucleotide, preferably a transgene, or a polypeptide contained in a vector encoding RNA, the guide RNA, split Cas9, Cas9, exogenous nucleic acid, DNA end processing enzyme, or others of interest Molecules may be synthesized intracellularly in situ. Alternatively, the molecule of interest may be produced extracellularly and then introduced into the cell.

  We use, as non-limiting examples, liposome delivery vehicles, polymeric carriers, chemical carriers, to deliver drugs / chemicals and molecules (proteins and nucleic acids) into cells or subcellular compartments. It was thought that any means known in the art could be used including physical methods such as lipoplex, polyplex, dendrimer, nanoparticles, emulsion, natural endocytosis or phagocytic pathways as well as electroporation .

In a preferred embodiment of the present invention, the polynucleotide encoding the RNA guide endonuclease of the present invention is transfected, for example, in the form of mRNA directly introduced into cells by electroporation. The inventors confirmed various conditions optimal for mRNA electroporation in T cells as shown in Table 1. The inventor used cytoPulse technology, which allows transient permeabilization of live cells to deliver material to cells using a pulsed electric field. This technique is based on the use of PulseAgile (Harvard Apparatus, Holliston, Mass. 01746 USA) electroporation waveform, which gives precise control of pulse duration, intensity, and spacing between pulses (US Pat. No. 6,010,613 and WO2004083379). All these parameters can be modified to reach the highest conditions for high transfection efficiency and minimal death. Basically, the first high electric field pulse forms a pore, while the subsequent low electric field pulse moves the polynucleotide into the cell. In one aspect of the invention, the present inventors have developed a process leading to the achievement of> 95% transfection efficiency of mRNA in T cells, and an electroporate for transiently expressing various types of proteins in T cells. Describe the use of the translation protocol. In particular, the invention relates to a method of transforming T cells, the method comprising contacting said T cells with RNA;
(A) the voltage range is 2250 to 3000 V / cm, the pulse width is 0.1 ms, and the pulse interval between the electrical pulse of step (a) and the electrical pulse of (b) is 0.2 to 10 ms, One electric pulse,
(B) a single pulse having a voltage range of 2250 to 3000 V, a pulse width of 100 ms, and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c) And (c) voltage of 325 V, pulse width of 0.2 ms, pulse interval between 4 electric pulses of 2 ms between each pulse, a quick pulse sequence consisting of 4 electric pulses T cell Apply to

In a particular embodiment, a method of transforming T cells comprises contacting said T cells with RNA;
(A) The voltage is 2250 V, 2300 V, 2350 V, 2400 V, 2450 V, 25 50 V, 2400 V, 2450 V, 2500 V, 2600 V, 2700 V, 2800 V, 2900 V, or 3000 V / cm and the pulse width is 0.1 ms. The pulse interval between the electrical pulse of (a) and the electrical pulse of (b) is 0.2 ms, 0.5 ms, 1 ms, 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms or 10 ms, 1 Times of electrical pulse,
(B) The voltage range is 2250 V, 2250 V, 2300 V, 2400 V, 2450 V, 2500 V, 2550 V, 2450 V, 2450 V, 2500 V, 2600 V, 2700 V, 2800 V, 2900 V, or 3000 V, and the pulse width is 100 ms. One electrical pulse with a pulse interval of 100 ms between the electrical pulse of b) and the first electrical pulse of step (c), and (c) a voltage of 325 V and a pulse width of 0.2 ms, Applying a quick pulse sequence consisting of 4 electrical pulses to the T cells, the pulse interval between each of the 4 electrical pulses being 2 ms.

  Any value falling within the aforementioned range of values is disclosed herein. The electroporation medium may be any suitable medium known in the art. Preferably, the conductivity of the electroporation medium is in the range of 0.01 to 1.0 millisiemens.

TABLE 1 Various cytopulse programs used to determine the lowest voltage required for electroporation in PBMC-derived T cells

Viral Transduction According to the invention, it is preferred to use the viral vector defined below for transduction of transcripts used as nucleic acid encoding RNA guide endonuclease and / or as guide RNA. Was found to be a conceivable alternative to Methods for viral transduction are well known in the art. However, such a method is, due to the stable integration of the retroviral vector or lentiviral vector into the T cell genome and the resulting long-term expression of the endonuclease, at present a number of limitations. There is a possibility of encounter. This can adversely affect T cells depending on the level of specificity imparted by the RNA guide.

Pseudotransduction using viral encapsidation of RNA-guided endonucleases
Given some of the shortcomings of the existing means and materials for delivering RNA-guided endonucleases to the site of action in T cells, we fuse with alternative delivery methods, in particular the said polypeptide virus component. We have sought a delivery method by which RNA-guide endonucleases are introduced in the form of polypeptides. As an example, an RNA guide endonuclease may be fused to an HIV preintegration complex protein such as Vpr or Vpx. Thus, RNA-guide endonucleases in the form of polypeptides can act on specific targets in the host cell genome as soon as they are released into cells after viral entry and decapsulation.

  For therapeutic use, its use may pose a problem as the aforementioned retroviral component is a "helper protein" for opportunistic viral infections. The inventors were able to find an alternative unexpectedly. We are able to incorporate RNA guide endonucleases, in particular Cas9, into the lentiviral vector particle during virion assembly and to serve to modify the target genome after infection of target cells with such viral vectors: It was observed that such "self" incorporation endonuclease was sufficient.

According to this aspect of the invention, the following steps:
(A) assembling at least one viral vector in the presence of an endonuclease, wherein the endonuclease is preferably not fused to any lentiviral component,
(B) contacting at least one target cell with said viral vector,
(C) one or several steps of the nuclease recognizing and cleaving at least one specific target within the genome of the at least one target cell after internalization of the viral vector by the target cell An accompanying genome manipulation method is provided.

  According to the invention, viral vectors include viruses such as retroviruses, adenoviruses, parvoviruses (e.g. adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g. influenza virus) Rhabdovirus (eg, rabies virus and vesicular stomatitis virus), paramyxoviruses (eg, measles and Sendai), plus-strand RNA viruses such as picornavirus and alphavirus, and adenovirus, herpesvirus (eg, simplex virus) A virus derived from double-stranded DNA virus, including herpesvirus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (eg vaccinia, fowlpox and canarypox) Scan vectors are included. Other viruses include, for example, Norwalk virus, toga virus, flavivirus, reovirus, papova virus, hepadna virus, and hepatitis virus. Examples of retroviruses include avian leukaemic sarcoma virus, mammalian C, B, D, HTLV-BLV, lentivirus, spuma virus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). The terms "viral vector" or "vector" refer to a modified viral particle that can be used to introduce nucleic acid molecules and / or peptides or other molecules into target cells.

  We have shown that after the RNA guide endonuclease has been introduced into the cytoplasm, it retains its activity and can easily bind to the guide RNA. The guide RNA may also be encapsidated during virion assembly and thereby released simultaneously into the cytoplasm of T cells.

  According to a further aspect of the invention, the viral vector is a lentiviral or retroviral based vector (LV), preferably a non-integrating viral vector. LV is a replication deficient viral particle that contains viral genetic material and includes an inner protein core and an outer lipid membrane, commonly referred to as the nucleocapsid core. These replication defective viral particles are assembled by expressing the proteins encoded by lentiviral gag and pol genes in packaging cells. The gag and pol genes encode polyproteins and proteases that process these polyproteins into individual components of the viral particle.

  The NLS is an amino acid sequence that acts to transfer the protein through the nuclear pore complex to the cell nucleus and may be fused with the RNA guide endonuclease to improve the delivery efficiency of the RNA guide endonuclease. Typically, the NLS is one or more short sequences consisting of positively charged amino acids such as lysine or arginine, such as the short sequence derived from the known protein SV40 large T antigen-PKKKRKV-, short sequence derived from nucleoplasmin -KR [PAATKKAGQA] KKKK-, consists of the short sequence -RIRKKLR- derived from p54, the short sequence -PRRRK- derived from SOX9, the short sequence -PPRKKRTVV- derived from NS5A.

  Independent of this use of the RNA-guide endonuclease according to the invention, the virus encapsidation method described above can be applied to TAL-nucleases, zinc finger nucleases, or homing endonucleases for any type of cell that the viral vector can infect. And can be extended to other types of rare cutting endonucleases.

Cell Permeable Peptide Delivery Methods As part of the present invention, we use additional delivery methods, in particular the use of cell permeable peptides (CPP) to introduce RNA guides and / or RNA guide endonucleases into T cells. I examined.

  Thus, the method of the invention may comprise the step of preparing a composition comprising a cell penetrating peptide linked to an RNA guide and / or an RNA guide endonuclease. These molecules can also be linked to the CPP, preferably the N-terminus or C-terminus of the CPP. This bond may be covalent or non-covalent. CPPs can be further divided into two major classes. The first class requires chemical linkage with the molecule, and the second class involves the formation of a stable non-covalent complex. Covalently linked CPPs form covalent conjugates by chemical crosslinking (eg, disulfide linkage) or by expressing CPP-RNA guide endonuclease fusion proteins after cloning. In a preferred embodiment, the CPP has a pyridyl disulfide function such that the thiol modifying molecule forms a disulfide bond with CPP. The disulfide bond can be cleaved, particularly in a reducing environment such as cytoplasm. Non-covalently linked CPPs are preferentially amphiphilic peptides.

  The definition of CPP is constantly evolving, but generally short peptides of less than 35 amino acids that are derived from proteins or chimeric sequences and can transport polar hydrophilic biomolecules across cell membranes in a receptor independent manner It is stated. The CPP may be a cationic peptide, a peptide having a hydrophobic sequence, an amphipathic peptide, a peptide having a proline rich and antimicrobial sequence, and a chimeric peptide or bipartite peptide (Pooga and Langel 2005). In certain embodiments, the cationic CPP may comprise more than one basic or cationic CPP (eg, arginine and / or lysine). Preferably, CCPs are amphiphilic and carry a net positive charge. CPPs penetrate biological membranes, induce various biomolecules to cross the cell membrane and into the cytoplasm, and improve the intracellular transport of various biomolecules, thereby facilitating interaction with targets Can be Examples of CPPs are the nuclear transcription activator protein Tat, a 101 amino acid protein required for viral replication by human immunodeficiency virus type 1 (HIV-1), and the homeoprotein antennapedia of Drosophilia (Drosophilia) Penetratin corresponding to the third helix of Antennapedia, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, guanine rich molecule transporter, MPG, pep-1, sweet arrow peptide , Dermaceptin, transportan, pVEC, human calcitonin, mouse prion protein (mPrPr), polyarginine peptide Args sequence, VP22 protein derived from herpes simplex virus, antibacterial peptide Buforin I, and SynB (US 2013/0065314) May be included. New CPP variants can be linked to various transduction domains.

Non alloreactive T cells
T cell receptors (TCRs) are cell surface receptors involved in T cell activation in response to antigen presentation. The TCR is generally made up of two alpha and beta chains that assemble to form a heterodimer and combine with the CD3 transduction subunit to form the T cell receptor complex present on the cell surface. The α and β chains of the TCR consist of an immunoglobulin-like N-terminal variable (V) region and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region, respectively. Similar to immunoglobulin molecules, the variable regions of the alpha and beta chains are generated by V (D) J recombination, which produces a large diversity of antigen specificities within the T cell population. However, in contrast to immunoglobulins that recognize intact antigens, T cells are activated by processing peptide fragments bound to MHC molecules. This introduces an additional element to antigen recognition by T cells, known as MHC restriction. When MHC mismatches between donor and recipient are recognized via T cell receptors, T cells proliferate, and in some cases, GVHD develops. Normal TCR surface expression has been shown to be dependent on coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusnor 1990). Inactivation of TCRα or TCRβ results in the loss of the TCR from the T cell surface, thereby blocking the recognition of alloantigens and thus blocking GVHD. However, destruction of the TCR generally abolishes the CD3 signaling component and alters the means of further T cell proliferation.

  One of the main objects of the present invention is to use the RNA guide endonuclease described above in a method for engineering non-alloreactive T cells for use in immunotherapy.

  The method further specifically modifies said T cells by inactivating at least one component of the T cell receptor (TCR) by using an RNA guide endonuclease linked to a specific guide RNA. Including the step of

  Engraftment of allogeneic T cells is made possible by inactivating at least one gene encoding a TCR component. By inactivating the TCRα gene and / or the TCRβ gene, the TCR becomes nonfunctional in the cell. TCR inactivation in allogeneic T cells aims to block or reduce GvHD.

  Inactivating a gene is intended that the gene of interest is not expressed in the form of a functional protein. In a particular embodiment, the genetic recombination of the method catalyzes the cleavage of a gene of interest, thereby inactivating the gene of interest such as RNA in a cell ready to be manipulated Rely on the expression of guide endonucleases. Nucleic acid strand breaks caused by the endonuclease are generally repaired by separate mechanisms of homologous recombination end-joining or non-homologous end-joining (NHEJ). However, NHEJ is an incomplete repair process that often changes the DNA sequence at the cleavage site. The mechanism is by direct religation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003) the remaining of the two DNA ends It involves rejoining things. Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used to create specific gene knockouts. The recombination may be substitution, deletion or addition of at least one nucleotide. Mutagenesis events induced by cleavage, ie cells in which a mutagenic event following the NHEJ event has taken place can be identified and / or selected by methods well known in the art.

  We determined appropriate target sequences in the three exons encoding the TCR. This has made it possible to significantly reduce the toxicity while maintaining the cutting efficiency. Preferred target sequences are shown in Table 2 (low ratio of TCR negative cells +, ++ at medium ratio, + + at high ratio).

TABLE 2 Appropriate Target Sequences of Guide RNA Using Cas9 in T Cells

Methods for Manipulating Drug-Resistant T Cells In order to improve cancer therapy and selective engraftment of allogeneic T cells, the engineered T cells are rendered drug resistant to chemo-treat the engineered T cells. Or may be protected from the toxic side effects of the immunosuppressant. In fact, we observed that most patients had received treatment with chemotherapy and immune depleting agents as a standard of treatment prior to testing with T cell immunotherapy. We added chemotherapeutic drugs in the culture medium to expand the cells ex vivo prior to treatment, or in vivo engineered T cells in patients undergoing chemotherapy or immunosuppressive treatment It has also been discovered that these treatments can be used to aid in the selection of engineered T cells by either obtaining selective outgrowths.

T cell drug resistance also allows T cell enrichment in vivo or ex vivo as T cells expressing drug resistant genes survive and proliferate compared to drug sensitive cells. In particular, the present invention
(A) preparing T cells,
(B) selecting at least one drug,
(C) modifying T cells to impart drug resistance to the T cells;
(D) proliferating the engineered T cells in the presence of the drug, to a method of engineering allogeneic and drug resistant T cells for immunotherapy. Optionally, the previous step was previously mentioned,
(E) modifying the T cell by inactivating at least one gene encoding a T cell receptor (TCR) component,
(F) It may be combined with the step of differentiating transformed T cells which do not express TCR on the cell surface.

  Thus, according to one aspect of the present invention, the method comprises the steps of inactivating at least one gene encoding a T cell receptor (TCR) component, resistance to a drug, more particularly to a chemotherapeutic agent. Further modifying the T cells to provide Drug resistance can be imparted to T cells by expressing a drug resistance gene. Several genes have been identified that confer drug resistance to cells, such as variant alleles of dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin, or methylguanine transferase (MGMT) . The drug resistance gene may be expressed in cells by introducing a transgene encoding the gene into cells, and may be expressed in cells by integrating the drug resistance gene into the cell genome by homologous recombination It is also good.

  Drug resistance can be achieved by inactivating one or more genes responsible for the sensitivity of the cells to the drug (drug sensitive genes), such as the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (GenBank: M26434.1). , T cells can be given. In particular, HPRT can be inactivated in engineered T cells to confer resistance to the cytostatic metabolite 6-thioguanine (6TG). 6-Thioguanine (6TG) is converted by HPRT into the cytotoxic thioguanine nucleotide currently used to treat cancer patients, particularly leukemia patients (Hacke, Treger et al. 2013). In another example, inactivation of CD3 normally expressed on the surface of T cells can confer resistance to anti-CD3 antibodies such as teplizumab.

  In other cases, the drug resistance can be conferred to T cells by expressing at least one drug resistance gene. The drug resistant gene refers to a nucleic acid sequence encoding "resistance" to an agent such as a chemotherapeutic agent (eg, methotrexate). In other words, expressing a drug resistance gene in cells allows the cells to be grown in the presence of the drug in greater amounts than growing the corresponding cells without the drug resistance gene. The drug resistance gene of the present invention may encode resistance to antimetabolites, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, analogs or derivatives thereof and the like.

  Several drug resistance genes have been identified that can potentially be used to confer drug resistance on targeted cells (Takebe, Zhao et al. 2001; Sugimoto, Tsukahara et al. 2003; Zielske , Reese et al. 2003; Nivens, Felder et al. 2004; Bardenheuer, Lehmberg et al. 2005; Kushman, Kabler et al. 2007).

  An example of a drug resistant gene may also be a mutated or modified form of dihydrofolate reductase (DHFR). DHFR is involved in the regulation of the amount of tetrahydrofolate in cells and is an enzyme essential for DNA synthesis. Folate analogues such as methotrexate (MTX) inhibit DHFR and are therefore used as antineoplastic agents in the clinic. Various DHFR variants have been described which make them more resistant to inhibition by antifolates used in therapy. In a particular embodiment, the drug resistance gene according to the invention is a nucleic acid sequence encoding a mutant form of human wild-type DHFR, comprising at least one mutation that confers resistance to antifolate treatment, eg methotrexate (GenBank: AAH71996 .1) may be used. In a particular embodiment, the DHFR variant comprises at least one variant amino acid at position G15, L22, F31 or F34, preferably at position L22 or F31 ((Schweitzer, Dicker et al. 1990); International Application W094 / U.S. Patent No. 6,642,043).

  As used herein, "folate antagonists" or "folate analogs" refer to molecules directed to interfere with the folate metabolic pathway at some level. Examples of antifolates include, for example, methotrexate (MTX); aminopterin; trimetrexate (Neutrexin®); edatrexate; 5,8-dideazaisofolic acid (IAHQ); 5,10-dideazatetrahydrofolic acid (DDARTF); 5-deazafolic acid; Ornithine); 10-ethyl-10-deazaaminopterin (DDATHF, lomatrexol); pyritrexime; 10-EDAM; ZD1694; GW1843; Is included.

  Another example of a drug resistant gene may also be a mutated or modified version of ionisine-5'-monophosphate dehydrogenase II (IMPDH2), which is the rate-limiting enzyme in guanosine nucleotide de novo synthesis. A mutated or modified version of IMPDH2 is the IMPDH inhibitor resistance gene. The IMPDH inhibitor may be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). The mutant IMPDH2 may comprise at least one mutation, preferably two mutations, at the MAP binding site of wild-type human IMPDH2 (NP_000875.2), which greatly enhances IMPDH inhibitor resistance. The mutations are preferably at position T 333 and / or S 351 (Yam, Jensen et al. 2006; Sangiolo, Lesnikova et al. 2007; Jonnalagadda, Brown et al. 2013). In a particular embodiment, the threonine residue at position 333 is replaced with an isoleucine residue and the serine residue at position 351 is replaced with a tyrosine residue.

  Another drug resistant gene is mutant calcineurin. Calcineurin (PP2B) is a widely expressed serine / threonine protein phosphatase that is involved in many biological processes and is central to T cell activation. Calcineurin is a heterodimer consisting of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). After binding to the T cell receptor, calcineurin dephosphorylates the transcription factor NFAT, allowing transcription factor NFAT to be transferred to the nucleus and active key target genes such as IL2. FK506 complexed with FKBP12, or cyclosporin A (CsA) complexed with CyPA, blocks NFAT access to the calcineurin active site and blocks NFAT dephosphorylation, thereby activating T cells (Brewin, Mancao et al. 2009). The drug resistant gene of the present invention may be a nucleic acid sequence encoding a calcineurin variant resistant to calcineurin inhibitors such as FK506 and / or CsA. In a particular embodiment, said variant has at least one mutated amino acid at position V314, Y341, M347, T351, W352, L354, K360 of wild type calcineurin heterodimer a, preferably position T351 and L354 or V314. And Y341 may contain double mutations. The correspondence of amino acid positions described herein is often represented by the amino acid position of wild type human calcineurin heterodimer (GenBank: ACX34092.1).

  In another particular embodiment, said variant has a double mutation at the position of wild-type calcineurin heterodimer b: at least one mutated amino acid at V120, N123, L124 or K125, preferably at positions L124 and K125. May be included. The correspondence of amino acid positions described herein is often represented by the amino acid position of wild type human calcineurin heterodimer b polypeptide (GenBank: ACX34095.1).

  Another drug resistant gene is O (6) -methyl guanine methyltransferase (MGMT), which encodes human alkyl guanine transferase (hAGT). AGT is a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an AGT inhibitor co-administered with TMZ to enhance the toxicity of nitrosoureas and to enhance the cytotoxic effect of this drug. Several MGMT variants encoding AGT variants are highly resistant to inactivation by 6-BG but retain the ability to repair DNA damage (Maze, Kurpad et al. 1999). . In a particular embodiment, the AGT variant may comprise a mutated amino acid at wild-type AGT position P140 (UniProtKB: P16455).

  Another drug resistance gene may be the multidrug resistance protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein known as P-glycoprotein (P-GP), which is involved in the transport of metabolic byproducts across cell membranes. P-Gp proteins show broad specificity to several structurally unrelated chemotherapeutic agents. Thus, drug resistance can be conferred to cells by expressing a nucleic acid sequence encoding MDR-1 (NP_000918).

  The drug resistant gene may also be a cytotoxic antibiotic such as the ble gene or mcrA gene. Ectopic expression of ble gene or mcrA in immune cells provides a selection advantage when exposed to the chemotherapeutic agents bleomycin or mitomycin C, respectively.

With regard to the immunosuppressive agent, the present invention provides any possible steps:
(A) preparing T cells, preferably from cell cultures or blood samples,
(B) selecting a gene expressing an immunosuppressive agent target in the T cell,
(C) introducing into the T cell an RNA guide endonuclease capable of selectively inactivating the gene encoding the target of the immunosuppressive agent by DNA cleavage, preferably double strand cleavage;
(D) optionally providing the step of growing the cells in the presence of the immunosuppressant.

  In a further preferred embodiment, the method comprises the step of inactivating a component of a T cell receptor (TCR).

  Immunosuppressive agents are agents that suppress immune function by one of several mechanisms of action. In other words, the immunosuppressive agent is the role played by the compound indicated by the degree of immune response and / or the ability to reduce phagocytosis. As non-limiting examples, the immunosuppressant may be a calcineurin inhibitor, rapamycin target, interleukin-2 alpha chain blocker, inosine monophosphate dehydrogenase inhibitor, dihydrofolate reductase inhibitor, corticosteroids, or immunosuppressive It may be an antimetabolite. Classical cytotoxic immunosuppressants act by inhibiting DNA synthesis. Other immunosuppressive agents may act by activating T cells or may act by inhibiting the activation of helper cells. By using the method according to the present invention, it is possible to confer immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent present in T cells. As non-limiting examples, the target of the immunosuppressive agent may be a receptor of an immunosuppressive agent, such as CD52, glucocorticoid receptor (GR), FKBP family gene member, and cyclophilin family gene member.

  In an immunocompetent host, allogeneic cells are usually rapidly rejected by the host immune system. It has been demonstrated that allogeneic leukocytes present in non-irradiated blood products remain for only 5 to 6 days (Boni, Muranski et al. 2008). Thus, in order to prevent rejection of allogeneic cells, the host immune system must be effectively suppressed. Glucocorticoid steroids are widely used in therapy for immunosuppression (Coutinho and Chapman 2011). This class of steroid hormones binds to the glucocorticoid receptor (GR) present in the T cell cytosol and, as a result, translocates to the nucleus and specific DNA motifs that regulate the expression of many genes involved in the immune process Bond to Treatment of T cells with glucocorticoid steroids reduces levels of cytokine production, thereby generating T cell anergy and interfering with T cell activation. Alemtuzumab, also known as CAMPATH1-H, is a humanized monoclonal antibody that targets CD52, a 12 amino acid glycosylphosphatidyl-inositol (GPI) linked glycoprotein (Waldmann and Hale 2005). CD52 is expressed at high levels in T and B lymphocytes and at low levels in monocytes, whereas it is absent in granulocytes and bone marrow precursors. Treatment with alemtuzumab, a humanized monoclonal antibody directed against CD52, has been shown to induce rapid depletion of circulating lymphocytes and monocytes. Alemtuzumab is frequently used in the treatment of T-cell lymphomas, and in certain cases as part of pre-transplant treatment for transplantation. However, in the case of adoptive immunotherapy, the use of immunosuppressive drugs can also adversely affect the introduced therapeutic T cells. Thus, to effectively use the adoptive immunotherapy approach in these conditions, the introduced cells will need to be resistant to the immunosuppressive treatment.

  In a preferred embodiment of the above steps, the gene of step (b) specific for immunosuppressive treatment is CD52, and the immunosuppressive treatment of step (d) comprises a humanized antibody that targets the CD52 antigen. In another embodiment, said gene of step (b) specific for immunosuppressive treatment is glucocorticoid receptor (GR) and the immunosuppressive treatment of step (d) comprises a corticosteroid such as dexamethasone . In another embodiment, said gene of step (b) specific for immunosuppressive treatment is a FKBP family gene member or a variant thereof and the immunosuppressive treatment of step (d) is also as tacrolimus or fujimycin It contains known FK506. In another embodiment, the FKBP family gene member is FKBP12 or a variant thereof. In another embodiment, said gene of step (b) specific for immunosuppressive treatment is a cyclophilin family gene member or a variant thereof, and the immunosuppressive treatment of step (d) comprises cyclosporin.

  In a specific embodiment of the present invention, the genetic recombination step of the above method comprises two kinds selected from the group consisting of CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ Rely on gene inactivation. In another embodiment, the genetic recombination step of the method relies on the inactivation of more than two genes. Genetic recombination is performed ex vivo, preferably using at least two RNA guides targeting different genes.

  By inactivating the gene, it is intended that the gene of interest is not expressed in the form of a functional protein.

Engineering highly active T cells for immunotherapy The scope of the present invention also encompasses isolated T cells obtained according to any one of the previously described methods. Said T cells according to the invention may be derived from stem cells. The stem cells may be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human cells are CD34 + cells. The T cells of the present invention may be selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cells may be derived from the group consisting of CD4 + T lymphocytes and CD8 + T lymphocytes. Prior to growth of the cells of the present invention and genetic recombination, cell sources can be obtained from the subject by various non-limiting methods. T cells are obtained from a number of non-limiting sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue derived from the site of infection, ascites fluid, pleural fluid, spleen tissue and tumors. be able to. In certain embodiments of the invention, any number of T cell lines available and known to those skilled in the art can be used. In another embodiment, the cells may be obtained from a healthy donor, or may be obtained from a patient diagnosed with cancer or a patient diagnosed with an infection. In another embodiment, the cells may be part of a mixed population of cells exhibiting different phenotypic characteristics. Also included within the scope of the present invention are cell lines obtained from transformed T cells by the methods previously described. Modified cells resistant to immunosuppressive treatment and obtainable by the methods described above are encompassed within the scope of the present invention.

Immune Checkpoint It is understood by those skilled in the art that the term "immune checkpoint" refers to a group of molecules that T cells express. These molecules effectively act as a "brake" to downregulate or inhibit the immune response. The immune checkpoint molecule directly inhibits immune cells, Programmed Death 1 (PD-1; also known as PDCD1 or CD279. Accession No: NM — 005018), cytotoxic T lymphocyte antigen 4 (CTLA-4. CD152) Also known as GenBank Accession No. AF414120.1), LAG3 (also known as CD223. Accession No .: NM_002286.5), Tim3 (also known as HAVCR2, GenBank Accession No .: JX049979.1), BTLA (Also known as CD 272. Accession number: NM — 181780.3), BY 55 (also known as CD 160. GenBank accession number: CR 541888.1), TIGIT (also known as IVSTM 3; accession number: NM — 173799), LAIR1 (Also known as CD305. GenBank Accession Number: CR542051.1), SIGLEC10 (GeneBank Accession Number: AY358337.1), 2B4 (also known as CD244. Accession Number: NM_001166664.1), P. PP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7, SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP6, CASP7, FADD, FAS, TGFBRI, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SMAD10, These include, but are not limited to: TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3. For example, CTLA-4 is a cell surface protein expressed on certain CD4 T cells and on CD8 T cells, and when its ligands (B7-1 and B7-2) bind on antigen presenting cells, T Cell activation and effector functions are inhibited. Thus, the invention relates to a method of engineering T cells, in particular for immunotherapy, which comprises at least one protein involved in the immune checkpoint, in particular PD1 and / or CTLA-4, or Table 3 Recombine T cells by inactivating any of the immune checkpoint proteins mentioned in.

  In a preferred embodiment, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUTC1 At least two genes encoding an immune checkpoint protein selected from the group consisting of GUCY1A3, GUCY1B2, GUCY1B3 are inactivated.

  In a further preferred embodiment, the inactivation of the above mentioned immune checkpoint proteins is combined with the previously proposed inactivation, in particular of the TCR component.

  In another preferred embodiment, the engineered T cells according to the invention comprise CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD1 and TCRα, PD1 and TCRβ, CTLA- 4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRα, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, including two inactivated genes selected from the group consisting of And / or express CAR or multi-chain CAR.

Engineered T-cell single-chain CAR expressing chimeric antigen receptor for malignant cells
Adoptive immunotherapy involves the introduction of antigen-specific autologous T cells generated ex vivo and is a promising strategy for treating viral infections and cancer. T cells used for adoptive immunotherapy may be generated by proliferation of antigen-specific T cells, or may be generated by reinduction of T cells by genetic engineering (Park, Rosenberg et al. 2011). Introduction of viral antigen-specific T cells is a well-established procedure used to treat viral infections associated with transplantation and rare virus-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in the treatment of melanoma.

(Table 3) List of genes encoding immune checkpoint proteins

  New specificity in T cells has been successfully generated by transfecting transgenic T cell receptors or chimeric antigen receptors (CAR) (Jena, Dotti et al. 2010). CAR is a synthetic receptor consisting of a targeting moiety linked to one or more signal transduction domains in the form of a fusion molecule. Generally, the binding part of CAR consists of a single chain antibody (scFv) antigen binding domain comprising the light chain variable fragment of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptors or ligand domains have also been successfully used. The signaling domain for first generation CAR is derived from the cytoplasmic region of CD3ζ or Fc receptor γ chain. First generation CARs have been shown to successfully reinduce T cell cytotoxicity, but have not resulted in long-term growth and antitumor activity in vivo. Signal transduction domains derived from costimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) alone (secondary) to improve survival and increase proliferation of CAR modified T cells Generations) or in combination (third generation) have been added. With CAR, T cells could be successfully reintroduced to antigens expressed on the surface of tumor cells derived from a variety of malignancies, including lymphomas and solid tumors (Jena, Dotti et al. 2010). .

  The invention further includes the possibility to express the chimeric antigen receptor described in the literature in engineered T cells described herein.

  Although CD19 is an attractive target for immunotherapy, as non-hematopoietic cells, as well as myeloid cells, as the vast majority of B acute lymphoblastic leukemia (B-ALL) uniformly express CD19. There is no expression in erythroid cells and T cells, and in bone marrow stem cells. Clinical trials targeting CD19 on B-cell malignancies are underway and have shown promising anti-tumor responses. Inject T cells genetically engineered to express a chimeric antigen receptor (CAR) with specificity derived mostly from the scFv region of the CD19-specific mouse monoclonal antibody FMC63 (Nicholson, Lenton et al. 1997 Cooper, Topp et al. 2003; Cooper, Jena et al. 2012) (international application: WO 2013/126712).

  An example of a single chain chimeric antigen receptor expressed in genetically engineered T cells according to the present invention is a single polypeptide comprising at least one extracellular ligand binding domain, a transmembrane domain, and at least one signaling domain The extracellular ligand binding domain comprises scFV derived from the specific anti-CD19 monoclonal antibody 4G7. For example, when T cells are transduced by using retroviral transduction or lentiviral transduction, this CAR contributes to the recognition of the CD19 antigen present on the surface of malignant B cells involved in lymphoma or leukemia.

  Multiple myeloma or lymphoblastic leukemia antigen markers such as TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9 NQ25), GPRC5D (UNIPROT Q9 NZD1), FKBP11 (UNIPROT Q9 NYL4), KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 Other examples of chimeric antigen receptors such as CAR with antigen receptors directed against UNIPROT Q68 SN8) can also be introduced into T cells according to the invention.

  As a further example, the target antigen may be a differentiation molecule (eg, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD71, CD71, CD123, and CD138), a tumor associated surface antigen such as ErbB2 (HER2) / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFR vIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal epithelium Mucin, gp36, TAG-72, glycosphingolipids, glioma-related antigen, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX , Human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA- 1a, p53, prostein, PSMA, surviving and Telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1) -I, IGF-II, IGFI receptor, mesothelin, tumor specific Histocompatibility complex (MHC) molecules presenting 5 specific peptide epitopes, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, fibronectin extra domain A (EDA) and extra domain B (EDB), And A1 domain (TnCA1) of tenascin-C and fibroblast related protein (fap), a cell lineage specific antigen or a tissue specific antigen, such as CD3, CD4, CD8, CD24, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptor, endoglin, major histocompatibility complex (MHC) molecules, BCMA (CD269, TNFRSF17), or Virus specific surface antigens, eg For example, HIV-specific antigens (eg, HIV gp120), EBV-specific antigens, CMV-specific antigens, HPV-specific antigens, Lasse virus-specific antigens, influenza virus-specific antigens, and any of these surface markers It may be derived from any cluster of derivatives or variants of The antigen is not necessarily a surface marker antigen, and may be an endogenous small antigen presented by HLA class I on the cell surface.

Multi-subunit CAR
Prior art chimeric antigen receptors introduced into T cells were formed from single chain polypeptides that required the sequential addition of a signaling domain. However, movement of the signaling domain from its natural near-membrane location may interfere with its function. To overcome this shortcoming, Applicants recently designed multi-chain CAR derived from FcεRI to allow normal near-membrane location of all relevant signaling domains. In this new structure, the high affinity IgE binding domain of the FcεRI α chain is exchanged with an extracellular ligand binding domain such as a scFv to reinduce T cell specificity to cellular targets, and costimulatory signals close to the normal membrane. The N-terminal and / or C-terminal tails of the FcεRI β chain are used to position them.

Thus, the CAR expressed by the engineered T cells according to the invention may in particular be a multi-chain chimeric antigen receptor (CAR) adapted to the generation and proliferation of the engineered T cells of the invention. Such multi-chain CAR has the following components:
(A) one polypeptide comprising the transmembrane domain of the FcεRI α chain and the extracellular ligand binding domain,
(B) a polypeptide comprising an N-terminal cytoplasmic tail and a portion of a C-terminal cytoplasmic tail of the FcεRI β chain and a transmembrane domain, and / or (c) a portion of the cytoplasmic tail of the FcεRI γ chain and a transmembrane, respectively At least two of the at least two polypeptides comprising domains, wherein different polypeptides spontaneously multimerize to form dimeric CAR, trimeric CAR or tetrameric CAR.

  According to such a structure, the ligand binding domain and the signaling domain are carried on separate polypeptides. The different polypeptides are immobilized in close proximity in the membrane, allowing them to interact with one another. In such structures, the signaling domain and the costimulatory domain may be located near the membrane (ie, adjacent to the cell membrane and inside the cell membrane). This is believed to allow improvement of the function of the costimulatory domain. This multi-subunit structure also offers greater flexibility and the possibility of designing a CAR to more closely control T cell activation. For example, to obtain multispecific CAR structures, it is also possible to include several extracellular antigen recognition domains with different specificities. It is also possible to control the relative ratio between the different subunits that fall within multichain CAR. This type of structure has recently been described by the applicant in PCT / US2013 / 058005.

  Assembling different chains as part of one multichain CAR, for example, of the high affinity receptor for IgE (FcεRI) (Metzger, Alcaraz et al. 1986) fused to a signaling domain and a costimulatory domain. It is made possible by using different alpha, beta and gamma chains. The gamma chain contains a transmembrane region and a cytoplasmic tail containing one immunoreceptor tyrosine-based activation motif (ITAM) (Cambier 1995).

  Multichain CARs may contain several types of extracellular ligand binding domains in order to simultaneously bind different elements within the target, thereby enhancing immune cell activation and function. In one aspect, the extracellular ligand binding domains may be arranged in tandem on the same transmembrane polypeptide, optionally separated by a linker. In another embodiment, the different extracellular ligand binding domains may be arranged in different transmembrane polypeptides that make up a multi-chain CAR. In another aspect, the invention relates to a population consisting of multi-chain CARs, each comprising one different extracellular ligand binding domain. In particular, the present invention relates to a method of engineering an immune cell, the method comprising the steps of providing an immune cell and expressing on the surface of said cell a population consisting of multi-chain CARs each comprising a different extracellular ligand binding domain Including the step of In another particular aspect, the invention relates to a method of engineering an immune cell, the method comprising the steps of providing an immune cell, and constructing a population consisting of multi-chain CARs, each comprising a different extracellular ligand binding domain. Introducing a polynucleotide encoding the polypeptide of interest into said cell. In a particular embodiment, the method of engineering the immune cell comprises at least a portion of FcεRI β chain and / or γ chain fused to a signaling domain and FcεRIα fused to a different extracellular ligand binding domain on the surface of the cell. Including the step of expressing some portion of the chain. In a further detailed aspect, the method comprises at least one polynucleotide encoding a portion of FcεRI β chain and / or γ chain fused to a signaling domain and several FcεRI α chains fused to different extracellular ligand binding domains Introducing the cells into the cells. A population consisting of multichain CARs means at least two, three, four, five, six or more multichain CARs, each comprising a different extracellular ligand binding domain. Different extracellular ligand binding domains according to the invention may preferably simultaneously bind to different elements within the target, thereby enhancing immune cell activation and function.

  The present invention also relates to isolated immune cells comprising multiple chain CAR populations each comprising a different extracellular ligand binding domain.

  The signaling domain or intracellular signaling domain of the multichain CAR of the present invention is responsible for intracellular signaling that results in activation of immune cells and immune responses after the extracellular ligand binding domain is bound to a target. In other words, the signaling domain is responsible for at least one activation of normal effector function of immune cells expressing multi-chain CAR. For example, the effector function of T cells may be cytolytic activity or helper activity including cytokine secretion.

  As used herein, the term "signal transduction domain" refers to a portion of a protein that induces effector signal function signals and directs cells to perform specialized functions.

  Preferred examples of signal transduction domains for use in single chain CAR or multiple chain CAR are Fc and T cell receptors and co-receptors that work in concert to initiate signal transduction after antigen receptor binding. It may be a cytoplasmic sequence, as well as any derivative or variant of these sequences, and any synthetic sequence having the same function. The signaling domains are two distinct classes of cytoplasmic signaling sequences that initiate antigen-dependent primary activation, and cytoplasmic signaling sequences that function in an antigen-independent manner to provide secondary or costimulatory signals. Contains cytoplasmic signaling sequences. The primary cytoplasmic signaling sequence may comprise a signaling motif known as the immunoreceptor tyrosine-based activation motif of ITAM. ITAMs are well-defined signaling motifs found in the intracytoplasmic tail of various receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs used in the present invention may include, as non-limiting examples, ITAMs derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d . In a preferred embodiment, the signaling domain of multichain CAR may comprise the CD3ζ signaling domain and may comprise the cytoplasmic domain of FcεRIβ chain or γ chain.

  In certain embodiments, the signaling domain of multi-chain CARs of the invention comprises a costimulatory signal molecule. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligands that are required for an efficient immune response.

  The ligand binding domain may be any antigen receptor previously used and mentioned for single chain CAR mentioned in the literature, in particular a scFv derived from a monoclonal antibody.

  Also within the scope of the present invention is a bispecific CAR or multispecific CAR as described in WO 2014/4011988.

T cell activation and proliferation
Before or after genetic recombination of T cells, T cells generally have, for example, US Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; No. 6,887,466; No. 6,905,681; No. 7,144,575; No. 7,067,318; No. 7,172,869; No. 7,232,566; No. 7,175,843; No. 5,883,223; No. 6,905,874; No. 6,867,041; and U.S. Patent Application Publication No. 20060121005 can be used for activation and propagation. T cells may be grown in vitro or in vivo.

  In general, the T cells of the invention proliferate by contacting an agent that stimulates a CD3 TCR complex related signal with a surface to which a ligand that stimulates a costimulatory molecule on the T cell surface is attached.

  In particular, the T cell population may be stimulated in vitro, for example by contact with a surface-immobilized anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody, protein kinase C activator (e.g. bryostatin) ) May be stimulated by contact with a calcium ionophore. In order to co-stimulate accessory molecules on the surface of T cells, ligands that bind to the accessory molecules are used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. Anti-CD3 and anti-CD28 antibodies to stimulate proliferation of CD4 + T cells or CD8 + T cells. For example, agents that provide the respective signals may be in solution or may be bound to the surface. As one skilled in the art can readily appreciate, the ratio of particles to cells may depend on the particle size relative to the target cells. In a further aspect of the invention, cells, such as T cells, are combined with drug coated beads, the beads and cells are later separated and then the cells are cultured. In another aspect, drug-coated beads and cells are not separated prior to culture and are cultured together. Cell surface proteins may be linked by allowing paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached to contact T cells. In one aspect, cells (eg, 4 to 10 T cells) and beads (eg, DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads in a 1: 1 ratio) are preferably contained in a buffer. , PBS (without divalent cations such as calcium and magnesium). Again, one skilled in the art can readily appreciate that any cell concentration can be used. The mixture may be cultured for several hours (about 3 hours) to about 14 days, and may be cultured for any integer value of time in between. In another aspect, the mixture may be cultured for 21 days. Conditions suitable for T cell culture include serum (eg, fetal calf serum bovine or fetal human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM- It may contain factors necessary for growth and survival, including CSF, -10, -2, 1L-15, TGFp, and TNF-, or any other additives known to those of skill in the art for cell growth. Medium (eg, minimal essential medium or RPMI medium 1640 or X-vivo 5 (Lonza)). Other additives for cell growth include, but are not limited to, detergents, plasmanates, and reducing agents, such as N-acetyl-cysteine and 2-mercaptoethanol. The medium is supplemented with amino acids, sodium pyruvate and vitamins and is serum-free, or in a suitable amount of serum (or plasma), or a defined set of hormones, and / or sufficient for T cell growth and proliferation. RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer to which amounts of cytokines have been added may be included. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into the subject. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (eg, 37 ° C.) and an atmosphere (eg, air + 5% CO 2). T cells exposed to different stimulation times may exhibit different characteristics.

  In another particular embodiment, the cells can also be grown by co-culturing with tissue or cells. The cells may also be grown in vivo, for example, they can be grown in the blood of a subject after being administered to the subject.

Therapeutic Applications The T cells obtained by the various methods described above are intended for use as a medicament for treating cancer, infections or immune diseases, among others, in patients in need.

  The treatment may be a remission treatment, a curative treatment, or a prophylactic treatment. The treatment may be part of an autologous immunotherapy or may be part of an allogeneic immunotherapy treatment. By autologous is meant that the cells, cell lines, or cell populations used to treat the patient are derived from said patient or human leukocyte antigen (HLA) matched donor. By allogeneic is meant that the cells or cell populations used to treat the patient are not from said patient but from a donor.

  The present invention is particularly suitable for allogeneic immunotherapy, as long as it can transform T cells, typically T cells obtained from a donor, into non-alloreactive cells. This is done under standard protocols and may be repeated as many times as needed. The resulting modified T cells may be pooled and administered to one or several patients, thereby making them available as "off-the-shelf" therapeutic products. Cells that can be used with the disclosed methods are described in the previous section.

  The treatment is primarily for treating patients diagnosed with cancer, viral infections, autoimmune disorders, or graft versus host disease (GvHD). The cancer is preferably leukemia and lymphoma with humoral tumors but may be solid tumors of interest. The types of cancer treated with the CAR of the present invention include carcinoma, blastoma and sarcoma, as well as certain leukemia or lymphoid tumors, benign and malignant tumors, and malignant diseases such as sarcomas, carcinomas, And melanoma, but is not limited thereto. Adult tumors / cancers and pediatric tumors / cancers are also included.

  When loaded with a specific CAR directed against the patient's own immune cells, the T cells of the present invention can be used to treat rheumatoid polyarthritis, systemic lupus erythematosus, Sjögren's syndrome, scleroderma, fibromuscular muscle. Pain, myositis, ankylosing spondylitis, insulin-dependent type I diabetes, Hashimoto's thyroiditis, Addison's disease, Crohn's disease, celiac disease, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS) etc. Enables inhibition or modulation of the cells, which is an important step for treating autoimmune diseases of

  The treatment may be performed in combination with one or more treatments selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser beam therapy and radiation therapy.

  According to a preferred embodiment of the invention, the treatment is particularly suitable for patients undergoing immunosuppressive or chemotherapeutic treatment. In fact, the invention preferably relates to a cell or a population of cells rendered resistant to at least one immunosuppressive agent, preferably due to the inactivation of the gene encoding the receptor of such an immunosuppressive agent. Rely on. In this aspect, the immunosuppressive treatment should support T cell selection and expansion according to the invention in the patient.

  According to one aspect, said T cells of the invention can actively proliferate T cells in vivo when administered to a patient, preferably over a period of time, preferably one week, more preferably two weeks, even more preferably It can be sustained in body fluid for at least one month. Although T cells according to the invention are expected to persist over these periods, the lifespan in the body of the patient does not exceed 1 year, preferably 6 months, more preferably 2 months, even more preferably 1 month Not intended.

  Administration of the cells or populations of cells according to the present invention may be performed in a convenient manner, including aerosol inhalation, injection, oral, infusion, infusion, or transplantation. The compositions described herein are administered to the patient subcutaneously, intradermally, intratumorally, intrathoracically, intramuscularly, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally to the patient. It can be done. In one embodiment, the cell composition of the present invention is preferably administered by intravenous injection.

Administration of a cell or a population of cells comprises 10 ⁴ to 10 ⁹ cells per kg of body weight, preferably 10 ⁵ to 10 ⁶ cells per kg of body weight, including all integer values of cell numbers within these ranges Administration of The cell or population of cells may be administered once or multiple times. In another embodiment, an effective amount of cells is administered in a single dose. In another embodiment, an effective amount of cells is administered multiple times over a period of time. The timing of administration is at the discretion of the managing physician and depends on the patient's clinical condition. The cells or populations of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of the optimal range of effective amounts for a given cell type for a particular disease or condition is within the skill of the art. By effective amount is meant an amount that provides a therapeutic or prophylactic benefit. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

  In another embodiment, an effective amount of cells or compositions comprising those cells are administered parenterally. Administration may be intravenous. Administration may be directly by injection into the tumor.

  In certain embodiments of the invention, cells are treated with drugs such as antiviral therapy, cidofovir, and interleukin 2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or psoriasis The patient is administered with (e.g., before, simultaneously with or after) a number of related treatment modalities including, but not limited to, efalizumab treatment for patients or other treatments for PML patients. In a further embodiment, the T cells of the invention may be used for chemotherapy, radiation, cyclosporin, azathioprine, methotrexate, mycophenolic acid, and immunosuppressants such as FK506, antibodies, or CAMPATH, anti-CD3 antibodies, or other antibody therapeutics. Such other immunoablation agents, cytotoxins, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, as well as radiation may be used in combination. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporin and FK506) or inhibit p70 S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66: 807- Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Citrr. Opin. Mm n. 5: 763-773, 93). In a further embodiment, the cell composition of the invention is T using bone marrow transplantation, fludarabine, external radiation therapy (XRT), a chemotherapeutic agent such as cyclophosphamide, or an antibody such as OKT3 or CAMPATH. It is administered to the patient along with (eg, before, simultaneously with, or after) cell removal therapy. In another embodiment, the cellular composition of the present invention is administered after an agent that reacts with CD20, for example, a B cell ablation therapy such as Rituxan. For example, in one embodiment, a subject can receive peripheral blood stem cell transplantation after receiving standard treatment with high dose chemotherapy. In certain embodiments, after transplantation, the subject receives an injection of increased immune cells of the invention. In additional embodiments, the expanded cells are administered before or after surgery. The modified cells obtained by any one of the methods described herein treat patients in need of treatment for host versus graft (HvG) rejection and graft versus host disease (GvHD) Can be used in certain aspects of the invention. Thus, a method of treating a patient in need of treatment for host versus graft (HvG) rejection and graft versus host disease (GvHD), comprising an inactivated TCRα gene and / or a TCRβ gene Methods within the scope of the invention include treating the patient by administering an amount of modified cells to the patient.

Other Definitions Amino acid residues within polypeptide sequences are referred to herein by the one letter code, eg, Q means Gln, ie glutamine residue, R means Arg, ie arginine residue, D means Asp, ie an aspartic acid residue.

  Amino acid substitution refers to the exchange of one amino acid residue for another, for example, the exchange of an arginine residue for a glutamine residue in a peptide sequence is an amino acid substitution.

  Nucleotides are written as follows: One letter code is used to denote the base of the nucleoside: a is adenine, t is thymine, c is cytosine and g is guanine. For degenerate nucleotides, r represents g or a (purine nucleotide), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a, or t, v represents g, a, or c, b represents g, t, or c, h is a, t Or c represents, n represents g, a, t or c.

  As used herein, "nucleic acid" or "polynucleotide" refers to nucleotides and / or polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, polymerase chain reaction (PCR) ) And fragments produced by any of ligation, cleavage, endonuclease action, and exonuclease action. The nucleic acid molecule is composed of naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (eg, enantiomers of naturally occurring nucleotides), or monomers that are a combination of both It can be done. Modified nucleotides can have alterations in sugar moieties and / or pyrimidine base moieties or purine base moieties. Sugar modifications include, for example, exchange of one or more hydroxyl groups for halogens, alkyl groups, amines, and azido groups, or sugars may be functionalized as ethers or esters. Furthermore, the entire sugar moiety may be exchanged into sterically and electronically similar structures such as azasugars and carbocyclic sugar analogues. Examples of base moiety modifications include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. The nucleic acid can be either single stranded or double stranded.

  "A first region homologous to the sequence upstream of said double strand break, a sequence to be inserted into the genome of said cell, and a second region homologous to a sequence downstream of said double strand break A polynucleotide comprising "continuously" is intended to mean a DNA construct or matrix comprising a first portion and a second portion homologous to the 5 'and 3' regions of the DNA target in situ. Be done. The DNA construct may also contain some homology with the corresponding DNA sequence in situ, or no homology with the 5 'and 3' regions of the DNA target in situ, the first part and the first It also includes a third portion disposed between the two portions. After cleavage of the DNA target, a homologous recombination event is stimulated between the genome containing the target gene contained in the locus of interest and this matrix, and the genomic sequence containing the DNA target is a matrix And the variable part of the first and second parts of the matrix.

  The "DNA target", "DNA target sequence", "target DNA sequence", "nucleic acid target sequence", "target sequence" or "processing site" are targeted and processed by the rare cutting endonuclease according to the invention Intended is a polynucleotide sequence that can These terms refer not only to the location of a particular DNA within a cell, preferably to the location of the genome, but also to genetic material parts that can exist independently of the body of the genetic material, eg, non-limiting examples It also refers to the portion of the genetic material that is in a organelle such as a plasmid, episome, virus, transposon, or mitochondria. Non-limiting examples of RNA guide target sequences include genomic sequences that can be hybridized to guide RNA that directs the RNA guide endonuclease to the desired locus.

  A "delivery vector" is for contacting (i.e., "contacting") cells / agents and molecules (proteins or nucleic acids) required in the present invention with cells, or for delivering cells or intracellular compartments. (Ie, "introduction") refers to a delivery vector that can be used in the present invention. Liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other suitable Transfer vectors, but not limited thereto. These delivery vectors allow the delivery of molecules, chemicals, macromolecules (genes, proteins) or other vectors such as plasmids, or permeable peptides. In these latter cases, the delivery vector is a molecular carrier.

  The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The "vector" in the present invention includes a linear or circular DNA or RNA molecule which may consist of viral vector, plasmid, RNA vector or chromosomal nucleic acid, non-chromosomal nucleic acid, semi-synthetic nucleic acid or synthetic nucleic acid. Not limited to these. Preferred vectors are those capable of autonomous replication of the nucleic acid to which they are linked (episomal vector) and / or those capable of expression (expression vector). Many suitable vectors are known to those skilled in the art and are commercially available.

  Viral vectors include retrovirus, adenovirus, parvovirus (eg, adeno-associated virus), coronavirus, orthomyxovirus (eg, influenza virus), rhabdovirus (eg, rabies virus and vesicular stomatitis virus), paramyxo Negative-strand RNA viruses such as viruses (eg measles and Sendai), positive-strand RNA viruses such as picornavirus and alphavirus, and adenoviruses, herpesviruses (eg herpes simplex virus types 1 and 2, Epstein-Barr Included are double-stranded DNA viruses, including viruses, cytomegalovirus), and poxviruses (eg, vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, toga virus, flavivirus, reovirus, papova virus, hepadna virus, and hepatitis virus. Examples of retroviruses include avian leukaemic sarcoma, mammalian C, B, D, HTLV-BLV, lentivirus, spuma virus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

  "Lentiviral vectors" are extremely promising for gene delivery because of their relatively large packaging capacity, reduced immunogenicity, and ability to stably transduce a wide range of different cell types with high efficiency , Means a lentiviral vector based on HIV. Lentiviral vectors are generally generated after transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. After entry, viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA which serves as a substrate for viral integration into the DNA of infected cells. By "integrative lentiviral vector (or LV)" is meant, by way of non-limiting example, such vectors that can be integrated into the genome of the target cell. Conversely, "non-integrating lentiviral vector (or NILV)" means an efficient gene delivery vector that does not integrate into the target cell's genome through the action of viral integrase.

  Delivery vectors and vectors may be associated or combined with cell permeabilization techniques such as sonoporation or electroporation, or variations of these techniques.

  Cells represent eukaryotic viable cells, primary cells, and cell lines derived from these organisms for in vitro culture.

  "Primary cells" are those that have undergone very few population doublings and are thus the main function of the tissue from which they are derived as compared to tumorigenic continuous or artificially immortalized cell lines The cells are taken directly from living tissue (i.e. biopsy material) and are established for growth in vitro, better representing the components and characteristics.

  As non-limiting examples, cell lines include CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells;

  All these cell lines can be modified by the method of the present invention to provide a cell line model for producing, expressing, quantifying, detecting and studying the gene or protein of interest; The model can also be used to screen bioactive molecules of interest in various areas such as research and production, and as non-limiting examples chemicals, biofuels, therapeutics, and agronomy.

  The “mutation” refers to one, two, three, four, five, six, seven, eight, nine, ten, 11 in the polynucleotide (cDNA, gene) or polypeptide sequence. It represents substitution, deletion, insertion of up to 12, 12, 13, 15, 20, 25, 30, 40, 50 or more nucleotides / amino acids. Mutations can affect the coding sequence of a gene or its regulatory sequences. It may also affect the structure of the genomic sequence or the structure / stability of the encoded mRNA.

  "Variant" refers to a repeat variant, variant, DNA binding variant, TALE nuclease variant, polypeptide variant obtained by mutation or exchange of at least one residue in the amino acid sequence of the parent molecule.

  "Functional variant" refers to a catalytically active variant of a protein or protein domain; such variant has the same activity, or additional properties, or relative to its parent protein or protein domain, or It may have higher or lower activity.

  By "gene" is meant a genetic unit of DNA consisting of DNA segments aligned linearly along a chromosome, encoding a particular protein or protein segment. The gene typically includes a promoter, 5 'untranslated region, one or more coding sequences (exons), optionally, introns, 3' untranslated region. The gene may further comprise a terminator, an enhancer, and / or a silencer.

  The term "locus" as used herein is a specific physical location of a DNA sequence (e.g., a gene) located on a chromosome. The term "locus" may refer to a specific physical location of a rare cutting endonuclease target sequence on a chromosome. Such loci may comprise target sequences that are recognized and / or cleaved by the rare cutting endonuclease according to the invention. The locus of interest of the present invention is not only a nucleic acid sequence present in the body (ie chromosome) of the genetic material of the cell, but also a genetic material part that can be present independently of said body of genetic material, eg It is understood that genetic material parts that are in organelles such as plasmids, episomes, viruses, transposons, or mitochondria, as non-limiting examples, also apply.

  The term "cleavage" refers to the disruption of the covalent backbone of a polynucleotide. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single and double strand breaks are possible, and double strand breaks may occur as a result of two separate single strand break events. Double stranded DNA, RNA, or DNA / RNA hybrid cleavage can result in either blunt or cohesive ends.

  By "fusion protein" is intended the result of a process well known in the art that combines two or more genes originally encoding separate proteins or parts thereof. When the “fusion gene” is translated, a single polypeptide having functional properties derived from each of the original proteins is generated.

  "Identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity may be determined by comparing the position of each sequence which may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and / or programs, including FASTA or BLAST, available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), Calculate the identity between two sequences May be used, for example, with default settings. For example, it has at least 70%, 85%, 90%, 95%, 98%, or 99% identity to a particular polypeptide described herein, preferably substantially identical. Polypeptides exhibiting the function of and polynucleotides encoding such polypeptides are contemplated.

  A "signaling domain" or "costimulatory ligand" specifically binds to a cognate costimulatory molecule on T cells, thereby, for example, binding of the TCR / CD3 complex to a peptide loaded MHC molecule In addition to the primary signals provided by, the molecules on antigen presenting cells that provide signals that mediate T cell responses, including but not limited to proliferation activation, differentiation, and the like. Costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1 BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion Molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, agonist or antibody that binds to the Toll ligand receptor, and B7-H3 Costimulatory ligands may include, but are not limited to, among others, but are not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1,, but not limited to: Specific to costimulatory molecules present on T cells, such as ligands specifically binding to ICOS, lymphocyte function associated antigen 1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, CD83 Also included are antibodies that bind to

  "Costimulatory molecule" refers to a cognate binding partner on T cells that specifically binds a costimulatory ligand and thereby mediates a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and toll ligand receptors.

  "Co-stimulatory signal" as used herein refers to a signal that results in T-cell proliferation and / or upregulation or downregulation of a key molecule in combination with a primary signal such as TCR / CD3 ligation. .

  "Bispecific antibody" refers to an antibody that has binding sites for two different antigens in one antibody molecule. It will be appreciated by those skilled in the art that, in addition to standard antibody structures, other molecules with two binding specificities can be constructed. It will be further understood that antigen binding by bispecific antibodies may occur simultaneously or sequentially. Bispecific antibodies may be made by chemical techniques (see, eg, Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5807), the "polydoma" method. (See US Pat. No. 4,474,893) or may be made by recombinant DNA methods. All these techniques are known per se. As a non-limiting example, each binding domain comprises at least one variable region ("VH region or H region") derived from an antibody heavy chain, wherein the VH region of the first binding domain is a lymphocyte such as CD3. It specifically binds to the marker and the VH region of the second binding domain specifically binds to the tumor antigen.

  The term "extracellular ligand binding domain" as used herein is defined as an oligopeptide or polypeptide capable of binding a ligand. Preferably, the domain will be able to interact with cell surface molecules. For example, the extracellular ligand binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands include viral, bacterial and parasitic infections, autoimmune diseases, and those associated with cancer cells.

  The terms "subject" or "patient", as used herein, include all members of the animal kingdom including non-human primates and humans.

  The above description of the present invention provides the manner and process of making and using the present invention so as to enable those skilled in the art to make and use the present invention, the possibility of which is initially described. It is provided in particular for the subject matter of the attached claims forming part of

  Where numerical limits or ranges are stated herein, the endpoints are included. All values and subranges included within the numerical limits or ranges are specifically included as if explicitly stated.

  Although the invention has been generally described, further understanding can be obtained by reference to certain embodiments. These specific examples are provided herein for illustrative purposes only, and are not intended to be limiting, unless otherwise specified.

Example 1: A schematic method of manipulating human allogeneic cells for immunotherapy In order to further understand the present invention, a method of manipulating human allogeneic cells for immunotherapy is illustrated in FIG. did. The method comprises a combination consisting of one or several of the following steps:
1. Providing T cells from cell cultures or blood samples obtained from an individual patient or from a blood bank, and activating the T cells using anti-CD3 / C28 activator beads. The beads provide both primary and costimulatory signals necessary for T cell activation and proliferation.
2. Transducing the cells with multi-chain CAR, which express T cells against antigens expressed on the surface of target cells derived from various malignancies including lymphomas and solid tumors, particularly antigen CD19. A process that makes it possible to reinduce. In order to improve the function of the costimulatory domain, we designed multichain CAR derived from FcεRI as described previously. Transduction can be accomplished before or after gene inactivation with RNA-guided endonucleases.
3. Manipulating non-alloreactive, optionally resistant T cells:
(A) It is possible to inactivate TCRα in said cells in order to eliminate TCRs from the cell surface and to prevent allogeneic TCRs from recognizing host tissue as foreign and thus to avoid GvHD. .
(B) an immunosuppressive agent or chemotherapy drug such that the cells become resistant to the immunosuppressive treatment or the chemotherapy treatment to prevent graft rejection without affecting the transplanted T cells It is also possible to inactivate one type of gene encoding the target of In this example, the target of the immunosuppressant is CD52, and the immunosuppressant is a humanized monoclonal anti-CD52 antibody.
As shown by the present inventors below, it is merely possible in T cells to introduce two separate guide RNAs (gRNAs) each targeting one type of gene, for example the TCRα gene and the CD52 gene. It is particularly advantageous to use an RNA guide endonuclease to achieve heavy gene inactivation. This can be done by electroporation of T cells with mRNA encoding Cas9 and simultaneously transcribing a DNA plasmid encoding a specific gRNA. Alternatively, gRNA may be transcribed from DNA sequences stably integrated into the genome using a retroviral vector. It has been found by the present inventors that transient expression of Cas9 from mRNA results in high transformation rates and is not harmful to T cells. The inactivated T cells are then fractionated using magnetic beads. For example, T cells that still express the target gene (eg, CD52) can be removed by being immobilized on a solid surface, and inactivated cells are not exposed to stress through the column. This gentle method raises the concentration of correctly engineered T cells.
4. Expand the engineered T cells in vitro prior to administration to the patient via CD3 complex stimulation, or in vivo after administration to the patient. Prior to the administration step, the patient may be subjected to an immunosuppressive treatment such as CAMPATH1-H, which is a humanized monoclonal antibody anti-CD52.
5. Optionally, expose the engineered cells ex vivo to a bispecific antibody prior to administration to the patient to bring the engineered cells into proximity to the target antigen, or in vivo after administration of the cells to the patient Exposing to the bispecific antibody.

Example 2: Specific inactivation of TCR gene and CD52 gene in T cells
1- CAS 9 and gRNA plasmid construction
The sequence encoding Cas9 derived from S. pyogenes was de novo synthesized (GeneCust) and flanked by 3x NLS and HA tag at the C-terminus (pCLS 22972 (SEQ ID NO: 53)). Sequences encoding gRNA targeting the respective 20 bp sequences (5 'to 3') in the TCRa gene (SEQ ID NO: 54) and the CD52 gene (SEQ ID NO: 55) are designated U6 of the pUC57-derived plasmid. Cloning downstream of the promoter yielded plasmid vectors pCLS24029 (SEQ ID NO: 56) and pCLS 24027 (SEQ ID NO: 57), respectively.

2- CAS9 mRNA synthesis
The mRNA encoding CAS9 was made and polyadenylated using mMessage mMachine T7 Ultra kit (Life technologies) according to the manufacturer's instructions. The RNA was then purified on a RNeasy column (Qiagen), eluted in electroporation buffer and quantified by measuring the absorbance at 260 nm (Nanodrop ND-1000 spectrophotometer). The quality of the RNA was verified on a denatured formaldehyde / MOPS agarose gel.

3-Transfection Four days after activation (Dynabeads® Human T-Activator CD3 / CD28, Life technologies), encode mRNA and gRNA encoding CAS9 using AgilePulse MAX system (Harvard Apparatus) T lymphocyte cells were transfected by electrophoretic transfer of plasmids. Briefly, T cells were pre-activated for several days (3-5 days) by pretransfection with anti-CD3 / CD28 coated beads and IL2. The culture medium was changed one day prior to electroporation, cells were seeded at ¹⁰⁶ cells / ml. After 24 hours, the T cells were washed with cytoporation medium T (Harvard Apparatus) and the activated beads were removed by decanting the T cell suspension from tubes inserted into EasySep magnets (StemCell technologies). The T cells were then pelleted and resuspended in cytoporation medium T at 25 × 10 ⁶ cells / ml. 5 × 10 ⁶ cells were mixed with 10 μg of mRNA encoding CAS9 and a plasmid encoding gRNA targeted to TCRa in a 0.4 cm cuvette. Electroporation consisted of two 0.1 V pulses at 1200 V followed by four 0.2 V pulses at 130 V. After electroporation, T cells were diluted in culture medium and incubated for 24 hours at 37 ° C./5% CO ₂ before changing the medium one more time. Three days after electroporation, viability discrimination dye eFluor-780 capable of fixing T cells, and specific PE-conjugated goat anti-mouse IgG F (ab ') to evaluate cell surface expression of TCRa Two fragments were stained with a specific APC-conjugated goat anti-mouse IgG F (ab ') 2 fragment to assess cell surface expression of CD52. Knockout of the TCRα gene or CD52 gene was analyzed by flow cytometry (MACSQuant). The results are shown in FIG. 5 and FIG. It can be seen from FIGS. 5 and 6 that the TCR gene and the CD52 gene were inactivated, respectively, in a large percentage of analyzed cells.

Example 3: Functional analysis of engineered T cells electroporated with monocistronic mRNA encoding anti-CD 19 single chain chimeric antigen receptor (CAR) A chimeric antigen receptor (CAR CD 19) was provided Occasionally, in order to verify that the ability of the engineered T cells to exhibit anti-tumor activity was not affected by genomic manipulation, we previously targeted it with Cas9 and a specific guide RNA that targets TCRα T cells were further transfected with 10 μg of RNA encoding anti-CD19 CAR. After 24 hours, T cells were incubated with CD19 expressing Daudi cells for 4 hours. Cell surface upregulation of CD107a, a marker of cytotoxic granule release (referred to as degranulation) by T lymphocytes, was measured by flow cytometric analysis (Betts, Brenchley et al. 2003).

Resuspend 5 × 10 ⁶ T cells pre-activated for several days (3-5 days) with anti-CD3 / CD28 coated beads and IL2 in cytoporation buffer T and place in 0.4 cm cuvette, no mRNA or single chain Electroporation was performed using 10 μg of mRNA encoding CAR.

Twenty-four hours after electroporation, cells can be fixed with the viable discrimination dye eFluor-780, and a specific PE-conjugated goat anti-mouse IgG F (ab ') to assess cell surface expression of CAR on live cells. Stained with 2 fragments. The data are shown in FIG. A shows that the overwhelming majority of live T cells electroporated with the monocistronic mRNA described above express CAR on the surface. Twenty-four hours after electroporation, T cells were co-cultured with daudi (CD19 ⁺ ) cells for 6 hours and analyzed by flow cytometry to detect expression of the degranulation marker CD107a on the surface (Betts, Brenchley et al. 2003).

  These results indicate that the TCRα negative cells and the TCRα positive T cells have the same ability to degranulate in response to PMA / ionomycin (positive control) or CD19 + Daudi cells. CD107 upregulation depends on the presence of CD19 +. These data suggest that the ability of T cells to initiate a controlled anti-tumor response was not adversely affected by genomic manipulation with Cas9.

List of references cited in the description

As a result of the present invention, the modified T cells are ideally "off the shelf" as a therapeutic product in a method for treating or preventing cancer, infection or autoimmune disease. It can be used as a product.
[Invention 1001]
(A) At least
-RNA guide endonucleases, and
-A specific guide RNA which directs the endonuclease to at least one target locus within the T cell genome
Recombining T cells by introduction and / or expression of
(B) A method of preparing T cells for immunotherapy, which comprises the steps of growing the resulting cells.
[Invention 1002]
An amino acid sequence showing at least 80% amino acid sequence identity with the RuvC domain or HNH domain (SEQ ID NO: 1 or SEQ ID NO: 2) derived from Cas9 of S. pyogenes (S. Pyogenes) The method of the present invention 1001, comprising
[Invention 1003]
The method of the invention 1001 or 1002, wherein the RNA guide endonuclease is Cas9.
[Invention 1004]
The method of the invention 1002, wherein the RNA guide endonuclease is split into at least two polypeptides, one polypeptide comprising RuvC and another polypeptide comprising HNH.
[Invention 1005]
RNA-guided endonuclease is expressed in stabilized or inactive form,
The method of the invention 1004, wherein the endonuclease is activated when it is activated by an enzyme produced by T cell or its polypeptide structure is destabilized in T cell.
[Invention 1006]
The method of any of the inventions 1001-1005, wherein the guide RNA is generated by peptide nucleic acid (PNA) or locked nucleic acid (LNA).
[Invention 1007]
The method of any of the inventions 1001-1006, wherein the RNA guide endonuclease is expressed from the transfected mRNA.
[Invention 1008]
The method of the invention 1007, wherein the RNA guide endonuclease is expressed from the transfected mRNA and the guide RNA is expressed from plasmid DNA.
[Invention 1009]
The method of the invention 1007 or 1008, wherein the transfected mRNA is stabilized by including a modified nucleobase or a polyadenylation sequence.
[Invention 1010]
The method of the invention 1007, wherein the transfected mRNA is stabilized by the addition of ARCA (anti-reverse Cap analog).
[Invention 1011]
The method of the invention 1007, wherein the transfected mRNA is stabilized by the addition of a 5 'UTR or a 3' UTR.
[Invention 1012]
The method of the invention 1007, wherein the transfected mRNA is conjugated to at least one cell penetrating peptide.
[Invention 1013]
The method of the invention 1012 wherein the cell penetrating peptide is selected from penetratin, TAT, polyarginine peptide, pVEC, MPG, transportan, guanidinium rich molecule transporter.
[Invention 1014]
The method of the invention 1013 wherein the penetrating peptide comprises an amino acid sequence selected from RQIRIWFQNRRMRWRR, RQIRIWFQNRRMRWRRC, and / or RQIKIWFQNRRMKWKKC.
[Invention 1015]
The method of any of the inventions 1001 to 1014, wherein the guide RNA and / or the mRNA is introduced into the cells by electroporation.
[Invention 1016]
The method of any of the inventions 1001-1006, wherein the endonuclease is expressed from a DNA vector.
[Invention 1017]
The method of the invention 1016, wherein expression of the endonuclease is induced when the guide RNA is produced in the cell.
[Invention 1018]
The method of the invention 1016, wherein the DNA vector is not integrated into the genome.
[Invention 1019]
The method of the invention 1016, wherein the DNA vector is an episomal vector.
[Invention 1020]
The method of the invention 1016, wherein the DNA vector is a transposon.
[Invention 1021]
The method of any of the inventions 1001-100, wherein the guide RNA is a transcript from a DNA vector.
[Invention 1022]
The method of the invention 1021, wherein the DNA vector comprises a U6 promoter in order to cause guide RNA to be transcribed more efficiently in T cells.
[Invention 1023]
The method of the invention 1001, wherein the endonuclease is expressed from a non-integrating viral vector.
[Invention 1024]
The method of the invention 1001, wherein the endonuclease is expressed when the retroviral vector or lentiviral vector is integrated into the chromosome.
[Invention 1025]
The method of the present invention 1001, wherein the nuclease and / or the guide RNA is introduced into T cells upon formation of a viral capsid by mock transduction.
[Invention 1026]
The method of the invention 1001, wherein the endonuclease and / or the guide RNA is transfected using a liposomal vector.
[Invention 1027]
The method of any of the inventions 1001-1002 wherein at least one target locus encodes a component of a T cell receptor (TCR).
[Invention 1028]
The method of the invention 1027 wherein the component of the T cell receptor is TCRα.
[Invention 1029]
The method of the invention 1028, wherein the guide RNA specifically hybridizes to a target sequence of TCRα selected from SEQ ID NO: 6 to SEQ ID NO: 52 listed in Table 2.
[Invention 1030]
The method of any of the inventions 1001-1029, wherein the at least one target locus encodes an HLA gene.
[Invention 1031]
The method of any of the claims 1001-1026, wherein at least one target locus is involved in the regulation of expression of a component of the T cell receptor (TCR).
[Invention 1032]
At least one type of target locus is CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8 , CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIL, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, FOXP3, SIF1 The method according to any of the present inventions 1001 to 1026, which is involved in the expression of an immune checkpoint protein selected from PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.
[Invention 1033]
The method of the invention 1032 wherein the locus is involved in expression of PD1 gene or CTLA-4 gene.
[Invention 1034]
At least two inactivated genes are PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ2B4 and TCRα, The method of the invention 1032 selected from the group consisting of 2B4 and TCRβ.
[Invention 1035]
The method of any of the claims 1001-1026, wherein the at least one target locus confers T cell sensitivity to a chemotherapeutic or immunosuppressive drug.
[Invention 1036]
The method of the invention 1035, wherein the targeted locus is CD52.
[Invention 1037]
The method of the invention 1035, wherein the targeted locus is hypoxanthine-guanine phosphoribosyltransferase (HPRT).
[Invention 1038]
The method of the invention 1035, wherein the targeted locus encodes a glucocorticoid receptor (GR).
[Invention 1039]
The method of any of the inventions 1001 to 1038, wherein homologous recombination is induced at the targeted locus so as to introduce exogenous DNA or a mutation into the T cell genome.
[Invention 1040]
The method of any of the inventions 1001 to 1038, further comprising the step of introducing a chimeric antigen receptor (CAR) into T cells.
[Invention 1041]
The method of any of the present invention 1001 to 1040, wherein the T cells of step (a) are derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes.
[Invention 1042]
The method of the invention 1041 wherein the T cells of step (a) are derived from CD4 + T lymphocytes and / or CD8 + T lymphocytes.
[Invention 1043]
The method of any of the inventions 1001 to 1042, wherein the transformed T cells grow in vitro.
[Invention 1044]
The method of any of the inventions 1001 to 1042, wherein the transformed T cells proliferate in vivo.
[Invention 1045]
The method of any of the present invention 1001 to 1044 for preparing T cells to be used as a medicine.
[Invention 1046]
The method of the invention 1014 for preparing T cells for treating cancer or viral infection in a patient in need.
[Invention 1047]
The method of the invention 1046 for treating a lymphoma.
[Invention 1048]
An engineered T cell obtainable by the method of any of the present invention 1001 to 1047.
[Invention 1049]
Treating the patient, comprising the steps of: (a) preparing a population of modified T cells according to any of the methods of the invention 1001 to 1047; and (b) administering the transformed T cells to the patient. How to do it.
[Invention 1050]
The method of the invention 1049 wherein the patient is diagnosed with cancer, viral infection, autoimmune disorder, or graft versus host disease (GvHD).
[Invention 1051]
The method of the invention 1050 wherein the T cells are from a patient to be treated.
[Invention 1052]
The method of the invention 1051 wherein the T cells are derived from a donor.

Claims (52)

(A) At least
-RNA guide endonucleases, and
-A specific guide RNA which directs the endonuclease to at least one target locus within the T cell genome
Recombining T cells by introduction and / or expression of
(B) A method of preparing T cells for immunotherapy, which comprises the steps of growing the resulting cells.   An amino acid sequence showing at least 80% amino acid sequence identity with the RuvC domain or HNH domain (SEQ ID NO: 1 or SEQ ID NO: 2) derived from Cas9 of S. pyogenes (S. Pyogenes) The method of claim 1, comprising:   The method according to claim 1 or 2, wherein the RNA guide endonuclease is Cas9.   3. The method of claim 2, wherein the RNA guide endonuclease is split into at least two polypeptides, one polypeptide comprising RuvC and another polypeptide comprising HNH. RNA-guided endonuclease is expressed in stabilized or inactive form,
5. The method according to claim 4, wherein said endonuclease becomes active when it is activated by an enzyme produced by T cell or its polypeptide structure is destabilized in T cell.   6. The method according to any one of claims 1 to 5, wherein the guide RNA is carried by a peptide nucleic acid (PNA) or a locked nucleic acid (LNA).   7. The method of any one of claims 1 to 6, wherein the RNA guide endonuclease is expressed from the transfected mRNA.   The method according to claim 7, wherein the RNA guide endonuclease is expressed from the transfected mRNA and the guide RNA is expressed from plasmid DNA.   9. The method of claim 7 or 8, wherein the transfected mRNA is stabilized by the inclusion of modified nucleobases or polyadenylation sequences.   8. The method of claim 7, wherein the transfected mRNA is stabilized by the addition of ARCA (anti-reverse Cap analog).   8. The method of claim 7, wherein the transfected mRNA is stabilized by the addition of a 5 'UTR or a 3' UTR.   8. The method of claim 7, wherein the transfected mRNA is conjugated to at least one cell penetrating peptide.   The method according to claim 12, wherein the cell permeable peptide is selected from penetratin, TAT, polyarginine peptide, pVEC, MPG, transporter, guanidinium rich molecule transporter.   14. The method according to claim 13, wherein the permeable peptide comprises an amino acid sequence selected from RQIRIWFQNRRMRWRR, RQIRIWFQNRRMRWRRC, and / or RQIKIWFQNRRMKWKKC.   The method according to any one of claims 1 to 14, wherein the guide RNA and / or mRNA is introduced into the cells by electroporation.   7. The method of any one of claims 1 to 6, wherein the endonuclease is expressed from a DNA vector.   17. The method of claim 16, wherein expression of the endonuclease is induced when the guide RNA is produced intracellularly.   17. The method of claim 16, wherein the DNA vector is not integrated into the genome.   17. The method of claim 16, wherein the DNA vector is an episomal vector.   17. The method of claim 16, wherein the DNA vector is a transposon.   21. The method of any one of claims 1 to 20, wherein the guide RNA is a transcript from a DNA vector.   22. The method of claim 21, wherein the DNA vector comprises a U6 promoter to more efficiently transcribe the guide RNA in T cells.   The method of claim 1, wherein the endonuclease is expressed from a non-integrating viral vector.   The method according to claim 1, wherein the endonuclease is expressed when the retroviral vector or lentiviral vector is integrated into the chromosome.   The method according to claim 1, wherein the nuclease and / or the guide RNA are introduced into T cells upon formation of a viral capsid by mock transduction.   The method according to claim 1, wherein the endonuclease and / or the guide RNA is transfected using a liposomal vector.   27. The method of any one of claims 1-26, wherein the at least one target locus encodes a component of a T cell receptor (TCR).   28. The method of claim 27, wherein the component of the T cell receptor is TCRα.   29. The method of claim 28, wherein the guide RNA specifically hybridizes to a target sequence of TCRα selected from SEQ ID NO: 6 to SEQ ID NO: 52 listed in Table 2.   30. The method of any one of claims 1-29, wherein at least one target locus encodes an HLA gene.   27. The method according to any one of the preceding claims, wherein at least one target locus is involved in the regulation of expression of a component of the T cell receptor (TCR).   At least one type of target locus is CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8 , CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIL, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, FOXP3, SIF1 The method according to any one of claims 1 to 26, which is involved in the expression of an immune checkpoint protein selected from PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.   33. The method of claim 32, wherein the locus is involved in expression of a PD1 gene or a CTLA-4 gene.   At least two inactivated genes are PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ2B4 and TCRα, 33. The method of claim 32, wherein the method is selected from the group consisting of 2B4 and TCRβ.   27. The method of any one of claims 1-26, wherein the at least one target locus confers T cell sensitivity to a chemotherapeutic or immunosuppressive drug.   36. The method of claim 35, wherein the targeted locus is CD52.   36. The method of claim 35, wherein the targeted locus is hypoxanthine-guanine phosphoribosyltransferase (HPRT).   36. The method of claim 35, wherein the targeted locus encodes a glucocorticoid receptor (GR).   39. The method of any of claims 1-38, wherein homologous recombination is induced at the targeted locus so as to introduce exogenous DNA or a mutation into the T cell genome.   39. The method of any one of claims 1-38, further comprising the step of introducing a chimeric antigen receptor (CAR) into T cells.   41. The method of any one of claims 1-40, wherein the T cells of step (a) are derived from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. .   42. The method of claim 41, wherein the T cells of step (a) are derived from CD4 + T lymphocytes and / or CD8 + T lymphocytes.   43. The method of any one of claims 1-42, wherein the transformed T cells are grown in vitro.   43. The method of any one of claims 1-42, wherein the transformed T cells proliferate in vivo.   45. A method according to any one of claims 1 to 44 for preparing T cells for use as a medicament.   The method according to claim 14, for preparing T cells for treating cancer or viral infection in a patient in need.   47. The method of claim 46 for treating a lymphoma.   48. Engineered T cells obtainable by the method of any one of claims 1-47. (A) preparing a population of modified T cells according to the method of any one of claims 1 to 47, and (b) administering the transformed T cells to a patient, Methods for treating a patient.   50. The method of claim 49, wherein the patient is diagnosed with cancer, viral infection, autoimmune disorders, or graft versus host disease (GvHD).   51. The method of claim 50, wherein the T cells are from a patient to be treated.   52. The method of claim 51, wherein the T cells are from a donor.

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