RU2824204C2 - Insertion of target gene for improved cellular immunotherapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a method of producing engineered T-cells or natural killer (NK) cells for cellular immunotherapy, engineered T-cell or NK-cell for use in cellular immunotherapy, a population of cells for use in immunotherapy and a pharmaceutical composition for use in treating cancer, where the pharmaceutical composition contains an engineered T-cell or NK-cell or a population of immune cells. In one embodiment, the method comprises the steps of producing a population of primary T cells or NK cells and introducing a nucleic acid into said cells, containing an exogenous polynucleotide sequence coding a chemokine or cytokine to be integrated into an endogenous gene, and one sequence-specific endonuclease reagent.

EFFECT: invention extends the range of products for cellular immunotherapy.

20 cl, 16 dwg, 13 tbl, 2 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of adaptive cellular immunotherapy. Its purpose is to increase the functionality of primary immune cells against pathologies such as tumors, which leads to the development (resistance of the immune system) of immunity, thereby increasing the therapeutic potential of said immune cells. The method proposed in the invention is based on the genetic insertion of exogenous coding sequence(s) that help immune cells direct their immune response against infected or malignant cells. Said exogenous coding sequences are more preferably inserted under the transcriptional control of promoters of endogenous genes that are up- or down-regulated after activation of immune cells, under the influence of the tumor microenvironment or in life-threatening inflammatory conditions, or promoters that are insensitive to immune cell activation. The invention also relates to reagents that are sequence-specific endonucleases and donor DNA vectors, such as AAV vectors, for carrying out said targeted insertions into said specific loci. The method proposed in the invention contributes to increasing the therapeutic potential and safety of engineered primary immune cells for their effective use in cell therapy.

Prerequisites for the creation of an invention

The effective clinical application of primary immune cell populations, including hematopoietic cell lineages, has been confirmed in numerous clinical trials over the past decade in a wide variety of pathologies, in particular HIV infection and leukemia (Tristen S.J. et al., Treating cancer with genetically engineered T cells. Trends in Biotechnology. 29(11), 2011, pp. 550-557).

However, most of these clinical trials used immune cells, mainly NK and T cells, derived from the patients themselves or from compatible donors, which introduced some limitations regarding the number of immune cells available, their suitability, and their effectiveness in overcoming diseases that have already developed strategies to evade or reduce the patient's immune system's impact.

As a major advance in the development of allogeneic immune cells, universal immune cells have been produced as "off-the-shelf" therapeutic products using gene editing (Poirot et al., Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies Cancer Res. 75, 2015, pp. 3853-3864). These universal immune cells can be produced by expressing a specific rare-cleaving endonuclease in donor-derived immune cells with the effect of disintegration by double-strand break of their cognate antigenic determinants.

Since the introduction of the first programmable sequence-specific reagents at the turn of the century, initially referred to as meganucleases (Smith et al., A combinatorial approach to create artificial homing endonucleases by cleaving chosen sequences. Nucl. Acids Res. 34 (22), 2006, e149.), various types of sequence-specific endonuclease reagents have been developed that have improved specificity, safety, and robustness.

TALE nucleases (WO 2011/072246), which are fusions of a TALE binding domain with a catalytic cleavage domain, have been successfully applied to primary immune cells, in particular T cells from peripheral blood mononuclear cells (PBMCs). These TALE nucleases, marketed under the trade name TALEN ^® , are nucleases that are currently used to simultaneously inactivate gene sequences in T cells obtained from donors, in particular to generate allogeneic therapeutic T cells in which the genes encoding TCR (T cell receptor) and CD52 are disrupted. These cells can be endowed with chimeric antigen receptors (CARs) for the treatment of cancer patients (US 2013/0315884). TALE nucleases are highly specific reagents because they must bind DNA in pairs in an obligately heterodimeric form to dimerize the Fok-1 cleavage domain. The left and right elements of the heterodimer can each recognize different nucleotide sequences of approximately 14-20 base pairs (bp), which determines their specificity for target sequences of 30-50 bp overall.

Other endonuclease reagents are based on prokaryotic CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) type II components from the adaptive immune system of the bacteria S. pyogenes. This multicomponent system, which is referred to as the RNA-guided nuclease system (Gasiunas, Barrangou et al., 2012; Jinek, Chylinski et al., 2012), includes members of the Cas9 or Cpf1 endonuclease families linked to guide RNA molecules that are capable of transporting the nuclease to specific genomic sequences (Zetsche et al., Cpf1 is a single RNA-guided endonuclease that provides immunity in bacteria and can be adapted for genome editing in mammalian cells. Cell 163, 2015, pp. 759–771). These RNA-guided programmable endonucleases are easy to obtain because their cleavage specificity is determined by the guide RNA sequence, which is easy to create and obtain at low cost. In this case, CRISPR/Cas9 has specificity for shorter sequences than TAL nucleases, consisting of approximately 10 bp, which must be localized near a specific PAM motif in the target genetic sequence. Similar systems have been described that use a single-stranded DNA oligonucleotide (DNA guide) in combination with Argonaute proteins (Gao F. et al., DNA-guided genome editing using the Natronobacterium gregoryi Argonaut, 2016, doi: 10.1038/nbt.3547).

Other endonuclease systems derived from homing endonucleases (e.g. I-OnuI or I-CreI), combined or not with a TAL nuclease (e.g. MegaTAL) or zinc finger nucleases, also exhibit specificity, but to a lesser extent.

In addition, new specificities can be conferred on immune cells by genetic transfer of transgenic T-cell receptors or so-called chimeric antigen receptors (CARs) (Jena et al., Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor. Blood. 116, 2010, pp. 1035-1044). CARs are recombinant receptors consisting of a targeting moiety associated with one or more signaling domains in a single fusion molecule. In general, the CAR binding moiety consists of the antigen-binding domain of a single-chain antibody (scFv), which contains fragments of a monoclonal antibody containing the light and heavy chain variable domains connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for the first generation of CARs were derived from the cytoplasmic region of CD3 zeta chains or Fc receptor gamma chains. It was demonstrated that the first generation of CARs successfully redirected T cell cytotoxicity, but they failed to provide prolonged expansion and antitumor activity in vivo. To improve the survival and proliferation of CAR-modified T cells, signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137), were added individually (second generation) or in combination (third generation). CARs successfully redirected T cells to antigens expressed on the surface of tumor cells in a variety of malignancies, including lymphomas and solid tumors.

Currently, T-cells with their T-cell receptor (TCR) disrupted by TALE nucleases, endowed with a chimeric antigen receptor (CAR) targeting the malignant antigen CD19, which are designated the product "UCART19", have been described to have therapeutic potential when studied in at least two children with refractory leukemia (Leukaemia success heralds wave of gene-editing therapies. Nature 527, 2015, pp. 146-147). To generate these UCART19 cells, TALE nuclease was transiently expressed in the cells by electroporation of capped mRNA to ensure TCR gene disruption, while a cassette encoding the chimeric antigen receptor (CAR CD19) was randomly introduced into the genome using a retroviral vector.

Using this latter approach, the steps of gene inactivation and chimeric antigen receptor expression are performed independently after induction of T cell activation ex vivo.

However, engineering primary immune cells is not without consequences for the growth/physiology of these cells. In particular, one of the main challenges is to prevent exhaustion/anergy, which significantly reduces their immune response and lifespan. This is most likely to occur when the cells are artificially activated before infusing them into a patient. This is also the case when a cell is endowed with a CAR that is too reactive.

To avoid these pitfalls, the invention takes advantage of the transcriptional regulation of certain key genes during T-cell activation to express exogenous genetic sequences that enhance the therapeutic potential of immune cells. Exogenous genetic sequences to be expressed or co-expressed following immune cell activation are introduced by targeted gene insertion using sequence-specific endonuclease reagents such that their coding sequences are transcribed under the control of endogenous promoters present at these loci. Alternatively, loci that are not expressed during immune cell activation can be used as safe harbor loci for integrating expression cassettes without any deleterious effects on the genome.

The said cell engineering strategies proposed in the present invention tend to provide an increase in the therapeutic potential of primary immune cells in general, in particular by increasing their lifespan, persistence and immune activity, as well as by limiting cell exhaustion. To implement the invention, primary cells obtained from the body of patients can be used as a component of autologous treatment strategies, as well as primary cells obtained from the body of donors as a component of allogeneic treatment strategies.

Brief summary of the invention

Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are the two main pathways used to repair DNA breaks in vivo. The latter pathway repairs the break in a template-dependent manner (in vivo, HDR uses the sister chromatid as a template for DNA repair). Homologous recombination has been used for several decades to precisely edit genomes by targeted DNA modifications using an exogenous donor template. Artificial creation of double-strand breaks (DSBs) in the target region using rare cleaving endonucleases significantly increases the efficiency of homologous recombination (see, e.g., US 8921332). In addition, co-delivery of a rare cleaving endonuclease together with a donor template containing DNA sequences homologous to the breakpoint site enables HDR-based gene editing such as gene correction or gene insertion. However, these technologies have not found wide application in primary immune cells, especially CAR T cells, due to several technical limitations, such as: difficulty in transfecting DNA into such cell types that leads to apoptosis, limited lifespan of immune cells, and limited number of generations, resulting in homologous recombination occurring at a low frequency in general.

Until now, sequence-specific endonuclease reagents have been used mainly for gene inactivation in primary immune cells (e.g. WO 2013/176915) using the NHEJ pathway.

The main object of the present invention is to carry out site-directed gene editing, in particular for inserting a gene (or insertions of several genes) into a target cell in such a way that transcription of the integrated gene is under the control of an endogenous promoter.

The main object of the present invention is to perform gene editing in primary immune cells such that transcription of the integrated gene is under the control of an endogenous promoter, while maintaining expression of the native gene, by using cis-regulatory elements (e.g. cis-acting elements of hydrolase 2A) or an internal ribosome entry site (IRES) in the donor template.

The main object of the present invention is, by way of example (but not limited to), the control of expression in primary T cells of chimeric antigen receptors (CARs), cytokines that are crucial for triggering an antitumor response, stimulatory cytokines for increasing proliferative potential, chemokine receptors for stimulating directed migration to a tumor, or various protective or inhibitory genes for blocking the inhibition of immunity carried out by a tumor. In fact, one of the main advantages of the present invention is the placement of said exogenous sequences under the control of endogenous promoters, the transcriptional activity of which is not reduced by the action of immune cell activation.

In contrast to the previously used method of constructing therapeutic immune cells, in which, for example, an exogenous coding sequence was integrated and expressed in a TCR locus for constitutive gene expression, in the creation of the invention, the coding sequence was integrated into loci that are specifically transcribed during T cell activation, preferably in a CAR-dependent manner.

One aspect of the invention is the expression of a chimeric antigen receptor (CAR) at selected gene loci that are up-regulated upon activation of immune cells. The exogenous CAR encoding sequence(s) and the endogenous gene encoding sequence(s) can be co-transcribed, for example, if separated, by cis-regulatory elements (e.g., hydrolase 2A cis-acting elements) or by an internal ribosome entry site (IRES) that are also introduced. For example, the exogenous CAR encoding sequences can be placed under the transcriptional control of a promoter of endogenous genes that are activated by the tumor microenvironment, such as HIF1a, i.e. hypoxia-inducible transcription factor or aryl hydrocarbon receptor (AhR), which are sensory genes induced by hypoxia and xenobiotics, respectively, in the immediate environment of tumors.

Thus, the present invention can be used to improve the therapeutic outcome of CAR T cell-based therapies by integrating exogenous genetic signatures/patterns under the control of endogenous T cell promoters influenced by the tumor microenvironment (TME). It is known that hallmarks of the TME, including (but not limited to) such factors as arginine, cysteine, tryptophan and oxygen deprivation, as well as extracellular acidosis (lactate accumulation), lead to up-regulation of specific endogenous genes. According to the invention, the up-regulation of endogenous genes can be "borrowed" to re-express relevant exogenous coding sequences in order to enhance the anti-tumor activity of CAR T cells in the microenvironment of certain tumors.

In preferred embodiments, the method of the invention comprises the step of creating a double-strand break at a locus that is highly transcribed in the tumor microenvironment by expressing reagents that are sequence-specific nucleases, such as, but not limited to, TALENs, ZFNs or RNA-directed endonucleases, in the presence of a DNA repair matrix, preferably housed in an AAV6 vector. The DNA-containing donor matrix typically comprises two homologous arms, with integrated unique or multiple open reading frames and regulatory genetic elements (stop codon and polyA sequences), referred to herein as exogenous coding sequences.

According to another aspect of the invention, said exogenous sequence is introduced into the genome by deleting or modifying the endogenous coding sequence(s) present in the locus (knock-out by knock-in), so as to combine gene inactivation with transgenesis.

Depending on the target locus and its involvement in immune cell activity, the target endogenous gene may be inactivated or its original function may be maintained. If the target gene is important for immune cell activity, the insertion procedure may result in a single knockin (KI) without inactivating the gene. In contrast, if the target gene is suspected of being involved in immune cell inhibition/depletion, the insertion procedure is designed to interfere with expression of the endogenous gene, preferably by knocking out the endogenous sequence, while allowing expression of the introduced exogenous coding sequence(s).

More specific objects of the invention relate to up-regulation with different kinetics of target gene expression following activation of CAR signaling pathways by targeted integration (with or without disruption of the native gene) into specific loci, such as, for example, (but not limited to) PD1, PDL1, CTLA-4, TIM3, LAG3, TNFa or IFNg.

An even more specific subject of the invention relates to engineered immune cells as described herein, and preferably primary immune cells intended for infusion into patients, which comprise exogenous sequences encoding IL-15 or IL-12 polypeptide(s) integrated into an endogenous PD1, CD25 or CD69 locus for their expression under the control of endogenous promoters present in said loci.

The immune cells according to the present invention may be [CAR] ^positive , [CAR] ^negative , [TCR] ^positive or [TCR] ^negative cells depending on the therapeutic indication and the recipient patients. According to one preferred aspect of the invention, the immune cells are further engineered as [TCR] ^negative for allogeneic transplantation. This is achieved primarily by genetically disrupting at least one endogenous sequence encoding at least one TCR component, such as TRAC (the locus encoding TCRalpha), preferably by integrating an exogenous sequence encoding a chimeric antigen receptor (CAR) or a recombinant TCR, or component(s) thereof.

According to a further aspect of the invention, immune cells are transfected with an exogenous sequence encoding a polypeptide that can associate (bind) and preferably interact with a cytokine receptor of the IL-6 receptor family, such as mutant GP130 (glycoprotein 130). In particular, the invention provides immune cells, preferably T cells, that secrete soluble mutant GP130, for the purpose of reducing cytokine release syndrome (cytokine storm) (CRS) by affecting and ideally blocking interleukin-6 (IL-6) signal transduction. CRS is a well-known complication of cellular immunotherapy, leading to autoimmunity, which occurs when transduced immune cells become activated in vivo. Upon binding of IL-6 to its receptor IL-6R, the complex associates with the GP130 subunit, initiating signal transduction and an inflammatory response cascade. According to a specific aspect of the invention, a dimeric protein comprising the extracellular domain of GP130 fused to the Fc region of an antibody as IgG1 (sgp130Fc) is expressed in engineered immune cells to specifically bind to the soluble IL-R/IL-6 complex to achieve partial or complete blockade of IL-6 signaling in trans. Thus, the present invention provides a method for limiting CRS in immunotherapy, wherein immune cells are genetically modified to express a soluble polypeptide that can bind and preferentially interact with a cytokine receptor of the IL-6 receptor family, such as sgp130Fc. According to a preferred aspect of the invention, said sequence encoding said soluble polypeptide that can bind and preferably interact with a cytokine receptor of the IL-6A receptor family is integrated under the control of an endogenous promoter, preferably into one locus responsible for T-cell activation, such as one selected from those listed in Tables 6, 8 or 9, more particularly into PD1, CD25 or CD69. The polynucleotide sequences of the vectors, donor matrices that contain exogenous coding sequences and/or sequences homologous to the endogenous loci, sequences related to the resulting engineered cells, as well as sequences allowing the detection of said engineered cells, are all part of the present application.

The main object of the present invention is, for example (but not limited to), the control of expression of components of biological "logic gates" ("AND" or "OR" or "NOT" or any combination thereof) by targeted gene integration. Similar to logic gates in electronics, cellular components expressed at different loci can alter negative and positive signals that control, for example, the activation states of an immune cell. Said component includes, for example (but not limited to), positive and negative chimeric antigen receptors that can be used to control T-cell activation and the resulting cytotoxicity of engineered T-cells in which they are expressed.

A preferred embodiment of the invention is the introduction of a sequence-specific endonuclease reagent and/or a donor template comprising a gene of interest and a sequence homologous to the target gene by transfection with ssDNA (e.g., but not limited to, oligonucleotides), dcDNA (e.g., but not limited to, plasmid DNA), and most preferably, e.g., but not limited to, adeno-associated virus (AAV).

The invention also relates to vectors, donor matrices, reagents and engineered cells obtained using the methods described above, as well as to their use in therapy.

Brief description of drawings and tables:

The drawings show:

Fig. 1 shows strategies for engineering hematopoietic stem cells (HSCs) by introducing exogenous sequences into specific loci under the transcriptional control of endogenous promoters that are specifically activated in particular immune cell types. The figure shows examples of specific endogenous genes into whose loci exogenous coding sequence(s) can be introduced for expression in the desired hematopoietic lineages according to the present invention. The object was to obtain ex vivo engineered HSCs that can be transplanted into patients so that they can produce immune cells in vivo that will express the selected transgenes when they differentiate into the desired lineage;

Fig. 2 is a schematic representation of the donor sequences, the use of which is described in the Experimental Section, for insertion of an exogenous IL-15 coding sequence into the CD25 and PD1 loci, and an exogenous anti-CD22 CAR coding sequence into the TRAC locus. A: A donor template (designated IL-15m-CD25) designed for site-directed insertion of IL-15 into the CD25 locus to ensure co-transcription of CD25 and IL-15 polypeptides in an immune cell. Sequence details are provided in the Examples. B: A donor template (designated IL-15m-PD1) designed for site-directed insertion of IL-15 into the PD1 locus to ensure transcription of IL-15 under the control of the transcriptional activity of the endogenous PD1 gene promoter. The right and left border sequences of PD1 can be selected to leave the endogenous PD1 coding sequence intact or disrupted. In the latter case, PD1 is knocked down when IL-15 is added (knocked in) and transcribed. B: A donor template designed for site-directed insertion of a chimeric antigen receptor (e.g., an anti-CD22 CAR) into the TCR locus (e.g., TRAC). In general, the left and right border sequences are selected to disrupt the TCR to generate [TCR] ^neg [CAR] ^pos engineered immune cells suitable for allogeneic transplantation into patients;

Fig. 3: Flow cytometry data on the frequency of targeted integration of IL-15m into either the PD1 or CD25 locus using PD1- or CD25- ^TALEN® , respectively, when the anti-CD22 CAR was also integrated into the TRAC locus using TRAC- ^TALEN® . These results demonstrate efficient targeted integration in both cases when the anti-CD22 CAR is integrated into the TRAC locus in combination with integration of the IL-15 coding sequence into the PD1 or CD25 loci. A: mimic-transfected primary T cells. B: Primary T cells transfected with the donor sequences described in Fig. 1 (B and C) and a specific ^TALEN® for dual integration into the TCR and PDI loci. B: Primary T cells transfected with the donor sequences described in Fig. 1 (A and B) and a specific ^TALEN® for dual integration into the TCR and CD25 loci;

Fig. 4 is a schematic representation of the exogenous sequences used in the experimental section to transfect primary immune cells to obtain the results shown in Figs. 5 and 6;

Fig. 5 and 6 show flow cytometric data on LNGFR expression in viable T cells transfected with the donor matrices shown in Fig. 4 and specific TALENs.^®(TCR and CD25) after non-specific activation with anti-CD3/CD28 (Dynabeads^®) and after CAR-dependent activation by tumor cells (Raji tumor cells). As shown in Fig. , LNGFR expression was specifically induced in [CAR anti-CD22]^positivecells after CAR/tumor contact;

Fig. 7 and 8 show flow cytometric data of CD25 expression in viable T cells transfected with the donor matrices shown in Fig. 4 and specific TALENs ^® (TCR and CD25) after non-specific activation with anti-CD3/CD28 (Dynabeads ^® ) and activation by tumor cells (Raji tumor cells). As shown in Fig. 8, CD25 expression was specifically induced in [CAR anti-CD22] ^positive cells after CAR/tumor contact;

Fig. 9 is a schematic representation of the exogenous sequences used in the experimental section to transfect primary immune cells to obtain the results shown in Figs. 11 and 12;

Fig. 10 and 11 show flow cytometric data of LNGFR expression in viable T cells transfected with the donor matrices shown in Fig. 9 and specific TALENs ^® (TCR and PD1) after non-specific activation with anti-CD3/CD28 (Dynabeads ^® ) and after activation by tumor cells (Raji tumor cells). As shown in Fig. 11, LNGFR expression was specifically induced in [CAR anti-CD22] ^positive [cells after CAR/tumor contact;

Fig. 12 shows flow cytometric data on endogenous PD1 expression in viable T cells transfected with the donor matrices shown in Fig. 9 and specific TALEN ^® (TCR and PD1) after non-specific activation with anti-CD3/CD28 (Dynabeads ^® ) and activation by tumor cells (Raji tumor cells) with or without TALEN ^® (TCR and PD1). PD1 was efficiently “switched off” by TALEN treatment (residual PD1 expression level was 8% compared to 54%);

Fig. 13 is a graph showing IL-15 production data in [CAR] ^positive (CARm) and [CAR] ^negative engineered immune cells of the invention that were transfected with the donor template shown in Fig. 2(B) and ^TALEN® to insert exogenous IL-15 coding sequences into the PD1 locus. IL-15, transcriptionally controlled by the endogenous PD1 promoter, was efficiently induced following non-specific activation with anti-CD3/CD28 ( ^Dynabeads® ) and tumor cell activation (Raji tumor cells) and was secreted into the culture media;

Fig. 14 is a graph showing the amount of IL-15 secreted over time (in days) following activation by engineered immune cells of the invention. A: Cells engineered by integrating the IL-15 coding sequence into the CD25 locus using the donor DNA templates shown in Fig. 2A (IL-15m_CD25) and/or 2B (CARm). B: Cells engineered by integrating the IL-15 coding sequence into the PD1 locus using the donor DNA templates shown in Fig. 2B (IL-15m_PD1) and/or 2B (CARm). Integration into both loci was shown to result in similar IL-15 secretion profiles. IL-15 secretion was significantly increased upon tumor-specific CAR activation;

Fig. 15 is a graph showing the number of Raji-Luc tumor cells expressing the CD22 antigen (luciferase signal) over time in a survival assay (serial killing assay) as described in Example 2. Immune cells (PBMCs) were engineered to integrate IL-15 coding sequences into the PD1 locus (A) or the CD25 locus (B) and to express an anti-CD22 CAR at the TCR locus (thereby disrupting TCR expression). In this assay, tumor cells were regularly added to the culture medium because they were partially or completely eliminated by CAR-positive cells. Re-expression of IL-15 on either PD1 or CD25 cells greatly facilitated the elimination of tumor cells by CAR-positive cells.

Fig. 16 is a schematic representation of the donor sequences used in the experimental section to insert into the PD1 locus the endogenous sequences encoding IL-12 and gp130Fc. A: A donor template (designated IL-12m-PD1) designed for site-directed insertion of the IL-12a and IL-12b coding sequences (SEQ ID NOs: 47 and 48) into the PD1 locus to achieve co-transcription of IL-12a and IL-12b while disrupting the endogenous PD1 coding sequence. The right and left border sequences homologous to the PD1 locus sequences are at least 100 bp in length, preferably at least 200 bp in length, and more preferably at least 300 bp in length, and comprise SEQ ID NOs: 45 and 46. A detailed description of the sequences is provided in Table 5. B: donor template (designated gp130Fcm-PD1) designed for site-directed insertion of the gp130Fc coding sequences (SEQ ID NO: 51) to achieve transcription at the PD1 locus under the control of the PD1 promoter, thereby disrupting the endogenous PD1 coding sequence. The right and left border sequences homologous to the PD1 locus sequences are at least 100 bp in length, preferably at least 200 bp in length, and more preferably at least 300 bp in length, and comprise SEQ ID NOs: 45 and 46. A detailed description of the sequences is provided in Table 5.

Table 1: ISU domain variants from different viruses.

Table 2: Amino acid sequences of FP polypeptide from natural and artificial sources.

Table 3: List of genes involved in inhibitory pathways in immune cells that can be successfully modified or inactivated by inserting an exogenous coding sequence according to the invention.

Table 4: Sequences mentioned in Example 1.

Table 5: Sequences mentioned in Example 2.

Table 6: List of human genes that are up-regulated upon T cell activation (activation-responsive CAR promoters) in which targeted gene insertion according to the present invention can enhance the therapeutic potential of immune cells.

Table 7: Selected genes that are stably transcribed during immune cell activation (with or without T cell activation).

Table 8: Selected genes characterized by transient up-regulation upon T cell activation.

Table 9: Selected genes up-regulated upon T cell activation for more than 24 h.

Table 10: Selected genes that are down-regulated during immune cell activation.

Table 11: Selected genes that are silenced upon T cell activation (safe haven loci for targeted gene integration).

Table 12: List of gene loci up-regulated in tumor-derived infiltrating lymphocytes (derived from multiple tumors) that can be used for gene integration of exogenous coding sequences of the present invention.

Table 13: List of gene loci characterized by up-regulation under hypoxic conditions in tumors that can be used for gene integration of exogenous coding sequences proposed in the present invention.

Detailed description of the invention

Unless otherwise specifically defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art of gene therapy, biochemistry, genetics and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned in this specification are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, controls. In addition, the materials, methods, and examples are provided for illustration only and are not intended to limit the scope of the invention unless otherwise indicated.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology within the skill in the art. Such techniques are described in detail in the literature, see, for example, Frederick M. Ausubel, Current Protocols in Molecular Biology, Wiley and son Inc, Library of Congress, USA, 2000; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; Oligonucleotide Synthesis, M.J. Gait, 1984; Mullis et al., US Patent Application No. 4,683,195; Nucleic Acid Hybridization, B.D. Harries and S.J. Higgins, 1984; Transcription And Translation, ed. V.D. Hames and S.J. Higgins, 1984; R.I. Freshney, Culture Of Animal Cells, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 198; B. Perbal, A Practical Guide To Molecular Cloning, 1984; series: Methods In ENZYMOLOGY, under. ed. J. Abelson and M. Simon, Academic Press, Inc., New York, especially vol. 154 and vol. 155, ed. Wu et al., et al. 185, Gene Expression Technology, ed. D. Goeddel; Gene Transfer Vectors For Mammalian Cells, ed. J.H. Miller and M.P. Calos, 1987, Cold Spring Harbor Laboratory; Immunochemical Methods In Cell And Molecular Biology, ed. Mayer and Walker, Academic Press, London, 1987; Handbook Of Experimental Immunology, volumes I-IV, ed. D.M. Weir and S.S. Blackwell, 1986; and Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986.

The present invention relates to a general method for producing primary immune cells for cellular immunotherapy, comprising targeted gene integration of an exogenous coding sequence into the chromosomal DNA of said immune cells. According to some aspects of the invention, said integration is carried out in such a way as to place said coding sequence under the transcriptional control of at least one promoter endogenous to said cells, wherein said endogenous promoter is preferably not a constitutive promoter, such as the promoter providing transcription of the constant domain of the alpha chain of the T cell receptor (TRAC-NCBI Gene ID No. 28755). A constitutive promoter according to the present invention is, for example, a promoter that is active regardless of CAR activation, for example when the T cells are not yet activated.

Enhancing the Therapeutic Potential of Immune Cells through Targeted Gene Integration

Gene editing techniques using polynucleotide sequence-specific reagents such as rare cleavage endonucleases have become accepted in the field for introducing genetic modifications into primary cells. However, to date they have not been used in immune cells to introduce exogenous coding sequences under the transcriptional control of endogenous promoters.

The objective of the present invention was to enhance the therapeutic potential of immune cells using gene editing techniques, primarily targeted gene integration.

By "targeted gene integration" is meant any known site-specific methods that allow insertion, replacement or correction of a genomic sequence in a living cell. According to one of the preferred aspects of the present invention, said targeted gene integration comprises homologous recombination of a gene at the locus of the target gene, to achieve insertion of at least one exogenous nucleotide, preferably a sequence consisting of several nucleotides (i.e. a polynucleotide), and more preferably a coding sequence, or substitution by said nucleotide.

By "sequence-specific reagent" is meant any active molecule which has the ability to specifically recognize a selected polynucleotide sequence in a genomic locus, preferably at least 9 bp in length, more preferably at least 10 bp and even more preferably at least 12 bp, in the context of modifying said genomic locus. According to a preferred aspect of the invention, said sequence-specific reagent is a reagent in the form of a sequence-specific nuclease.

By "immune cell" is meant a cell of hematopoietic origin, functionally involved in the initiation and/or implementation of the innate and/or adaptive immune response, as a rule, such as CD3- or CD4-positive cells. According to the present invention, the immune cell can be a dendritic cell, a dendritic killer cell, a mast cell, an NK cell, a B cell or a T cell selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes or helper T lymphocytes. The cells may be obtained from a variety of sources (but not limited to), including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors, such as tumor-infiltrating lymphocytes. In some embodiments, the immune cell may be obtained from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In another embodiment, the cell is part of a mixed immune cell population that includes cells with different phenotypic characteristics, such as CD4, CD8, and CD56 positive cells.

By "primary cell" or "primary cells" is meant cells that are obtained directly from living tissue (e.g. biopsy material) and engineered to grow in vitro for a limited period of time, meaning that they can undergo time-limited population doubling. Primary cells are in contrast to continuously expanding tumorigenic or artificially immortalized cell lines. Examples of such cell lines include, but are not limited to, CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells. Primary cells are generally used in cell therapy because they are thought to be more functional and less oncogenic.

In general, primary immune cells are obtained from donors or patients using various methods known in the art, such as leukapheresis methods, which are summarized in Schwartz J. et al. (Guidelines on the use of therapeutic apheresis in clinical practice-evidence-based approach from the Writing Committee of the American Society for Apheresis: 6th special issue of J Clin Apher. 28(3), 2013, pp. 145-284).

The primary immune cells according to the present invention may also be differentiated stem cells, such as cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSC) and induced pluripotent stem cells (iPS).

By "nuclease reagent" is meant a nucleic acid molecule that participates in a nuclease-catalyzed reaction in a target cell, preferably an endonuclease reaction, either alone or as a subunit of a complex such as guide RNA/Cas9, preferably resulting in cleavage of a target nucleotide sequence.

The nuclease reagents of the invention are typically "sequence-specific reagents", meaning that they can induce DNA cleavage in cells at predetermined loci, broadly considered as a "target gene". The nucleotide sequence that is recognized by the sequence-specific reagents is referred to as the "target sequence". Said target sequence is typically selected from a rare or unique sequence for the cellular genome, and more broadly for the human genome, which can be determined using software and data available in human genome databases such as http://www.ensembl.org/index.html.

"Rarely cleaving endonucleases" are reagents such as sequence-specific endonucleases, wherein the sequences they recognize typically consist of 10-50 consecutive nucleotide pairs, preferably from 12 to 30 bp and more preferably from 14 to 20 bp.

According to a preferred aspect of the invention, said endonuclease reagent is a nucleic acid which encodes an "engineered" or "programmable" rare cleaving endonuclease, such as the homing nuclease described, for example, by Arnould S. et al. (WO 2004/067736), the zinc finger nuclease (ZFN) described, for example, by Urnov F. et al. (Highly efficient endogenous human gene correction using designed zinc-finger nucleases Nature 435, 2005, pp. 646-651), the TALE nuclease described, for example, by Mussolino et al. (A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity Nucl. Acids Res. 39(21), 2011, pp. 9283-9293), or Mega-TAL nuclease, described, for example, by Boissel et al. (MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering Nucleic Acids Research 42 (4), 2013, pp. 2591-2601).

According to another embodiment of the invention, the endonuclease reagent is a guide RNA intended for use in combination with an RNA-guided endonuclease such as Cas9 or Cpf1, as described, inter alia, by Doudna J. and Chapentier E. (The new frontier of genome engineering with CRISPR-Cas9 Science 346 (6213), 2014, p. 1077) (the publication is incorporated herein by reference).

According to a preferred aspect of the invention, the endonuclease reagent is expressed transiently in cells, meaning that said reagent is not intended to be integrated into the genome or to be maintained for a long period of time, which is the case for RNA, more particularly mRNA, proteins or complexes in the form of a mixture of proteins and nucleic acids (for example, ribonucleoproteins).

Typically, 80% of the endonuclease reagent is cleaved within 30 h, preferably 24, more preferably 20 h after transfection.

The endonuclease in the form of mRNA is preferably synthesized with a cap to increase its stability using methods well known in the art, which are described, for example, by Kore A.L. et al. (Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization J Am Chem Soc. 131(18), 2009, pp. 6364-6365).

Typically, the electroporation step used for transfection of immune cells is usually carried out in closed chambers containing parallel plate electrodes that generate a pulsating electric field between said parallel plate electrodes, the intensity of which is higher than 100 V/cm and lower than 5000 V/cm, substantially uniform throughout the volume being treated, as described in WO/2004/083379, which is incorporated herein by reference, particularly at page 23, line 25 to page 29, line 11. One of said electroporation chambers preferably has a geometric factor (cm ^-1 ) defined as the quotient obtained by dividing the square of the interelectrode gap (cm ² ) by the volume of the chamber (cm ³ ), where the geometric factor is less than or equal to 0.1 cm ^-1 where the cell suspension and the sequence-specific reagent are in a medium that is controlled so that the conductivity of the medium is in the range of 0.01 to 1.0 mS. Typically, the cell suspension is exposed to one or more electric field pulses. According to the method, the volume of the suspension to be processed can be scaled, and the time of processing the cells in the chamber is maintained substantially constant.

It has been established that due to their higher specificity, TALE nucleases are the most suitable sequence-specific nuclease reagents for therapeutic use, especially in heterodimeric forms - i.e. when they "work" in pairs comprising a "right" monomer (also designated as "5'" or "forward") and a "left" monomer (also designated as "3'" or "reverse"), as described, for example, by Mussolino et al. (TALEN ^® facilitate targeted genome editing in human cells with high specificity and low cytotoxicity Nucl. Acids Res. 42(10), 2014, pp. 6762-6773).

As indicated previously, the sequence specific reagent is preferably in the form of nucleic acids, for example in the form of DNA or RNA encoding its subunit, which is a rare cleaving endonuclease, but they can also be a component of a conjugate comprising polynucleotide(s) and polypeptide(s), such as so-called "ribonucleoproteins". These conjugates can be formed in combination with reagents such as Cas9 or Cpf1 (RNA-guided endonucleases) or Argonaute (DNA-guided endonucleases), which are currently described, respectively, by Zetsche B. et al. (Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System Cell 163(3), 2015, pp. 759-771) and by Gao F. et al. (DNA-guided genome editing using the Natronobacterium gregoryi Argonaute Nature Biotech, 2016), which include guide RNAs or DNAs that can form complexes with their corresponding nucleases.

The term "exogenous sequence" refers to any nucleotide or nucleotide sequence that is not naturally present at the selected locus. This sequence may be homologous to or a copy of a genomic sequence, or may be a foreign sequence introduced into the cell. In contrast, an "endogenous sequence" means a genomic sequence that is naturally present at a locus in a cell. The exogenous sequence preferably encodes a polypeptide, the expression of which provides a therapeutic advantage over sister cells that do not have the exogenous sequence integrated into the locus. An endogenous sequence in which the genome is edited by inserting a nucleotide or polynucleotide in the manner of the present invention, resulting in the expression of a different polypeptide, is broadly referred to as an exogenous coding sequence.

The method of the present invention can be combined with other methods, including physical methods of genetic transformation, such as viral transduction or transfection using nanoparticles, and they can also be combined with other methods of gene inactivation and/or transgene insertion.

According to one of the objects of the invention, the method proposed in the invention includes the stages of:

- obtain a population of primary immune cells;

- are introduced into a portion of the specified primary immune cells:

I) at least one nucleic acid containing an exogenous nucleotide or polynucleotide sequence subject to integration into a selected endogenous locus in order to encode at least one molecule that enhances the therapeutic potential of said population of immune cells;

II) at least one sequence-specific reagent that specifically targets said selected endogenous locus, wherein said nucleotide or polynucleotide sequence is inserted by integrating a target gene into said endogenous locus such that said exogenous nucleotide or polynucleotide sequence is under the transcriptional control of an endogenous promoter present at said locus.

According to one embodiment of the method, the sequence-specific reagent is a nuclease and integration of the target gene is achieved by homologous recombination or NHEJ in said immune cells.

According to another aspect of the invention, said endogenous promoter is selected from promoters that are active in the process of activation of immune cells and are preferably subject to up-regulation.

More specifically, the invention provides a method for producing engineered primary immune cells for immunotherapy, wherein said method comprises:

- obtaining a population of primary immune cells;

- introduction into a portion of the specified primary immune cells:

I) at least one exogenous nucleic acid containing an exogenous coding sequence that encodes at least one molecule that increases the therapeutic potential of said population of immune cells;

II) at least one sequence-specific reagent that is a nuclease that specifically targets a gene under the control of an endogenous promoter active in the process of immune cell activation;

wherein said coding sequence is introduced into the genome of a primary immune cell by targeted homologous integration so as to place said coding sequence under the transcriptional control of at least one endogenous promoter of said gene.

By "increased therapeutic potential" is meant that the engineered immune cells acquire at least one advantageous property when used in cell therapy compared to their sister non-engineered immune cells. Therapeutic properties in the present invention are any of the measurable properties mentioned in the relevant scientific literature.

Increased therapeutic potential may be more specifically reflected in the resistance of immune cells to the drug, increased persistence in vitro or in vivo, or safety/greater ease of processing during the manufacturing of therapeutic compositions and during treatments.

In general, the molecule that enhances therapeutic potential is a polypeptide, but may also be a nucleic acid that has the ability to direct or suppress the expression of other genes, such as interfering RNA or guide RNA. Polypeptides may act directly or indirectly as signal transducers or transcriptional regulators.

According to one embodiment of the present method, the exogenous sequence is introduced into the endogenous chromosomal DNA by targeted homologous recombination. Thus, the exogenous nucleic acid introduced into the immune cell comprises at least one coding sequence(s) along with sequences that can hybridize with the endogenous chromosomal sequences under physiological conditions. Typically, said homologous sequences have at least 70%, preferably 80% and more preferably 90% sequence identity with the endogenous gene sequences localized in the locus into which the insertion is carried out. These homologous sequences may flank the coding sequence to improve the fidelity of recombination, as already described, for example, in US 6,528,313. Using available software and online genome databases, vectors can be created that include said coding sequence(s), such that said sequence(s) are(are) introduced into the correct locus under the transcriptional control of at least one endogenous promoter, which is a promoter of an endogenous gene. The exogenous coding sequence(s) is(are) then preferably inserted in "reading frame" with said endogenous gene. The sequences resulting from the integration of the exogenous polynucleotide sequence(s) can encode many different types of proteins, including fusion proteins, a tagged protein, or mutant proteins. Fusion proteins allow the addition of new functional domains to proteins expressed in a cell, such as a dimerization domain that can be used to "switch on" or "switch off" the activity of a given protein, such as switching caspase-9. Tagged proteins may provide an advantage in detecting engineered immune cells and tracking them in patients treated with the cells. Introducing mutations into proteins may confer resistance to drugs or immune-depleting agents, as will be discussed further below.

Conferring resistance to drugs or immune system depleting agents

According to one embodiment of the method proposed in the present invention, the exogenous sequence, which is integrated into the genomic locus of immune cells, encodes a molecule that imparts resistance to the drug to said immune cells.

Examples of preferred exogenous sequences include dihydrofolate reductase (DHFR) variants conferring resistance to folate analogues such as methotrexate, inosine monophosphate dehydrogenase 2 (IMPDH2) variants conferring resistance to IMPDH inhibitors such as mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF), calcineurin or methylguanine transferase (MGMT) variants conferring resistance to a calcineurin inhibitor such as FK506 and/or CsA, mTOR variants such as mTORmut conferring resistance to rapamycin, and Lck variants such as Lckmut conferring resistance to imatinib and Gleevec.

The term "drug" as used herein refers to a compound or derivative thereof, preferably a standard chemotherapeutic agent, which is typically used to interact with a cancer cell, thereby reducing the proliferative or viable status of the cell. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide), metabolic antagonists (e.g., a purine nucleoside antimetabolite such as clofarabine, fludarabine or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil or its derivatives), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vindesine, taxol), cisplatin, carboplatin, etoposide, etc. Said agents may also include (but are not limited to) the anticancer agents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU), and CAMPTOTHECIN™, or a therapeutic derivative of any of them.

In the context of the present description, an immune cell is rendered "resistant or tolerant" to a drug when said cell or population of cells is modified so that it can proliferate, at least in vitro, in a culture medium containing half the maximum inhibitory concentration (IC ₅₀ ) of said drug (said IC ₅₀ is determined relative to unmodified cell(s) or population of cells).

In a particular embodiment of the invention, resistance to said drug can be conferred on immune cells by expressing at least one "drug resistance coding sequence". Said term "drug resistance coding sequence" refers to a nucleotide sequence that confers "resistance" to an agent, such as one of the chemotherapeutic agents mentioned above. The drug resistance coding sequence of the invention may encode resistance to an antimetabolite, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, their analogs or derivatives, etc. (Takebe N., S.C. Zhao et al. "Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene". Mol. Ther. 3(1), 2001, pp. 88-96, Zielske S.P., J.S. Reese et al. "In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning." J. Clin. Invest. 112(10), 2003, pp. 1561-1570, Nivens M.S., T. Felder et al. "Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase" Cancer Chemother Pharmacol 53(2), 2004, pp. 107- 115, Bardenheuer W., K. Lehmberg et al. "Resistance to cytarabine and gemcitabine and in vitro selection of transduced cells after retroviral expression of cytidine deaminase in human hematopoietic progenitor cells." Leukemia 19(12), 2005, cc. 2281-2288, Kushman M.E., S.L. Kabler et al. "Expression of human glutathione S-transferase P1 confers resistance to benzo[a]pyrene or benzo[a]pyrene-7,8-dihydrodiol mutagenesis, macromolecular alkylation and formation of stable N2-Gua-BPDE adducts in stably transfected V79MZ cells co-expressing hCYP1A1" Carcinogenesis 28(1), 2007, cc. 207-214).

Expression of said drug resistance-conferring exogenous sequences in immune cells according to the present invention more particularly enables use of said immune cells in cell therapy-based treatment regimens in which cell therapy is combined with chemotherapy or use in patients previously treated with said drugs.

Several drug resistance coding sequences have been identified that are potentially useful for conferring resistance to the drug of the invention. One example of a drug resistance coding sequence may be, in particular, a mutant or modified form of dihydrofolate reductase (DHFR). DHFR is an enzyme involved in regulating the amount of tetrahydrofolate in the cell and is important for DNA synthesis. Folate analogues such as methotrexate (MTX) inhibit DHFR and are therefore used as antineoplastic agents in clinical settings. Various mutant forms of DHFR have been described that have increased resistance to inhibition by antifolate agents used in therapy. In a specific embodiment, the drug resistance coding sequence of the present invention may be a nucleotide sequence encoding a mutant form of wild-type human DHFR (GenBank: AAH71996.1) that contains at least one mutation that confer resistance to treatment with an antifolate agent, such as methotrexate. In a specific embodiment, the mutant form of DHFR contains at least one mutated amino acid at position G15, L22, F31 or F34, preferably at position L22 or F31 (Schweitzer et al., "Dihydrofolate reductase as a therapeutic target," Faseb J 4(8), 1990, pp. 2441-2452; International Patent Application WO 94/24277; U.S. Patent 6,642,043). In a specific embodiment, said mutant form of DHFR comprises two mutated amino acids at positions L22 and F31. The amino acid positions referred to herein are often expressed as amino acid positions in the wild-type DHFR polypeptide. In a specific embodiment, the serine residue at position 15 is preferably replaced with a tryptophan residue. In another specific embodiment, the leucine residue at position 22 is preferably replaced with an amino acid that can interfere with the binding of the mutant DHFR to antifolate agents, preferably uncharged amino acid residues such as phenylalanine or tyrosine. In another specific embodiment, the phenylalanine residue at positions 31 or 34 is preferably replaced with a low molecular weight hydrophilic amino acid residue such as alanine, serine or glycine.

Another example of a coding sequence conferring drug resistance may also be a mutant or modified form of inosine 5'-monophosphate dehydrogenase II (IMPDH2), the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides. A mutant or modified form of IMPDH2 is a gene conferring resistance to an IMPDH inhibitor. IMPDH inhibitors may be mycophenolic acid (MPA) or its prodrug mycophenolate mofetil (MMF). An IMPDH2 mutant may contain at least one, preferably two mutations in the MAP binding site of wild-type human IMPDH2 (Genebank: NP_000875.2) that result in significantly increased resistance to an IMPDH inhibitor. In this variant, mutations preferably affect positions T333 and/or S351 (Yam P., M. Jensen et al. "Ex vivo selection and expansion of cells based on expression of a mutated inosine monophosphate dehydrogenase 2 after HIV vector transduction: effects on lymphocytes, monocytes, and CD34+ stem cells", Mol. Ther. 14(2), 2006, pp. 236-244), (Jonnalagadda M. et al. "Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy", PLoS One 8(6), 2013, e65519).

Another coding sequence that confers drug resistance is a mutant form of calcineurin. Calcineurin (PP2B - NCBI: ACX34092.1) is a ubiquitously expressed serine/threonine protein phosphatase that is involved in many biological processes and plays a central role in T-cell activation. Calcineurin is a heterodimer consisting of a catalytic subunit (CnA; three isoforms) and a regulatory subunit (CnB; two isoforms). Upon engagement with the T-cell receptor, calcineurin dephosphorylates the transcription factor NFAT, resulting in its translocation to the nucleus where it activates critical genes such as IL2. FK506 in complex with FKBP12 or cyclosporin A (CsA) in complex with CyPA block access of NFAT to the active site of calcineurin, preventing its dephosphorylation and thereby inhibiting T-cell activation (Brewin et al., "Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease" Blood 114(23), 2009, pp. 4792-4803). In a specific embodiment of the invention, said mutant form may comprise at least one mutant amino acid in the wild-type calcineurin heterodimer at positions: V314, Y341, M347, T351, W352, L354, K360, preferably double mutations at positions T351 and L354 or V314 and Y341. In a specific embodiment, the valine residue at position 341 may be replaced by a lysine or arginine residue, the tyrosine residue at position 341 may be replaced by a phenylalanine residue; methionine at position 347 may be replaced by a glutamic acid, arginine or tryptophan residue; threonine at position 351 may be replaced by a glutamic acid residue; the tryptophan residue at position 352 may be replaced by a cysteine, glutamic acid or alanine residue, the serine at position 353 may be replaced by a histidine or asparagine residue, the leucine at position 354 may be replaced by an alanine residue; the lysine at position 360 may be replaced by an alanine or phenylalanine residue. In another specific embodiment of the invention, said mutant form may comprise at least one mutant amino acid in the wild-type calcineurin heterodimer at positions: V120, N123, L124 or K125, preferably double mutations at positions L124 and K125. In a specific embodiment, valine at position 120 may be replaced by a serine, aspartic acid, phenylalanine or leucine residue; asparagine at position 123 may be replaced by tryptophan, lysine, phenylalanine, arginine, histidine, or serine; leucine at position 124 may be replaced by a threonine residue; lysine at position 125 may be replaced by alanine, glutamic acid, tryptophan, or two residues such as leucine-arginine or isoleucine-glutamic acid may be added to the amino acid sequence after lysine at position 125. The amino acid positions described herein are often expressed as amino acid positions in the wild-type human calcineurin heterodimer b polypeptide (NCBI: ACX34095.1).

Another coding sequence that confers drug resistance is the O(6)-methylguanine methyltransferase (MGMT - UniProtKB: P16455) sequence encoding 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 that enhances nitrosourea toxicity and is co-administered with TMZ to enhance the cytotoxic effects of this agent. Several mutant forms of MGMT that encode variants of AGT are highly resistant to inactivation by 6-BG but retain their ability to repair DNA damage (Maze R. et al., "Retroviral-mediated expression of the P140A, but not P140A/G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment" J. Pharmacol. Exp. Ther. 290(3), 1999, pp. 1467-1474). In a specific embodiment, the mutant form of AGT may comprise a mutant amino acid at position P140 of wild-type AGT. In a preferred embodiment, said proline at position 140 is replaced by a lysine residue.

Another coding sequence that may confer drug resistance is the multidrug resistance confers protein 1 (MDR1) gene. This gene encodes a membrane glycoprotein known as P-glycoprotein (P-GP), which is involved in the transport of metabolic by-products across the cell membrane. P-GP has a broad spectrum of specificity for several structurally unrelated chemotherapeutic agents. Thus, drug resistance can be conferred on cells by expression of the nucleotide sequence that encodes MDR-1 (Genebank NP_000918).

Another coding sequence that confers drug resistance may be involved in the production of cytotoxic antibiotics, such as those synthesized by the ble or mcrA genes. Ectopic expression of the ble gene or the mcrA gene in an immune cell provides a selective advantage when exposed to the respective chemotherapeutic agents bleomycin and mitomycin C (Belcourt M.F., "Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA," PNAS. 96(18), 1999, pp. 10489-10494).

Other coding sequences conferring drug resistance may originate from genes encoding mutant versions of drug targets, such as the mTOR mutant variants (mTOR mut) conferring resistance to rapamycin, described in Lorenz M. C. et al., "TOR Mutations Confer Rapamycin Resistance by Preventing Interaction with FKBP12-Rapamycin," The Journal of Biological Chemistry 270 (1995): 27531-27537, or certain Lck mutant variants (Lckmut) conferring resistance to gleevec, described in Lee K. C. et al., "Lck is a key target of imatinib and dasatinib in T-cell activation," Leukemia, 24 (2010): 896-900.

As described above, the step of the method comprising genetic modification may be the step of introducing into the cells an exogenous nucleic acid comprising at least one sequence that encodes a coding sequence that confers drug resistance and a portion of an endogenous gene, whereby homologous recombination occurs between the endogenous gene and the exogenous nucleic acid. In a particular embodiment of the invention, said endogenous gene may be a wild-type gene "that confers drug resistance", whereby after homologous recombination the wild-type gene is replaced by a mutant form of the gene that confers drug resistance.

Increased persistence of immune cells in vivo

According to one variant of the method, the exogenous sequence, which is integrated into the genomic locus of immune cells, encodes a molecule that increases the persistence of immune cells, primarily persistence in vivo in the tumor environment.

By "increased persistence" is meant an extended survival of immune cells in terms of lifespan, especially when the engineered immune cells are injected into a patient. For example, persistence is increased if the average survival of the modified cells is significantly extended compared to unmodified cells, by at least 10%, preferably by 20%, more preferably by 30%, even more preferably by 50%.

This is especially important when the immune cells are allogeneic. They can be obtained by creating a local immune defense by introducing coding sequences that ectopically express and/or secrete immunosuppressive polypeptides on the cell membrane or through the cell membrane. Various panels of these polypeptides, in particular, are immune checkpoint antagonists, immunosuppressive peptides from the viral envelope or NKG2D ligand can increase the persistence and/or engraftment of allogeneic immune cells in patients.

According to one embodiment of the invention, the immunosuppressive polypeptide encoded by said exogenous coding sequence is a ligand for cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152, GenBank accession code AF414120.1). Said polypeptide ligand is preferably an anti-CTLA-4 immunoglobulin, such as CTLA-4a Ig and CTLA-4b Ig, or a functional variant thereof.

According to one embodiment of the invention, the immunosuppressive polypeptide encoded by said exogenous coding sequence is a PD1 antagonist, such as PD-L1 (other names: CD274, programmed cell death ligand 1; see UniProt human polypeptide sequence Q9NZQ7), which encodes a 290 amino acid type I transmembrane protein consisting of an Ig V-like domain, an Ig C-like domain, a hydrophobic transmembrane domain, and a 30 amino acid cytoplasmic tail. According to the present invention, said membrane-bound form of PD-L1 ligand may be a native form (wild type) or a truncated form, such as, for example, obtained by deletion of the intracellular domain, or a form with one or more mutation(s) (Wang S. et al., J Exp Med. 197(9), 2003, pp. 1083-1091). It should be noted that PD1 is not considered in the present invention as a membrane-bound form of PD-L1 ligand. According to another embodiment of the invention, said immunosuppressive polypeptide is in a secreted form. The said recombinant secreted PD-L1 (or soluble PD-L1) can be produced by fusing the extracellular domain of PD-L1 with the Fc region of immunoglobulin (Haile S. T. et al., Cancer Immunol. Res. 2(7), 2014, pp. 610-615; Song M. Y. et al., Gut. 64(2), 2015, pp. 260-271). The said recombinant PD-L1 can neutralize PD-1 and abrogate PD-1-mediated inhibition of T cells. PD-L1 ligand can be co-expressed with CTLA4 Ig to further enhance the persistence of both.

According to another embodiment of the invention, the exogenous sequence encodes a polypeptide that comprises a viral immunosuppressant env domain (ISU), which is obtained, for example, from HIV-1, HIV-2, SIV, MoMuLV, HTLV-I, -II, MPMV, SRV-1, syncytin 1 or 2, HERV-K or FELV.

Table 1 below shows ISU domain variants from various viruses that can be expressed according to the present invention.

According to another embodiment of the invention, the exogenous sequence encodes an FP polypeptide, such as gp41. Several FP polypeptides from naturally occurring and artificial sources are provided in Table 2 below.

According to another embodiment of the invention, the exogenous sequence encodes a non-human MHC homologue, especially a viral MHC homologue or a chimeric β2m polypeptide, such as described in Margalit, A. et al., "Chimeric β2 microglobulin/CD3ζ polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8+ T cells," Int. Immunol. 15 (11), 2008, pp. 1379-1387.

According to one embodiment of the invention, the exogenous sequence encodes an NKG2D ligand. Some viruses, such as cytomegaloviruses, have acquired mechanisms to evade NK cell-mediated immune surveillance and interact with the NKG2D pathway by secreting a protein that can bind to NKG2D ligands and prevent their surface expression (Welte S.A et al., "Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein," Eur. J. Immunol., 33 (2003): 194-203). In tumor cells, several mechanisms are involved in NKG2D evasion resulting from the secretion of NKG2D ligands such as ULBP2, MICB, or MICA (Salih H.R., Antropius H., Gieseke F., Lutz S.Z., Kanz L., et al., Functional expression and release of ligands for the activating immunoreceptor NKG2D in leukemia, Blood 102, 2003, pp. 1389-1396).

According to one embodiment of the invention, the exogenous sequence encodes a cytokine receptor, such as the IL-12 receptor. IL-12 is a well-known activator of immune cell activation (Curtis J.H., "IL-12 Produced by Dendritic Cells Augments CD8+ T Cell Activation through the Production of the Chemokines CCL1 and CCL171", The Journal of Immunology. 181 (12), 2008, pp. 8576-8584.

According to one embodiment of the invention, the exogenous sequence codes for an antibody directed against inhibitory peptides or proteins. Said antibody should preferably be secreted by immune cells in soluble form. Nanobodies from sharks and camels are preferred in this regard, since they have a structure in the form of single-chain antibodies (Muyldermans S., "Nanobodies: Natural Single-Domain Antibodies", Annual Review of Biochemistry 82, 2013, pp. 775-797). It is also believed that they are easier to fuse with secretory signal polypeptides and soluble hydrophilic domains.

The various aspects of enhancing cell persistence described above are particularly advantageous if the exogenous coding sequence is introduced by disruption of the endogenous gene encoding β2m or another MHC component, as will be described in detail below.

Increasing the therapeutic activity of immune cells

According to one embodiment of the method proposed in the present invention, the exogenous sequence, which is integrated into the genomic locus of immune cells, encodes a molecule that increases the therapeutic activity of immune cells.

By "increased therapeutic activity" is meant that the immune cells or cell population engineered according to the present invention become more aggressive compared to unengineered cells or cell population against a selected target cell type. Said target cells typically belong to a certain cell type or cell population, which are preferably characterized by common surface marker(s). As used herein, "therapeutic potential" reflects therapeutic activity measured in vitro experiments. Typically, sensitive cancer cell lines, such as Daudi cells, are used to determine whether immune cells are more or less active against said cells by assessing their ability to kill cells or reduce growth. This can be determined by measuring levels of immune cell degranulation or chemokine and cytokine production. Experiments can also be performed on mice injected with tumor cells and by monitoring the spread of the resulting tumor. Increased activity is considered significant when the number of cells developed in these experiments is reduced by immune cells by more than 10%, preferably more than 20%, more preferably more than 30%, even more preferably more than 50%.

According to one aspect of the invention, said exogenous sequence encodes a chemokine or cytokine, such as IL-12. It is most advantageous to express IL-12, since this cytokine is well known from the literature as an enhancer of immune cell activation (Colombo M.P. et al., "Interleukin-12 in anti-tumor immunity and immunotherapy", Cytokine Growth Factor Rev. 13(2), 2002, pp. 155-168).

According to one aspect of the invention, said exogenous sequence codes for or activates secreted factors that act on other populations of immune cells, such as regulatory T cells, to attenuate their inhibitory effect on said immune cells.

According to one aspect of the invention, said exogenous sequence encodes an inhibitor of regulatory T cell activity which is a polypeptide inhibitor of the transcription factor forkhead/winged helix 3 (FoxP3) family of proteins, and more preferably is a cell-penetrating peptide inhibitor of FoxP3, such as that designated P60 (Casares N. et al. "A peptide inhibitor of FoxP3 impairs regulatory T cell activity and improves vaccine efficacy in mice." J Immunol 185(9), 2010, pp. 5150-5159).

By "inhibitor of regulatory T cell activity" is meant a molecule or a precursor of said molecule secreted by T cells and allowing T cells to avoid down-regulation of their activity by regulatory T cells. In general, said inhibitor of regulatory T cell activity exerts its effect by reducing the transcriptional activity of FoxP3 in said cells.

According to one aspect of the invention, said exogenous sequence encodes a secreted inhibitor of tumor-associated macrophages (TAMs), such as a CCR2/CCL2 neutralizing agent. Tumor-associated macrophages (TAMs) are critical modulators of the tumor microenvironment.

Clinicopathological studies have suggested that accumulation of TAMs in tumors correlates with poor clinical outcome. In line with these data, experimental and animal studies have supported the idea that TAMs may provide a preferential microenvironment that enhances tumor development and progression (Theerawut C. et al., "Tumor-Associated Macrophages as Major Players in the Tumor Microenvironment", Cancers (Basel) 6(3), 2014, pp. 1670-1690). Chemokine (C-C motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP1 - NCBI NP_002973.1), is a small cytokine belonging to the C-C chemokine family secreted by macrophages that is a chemoattractive factor for monocytes, lymphocytes, and basophils. CCR2 (chemokine (C-C motif) receptor type 2 - NCBI NP_001116513.2) is the receptor for CCL2.

Increasing the specificity and safety of immune cells

Expression of chimeric antigen receptors (CARs) has become common in the field to control or enhance the specificity of primary immune cells such as T cells and NK cells to treat tumors or infected cells. CARs expressed by these immune cells specifically target antigenic markers on the surface of abnormal cells, which further helps immune cells destroy these cells in vivo (Sadelain M. et al., "The basic principles of chimeric antigen receptor design" Cancer Discov. 3(4), 2013, pp. 388-398). CARs are typically engineered to contain activating domains that stimulate immune cells in response to binding to a specific antigen (called positive CARs), but they can also contain an inhibitory domain with the opposite effect (called negative CARs) (Fedorov V. D., "Novel Approaches to Enhance the Specificity and Safety of Engineered T Cells", Cancer Journal 20 (2), 2014, pp. 160-165. Positive and negative CARs can be combined and co-expressed to fine-tune the immune specificity of cells depending on the different antigens that are present on the surface of target cells.

Genetic sequences encoding CARs are typically introduced into the genome of cells using retroviral vectors, which have increased transduction efficiency but integrate at random positions. According to the present invention, chimeric antigen receptor (CAR) components can be introduced into selected loci, more preferably under the control of endogenous promoters, using recombination of target genes.

According to one aspect of the invention, while the positive CAR is introduced into the immune cell by a viral vector, the negative CAR can be introduced by insertion of a target gene and vice versa, and preferably should have activity only during the activation of immune cells. Thus, the inhibitory (i.e. negative) CAR participates in increasing specificity, preventing the immune cells from attacking a specific cell type that is to be preserved. Furthermore, according to said aspect of the invention, said negative CAR can be an apoptosis-promoting CAR, i.e. The said CAR contains an apoptotic domain such as FasL (CD95 - NCBI: NP_000034.1) or its functional variant, which transduces a signal that induces cell death (Eberstadt M. et al., "NMR structure and mutagenesis of the FADD (Mort1) death-effector domain", Nature, 392 (6679), 1998, pp. 941-945).

Thus, the exogenous coding sequence that is inserted according to the invention may encode a factor that has the ability to induce cell death directly, in combination with another component(s), or by activating another component(s).

As another way to improve the safety of using primary immune cells, the exogenous coding sequence may encode molecules that confer sensitivity to immune cells to drugs or other exogenous substrates. Said molecules may be cytochrome(s), for example, of the P450 family (Preissner S. et al., "SuperCYP: a comprehensive database on Cytochrome P450 enzymes including a tool for analysis of CYP-drug interactions". Nucleic Acids Res 38, 2010 (Database Issue): D237-43), such as CYP2D6-1 (NCBI - NP_000097.3), CYP2D6-2 (NCBI - NP_001020332.2), CYP2C9 (), CYP3A4 (NCBI - NP_000762.2), CYP2C19 (NCBI - NP_000760.1) or CYP1A2 (NCBI - NP_000752.2.), which confer hypersensitivity to a drug such as such as cyclophosphamide and/or isophosphamide.

According to another aspect of the invention, the exogenous sequence is introduced into immune cells for expression, especially in vivo, to reduce IL-6 or IL-8 signaling in trans to control potential cytokine release syndrome (CRS).

Said exogenous sequence may encode, for example, antibodies directed against IL-6 or IL-8 or against their receptors IL-6R or IL-8R.

According to a preferred aspect of the invention, said exogenous sequence may encode a soluble extracellular domain of GP130, for example, characterized by at least 80% identity with SEQ ID NO: 61.

The said soluble extracellular domain of GP130, described, for example, in Rose-John S. ("The Soluble Interleukine Receptor: Advanced Therapeutic Options in Inflammation" Clinical Pharmacology & Therapeutics, 102(4), 2017, pp. 591-598), may be a fusion with immunoglobulin fragments, such as sgp130Fc (SEQ ID NO: 62). As described above, said exogenous sequence can be stably integrated into the genome by site-directed mutagenesis (i.e., using sequence-specific nuclease reagents) and placed under the transcriptional activity of an endogenous promoter at a locus that is active during immune cell activation, such as one of those listed in Tables 6, 8, or 9, and preferably that is characteristically up-regulated upon CAR activation or that is CAR dependent.

According to a more preferred embodiment of the invention, the exogenous sequence is introduced into a CAR-positive immune cell, such as a T cell, expressing an anti-CD22 CAR polynucleotide sequence such as SEQ ID NO: 31. According to some more specific embodiments of the invention, said exogenous sequence encoding a polypeptide that can bind, and preferably interacts, with a cytokine receptor of the IL-6 receptor family, such as said soluble extracellular domain of GP130, is integrated into the PD1, CD25 or CD69 locus. According to the present invention, the endogenous sequence encoding the PD1 locus is preferably disrupted by said exogenous sequence.

Thus, the invention provides a method for treating or reducing CRS in cellular immunotherapy, in which patients are administered cells or a therapeutic composition thereof, wherein said cells are genetically modified such that they secrete a polypeptide(s) comprising the soluble extracellular domain of GP130, sGP130Fc, an antibody to IL-6 or an antibody to IL6R, an antibody to IL-8 or an antibody to IL8R, or any fusion thereof.

Examples of preferred genotypes of engineered immune cells include:

[CAR] ^positive [GP130] ^positive

[CAR] ^positive [GP130] ^positive

[CAR] ^positive [TCR] ^negative [GP130] ^positive [PD1] ^negative

[CAR] ^positive [TCR] ^negative [GP130] ^positive [PD1] ^negative

[CAR] ^positive [GP130] ^positive [CD25] ^negative

[CAR] ^positive [TCR] ^negative [GP130] ^positive [CD25] ^negative

Enhancing the Efficiency of Targeted Gene Insertion into Primary Immune Cells Using AAV Vectors

The present disclosure provides donor templates and sequence-specific reagents, illustrated in the figures, that can be used to effect efficient in-frame insertion of coding sequence into endogenous promoters, in particular PD1 and CD25, as well as means and sequences for detecting correct insertion of said exogenous sequences into said loci.

The donor templates provided in the present invention are typically polynucleotide sequences that can be incorporated into various vectors known in the art that facilitate delivery of the donor templates into the nucleus at a time when endonuclease reagents become active to achieve site-directed insertion into the genome, typically by NHEJ or homologous recombination.

In particular, the present invention provides specific donor polynucleotides for the expression of IL-15 (SEQ ID NO: 59) at the PD1 locus, which comprise one or more of the following sequences:

- a sequence that encodes IL-15, for example, one having identity with SEQ ID NO: 50;

- sequences located in the reverse and forward directions (also referred to as left and right) homologous to the PD1 locus, which preferably contain the polynucleotide sequences SEQ ID NO: 45 and SEQ ID NO: 46;

- optionally a sequence that encodes a soluble form of the IL-15 receptor (sIL-15R), for example one having identity with SEQ ID NO: 50;

- optionally at least one peptide 2A cleavage site (peptide 2A recognition site), such as one of SEQ ID NO: 53 (F2A), SEQ ID NO: 54 (P2A) and/or SEQ ID NO: 55 (T2A).

In particular, the present invention provides specific donor polynucleotides for the expression of IL-12 (SEQ ID NO: 58) at the PD1 locus, which comprise one or more of the following sequences:

- a sequence that encodes IL-12a, for example, one having identity with SEQ ID NO: 47;

- sequences located in the reverse and forward directions (also referred to as left and right) homologous to the PD1 locus, which preferably contain the polynucleotide sequences SEQ ID NO: 45 and SEQ ID NO: 46;

- optionally a sequence that encodes IL-12b, for example one having identity with SEQ ID NO: 48;

- optionally at least one peptide 2A cleavage site such as one of SEQ ID NO: 53 (F2A), SEQ ID NO: 54 (P2A) and/or SEQ ID NO: 55 (T2A).

In particular, the present invention provides specific donor polynucleotides for the expression of soluble GP130 (comprising SEQ ID NO: 61) at the PD1 locus, which comprise one or more of the following sequences:

- a sequence that encodes soluble GP130, preferably soluble gp130 fused to Fc, for example one having identity with SEQ ID NO: 62;

- sequences located in the reverse and forward directions (also referred to as left and right) homologous to the PD1 locus, which preferably contain the polynucleotide sequences SEQ ID NO: 45 and SEQ ID NO: 46;

- optionally at least one peptide 2A cleavage site such as one of SEQ ID NO: 53 (F2A), SEQ ID NO: 54 (P2A) and/or SEQ ID NO: 55 (T2A).

In particular, the present invention provides specific donor polynucleotides for the expression of IL-15 (SEQ ID NO: 59) at the CD25 locus, which comprise one or more of the following sequences:

- a sequence that encodes IL-15, for example, one having identity with SEQ ID NO: 50;

- sequences located in the reverse and forward directions (also referred to as left and right) homologous to the CD25 locus, which preferably contain the polynucleotide sequences SEQ ID NO: 43 and SEQ ID NO: 44;

- optionally a sequence that encodes a soluble form of the IL-15 receptor (sIL-15R), for example one having identity with SEQ ID NO: 50;

- optionally at least one peptide 2A cleavage site, such as one of SEQ ID NO: 53 (F2A), SEQ ID NO: 54 (P2A) and/or SEQ ID NO: 55 (T2A).

In particular, the present invention provides specific donor polynucleotides for the expression of IL-12 (SEQ ID NO: 58) at the CD25 locus, which comprise one or more of the following sequences:

- a sequence that encodes IL-12a, for example, one having identity with SEQ ID NO: 47;

- sequences located in the reverse and forward directions (also referred to as left and right) homologous to the CD25 locus, which preferably contain the polynucleotide sequences SEQ ID NO: 43 and SEQ ID NO: 44;

- optionally a sequence that encodes IL-12b, for example one having identity with SEQ ID NO: 48;

- optionally at least one peptide 2A cleavage site such as one of SEQ ID NO: 53 (F2A), SEQ ID NO: 54 (P2A) and/or SEQ ID NO: 55 (T2A).

In particular, the present invention provides specific donor polynucleotides for the expression of soluble GP130 (comprising SEQ ID NO: 61) at the CD25 locus, which comprise one or more of the following sequences:

- a sequence that encodes soluble GP130, preferably soluble gp130 fused to Fc, for example one having identity with SEQ ID NO: 62;

- sequences located in the reverse and forward directions (also referred to as left and right) homologous to the CD25 locus, which preferably contain the polynucleotide sequences SEQ ID NO: 43 and SEQ ID NO: 44;

- optionally at least one peptide 2A cleavage site such as one of SEQ ID NO: 53 (F2A), SEQ ID NO: 54 (P2A) and/or SEQ ID NO: 55 (T2A).

As illustrated in the examples provided herein, the invention has substantially increased the level of targeted gene insertion into human cells using AAV vectors, particularly vectors from the AAV6 family.

Thus, in a broad sense, the object of the present invention is the transduction of AAV vectors into human primary immune cells in combination with the expression of sequence-specific endonuclease reagents, such as TALE endonucleases, which are more preferably introduced in mRNA form, to enhance homologous recombination events in said cells.

According to one aspect of the present invention, sequence-specific endonuclease reagents can be introduced into cells by transfection, more preferably by electroporation, of mRNA that encodes said sequence-specific endonuclease reagents, such as TALE nucleases.

Thus, in a broad sense, as a more specific object of the invention, there is provided a method for inserting an exogenous nucleotide sequence into an endogenous polynucleotide sequence in a cell, which comprises at least the steps of:

- transducing into said cell an AAV vector containing said exogenous sequence and sequences homologous to the targeted endogenous DNA sequence, and

- induce the expression of a sequence-specific reagent, which is an endonuclease, to cleave the endogenous sequence at the insertion locus.

The result of the insertion of an exogenous nucleotide sequence may be the introduction of genetic material, correction or replacement of an endogenous sequence, more preferably “in frame” with the corresponding endogenous gene sequences at a given locus.

According to another aspect of the invention, the cell is transduced using from 10 ⁵ to 10 ⁷ , preferably from 10 ⁶ to 10 ⁷ , more preferably about 5×10 ⁶ viral genomes per cell.

According to another aspect of the invention, cells can be treated with proteasome inhibitors, such as bortezomib, to further facilitate homologous recombination.

According to one aspect of the present invention, the AAV vector used in the method may comprise an exogenous promoter-less coding sequence corresponding to any of those described herein, so that it can be placed under the control of an endogenous promoter at one of the loci selected from those listed herein.

According to one aspect of the present invention, the AAV vector used in the method may comprise a peptide 2A cleavage site followed by cDNA (minus the start codon), resulting in the formation of an exogenous coding sequence.

According to one aspect of the invention, said AAV vector comprises an exogenous sequence that encodes a chimeric antigen receptor, in particular an anti-CD19 CAR, an anti-CD22 CAR, an anti-CD123 CAR, an anti-CS1 CAR, an anti-CCL1 CAR, an anti-HSP70 CAR, an anti-GD3 CAR or an anti-ROR1 CAR.

Thus, the invention relates to any AAV vectors created for carrying out the method specified in the present description, in particular vectors that contain a sequence homologous to the locus into which the insertion is carried out, localized in any of the endogenous genes responsible for T-cell activation, which are indicated in Table 4.

In accordance with the teaching of the present invention, many other vectors known in the art can also be used, such as plasmids, episomal vectors, linear DNA matrices, etc.

As stated above, the DNA vector used according to the invention preferably comprises: (1) said exogenous nucleic acid, which comprises an exogenous coding sequence to be inserted by homologous recombination, and (2) a sequence which codes for a sequence-specific reagent, which is an endonuclease, which facilitates said insertion. According to a more preferred aspect of the invention, the exogenous nucleic acid stated in subparagraph (1) does not comprise any promoter sequence, while the sequence stated in subparagraph (2) has its own promoter. According to an even more preferred aspect of the invention, the nucleic acid referred to in subparagraph (1) comprises an internal ribosome entry site (IRES) or "self-cleaving" 2A peptides, such as T2A, P2A, E2A or F2A, as a result of which the endogenous gene into which the exogenous coding sequence is inserted becomes polycistronic. The IRES of the 2A peptide may be located before or after said exogenous coding sequence.

According to the present invention, preferred vectors are AAV6-derived vectors that comprise the donor polynucleotides described above in the present description or illustrated in the experimental section and in the figures. Examples of vectors according to the invention comprise or consist of polynucleotides that are identical to the sequences of SEQ ID NO: 37 (template for integrating the IL-15 encoding sequence into the CD25 locus), SEQ ID NO: 38 (template for integrating the IL-15 encoding sequence into the PD1 locus), SEQ ID NO: 39 (template for integrating the IL-12 encoding sequence into the CD25 locus) and SEQ ID NO: 40 (template for integrating the IL-12 encoding sequence into the PD1 locus).

Targeted gene integration into immune cells under the transcriptional control of endogenous promoters

According to one of the main objects, the present invention is based on the advantage of the endogenous transcriptional activity of immune cells for the expression of exogenous sequences that enhance their therapeutic potential.

The invention provides several variants based on the transcriptional activity profile of endogenous promoters and on the selection of promoter loci that can be used to implement the invention. Preferred are loci whose transcriptional activity is generally high upon activation of immune cells, primarily in response to CAR activation (CAR-sensitive promoters), when the cells are endowed with CAR.

Thus, the invention provides a method for producing allogeneic therapeutic immune cells by expressing a first exogenous sequence encoding a CAR in a TCR locus, which disrupts TCR expression, and expressing a second exogenous coding sequence under the transcriptional activity of an endogenous locus, preferably dependent on any of the following causes:

- CD3/CD28 activation, for example using Dynabeads, which can be used, for example, to stimulate cell expansion;

- CAR activation, for example via the CD3zeta pathway, which can be used, for example, to activate immune cell functions on a target;

- transcriptional activity associated with the manifestation of a disease symptom or molecular marker, which can be used, for example, to activate cells in situ in diseased organs;

- cell differentiation that can be used to confer therapeutic activity to cells at a given differentiation level or to express a protein in a particular lineage (see Fig. 1), for example, at the time when hematopoietic cells acquire their immune functions; and/or

- TME (tumor microenvironment), which can be used to redirect cell activity and amplification under tumor-specific conditions (hypoxia, low glucose, etc.) or to prevent exhaustion and/or to maintain activation;

- CRS (cytokine release syndrome), which can be used to reduce the adverse effects associated with CAR T cell activity.

In creating the invention, a list of endogenous genes (Table 6) was created for the first time, for which the possibility of their use for recombination of the target gene in accordance with the present invention was established. To create this list, several mouse transcriptome databases were studied, in particular, those obtained within the framework of the international project on decoding the genome of mouse immune cells (Immunological Genome Project Consortium), which are described in Best J.A. et al., "Transcriptional insights into the CD8(+) T cell response to infection and memory T cell formation", Nat. Immunol. 14(4), 2013, pp. 404-142, which made it possible to compare the transcription levels of various genes during T-cell activation in response to treatment with ovalbumin antigens. In addition, since very little data are available regarding the activation of human T cells, in creating the invention some extrapolations were made and an analysis of these data was carried out and a comparison with the situation in humans was carried out by studying the available literature regarding human genes. The selected loci are particularly relevant for insertion of CAR coding sequences. Based on the first selection step, the results of which are presented in Table 6, subsequent gene selections were performed in the invention based on their predicted expression profiles (Tables 7-10).

On the other hand, in the creation of the invention, as a result of selection, transcriptional loci are identified that are the most inactive, which may be the most suitable for insertion of insertion cassette(s) for expression of exogenous coding sequence under transcriptional control of exogenous promoters. These loci are designated as "safe harbor loci" because they are the most inactive with respect to transcription, especially in the process of T-cell activation. They can be used for integration of the coding sequence by minimizing the risk of interference with the expression of the immune cell genome.

Targeted gene insertion under the control of endogenous promoters that are stably active during immune cell activation

The selected endogenous gene loci associated with this embodiment of the invention are listed in Table 7.

Thus, the present invention provides a method comprising a step of performing targeted insertion of a gene under the control of an endogenous promoter that is constantly active during the activation of immune cells, preferably from an endogenous gene selected from CD3G, Rn28s1, Rn18s, Rn7sk, Actg1, β2m, Rpl18a, Pabpc1, Gapdh, Rpl17, Rpl19, Rplp0, Cfl1 and Pfn1.

By "stable activity" we mean that the transcriptional activity detected at these promoters in the primary immune cell is not affected by down-regulation upon immune cell activation.

As is already known (Acuto, O. "Tailoring T-cell receptor signals by proximal negative feedback mechanisms". Nature Reviews Immunology 8, 2008, pp. 699-712), promoters present in the TCR locus are subject to various negative feedback mechanisms upon contact with the TCR and therefore may not be stably active or may be up-regulated during the implementation of the method proposed in the present invention. One of the objectives of the present invention is to avoid using the TCR locus as a possible insertion site for exogenous coding sequences that are to be expressed during T-cell activation. Thus, according to one of the aspects of the invention, the targeted insertion of an exogenous coding sequence is not carried out in the locus of the TCRalpha or TCRbeta gene.

Examples of exogenous coding sequences that may be usefully introduced into the loci under the control of stably active endogenous promoters include sequences encoding or upregulating the production of a cytokine, a chemokine receptor, a drug resistance molecule, a costimulatory ligand such as 4-1BRL and OX40L, or a secreted antibody.

Gene integration under the control of endogenous promoters that are dependent on immune cell activation or dependent on CAR activation

As indicated above, the method of the present invention includes a step of performing targeted insertion of a gene under the control of an endogenous promoter, the transcriptional activity of which is preferably up-regulated upon activation of immune cells, either briefly or for more than 10 days.

By "immune cell activation" is meant an immune response according to mechanisms that are generally described and are generally accepted in the literature for a given type of immune cell. For example, with respect to T cells, T cell activation is typically characterized by one of the changes that include cell surface expression resulting from their production of various proteins, including CD69, CD71, and CD25 (which is also a marker for Treg cells) and HLA-DR (a marker for human T cell activation), release of perforin, granzymes, and granulysin (degranulation), or production of the cytokine effectors IFN-γ, TNF, and LT-alpha.

According to a preferred embodiment of the invention, the transcriptional activity of an endogenous gene is up-regulated in an immune cell, especially in response to activation by a CAR. The CAR can be independently expressed in the immune cell. By "independently expressed" is meant that the CAR can be transcribed in the immune cell from an exogenous expression cassette introduced, for example, using a retroviral vector, such as a lentiviral vector, or by transfection by electroporation with capped RNAs encoding said CAR. Many methods for expressing a CAR in an immune cell are known in the art, as described, for example, in the referenced sources (REF).

The endogenous gene whose transcriptional activity is up-regulated is most suitable for the integration of exogenous sequences to encode a cytokine(s) such as IL-12 and IL-15, an immunogenic peptide(s), or a secreted antibody such as an anti-IDO1, anti-IL10, anti-PD1, anti-PDL1, anti-IL6, or anti-PGE2 antibody.

According to a preferred embodiment of the invention, the endogenous promoter is selected on the basis that its transcriptional activity is sensitive and more preferably dependent on CAR activation.

As demonstrated herein, CD69, CD25, and PD1 are loci most suitable for insertion of expression of exogenous coding sequences that are to be expressed when immune cells become activated, primarily in CAR-positive immune cells.

Thus, the present invention combines any methods of expressing a CAR in an immune cell with the step of performing a site-directed insertion of an exogenous coding sequence into a locus whose transcriptional activity is sensitive to or dependent on contact of said CAR with a tumor antigen. First of all, the method includes the step of introducing into a CAR-positive or recombinant TCR-positive immune cell an exogenous sequence encoding IL-12 or IL-15 under the transcriptional control of one promoter selected from the PD1, CD25 and CD69 promoters.

In particular, CAR-positive cells can be obtained by performing co-expression steps in an immune cell, preferably a primary cell and more preferably a primary T cell, of at least one exogenous sequence encoding a CAR and another exogenous sequence placed under the control of an endogenous promoter whose transcriptional activity is dependent on said CAR, such as the PD1, CD25 or CD71 promoter.

The expression "dependent on said CAR" means that the transcriptional activity of said endogenous promoter is necessarily increased by more than 10%, preferably more than 20%, more preferably more than 50% and even more preferably more than 80% as a result of contact of the CAR with its cognate antigen in a situation where, as a rule, the number of antigens exceeds the number of CARs that are present on the cell surface, and the number of CARs that are expressed on the cell surface is more than 10 molecules per cell, preferably more than 100 and more preferably more than 1000 molecules per cell.

Thus, according to the present invention, the expression of the CAR sequence, preferably integrated into the TCR locus, is constitutive expression, while another exogenous sequence integrated into another locus is co-expressed in response to or dependent on the contact of said CAR with its cognate antigen. Said other locus is, for example, the CD25, PD1 or CD71 locus or any loci specifically transcribed upon activation of the CAR.

In other words, the invention proposes to carry out joint expression of a CAR and at least one exogenous coding sequence, wherein the expression of said exogenous sequence is under the control of an endogenous promoter, the transcriptional activity of which is influenced by the activity of the CAR, in order to obtain engineered immune cells characterized by an improved immune response.

As described previously, this can be accomplished by transfecting cells with sequence-specific nuclease reagents that target the coding regions of the specified CAR-specific loci along with donor templates containing sequences homologous to the specified genomic regions. The sequence-specific nuclease reagents assist the donor templates in integrating by homologous recombination or NHEJ.

According to a preferred embodiment of the invention, the exogenous coding sequence is integrated in frame with the endogenous gene so as to maintain the expression of said endogenous gene. This is the case, for example, with CD25 and CD69, as demonstrated in one of the examples in the experimental section of the present description.

According to a preferred embodiment of the invention, the exogenous sequence disrupts the endogenous coding sequence of the gene, preventing the expression of one endogenous coding sequence, especially when said expression has a negative effect on the functions of immune cells, which is the case, for example, with PD1, as demonstrated in the experimental section herein.

According to even more preferred embodiments, the exogenous coding sequence that disrupts the endogenous gene sequence is placed in frame with the endogenous promoter so as to make its expression dependent on the endogenous promoter, as also demonstrated in the experimental section.

The present invention also relates to polynucleotide and polypeptide sequences that encode various TAL nucleases, examples of which are provided in the present description, especially those that provide site-directed insertion into the CD25 locus (SEQ ID NOs: 18 and 19), as well as to the sequences of the corresponding target and to RVD sequences.

The present invention also provides kits for transfection of immune cells that contain polynucleotides encoding sequence-specific reagents that are endonucleases, donor sequences designed to integrate an exogenous sequence into a locus that is the target of said reagents. Examples of such kits include a kit comprising an mRNA that encodes a rare cleaving endonuclease targeting the PD1 locus (e.g., a PD1 TALEN ^® ) and an AAV vector comprising an exogenous sequence encoding IL-12, a kit comprising an mRNA that encodes a rare cleaving endonuclease targeting the PD1 locus (e.g., a PD1 TALEN ^® ) and an AAV vector comprising an exogenous sequence encoding IL-15, a kit comprising an mRNA that encodes a rare cleaving endonuclease targeting the CD25 locus (e.g., a CD25 TALEN ^® ) and an AAV vector comprising an exogenous sequence encoding IL-12, a kit comprising an mRNA that encodes a rare cleaving endonuclease targeting the CD25 locus (e.g., CD25 ^TALEN® ), and an AAV vector comprising an exogenous sequence encoding IL-15, a kit comprising mRNA that encodes a rare cleaving endonuclease that targets the PD1 locus (e.g., PD1 ^TALEN® ), and an AAV vector comprising an exogenous sequence encoding soluble gp130, a kit comprising mRNA that encodes a rare cleaving endonuclease that targets the CD25 locus (e.g., CD25 ^TALEN® ), and an AAV vector comprising an exogenous sequence encoding soluble gp130, and any kits comprising reagents that are endonucleases that target the genes listed in Table 6 and a donor template for introducing the coding sequence described herein.

According to one aspect of the invention, the endogenous gene is selected for its weak up-regulation. The exogenous coding sequence introduced into said endogenous gene, the transcriptional activity of which is characterized by weak up-regulation, may preferably consist of an inhibitory CAR or an apoptosis-inducing CAR, the expression level of which, as a rule, remains lower compared to a positive CAR. Said combination of CAR expression, for example, one of which is transduced by a viral vector and the other is introduced according to the invention, can significantly increase the specificity or safety of CAR-bearing immune cells.

Some endogenous promoters are transiently up-regulated, sometimes for less than 12 h upon immune cell activation, such as those selected from the endogenous gene loci Spata6, Itga6, Rcbtb2, Cd1d1, St8sia4, Itgae, and Fam214a (Table 8). Other endogenous promoters are up-regulated, sometimes for less than 24 h upon immune cell activation, such as those selected from the endogenous gene loci IL3, IL2, Ccl4, IL21, Gp49a, Nr4a3, Lilrb4, Cd200, Cdkn1a, Gzmc, Nr4a2, Cish, Ccr8, Lad1, and Crabp2 (Table 9), and others for more than 24 h, generally more than 10 days, upon immune cell activation. The promoters indicated are selected from Gzmb, Tbx21, Plek, Chek1, Slamf7, Zbtb32, Tigit, Lag3, Gzma, Wee1, IL12rb2, Eea1, and Dtl (Table 9).

Alternatively, it has been found that an endogenous gene under the transcriptional control of promoters that are down-regulated upon activation of immune cells may also be of interest for use in the method of the present invention. Thus, it has been found that exogenous coding sequences encoding anti-apoptotic factors such as those belonging to the Bc12 family, as well as Bc1XL, NF-kB, survivin, as well as anti-FAP (fibroblast-activating protein), such as the CAR anti-FAP component, can be introduced into said loci. The specified endogenous gene under the transcriptional control of promoters that are characterized by down-regulation upon immune cell activation can be more specifically selected from Slc6a19, Cd55, Xkrx, Mturn, H2-Ob, Cnr2, Itgae, Raver2, Zbtb20, Arrb1, Abca1, Tet1, Slc16a5, and Ampd3 (Table 10).

Gene integration under the control of endogenous promoters activated in the tumor microenvironment (TME)

One of the objects of the present invention is more specifically methods for preventing the exhaustion of immune cells in the tumor microenvironment (TME). Often, the exhaustion of immune cells occurs in response to the depletion of nutrients or molecular signals present in the tumor microenvironment, which contributes to the resistance of tumors. The method includes the steps of engineering immune cells by integrating exogenous coding sequences under the control of endogenous promoters that are characterized by up-regulation in the presence of arginine, cysteine, tryptophan and oxygen deprivation, as well as extracellular acidosis (lactate accumulation).

Said exogenous sequences may encode chimeric antigen receptors, interleukins, or any polypeptide described anywhere in the present description to enhance the function or activation of immune cells and/or to impart a therapeutic benefit thereto.

The invention lists a number of loci that are found to be up-regulated in a large number of depleting tumor infiltrating lymphocytes (TILs), and they are listed in Tables 12 and 13. The invention provides a step of integrating exogenous coding sequences into said preferred loci to prevent depletion of immune cells, in particular T cells, in the tumor microenvironment.

For example, exogenous CAR encoding sequences can be placed under the transcriptional control of the promoter of endogenous genes that are activated by the tumor microenvironment, such as HIF1a, i.e., the hypoxia-inducible transcription factor, or the aryl hydrocarbon receptor (AhR). These genes are sensory genes induced by hypoxia and xenobiotics in the tumor environment, respectively.

Thus, the present invention can be used to improve the therapeutic outcome of CAR T cell-based therapies by integrating exogenous coding sequences and more specifically genetic attributes (traits)/circuits (patterns) under the control of endogenous T cell promoters influenced by the tumor microenvironment (TME).

According to the invention, up-regulation of endogenous genes can be “borrowed” to re-express relevant exogenous coding sequences in order to enhance the anti-tumor activity of CAR T cells in the microenvironment of certain tumors.

Insertion and expression of a target gene in hematopoietic stem cells (HSC)

More specifically, one of the objects of the present invention is the insertion of transgenes into hematopoietic stem cells (HSC).

Hematopoietic stem cells (HSCs) are multipotent, self-renewing progenitor cells that give rise to all differentiated blood cell types through the process of hematopoiesis. These cells include lymphocytes, granulocytes, and macrophages of the immune system, as well as circulating red blood cells and platelets. HSCs are classically considered to differentiate into two restricted lineages, lymphoid and myeloerythroid oligopotent progenitor cells. The mechanisms controlling HSC self-renewal and differentiation are thought to be influenced by a diverse array of cytokines, chemokines, receptors, and intracellular signaling molecules. HSC differentiation is regulated in part by growth factors and cytokines, including colony-stimulating factors (CSFs) and interleukins (ILs), which activate intracellular signaling pathways. The following factors are known to influence the multipotency, proliferation, and lineage specificity of HSCs. HSCs and their differentiated progeny can be identified by the expression of lineage-specific cell surface markers such as cluster of differentiation (CD) proteins and cytokine receptors in hematopoietic stem cells.

Gene therapy using HSCs has great potential for the treatment of diseases of the hematopoietic system, including immune diseases. In this approach, HSCs are isolated from a patient, genetically modified ex vivo using retroviral vector integration, and then infused into the patient. Retroviral vectors have been the only effective gene delivery system for gene therapy using HSCs to date. However, gene delivery to HSCs using integrating vectors ensures efficient introduction into HSC-derived mature hematopoietic cells. However, the genetically modified cells that are infused into the patient are a polyclonal population in which different cells have integrated proviral vectors at different chromosomal locations, which may result in many undesirable mutations that may be amplified as a result of some of the proliferation/survival benefits of these mutations (Powers and Trobridge, "Identification of Hematopoietic Stem Cell Engraftment Genes in Gene Therapy Studies," J Stem Cell Res Ther S3:004. doi:10.4172/2157-7633.S3-00, 2013).

HSCs are typically collected from peripheral blood after mobilization (patients receive recombinant human granulocyte colony-stimulating factor (G-CSF)). The patient's peripheral blood is collected and enriched for HSCs using the CD34 ⁺ marker. HSCs are then cultured ex vivo and treated with viral vectors. The ex vivo culture period ranges from 1 to 4 days. Before infusion of the genetically modified HSCs, patients may be treated with chemotherapeutic agents or radiation to enhance engraftment efficiency. The genetically modified HSCs are reinfused into the patient intravenously. The cells migrate to the bone marrow before their final invasion into the sinusoids and perivascular tissue. Homing and hematopoiesis are both components of engraftment. Cells that have reached the stem cell niche through homing must begin to produce mature myeloid and lymphoid cells from each blood lineage. Hematopoiesis continues through the action of long-lived HSCs, which have the capacity to self-renew throughout the patient’s long-lived generation of mature blood cells, in particular the production of common lymphoid progenitor cells such as T cells and NK cells, which are immune cells critical for the elimination of infected and malignant cells.

The present invention provides targeted gene insertion into HSCs to introduce exogenous coding sequences under the control of endogenous promoters, especially endogenous promoters of genes that are specifically activated in cells of a particular hematopoietic lineage or at a particular stage of differentiation, preferably at a late stage of differentiation. HSCs can be transduced with a polynucleotide vector (donor template), such as an AAV vector, during the ex vivo processing described in the previous paragraph, expressing a sequence-specific nuclease reagent to facilitate the insertion of coding sequences into the selected locus. The resulting engineered HSCs can then be transplanted into a patient in need thereof for the long-term in vivo production of engineered immune cells that will contain said exogenous coding sequences. Depending on the activity of the selected endogenous promoter, coding sequences can be selectively expressed in certain lineages or in response to the local environment of immune cells in vivo, thereby implementing adaptive immunotherapy.

According to one preferred aspect of the invention, the exogenous coding sequences are placed under the control of promoters of genes whose transcriptional activity is specifically induced in common lymphoid progenitor cells, such as CD34, CD43, Flt-3/Flk-2, IL-7R alpha/CD127 and neprilysin/CD10.

More preferably, the exogenous coding sequences are placed under the control of the promoters of genes whose transcriptional activity is specifically induced in NK cells, such as CD161, CD229/SLAMF3, CD96, DNAM-1/CD226, Fc gammaRII/CD32, Fc gamma RII/RIII (CD32/CD16), Fc gamma RIII (CD16), IL-2 R beta, integrin alpha 2/CD49b, KIR/CD158, NCAM-1/CD56, NKG2A/CD159a, NKG2C/CD159c, NKG2D/CD314, NKp30/NCR3, NKp44/NCR2, NKp46/NCR1, NKp80/KLRF1, Siglec-7/CD328 and TIGIT, or is induced in T cells such as CCR7, CD2, CD3, CD4, CD8, CD28, CD45, CD96, CD229/SLAMF3, DNAM-1/CD226, CD25/IL-2 R alpha, L-selectin/CD62b and TIGIT.

The invention provides as a preferred subject the introduction of an exogenous sequence encoding a CAR or a component thereof into an HSC, preferably under the transcriptional control of a promoter of a gene that is not expressed in the HSC, more preferably a gene that is expressed only in hematopoietic cells produced by said HSC, and even more preferably a gene that is expressed only in T cells or NK cells.

Conditional expression of CAR in HSCs to overcome the blood-thymus barrier

A particular object of the present invention is to produce in vivo, using the above-described engineered HSCs, hematopoietic immune cells, such as T cells or NK cells, which express exogenous coding sequences, in particular a CAR or a component thereof.

One of the main obstacles to obtaining hematopoietic CAR-positive cells using engineered HSCs is, for example, the rejection of CAR-positive cells by the immune system itself, primarily the thymus.

The blood-thymus barrier regulates the exchange of substances between the bloodstream and the thymus, which provides a sequestered environment for the development of immature T cells. The barrier also prevents immature T cells from coming into contact with foreign antigens (since contact with antigens at this stage can lead to T cell death by apoptosis).

One solution proposed in the present invention is to place the sequences encoding CAR components in HSCs under the transcriptional control of promoters that are not transcribed significantly in hematopoietic cells when they cross the thymus barrier. One example of a gene that provides conditional expression of CAR in hematopoietic cells with reduced or no significant transcriptional activity in the thymus is LCK (Uniprot: P06239).

According to a preferred aspect of the invention, an exogenous sequence encoding a CAR or a component thereof is introduced into HSCs under the transcriptional control of a gene known to be specifically expressed in T cells or NK cells, preferably only in said cell types.

Thus, the invention provides a method for producing HSCs that contain exogenous coding sequences that are to be expressed exclusively in selected hematopoietic lineage(s), said coding sequences encoding at least one CAR component or an antigen for stimulating the immune system.

In a broader sense, the invention provides a method for engineering HSCs by targeted insertion into the genome of exogenous coding sequences that are to be selectively expressed in hematopoietic cells produced by said HSCs. As a preferred embodiment of the invention, said hematopoietic cells produced by said engineered HSCs express said exogenous coding sequences in response to certain environmental factors or stimuli in vivo to enhance their therapeutic potential.

Combination of target insertion(s) of sequence into immune cells with inactivation of endogenous genomic sequences

One of the specific objects of the present invention is to perform gene inactivation in primary immune cells at a locus by integrating an exogenous coding sequence into said locus, the expression of which increases the therapeutic potential of said engineered cells. Examples of relevant exogenous coding sequences that can be inserted according to the invention are presented above in combination with their positive effects on the therapeutic potential of the cells. Below in the present description are presented endogenous genes that are preferably targeted by targeted gene insertion and the advantages associated with their inactivation.

According to a preferred aspect of the invention, the effect of inserting a coding sequence is to reduce or prevent the expression of genes involved in self-non-self recognition to reduce graft-versus-host disease (GVHD) or immune rejection of allogeneic cells in a recipient patient. For example, one or more sequence-specific reagents used in the method may reduce or prevent the expression of TCR in primary T cells, such as genes encoding TCR alpha or TCR beta.

As another preferred aspect of the invention, one step of gene editing is to reduce or prevent the expression of the β2m protein and/or another protein involved in its regulation, such as C2TA (Uniprot P33076), or in MHC recognition, such as HLA proteins. This ensures less alloreactivity of the engineered immune cells when infused into patients.

By "allogeneic therapeutic application" is meant that cells obtained from a donor are infused into patients with a different haplotype. In fact, the present invention provides an efficient method for obtaining primary cells that can be gene edited at various gene loci involved in the interaction and recognition of the graft-host.

Other loci can also be edited to enhance the potency, persistence, and therapeutic activity of the engineered primary cells, as described in more detail below:

Inactivation of checkpoint receptors and inhibitory pathways of immune cells:

According to a preferred aspect of the invention, the inserted exogenous coding sequence has the ability to reduce or prevent the expression of a protein that is involved in the inhibitory pathways of immune cells, which are in particular referred to in the literature as "immune checkpoint" (Pardoll D.M. The blockade of immune checkpoints in cancer immunotherapy, Nature Reviews Cancer, 12 (2012), pp. 252-264). In the context of the present invention, the expression "inhibitory pathways of immune cells" refers to any gene expression in immune cells that leads to a decrease in the cytotoxic activity of lymphocytes against malignant or infected cells. This may, for example, relate to a gene involved in the expression of FOXP3, which is known to increase Treg activity against T cells (reduction of T cell activity).

"Immune checkpoints" are molecules in the immune system that either increase the signal (costimulatory molecules) or decrease the signal of immune cell activation. According to the present invention, immune checkpoints are more preferably surface proteins involved in ligand-receptor interactions between T cells and antigen-presenting cells (APCs) that regulate the T cell response to antigen (mediated by peptide-major histocompatibility complex (MHC) molecule complexes that are recognized by the T cell receptor (TCR)). These interactions can occur during the initiation of T cell responses in lymph nodes (in which the main APCs are dendritic cells) or in peripheral tissues or tumors (in which they regulate effector responses). One important family of membrane-bound ligands that are associated with both costimulatory and inhibitory receptors is the B7 family. All members of the B7 family and their known ligands belong to the immunoglobulin superfamily. Many receptors recently identified as members of the B7 family are not fully characterized. Members of the tumor necrosis factor (TNF) family, which bind to cognate molecules of the TNF receptor family, constitute a second family of regulatory ligand-receptor pairs. These receptors primarily emit costimulatory signals upon contact with their cognate ligands. The other major category of signals that regulate T cell activation comes from soluble cytokines in the microenvironment. In other cases, activated T cells upregulate ligands such as CD40L, which engage cognate receptors on APC, e.g., A2aR, the adenosine A2a receptor; B7RP1, B7-related protein 1; BTLA, the B and T cell attenuator; GAL9, galectin 9; HVEM, herpes virus entry mediator; ICOS, inducible T-cell costimulator; IL, interleukin; KIR, killer cell immunoglobulin-like receptor; LAG3, lymphocyte activating gene 3; PD1, programmed cell death ligand 1; PDL, PD1 ligand; TGFβ, transforming growth factor-β; TIM3, T-cell membrane protein 3.

Examples of additional endogenous genes whose expression can be reduced or suppressed to enhance activation in the engineered immune cells of the present invention are listed in Table 3.

For example, the inserted exogenous coding sequence(s) may have the ability to reduce or prevent the expression in the engineered immune cell of at least one protein selected from PD1 (Uniprot Q15116), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG3 (Uniprot PI8627), HAVCR2 (Uniprot Q8TDQ0), BTLA (Uniprot Q7Z6A9), CD160 (Uniprot O95971), TIGIT (Uniprot Q495A1), CD96 (Uniprot P40200), CRTAM (Uniprot 095727), LAIR1 (Uniprot Q6GTX8), SIGLEC7 (Uniprot Q9Y286), SIGLEC9 (Uniprot Q9Y336), CD244 (Uniprot Q9BZW8), TNFRSF10B (Uniprot O14763), TNFRSF10A (Uniprot O00220), CASP8 (Uniprot Q1479 0), CASP10 (Uniprot Q92851), CASP3 (Uniprot P42574), CASP6 (Uniprot P55212), CASP7 (Uniprot P55210), FADD (Uniprot Q13158), FAS (Uniprot P25445), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), SMAD2 (Uniprot Q15796), SMAD3 (Uniprot P84022), SMAD4 (Uniprot Q13485), SMAD10 (Uniprot B7ZSB5), SKI (Uniprot P12755), SKIL (Uniprot P12757), TGIF1 (Uniprot Q15583), IL10RA (Uniprot Q13651), IL10RB (U niprot Q08334), HMOX2 (Uniprot P30519), IL6R (Uniprot P08887), IL6ST (Uniprot P40189), EIF2AK4 (Uniprot Q9P2K8), CSK (Uniprot P41240), PAG1 (Uniprot Q9NWQ8), SIT1 (Uniprot Q9Y3P8), Uniprot Q9BZS1), PRDM1 (Uniprot Q60636), BATF (Uniprot Q16520), GUCY1A2 (Uniprot P33402), GUCY1A3 (Uniprot Q02108), GUCY1B2 (Uniprot Q8BXH3) and GUCY1B3 (Uniprot Q02153). Gene editing introduced into the genes encoding the above proteins is preferably combined with TCR inactivation in CAR T cells.

Preferred is inactivation of PD1 and/or CTLA4 in combination with expression of a non-endogenous immunosuppressive polypeptide such as PD-L1 ligand and/or CTLA-4 Ig (see also peptides listed in Tables 1 and 2).

Inhibition of suppressor cytokines/metabolites

According to another aspect of the invention, the inserted exogenous coding sequence has the ability to reduce or prevent the expression of genes encoding or upregulating suppressor cytokines or metabolites or their receptors, in particular TGFbeta (Uniprot: P01137), TGFbR (Uniprot: P37173), IL10 (Uniprot: P22301), IL10R (Uniprot: Q13651 and/or Q08334), A2aR (Uniprot: P29274), GCN2 (Uniprot: P15442) and PRDM1 (Uniprot: O75626).

Preferred are engineered immune cells in which the sequence encoding IL-2, IL-12 or IL-15 replaces the sequence of at least one of the above-mentioned endogenous genes.

Induction of resistance to chemotherapeutic drugs

According to another embodiment of the method of the present invention, the inserted exogenous coding sequence has the ability to reduce or prevent the expression of a gene responsible for the sensitivity of immune cells to compounds used for standard cancer or infection treatment options, such as drugs that are purine nucleotide analogs (PNA) or 6-mercaptopurine (6MP) and 6-thioguanine (6TG), which are commonly used in chemotherapy. Reduction or inactivation of genes involved in the mechanism of action of these compounds (which are referred to as "genes that increase drug sensitivity") increases the resistance of immune cells to them.

Examples of genes that increase drug sensitivity include genes encoding DCK (Uniprot P27707), which influences the activity of PNAs such as clofarabine and fludarabine, HPRT (Uniprot P00492), which influences the activity of purine antimetabolites such as 6MP and 6TG, and GGH (Uniprot Q92820), which influences the activity of antifolate drugs, particularly methotrexate.

This allows the cells to be used after or in combination with conventional anti-cancer chemotherapies.

Resistance to immune suppressing treatments

According to another aspect of the present invention, the inserted exogenous coding sequence has the ability to reduce or prevent the expression of receptors or proteins that are drug targets, rendering said cells resistant to immune-depleting drug treatments. Said target may be glucocorticoid receptors or antigens, when engineering immune cells that are resistant to glucocorticoids or immune-depleting treatments using antibodies, such as alemtuzumab, which is used to deplete CD52-positive immune cells in many cancer treatments.

In addition, the method of the invention may include targeted gene insertion into endogenous gene(s) encoding or regulating the expression of CD52 (Uniprot P31358) and/or GR (glucocorticoid receptor, also referred to as NR3C1 - Uniprot P04150).

Increasing the activity and survival of CAR-positive immune cells

According to another aspect of the present invention, the inserted exogenous coding sequence may have the ability to reduce or prevent the expression of a surface antigen such as BCMA, CS1 and CD38, wherein said antigen is an antigen that is a target of a CAR that is expressed in said immune cells.

The said embodiment of the invention allows to solve the problem of antigens, which are targets for CAR, which are present on the surface of infected or malignant cells, but are also expressed to some extent by the immune cell itself.

According to a preferred embodiment of the invention, an exogenous sequence encoding a CAR or one of its components is integrated into a gene encoding an antigen that is the target of said CAR, in order to avoid self-destruction of immune cells.

Engineered immune cells and immune cell populations

The present invention also relates to a variety of engineered immune cells that can be obtained using one of the methods described above in isolated form or as part of cell populations.

According to a preferred aspect of the invention, the engineered cells are primary immune cells, such as NK cells or T cells, which are typically part of cell populations that may include different cell types. Typically, the population is isolated from patients or donors by leukapheresis from PBMCs (peripheral blood mononuclear cells).

According to a preferred aspect of the invention, more than 50% of the immune cells included in said population are TCR-negative T cells. According to a more preferred aspect of the invention, more than 50% of the immune cells included in said population are CAR-positive T cells.

The present invention relates to immune cells comprising any combinations of various exogenous coding sequences and inactivated genes, which are respectively and independently described above. Of these combinations, the most preferred are those that combine the expression of a CAR under the transcriptional control of an endogenous promoter, which is stably active during the activation of immune cells and preferably does not depend on said activation, and the expression of an exogenous sequence encoding a cytokine, such as IL-2, IL-12 or IL-15, under the transcriptional control of a promoter, which is characterized by up-regulation during the activation of immune cells.

Another preferred combination is the insertion of an exogenous sequence encoding CAR or one of its components under the transcriptional control of the hypoxia-inducible factor 1 gene promoter (Uniprot: Q16665).

The invention also relates to a pharmaceutical composition comprising the above-described engineered primary immune cell or population of immune cells for the treatment of infection or cancer and to a method for treating a patient in need thereof, wherein said method comprises:

- obtaining a population of engineered primary immune cells according to the above-described method of the invention;

- optionally purifying or sorting said engineered primary immune cells;

- activation of said population of engineered primary immune cells upon or following infusion of said cells into said patient.

Activation and proliferation of T cells

Both before and after genetic modification, the immune cells of the present invention can be activated or expanded even if their activation or proliferation is independent of antigen binding mechanisms. In particular, T cells can be activated and expanded using the methods described, for example, in US 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 and U.S. Patent Application Publication No. 2006/0121005. T cells can be expanded in vitro or in vivo. Typically, T cells are expanded by contacting an agent that stimulates the CD3 TCR complex and a costimulatory molecule on the surface of the T cell to create an activation signal for the T cell. For example, chemical compounds such as the calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

For example, but not limited to, T cell populations can be stimulated in vitro by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Co-stimulation of an accessory molecule on the surface of T cells involves the use of a ligand that binds to the accessory molecule. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions that stimulate T cell proliferation. Suitable conditions for culturing T cells include appropriate media (e.g., minimal essential medium or RPMI 1640 medium, or X-vivo 5, (Lonza)) which may contain factors necessary to ensure proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4, 1L-7, GM-CSF, -10, -2, 1L-15, TGFp and TNF, or any other cell growth supplements known to one of skill in the art. Other cell growth supplements include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. The media may be RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate and vitamins, they may be serum-free or supplemented with serum (or plasma) in a defined amount or a defined set of hormones and/or cytokine(s) in an amount sufficient to support the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures that are intended for administration by infusion to an individual. The target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO ₂ ). T cells that have been stimulated for different periods of time may have different characteristics.

In another specific embodiment of the invention, said cells can be propagated by co-cultivation with tissue or cells. Said cells can also be propagated in vivo, for example, in the blood of an individual after administration of said cell to the individual.

Therapeutic compositions and applications

The above-described method proposed in the present invention allows for the production of engineered primary immune cells in a limited time frame of approximately 15-30 days, preferably 15 to 20 days, and most preferably 18 to 20 days, which allows for the preservation of their full immune therapeutic potential, especially with regard to their cytotoxic activity.

These cells form a population of cells that are preferably derived from a single donor or patient. These populations of cells can be expanded in closed vessels specifically designed for culture in order to meet the highest requirements of manufacturing practices and can be frozen prior to infusion into a patient, thereby obtaining “off-the-shelf” or “ready-to-use” therapeutic compositions.

According to the present invention, a significant number of cells can be obtained by a single leukapheresis, which is critical for obtaining a sufficient number of doses for treating a patient. Although variations may be observed between cell populations obtained from different donors, the number of immune cells obtained by leukapheresis is typically about 10 ⁸ to 10 ¹⁰ PBMC cells. PBMCs contain several cell types: granulocytes, monocytes and lymphocytes, of which T cells account for 30 to 60%, which typically corresponds to 10 ⁸ to 10 ⁹ primary T cells from a single donor. The method of the present invention typically results in a population of engineered cells typically comprising greater than about 10 ⁸ T cells, more specifically greater than about 10 ⁹ T cells, even more specifically greater than about 10 ¹⁰ T cells, and typically greater than 10 ¹¹ T cells.

Thus, more particularly, the invention relates to a therapeutically effective population of primary immune cells, wherein at least 30%, preferably 50%, more preferably 80% of the cells in said population are modified by any method specified in the present description. Said therapeutically effective population of primary immune cells proposed in the present invention comprises immune cells into which at least one exogenous genetic sequence is integrated under the transcriptional control of an endogenous promoter of at least one of the genes listed in Table 6.

Thus, the said compositions or cell populations can be used as medicinal products; in particular for the treatment of cancer, in particular for the treatment of lymphoma, but also solid tumors such as melanomas, neuroblastomas, gliomas or carcinomas, for example tumors of the lung, breast, colon, prostate or ovary, in a patient in need thereof.

More particularly, the invention relates to populations of primary TCR-negative T cells from a single donor, wherein at least 20%, preferably 30%, more preferably 50% of the cells in said population are modified using sequence-specific reagents at at least two, preferably three different loci.

Another object of the present invention is methods of treating patients in need thereof, wherein said method comprises at least one of the following steps:

(a) identification of specific antigen markers present on the surface of patients' tumors based on biopsy data;

(b) obtaining a population of engineered primary immune cells, created using one of the above-described methods provided in the present invention, such that it expresses a CAR directed against said specific antigen markers;

(c) administering said engineered population of created primary immune cells to said patient.

Typically, these cell populations primarily contain CD4- and CD8-positive immune cells, such as T cells, which can be subjected to extensive T cell expansion in vivo and can be maintained for extended periods of time in vitro and in vivo.

Treatments using the engineered primary immune cells of the present invention may be ameliorating, curative or prophylactic. They may be either part of an autologous immunotherapeutic treatment or part of an allogeneic immunotherapeutic treatment. The term "autologous" means that the cells, cell line or population of cells used to treat patients are obtained from the body of said patient or from the body of a human leukocyte antigen (HLA)-compatible donor. The term "allogeneic" means that the cells or population of cells used to treat patients are not obtained from the body of said patient, but are obtained from the body of a donor.

In another embodiment of the invention, said isolated cell of the invention or a cell line derived from said isolated cell can be used to treat liquid tumors, preferably T-cell acute lymphoblastic leukemia.

The invention also includes adult and pediatric tumors/cancers.

Treatment using engineered immune cells according to the invention can be combined with one or more anti-cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormonal therapy, laser therapy and radiation therapy.

According to a preferred embodiment of the invention, said treatment can be used for patients undergoing immunosuppressive therapy. Thus, the present invention preferably relates to cells or a population of cells that are rendered resistant to at least one immunosuppressive agent by inactivating a gene encoding a receptor for said immunosuppressive agent. In this context, immunosuppressive therapy can promote the selection and expansion of T cells according to the invention in the patient's body.

The administration of the cells or cell population of the present invention may be by any conventional method, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions provided herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection or intraperitoneally. In one embodiment, the cellular compositions of the present invention are preferably administered by intravenous injection.

The administration of cells or a population of cells may comprise the administration of 10 ⁴ -10 ⁹ cells/kg body weight, preferably from 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer cell amounts in the ranges indicated. Thus, the present invention provides the use of more than 10, typically more than 50, more particularly more than 100 and typically more than 1000 doses containing from 10 ⁶ to 10 ⁸ gene-edited cells obtained from a sample taken from a single donor or patient.

The cells or population of cells can be administered in one or more doses. In another embodiment of the invention, said effective amount of cells is administered in a single dose. In another embodiment of the invention, said effective amount of cells is administered in more than one dose over a period of time. The administration schedule is within the discretion of the attending physician and depends on the clinical condition of the patient. The cells or population of cells can be obtained from any source, such as a blood bank or donor. Since individual needs vary, determining optimal ranges of effective amounts of the cell type in question for a particular disease or condition is within the skill of the art. An effective amount is an amount that provides a therapeutic or prophylactic benefit. The dose administered will depend on the age, health and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment and the nature of the desired effect.

In another embodiment of the invention, the cells or a composition containing these cells are administered in said effective amount parenterally. Said administration may be intravenous administration. Said administration may be carried out directly by injection into the tumor.

In some embodiments of the present invention, the cells are administered to the patient in combination with (e.g., before, simultaneously, or after) any number of appropriate forms of treatment, including (but not limited to) treatment with antiviral therapies such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for patients with MS (multiple sclerosis), or efalizumab treatment for patients with psoriasis, or other treatments for patients with PML (progressive multifocal leukoencephalopathy). In other embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunodestructive agents such as CAMPATH, CD3 antibodies, or other antibody-based therapies, cytotoxin, fludaribine, cyclosporine, FK506, rapamycin, mycophenolate acid, steroids, FR901228, cytokines and radiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase, which is important for growth factor-inducible signaling (rapamycin) (Henderson, Naya et al., 1991; Liu, Albers et al., 1992; Bierer, Hollander et al., 1993). In another embodiment, the cellular compositions of the present invention are administered to a patient in conjunction with (e.g., prior to, concurrently with, or following) bone marrow transplantation, T-cell ablation therapy using either chemotherapeutic agents such as fludarabine or external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cellular compositions of the invention are administered following B-cell ablation therapy using agents that interact with CD20, such as, for example, Rituxan. For example, in one embodiment, individuals may be treated with standard high-dose chemotherapeutic agents followed by peripheral blood stem cell transplantation. In some embodiments, the individuals are infused with expanded immune cells of the invention following transplantation. In another embodiment, the expanded cells are administered before or after surgery.

When CARs are expressed in immune cells or immune cell populations of the present invention, preferred CARs are those targeting at least one antigen selected from CD22, CD38, CD123, CS1, HSP70, ROR1, GD3, and CLL1.

The engineered immune cells of the present invention, which are endowed with a CAR or a modified TCR targeting CD22, are preferably used to treat leukemia such as acute lymphoblastic leukemia (ALL), cells with a CAR or a modified TCR targeting CD38 are preferably used to treat leukemia such as T-cell acute lymphoblastic leukemia (T-ALL) or multiple myeloma (MM), cells with a CAR or a modified TCR targeting CD123 are preferably used to treat leukemia such as acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasms (BPDCN), cells with a CAR or a modified TCR targeting CS1 are preferably used to treat multiple myeloma (MM).

The present invention also relates to means for detecting engineered cells of the present invention comprising the desired genetic insertions, especially by performing steps in which PCR methods are used to detect the insertion of exogenous coding sequences into the endogenous loci specified herein, especially into the PD1, CD25, CD69 and TCR loci, using probes or primers hybridizing to any of the sequences presented in SEQ ID NOs: 36-40.

Immunoassays can also be used to detect expression in engineered CAR cells, GP130, and to detect absence or decreased expression of TCR, PD1, IL-6, or IL-8 in cells in which the genes have been knocked out or have decreased expression.

Other definitions

- Amino acid residues in a polypeptide sequence are designated in the present description by a single-letter code, according to which, for example, Q designates Gln or a glutamine residue, R designates Arg or an arginine residue, and D designates Asp or an aspartic acid residue.

- An amino acid substitution refers to the replacement of one amino acid residue with another, for example, the replacement of an arginine residue with a glutamine residue in a peptide sequence is an amino acid substitution.

- Nucleotides are designated as follows: a single-letter code is used to designate the nucleoside base: a denotes adenine, t denotes thymine, c denotes cytosine, and g denotes guanine. For degenerate nucleotides, r denotes g or a (purine nucleotides), k denotes g or t, s denotes g or c, w denotes a or t, m denotes a or c, y denotes t or c (pyrimidine nucleotides), d denotes g, a or t, v denotes g, a or c, b denotes g, t, or c, h denotes a, t, or c, and n denotes g, a, t, or c.

- As used herein, the terms "nucleic acid" or "polynucleotides" refer to nucleotides and/or polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by ligation, cleavage, endonuclease treatment, and exonuclease treatment. Nucleic acid molecules may be composed of monomers that are naturally occurring nucleotides (such as DNA or RNA) or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination thereof. Modified nucleotides may have changes in sugar moieties and/or in moieties containing pyrimidine or purine bases. Modifications of sugars include, for example, the replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or the sugars can be functionalized to ethers or esters. In addition, the entire sugar moiety can be replaced by sterically and electronically similar structures, such as aza sugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. The nucleic acid monomers can be cross-linked by phosphodiester bonds or analogs of such bonds. Nucleic acids can be single-stranded or double-stranded.

- The term "endonuclease" refers to any wild-type enzyme or a variant thereof that has the ability to catalyze the hydrolysis (cleavage) of bonds between nucleic acids in a DNA or RNA molecule, preferably in a DNA molecule. Endonucleases do not cleave a DNA or RNA molecule regardless of its sequence, but they recognize and cleave a DNA or RNA molecule at specific polynucleotide sequences, hereinafter referred to as "target sequences" or "target sites". Endonucleases can be classified as rare cleaving endonucleases, for which, as a rule, the polynucleotide recognition site is more than 10 base pairs (bp) in length, more preferably 14-55 bp. Rarely, cleaving endonucleases significantly enhance homologous recombination by inducing DNA double-strand breaks (DSBs) at a specific locus, thereby enabling gene repair or gene insertion-based therapies (Pingoud A. and G.H. Silva. Precision genome surgery. Nat. Biotechnol. 25(7), 2007, pp. 743-744.).

- By "target DNA", "target DNA sequence", "target DNA sequence", "target nucleotide sequence", "target sequence" or "processing site" is meant a polynucleotide sequence that can be targeted and processed by the rare cleavage endonuclease of the present invention. These terms refer to a specific region of DNA, preferably a genomic region in a cell, but also to a region of genetic material that can exist independently of the main genetic material of an organism, such as plasmids, episomes, virus, transposons, or in organelles such as mitochondria (but not limited to them). For example (but not limited to them), RNA-guided target sequences are those genomic sequences that can hybridize to a guide RNA that directs the RNA-guided endonuclease to the desired locus.

- By "mutation" is meant a substitution, deletion, insertion of up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, forty, fifty or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. A mutation may affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.

- By "vector" is meant a nucleic acid molecule having the ability to transport another nucleic acid to which it is linked. "Vectors" according to the present invention include, but are not limited to, a viral vector, a plasmid, an RNA vector, or a linear or circular DNA or RNA molecule, which may consist of chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are vectors that are capable of autonomously replicating (episomal vector) and/or expressing the nucleic acids to which they are linked (expression vectors). A large number of suitable vectors are known to those skilled in the art and are commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated virus, AAV), coronavirus, negative-strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies virus and vesicular stomatitis virus), paramyxovirus (e.g., measles virus and Sendai virus), positive-strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and pox virus (e.g., cowpox virus, fowlpox virus, and canarypox virus). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovirus, hepadnavirus, and hepatitis virus. Examples of retroviruses are: avian leukemia-sarcoma virus, mammalian viruses of type C, type B, type D viruses, the HTLV-BLV group, lentivirus, spumavirus (Coffin J.M., Retroviridae: The viruses and their replication, in: Fundamental Virology, 3rd ed., edited by B.N. Fields et al., Lippincott-Raven Publishers, Philadelphia, 1996).

- As used herein, the term "locus" refers to a specific physical region (location) of a DNA sequence (e.g., a gene) in a genome. The term "locus" may refer to a specific physical region of a target sequence of a rare cleavage endonuclease on a chromosome or a genomic sequence of an infectious agent. Such a locus may comprise a target sequence that is recognized and/or cleaved by the sequence-specific endonuclease of the invention. It should be understood that a locus of interest according to the present invention may not only be a locus on a nucleotide sequence that exists in the main genetic material of an organism (i.e., in a chromosome) of a cell, but also a portion of the genetic material that may exist independently of the main genetic material of an organism, such as plasmids, episomes, a virus, transposons, or in organelles such as mitochondria (but not limited to these examples).

- The term "cleavage" refers to the disruption of a covalently linked polynucleotide backbone. Cleavage can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of the phosphodiester bond. Both single-stranded and double-stranded cleavage are possible, and double-stranded cleavage can result from two different single-stranded cleavage events. Cleavage of double-stranded DNA, RNA, or DNA/RNA hybrids can result in either blunt ends or staggered ends.

- The term "identity" refers to the sequence identity of two nucleic acid or polypeptide molecules. Identity can be assessed by comparing positions in each of the sequences that can be aligned for comparison purposes. If a position in the compared sequence is occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between two nucleotide or amino acid sequences is a function of the number of identical or matching nucleotides at the nucleotide sequence positions. Various sequence alignment algorithms and/or programs can be used to calculate the identity of two sequences, including FASTA or BLAST, which are available as a component of the GCG sequence analysis program package (University of Wisconsin, Madison, WI), and can be used, for example, using default parameters. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to the specific polypeptides described herein, and preferably having substantially the same functions, as well as polynucleotides encoding such polypeptides, can be considered.

As used herein, the term "individual" or "patient" includes all members of the animal kingdom, including non-human primates and humans.

The above description of the invention sets forth an approach and method for its implementation and application, so that any specialist in this field is able to implement and apply it, this possibility relates, first of all, to the essence of the invention, set forth in the attached claims, which are an integral part of the original description.

Where numerical limits or a numerical range are stated in this specification, they include their boundaries. In addition, all values and subranges within the stated limits or stated numerical range are deemed to be included as if they were specifically stated.

Having generally described the present invention, a more complete understanding can be obtained by reference to certain specific examples, which are presented herein for illustrative purposes only and are not intended to limit the scope.

Examples

Example 1: AAV-driven homologous recombination in human primary T cells at different loci under the control of endogenous promoters with knockdown of an endogenous gene

Introduction

Sequence-specific endonuclease reagents such as TALEN ^® (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France) provide site-specific induction of double-strand breaks (DSBs) in the genome at desired loci. DSB repair by cellular enzymes occurs primarily through two pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR). HDR uses a homologous region of DNA (template DNA) to repair DSBs by recombination and can be used to introduce any genetic sequence contained in the template DNA. As demonstrated in the present invention, said DNA template can be delivered using a recombinant adeno-associated virus (rAAV) along with an engineered nuclease, such as TALEN®, to introduce a site-specific DSB.

Creating matrices for integration

1.1 Insertion of an apoptosis-inducing CAR into an up-regulated locus via knockdown of the endogenous PD1 gene coding sequence

A region representing a TALEN target site was created, which should be localized in the targeted endogenous gene PDCD1 (programmed cell death protein 1, designated as PD1 - Uniprot No. Q15116). The 1000 bp upstream and downstream sequences of the TALEN targets are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The TALEN target sequences (SEQ ID NO: 3 and NO: 4) are shown in SEQ ID NO: 5. A template for integration was designed to consist of a 300 bp sequence homologous to the endogenous gene upstream of the TALEN site (SEQ ID NO: 1), followed by the 2A regulatory element (SEQ ID NO: 6), followed by a sequence encoding apoptosis-inducing CAR without a start codon (SEQ ID NO: 7), followed by a stop codon (TAG), followed by a polyadenylation sequence (SEQ ID NO: 8), followed by a 1000 bp sequence homologous to the endogenous gene downstream of the TALEN site. (SEQ ID NO: 2). The insertion template was then cloned into the promoter-less rAAV vector and used to generate AAV6.

1.2 Insertion of interleukin into an up-regulated locus via knockdown of the endogenous gene

A region representing a TALEN target site was created, which should be localized in the targeted endogenous gene PDCD1 (programmed cell death protein 1, PD1). The 1000 bp upstream and downstream sequences of the TALEN targets are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The TALEN target sequences (SEQ ID NO: 3 and NO: 4) are shown in SEQ ID NO: 5. A template for integration was created that consisted of a 300 bp sequence homologous to the endogenous gene upstream of the TALEN site (SEQ ID NO: 1), followed by the 2A regulatory element (SEQ ID NO: 6), followed by a sequence encoding an engineered single-chain fusion protein comprising the human IL-12 p35 subunit (SEQ ID NO: 9) and p40 subunit (SEQ ID NO: 10), followed by a stop codon (TAG), followed by a polyadenylation sequence (SEQ ID NO: 8), and then sequence (1000 bp) homologous to the endogenous gene, located downstream of the TALEN site (SEQ ID NO: 2). The template to be inserted was then cloned into the promoter-less rAAV vector and used to generate AAV6.

1.3 Insertion of an apoptosis-inducing CAR into a low-expression locus without endogenous gene knockdown - N-terminal insertion

A region representing a TALEN target site was created, which should be localized in the closest proximity to the start codon of the targeted endogenous LCK gene (LCK, LCK is a proto-oncogene from the Src family of tyrosine kinases [Homo sapiens (human)]). The 1000 bp upstream and downstream sequences of the start codon are shown in SEQ ID NO: 11 and SEQ ID NO: 12. An integration template was constructed to consist of 1000 bp of homology to the endogenous gene upstream of the start codon, followed by a sequence encoding the apoptosis-inducing CAR (SEQ ID NO: 13) containing the start codon, followed by the 2A regulatory element (SEQ ID NO: 8), followed by 1000 bp of homology to the endogenous gene downstream of the start codon (SEQ ID NO: 12). The insertion template was then cloned into the promoter-less rAAV vector and used to generate AAV6.

1.4 Insertion of an apoptosis-inducing CAR into a low-expression locus without endogenous gene knockdown - C-terminal insertion

A region representing a TALEN target site was created, which should be localized in the closest proximity to the stop codon of the targeted endogenous LCK gene (LCK, LCK is a proto-oncogene from the Src family of tyrosine kinases [Homo sapiens (human)]). The 1000 bp sequences upstream and downstream of the start codon are shown in SEQ ID NO: 14 and SEQ ID NO: 15. An integration template was constructed to consist of 1000 bp of sequence homologous to the endogenous gene upstream of the stop codon, followed by the 2A regulatory element (SEQ ID NO: 8), followed by a sequence encoding the apoptosis-inducing CAR without a start codon (SEQ ID NO: 7), followed by a stop codon (TAG), followed by 1000 bp of sequence homologous to the endogenous gene downstream of the stop codon (SEQ ID NO: 15). The insertion template was then cloned into the promoter-less rAAV vector and used to generate AAV6.

Expression of sequence-specific nuclease reagents in transduced cells

TALEN® mRNA was synthesized using the mMessage mMachine T7 Ultra kit (Thermo Fisher Scientific, Grand Island, NY), each TALEN was cloned downstream of the T7 promoter, purified using RNeasy columns (Qiagen, Valencia, CA), and eluted in T cytoporation medium (Harvard Apparatus, Holliston, MA). Human T cells were collected and activated from whole peripheral blood (ALLCELLS, Alameda, CA) in X-Vivo-15 medium (Lonza, Basel, Switzerland) supplemented with 20 ng/ml human IL-2 (Miltenyi Biotech, San Diego, CA), 5% human AB serum (Gemini Bio-Products, West San Francisco, CA), and Dynabeads® with human CD3/CD28 T cell activator at a bead:cell ratio of 1:1 (Thermo Fisher, Grand Island, NY). Beads were removed after 3 days and 5 x 10 ⁶ cells were transformed by electroporation of 10 μg of each of the two corresponding TALEN® mRNA using the apparatus (BTX Harvard Apparatus, Holliston, MA) using two 0.1 ms pulses at 3000 V/cm followed by four 0.2 ms pulses at 325 V/cm in cuvettes with an interelectrode distance of 0.4 cm in a final volume of 200 μl of T cytoporation medium (BTX Harvard Apparatus, Holliston, MA). Cells were immediately diluted in X-Vivo-15 medium supplemented with 20 ng/ml IL-2 and incubated at 37°C with 5% CO ₂ . After 2 h, cells were incubated with AAV6 particles using 3× ¹⁰⁵ viral genomes (vg) per cell (37°C, 16 h). Cells were passaged and maintained in X-Vivo-15 medium supplemented with 5% human AV serum and 20 ng/ml IL-2 until expression of the respective inserted gene sequences was assessed by flow cytometry.

Example 2: TALEN-mediated dual targeting of IL-15 and CAR encoding matrices into T cells

Materials

X-vivo-15 medium was purchased from Lonza (catalog #BE04-418Q), IL-2 was purchased from Miltenyi Biotech (catalog #130-097-748), human AB serum was purchased from Seralab (catalog #GEM-100-318), human CD3/CD28 T cell activator was purchased from Life Technology (catalog #11132D), QBEND10-APC was purchased from R&D Systems (catalog #FAB7227A), Vioblue®-labeled anti-CD3, PE-labeled anti-LNGFR, APC-labeled anti-CD25, and PE-labeled anti-PD1 were purchased from Miltenyi (catalog #130-094-363, 130-112-790, 130-109-021, and 130-104-892, respectively) were used in 48-well plates (CytoOne, catalog #CC7682-7548), human IL-15 Quantikine ELISA kit from R&D systems (catalog #S1500), ONE-Glo from Promega (catalog #E6110). AAV6 lots containing different templates were obtained from Virovek, PBMCs were obtained from Allcells (catalog #PB004F), and luciferase-expressing Raji cells were generated by transducing ATCC Raji cells (catalog #CCL-86) with firefly luciferase-encoding lentiviral particles.

Methods

2.1 Transfection-transduction

Dual targeting of TRAC and PD1 or CD25 loci was performed as follows. PBMCs were first thawed, washed, resuspended and cultured in X-vivo-15 complete medium (X-vivo-15, 5% AB serum, 20 ng/ml IL-2). After 1 day, cells were activated with Dynabeads® with CD3/CD28 human T cell activator (25 µl beads/ ^1U6 CD3-positive cells) and cultured at ^1U6 cells/ml for 3 days in X-vivo complete medium at 37°C in the presence of 5% _CO2 . Cells were then split using fresh complete medium and transduced/transfected the following day according to the following procedure. On the day of transduction-transfection, cells were first de-beaded by magnetic separation (EasySep kit), washed twice in cytoporation buffer T (BTX Harvard Apparatus, Holliston, MA), and resuspended at a final concentration of 28 x ¹⁰⁶ cells/ml in the same solution. The cell suspension was mixed with 5 μg mRNA encoding the TRAC ^TALEN® arms (SEQ ID NOS: 16 and 17) in the absence or presence of 15 μg mRNA encoding either the CD25- or PD1- ^TALEN® arms (SEQ ID NOS: 18 and 19 and SEQ ID NOS: 20 and 21, respectively) in a final volume of 200 μl. TALEN is a standard format TALE nuclease derived from the fusion of TALE with Fok-1. Transfection was performed using Pulse Agile technology using two 0.1 ms pulses at 3000 V/cm followed by four 0.2 ms pulses at 325 V/cm in cuvettes with an interelectrode distance of 0.4 cm in a final volume of 200 μl of T cytoporation medium (BTX Harvard Apparatus, Holliston, MA). Electroporated cells were immediately transferred to a 12-well plate containing 1 ml of prewarmed serum-free X-vivo-15 medium and incubated at 37°C for 15 min. Cells were then concentrated to 8 × 10 ⁶ cells/ml in 250 μl of the same medium in the presence of AAV6 particles (MOI = 3 × 10 ⁵ vg/cell) containing donor templates in 48-well routinely processed plates. After culturing for 2 h at 30°C, 250 μl of Xvivo-15 medium supplemented with 10% AB serum and 40 ng/ml IL-2 were added to the cell suspension, and the mixture was incubated for 24 h under the same culturing conditions. After 1 day, the cells were seeded at 1×10 ⁶ cells/ml in complete X-vivo-15 medium and cultured at 37°C in the presence of 5% CO ₂ .

2.2 Activation-dependent ΔLNGFR expression and IL-15 secretion

Engineered T cells were isolated following the transfection-transduction process described above and seeded at 1 x ¹⁰⁶ cells/ml alone or in the presence of Raji cells (E:T = 1:1) or Dynabeads (12.5 μl/1 x ¹⁰⁶ cells) in a final volume of 100 μl X-vivo-15 complete medium. Cells were cultured for 48 h before being isolated, labeled and analyzed by flow cytometry. Cells were labeled using two independent sets of antibodies. The first set of antibodies, designed to detect the presence of ΔLNGFR, CAR, and CD3 cells, consisted of QBEND10-APC (diluted 1/10), Vioblue®-labeled anti-CD3 (diluted 1/25), and PE-labeled anti-ΔLNGFR (diluted 1/25). The second set of antibodies, designed to detect endogenous CD25 and PD1 expression, consisted of APC-labeled anti-CD25 (diluted 1/25) and Vioblue®-labeled anti-PD1 (diluted 1/25).

The same experimental procedure was used to study the secretion of IL-15 into the medium. The cell mixture was maintained in co-culture for 2, 4, 7 and 10 days, after which it was collected and the supernatant was analyzed using an IL-15-specific ELISA kit.

2.3 Serial killing analysis

To evaluate the antitumor activity of the engineered CAR T cells, a serial killing assay was performed. The principle of this assay is that the antitumor activity of CAR T cells is induced daily by adding a constant amount of tumor cells every day. Monitoring of tumor cell proliferation, control, and relapse can be performed based on the detection of luciferase luminescence as a marker stably integrated into tumor cell lines.

Typically, CAR T cells were mixed with a suspension of 2.5 x 105 ^Raji -luc tumor cells using different E:T ratios (E:T = 5:1 or 1:1) in a total volume of 1 ml Xvivo 5% AB medium supplemented with 20 ng/μl IL-2. The mixture was incubated for 24 h, after which luminescence was detected in 25 μl of the cell suspension using the ONE-Glot reagent. The cell mixture was then centrifuged, the old medium was discarded and replaced with 1 ml fresh X-vivo-15 complete medium containing 2.5 x ¹⁰⁵ Raji-Luc cells, and the resulting cell mixture was incubated for 24 h. This protocol was repeated for 4 days.

Experiments and results

This example describes methods for enhancing the therapeutic outcome of CAR T cell-based therapies by integrating an expression cassette comprising the IL-15/soluble IL-15 receptor alpha (IL15/sIL15rα) heterodimer under the control of endogenous T cell promoters regulating the PD1 and CD25 genes. Since both genes are known to be up-regulated upon tumor contact with CAR T cells, they can be redirected to express IL-IL15/sIL15rα only in the vicinity of the tumor. This method helps to reduce potential side effects due to systemic secretion of IL15/sIL15rα, while maintaining the capacity for reduced activation-induced T cell death (AICD), improves T cell survival, enhances T cell antitumor activity, and reverses T cell anergy.

The method developed for IL15/sIL15rα integration into the PD1 and CD25 loci consists of creating a double-strand break at both loci using TALEN in the presence of a DNA repair template vectored with AAV6. The template consists of two homologous arms surrounding the IL15/sIL15rα coding regions separated by cis-acting 2A elements and regulatory elements (stop codon and polyA sequences). Depending on the locus being targeted and its involvement in T cell activity, the endogenous gene being targeted may or may not be inactivated using a specific template design. When the CD25 gene is considered as a targetable locus, the insertion template was designed for the IL15/sIL15rα knockin (KI) without inactivating CD25, since the protein product of this gene is considered important for T cell function. In contrast, since PD1 is involved in T cell inhibition/T cell exhaustion, the insertion template was designed to prevent its expression while maintaining the ability to express and secrete IL15/sIL15rα.

To illustrate this approach and demonstrate the feasibility of dual targeted insertion into primary T cells, three different arrays were generated (Figures 2A, 2B, and 2C). The first, designated CARm and shown in SEQ ID NO: 36, was designed to insert anti-CD22 CAR cDNA into the TRAC locus in the presence of TRAC ^TALENs (SEQ ID NOS: 16 and 17). The second, designated IL-15_CD25m (SEQ ID NO: 37), was designed to integrate IL15 cDNA, sIL15rα, and a surface marker designated ΔLNGFR, separated by cis-acting 2A elements, immediately upstream of the stop codon of the endogenous CD25 coding sequence, using CD25 ^TALENs (SEQ ID NOS: 18 and 19). The third, IL-15_PD1m (SEQ ID NO: 38), containing the same expression cassette, was designed to integrate into the middle of the PD1 open reading frame using PD1 TALEN ^® (SEQ ID NOs: 20 and 21). Three templates containing an additional cis-acting 2A element located upstream of the expression cassettes allowed co-expression of IL15/sIL15rα and CAR with the endogenous target gene.

In creating the invention, the efficiency of dual target insertion into T cells by transducing them with one of the AAV6 constructs encoding IL15/sIL15rα matrices (SEQ ID NO: 41; pCLS30519), along with one construct encoding CAR, and subsequent transfection of the corresponding TALEN ^® was assessed for the first time. The use of vectorization based on AAV6 matrices in the presence of mRNA encoding TRAC TALEN ^® (SEQ ID NO: 22 and 23) and PD1 TALEN ^® (SEQ ID NO: 24 and 25) or CD25 TALEN ^® (SEQ ID NO: 26 and 27) ensures expression of anti-CD22 CAR in up to 46% of the engineered T cells (Fig. 3).

To determine the level of IL15m integration at the CD25 and PD1 loci, engineered T cells were activated with either anti-CD3/CD28-coated beads or CD22-expressing Raji tumor cells. Two days after activation, cells were isolated and analyzed by FACS using LNGFR expression as a proxy for IL15/sIL15rα secretion (Figures 4 and 5). Our results demonstrated that anti-CD3/CD28-coated beads induced ΔLNGFR expression by T cells containing IL-15m_CD25 or IL-15m_PD1, regardless of the presence of anti-CD22 CAR (Figures 4A-B). However, tumor cells induced ΔLNGFR expression only by T cells treated with both CARm and IL-15m. This suggests that ΔLNGFR expression can be specifically induced by tumor cell contact with CAR (Figs. 5 and 6).

As expected, the endogenous CD25 gene was still expressed in activated treated T cells (Figs. 7 and 8), while PD1 expression was severely impaired (Fig. 12).

To confirm that ΔLNGFR expression correlated with IL-15 secretion into the medium, T cells expressing anti-CD22 CAR and ΔLNGFR were incubated in the presence of CD22-expressing Raji tumor cells (E:T ratio = 1:1) for a total of 10 days. Supernates were collected on days 2, 4, 7, and 10 and the presence of IL15 was quantified by ELISA. The results demonstrated that IL-15 was secreted into the medium only by T cells co-treated with both CARm and IL15m matrices along with their corresponding ^TALENs (Fig. 13). T cells treated with either matrices alone were unable to secrete any significant level of IL-15 relative to resting T cells. Importantly, it was found that the level of IL-15 secretion was transient with a maximum peak at day 4 (Fig. 14).

To address whether the level of secreted IL-15 (SEQ ID NO: 59) could influence CAR T cell activity, CAR T cells were co-cultured in the presence of tumor cells at an E:T ratio of 5:1 for 4 days. Their anti-tumor activity was induced daily by pelleting and resuspension in IL-2-deficient culture medium containing fresh tumor cells. The anti-tumor activity of CAR T cells was monitored daily by measuring the luminescence of the remaining luciferase-expressing Raji tumor cells. The results demonstrated that CAR T cells co-expressing IL-15 had higher anti-tumor activity than IL-15-deficient cells at all time points studied (Fig. 15).

Thus, taken together, the obtained results demonstrated that the invention provides a method for performing simultaneous targeted insertions of CAR cDNA and IL-15 into the TRAC and CD25 or PD1 loci. This dual targeted insertion resulted in strong expression of the anti-CD22 CAR and secretion of IL-15 in the media. The levels of secreted IL-15 were sufficient to enhance CAR T cell activity.

Claims (25)

1. A method for producing engineered T cells or natural killer (NK) cells for cellular immunotherapy, wherein the method comprises: - obtaining a population of primary T cells or NK cells; - introduction into the specified T cells or NK cells: I) a nucleic acid comprising an exogenous polynucleotide sequence encoding a chemokine or cytokine that is required to be integrated into an endogenous gene selected from a gene encoding PD1, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, CD25, CD69, TGFb, IL-10, IL-10R, GCN2, PRDM1, DCK, HPRT, GGH or CD52, or an endogenous gene selected from Spata6, Itga6, Rcbtb2, Cd1d1, St8sia4, Itgae, Fam214a, IL3, IL2, Ccl4, IL21, Gp49a, Nr4a3, Lilrb4, Cd200, Cdkn1a, Gzmc, Nr4a2, Cish, Ccr8, Lad1, Crabp2; Gzmb, Tbx21, Plek, Chek1, Slamf7, Zbtb32, Tigit, Lag3, Gzma, Wee1, IL12rb2, Eea1 and Dtl ; II) one sequence-specific endonuclease reagent selected from RNA- or DNA-guided endonucleases such as Cas9 or Cpf1, TAL endonucleases, zinc finger nucleases, a homing endonuclease, or any combination thereof, and which specifically targets said selected endogenous gene, wherein said exogenous polynucleotide sequence is inserted by integrating a target gene into said endogenous gene such that said exogenous polynucleotide sequence forms an exogenous coding sequence under the transcriptional control of an endogenous promoter present in said gene, and wherein the chimeric antigen receptor (CAR) or modified TCR is independently expressed in transfected T cells or NK cells. 2. The method of claim 1, wherein said sequence-specific reagent is an endonuclease reagent. 3. The method according to claim 1 or 2, wherein said integration of the target gene is carried out by homologous recombination or non-homologous end joining (NHEJ) in said T cells or NK cells. 4. The method according to one of claims 1-3, wherein said molecule encoded by said exogenous coding sequence is an RNA transcript, such as RNAi, or a polypeptide, such as a functional protein. 5. The method according to one of claims 1-4, wherein said exogenous sequence encodes interleukin-2 (IL-2), interleukin 12 (IL-12) or interleukin (IL-15). 6. The method according to one of claims 1-5, wherein said specific endonuclease reagent is introduced by electroporation in the form of a polypeptide or mRNA that is translated in the cell. 7. The method according to one of claims 1-6, wherein said exogenous nucleic acid containing said coding sequence is included in a DNA vector, preferably a viral vector, such as an AAV vector. 8. The method of claim 7, wherein a nucleic acid encoding said sequence-specific endonuclease reagent and said exogenous nucleic acid are both included in said DNA vector. 9. The method according to one of claims 1-8, wherein a sequence encoding the 2A peptide is placed before or after the exogenous sequence that is introduced into the T cells or NK cells to ensure transcription of said exogenous coding sequence together with at least one part of the endogenous gene. 10. The method according to one of claims 1-9, wherein an exogenous sequence is introduced that has the ability to inactivate the expression of at least one endogenous genomic sequence originally present in said endogenous gene. 11. The method according to claim 10, wherein said endogenous genomic sequence to be inactivated encodes TGFb, TGFbR, IL-10, IL-10R, GCN2 or PRDM1. 12. The method of claim 10, wherein said endogenous genomic sequence to be inactivated encodes PD1, PDL1, CTLA4, TIM3 or LAG3. 13. The method of claim 10, wherein said endogenous genomic sequence to be inactivated expresses DCK, HPRT or GGH. 14. The method according to one of claims 1-13, wherein said T cells or NK cells are primary T cells or NK cells, respectively. 15. An engineered T cell or NK cell for use in cellular immunotherapy, wherein the T cell or NK cell is equipped with a chimeric antigen receptor and comprises an exogenous polynucleotide sequence encoding a chemokine or cytokine under the transcriptional control of an endogenous gene promoter presented on an endogenous gene selected from a gene encoding PD1, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, CD25, CD69, TGFb, IL-10, IL-10R, GCN2, PRDM1, DCK, HPRT, GGH or CD52, or an endogenous gene selected from Spata6, Itga6, Rcbtb2, Cd1d1, St8sia4, Itgae, Fam214a, IL3, IL2, Ccl4, IL21, Gp49a, Nr4a3, Lilrb4, Cd200, Cdkn1a, Gzmc, Nr4a2, Cish, Ccr8, Lad1, Crabp2, Gzmb, Tbx21, Plek, Chek1, Slamf7, Zbtb32, Tigit, Lag3, Gzma, Wee1, IL12rb2, Eea1 and Dtl . 16. The engineered immune T cell or NK cell of claim 15, wherein said T cell or NK cell is a primary T cell or NK cell, respectively. 17. An engineered immune cell according to claim 15 or 16, wherein said cell expresses a first exogenous sequence encoding a CAR at the TCR locus, thereby disrupting TCR expression, and expresses a second exogenous sequence encoding a chemokine or cytokine under the transcriptional activity of a promoter of an endogenous gene selected from a gene encoding PD1, CD25, CD71 or CD69. 18. An engineered immune cell according to one of claims 15-17, wherein said transcriptional control of said endogenous gene promoter is responsive to an activation signal of said chimeric antigen receptor (CAR). 19. A population of cells according to one of paragraphs 15-18 for use in immunotherapy. 20. A pharmaceutical composition for use in the treatment of cancer, wherein said composition comprises an engineered T cell or NK cell according to one of claims 15-18 or a population of immunogenic cells according to claim 19, comprising from 10 ⁴ to 10 ⁹ cells for administration per kg of body weight to patients in need thereof.