EA042755B1 - IMMUNE CELLS WITH MODIFIED METABOLISM AND THEIR APPLICATIONS - Google Patents

Description

The present invention relates to T cells that perform their function and at the same time are adapted to a microenvironment with a low content of tryptophan or depleted in tryptophan, in particular to an environment in which tryptophan catabolism occurs, and T cells are modified so that the expression of amino acid transporters occurs, in particular glutamine and/or tryptophan transporters, eg SLC1A5 and its isoforms. The present invention also relates to methods for obtaining such T cells and their use.

The field of technology to which the invention belongs

Tryptophan destruction is a strategy used by a large number of tumors to evade immune surveillance.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxgenase (TDO) are the rate-limiting enzymes of the kynurenine pathway, in which the essential amino acid tryptophan is converted to kynurenine.

Conditions of low tryptophan levels, typically <5 μM, mediated by IDO activity, induce a massive rearrangement of tumor cells, amino acid metabolism, gene expression, and further upregulation of the expression of genes encoding amino acid transporters such as SLC7A11, SLC1A4, and SLC1A5, in including SLC1A5 splicing variants.

SLC1A5 is a sodium dependent high affinity glutamine transporter from the family of solute transporters. Increased expression of SLC1A5 (and its splicing variants) improves glutamine uptake into tumor cells. In addition to increasing glutamine uptake, increased expression of SLC1A5 also improves tryptophan transport by increasing the activity of the large neutral amino acid transporter (LAT1). LAT1 is a heterodimeric membrane transport protein that preferentially transports branched chain amino acids (valine, leucine, isoleucine) and aromatic (tryptophan, tyrosine) amino acids. The functional LAT1 transporter consists of two proteins encoded by two different genes:

the 4F2hc/CD98 heavy subunit protein encoded by the SLC3A2 gene; and the CD98 light subunit protein encoded by the SLC7A5 gene.

With the ability to switch obligate amino acid transport, LAT1 activity is largely dependent on the replacement of intracellular glutamine with branched chain amino acids and aromatic amino acids upon capture.

Constitutive expression of IDO and TDO has been reported in some human cancers leading to tryptophan catabolism in the tumor microenvironment. The limited availability of tryptophan has profound immunoregulatory effects leading to decreased proliferation and reduced effector functions of T cells. Malignant cells are protected by this hostile microenvironment due to increased activity of amino acid transporters, which gives malignant cells a selective advantage over other cells in the tumor.

Although tryptophan catabolism has been shown to have an immunosuppressive effect on T cells, the mechanism by which tryptophan catabolism affects T cells is poorly understood.

In recent years, attention has been focused on IDO because of its immunosuppressive effects on T lymphocytes, associated partly with tryptophan depletion and partly with the direct effects of tryptophan catabolites.

TDO, an enzyme that degrades tryptophan, has been shown to have an immunosuppressive effect.

TDO and IDO inhibitors have been proposed to enhance tumor immune rejection and improve the efficacy of cancer immunotherapy.

Brief description of the invention

IDO is a cytosolic enzyme, so the destruction of tryptophan by IDO occurs inside the cell. However, as tryptophan passes through the plasma membrane via specific transporters, the cells act as a drain for tryptophan, resulting in a low tryptophan microenvironment around the tumor cells. Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase (IDO) is an important mechanism of peripheral immune tolerance, promoting tumor evasion from the immune response due to tryptophan depletion in the tumor microenvironment. It is desirable to obtain T cells that are capable of targeting malignant cells and that are resistant to the immunoregulatory microenvironment of a tumor in which tryptophan is catabolized.

The present inventors have provided a method by which T cells resistant to proliferative blockade can be obtained after exposure to low tryptophan conditions, in particular those caused by a tumor expressing IDO or TDO enzyme(s), and the method involves obtaining T cells overexpressing SLC1A5 , the SLC1A5 isoform, or an alternative tryptophan or glutamine transporter.

Based on the components of the T cell receptor (TCR), T cells are divided into two groups. The heterodimeric TCR may contain an α- and β-chain. The α- and β-chain TCRs recognize foreign antigens via peptides presented by MHC molecules on antigen-presenting cells. Alternatively, a heterodimeric TCR may contain an α- and δ-chain. TCRs containing γ- and δ-chains (γδ TCR) are independent of the MHC.

Full activation of the T cell, resulting in effective killing of target cells, requires the production signal 1 and signal 2. After receiving signal 1 from TCR/CD3, signal 2 from co-stimulatory molecules, such as CD28, occurs.

A suitable T cell can be considered a cell that expresses αβ TCR or γδ TCR. Accordingly, the T cell may be a gamma delta (γδ) T cell that expresses a TCR with any pair of gamma delta TCRs, Ugamma (γ) 1-9 and Udelta (δ) 1-8. A T cell with γδ chains may have the Vγ9Vδ2 subtype.

Accordingly, in a first aspect, the present invention provides a T cell overexpressing SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter. Alternative transporters may include other members of the family of transporters with high affinity for glutamate and neutral amino acids (SLC1A1, SLC1A2, SLC1A3,

SLC1A4,

SLC1A5,

SLC1A6,

SLC1A7), heavy subunits of heterodimeric amino acid transporters (SLC3A1, SLC3A2), members of the family of sodium- and chloride-dependent sodium:neurotransmitter symporters (SLC6A1,

SLC6A2,

SLC6A3,

SLC6A4,

SLC6A5,

SLC6A6, SLC6A7, SLC6A8,

SLC6A9,

SLC6A10,

SLC6A11,

SLC6A12,

SLC6A13, SLC6A14, SLC6A15,

SLC6A16,

SLC6A17,

SLC6A18,

SLC6A19,

SLC6A20) or members of the glycoprotein-associated family of cationic amino acid transporters (SLC7A1,

SLC7A2, SLC7A3, SLC7A4, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A9,

SLC7A10, SLC7A11, SLC7A13, SLC7A14).

As one skilled in the art would understand, for example, as described in Timosenko et al., Nutritional Stress induced by tryptophan-degrading enzymes results in ATF4-dependent reprogramming of the amino acid transporter profile in tumor cells, Cancer Res. 2016 76 (21): 6193-6204, SLC1A5 is known to exist in full-length carrier form (long SLC1A5 (SLC1A5-L)) and in the form of shortened splicing variants, including medium SLC1A5 (SLC1A5-M) and short SLC1A5 (SLC1A5-S )).

Accordingly, T cells may express SLC1A5, an isoform of SLC1A5, or a tryptophan and glutamine transporter, optionally where the transporter is selected from the family of glutamate transporters and neutral amino acids (SLC1A1, SLC1A2, SLC1A3, SLC1A4,

SLC1A5,

SLC1A6,

SLC1A7);

heavy subunits of heterodimeric amino acid carriers (SLCA1, SLC3A2);

member of the sodium- and chloride-dependent family of sodium:neurotransmitter symporters (SLC6A1,

SLC6A2,

SLC6A3,

SLC6A4,

SLC6A5,

SLC6A6, SLC6A7, SLC6A8,

SLC6A9,

SLC6A10,

SLC6A11,

SLC6A12,

SLC6A13, SLC6A14, SLC6A15,

SLC6A16,

SLC6A17,

SLC6A18,

SLC6A19,

SLC6A20) or a member of the glycoprotein-associated cationic amino acid transporter family (SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A5, SLC7A6, SLC7A7, SLC7A8,

SLC7A9, SLC7A10, SLC7A11, SLC7A13, SLC7A14) with an expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 50, at least 100 times the level of expression normally seen in a T cell. Expression levels of endogenous SLC1A5 or alternative tryptophan or glutamine transporters in unmodified T cells can be determined using techniques such as Western blotting or flow cytometry and compared with levels in genetically modified T cells.

Accordingly, T cells can express SLC1A5, an isoform of SLC1A5, or a tryptophan and glutamine transporter, optionally where the transporter is selected from the family of glutamate transporters and neutral amino acids (SLC1A1, SLC1A2, SLC1A3, SLC1A4 ,

SLC1A5,

SLC1A6,

SLC1A7);

heavy subunits of heterodimeric amino acid carriers (SLCA1, SLC3A2);

a member of the sodium- and chloride-dependent family of sodium:neurotransmitter symporters

- 2 042755 (SLC6A1, SLC6A2, SLC6A3,

SLC6A4, SLC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, SLC6A10,

SLC6A11, SLC6A12, SLC6A13, SLC6A14, SLC6A15, SLC6A16, SLC6A17,

SLC6A18, SLC6A19, SLC6A20) or a member of the glycoprotein-associated cationic amino acid transporter family (SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A5, SLC7A6, SLC7A7, SLC7A8,

SLC7A9, SLC7A10, SLC7A11, SLC7A13, SLC7A14) with an expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 20, at least 50, at least 100 times the level of expression normally seen in an activated T cell.

Accordingly, the T cell may be a gamma delta T cell. In embodiments, gamma delta T cells can be activated (ie, they proliferate more rapidly and secrete cytokines). The T cell may be an alpha-beta T cell. The alpha beta T cell can be activated. The cell may be a gamma-delta or alpha-beta T cell containing an SLC1A5 transporter or isoform thereof and/or a glutamine or tryptophan transporter together with a chimeric antigen receptor (CAR) capable of binding to a tumor antigen. Accordingly, a CAR may be a CAR responding to signal 1 only, such as the CD3zeta domain, or a signal 1 and signal 2 response, such as the CD3zeta domain and co-stimulatory domain, if the extracellular portion of the CAR binds to an antigen. Such CARs can be used in conjunction with alpha-beta T cells. The CAR may be a co-stimulatory CAR and only respond to signal 2 when bound to an antigen, as described in WO2016/166544. A CAR that only responds to signal 2, such as a co-stimulatory domain, can be used effectively with gamma-delta T cells when signal 1 can be elicited by T-cell receptor (TCR) binding to the gamma-delta T cell at recognition of the TCR antigen.

Accordingly, the T cell may be an alpha-beta or gamma-delta T cell that overexpresses SLC1A5 or an isoform thereof and/or a glutamine or tryptophan transporter together with a chimeric antigen receptor (CAR) that is capable of specifically binding to a disease antigen.

Accordingly, the T cell may be a gamma-delta (γδ) T cell that expresses the TCR of any pair of gamma-delta TCRs, Ugamma (γ) 1-9 and Ugamma (δ) 1-8, and which expresses SLC1A5, or an isoform thereof and/or a glutamine and tryptophan transporter together with a chimeric antigen receptor (CAR) that is capable of specifically binding to a disease antigen. The γδ T cell may have the νγ9νδ2 subtype.

Gamma delta T cells may contain a glutamine and/or tryptophan transporter such as SLC1A5 and CAR. A suitable CAR may be a classic or non-tunable CAR (a CAR that can produce signal 1 and signal 2). Classical CAR consists of an extracellular antigen-binding domain, a hinge region, a transmembrane domain, one or more co-stimulatory domains (signal 2), and an activation domain causing signal 1, such as CD3zeta. In embodiments, the CAR may be a co-stimulatory CAR, including only co-stimulatory domains, but not including the activation domain causing signal 1 (so that when bound to the CAR, only a co-stimulatory signal (signal 2) occurs (i.e., signal 1 does not occur when only the co-stimulatory CAR is activated)). In such embodiments, a second receptor present on the T cell, such as the T cell receptor (TCR), may signal 1 for signal 1 and signal 2 synergism for T cell activation.

SLC1A5 can be overexpressed alone or in combination with SLC7A5 and SLC3A2 to form the LAT1 transporter, further activating tryptophan uptake by the T cell.

Accordingly, the T cell can express SLC1A5 and/or a glutamine or tryptophan transporter at an expression level of at least two, at least three, at least four, at least five, at least six, at least at least seven, at least eight, at least nine, at least ten, at least 20, at least 50, at least 100 times the level of expression normally seen in a T cell .

Accordingly, the T cell can express SLC1A5 and/or a glutamine or tryptophan transporter at an expression level of at least two, at least three, at least four, at least five, at least six, at least at least seven, at least eight, at least nine, at least ten, at least 20, at least 50, at least 100 times the level of expression normally seen in an activated T cell. Overexpression can be caused by any method known in the art. Accordingly, overexpression allows the modified T cell to function efficiently in a low tryptophan microenvironment, such as a low tryptophan microenvironment as found around some tumor cells.

- 3 042755

A modified γδ T cell adapted to operate in a low-tryptophan microenvironment in which tryptophan catabolism occurs may contain a chimeric antigen receptor that contains an extracellular antigen-binding domain with disease antigen-binding specificity, a transmembrane domain, and (i) at least at least one co-stimulatory signaling region (capable of signaling 2 but not capable of signaling 1) and a signaling region not signaling 1, such as CD3zeta (to produce a co-stimulatory or tunable CAR), or (ii) an activation domain a CD3zeta signaling domain (capable of signaling 1), or (iii) at least one co-stimulatory signaling region and an activation domain of CD3zeta (classic CAR capable of signaling 1 and signaling 2)/signaling domain.

Accordingly, if a CAR nucleic acid sequence contains a CD3zeta domain, then the CAR is considered classical or non-configurable. In embodiments where a CAR contains only co-stimulatory domains, it can be considered a co-stimulatory or TCR-tuned CAR.

A nucleic acid sequence encoding a CAR, classical or co-stimulatory, may comprise a single-stranded variable region-based (scFv) fragment recognizing a disease-associated antigen or tumor antigen, or protein, or carbohydrate, or lipid, or small molecule.

The antigen binding domain of a CAR can take many forms, including (but not limited to) the form of a single-chain variable region-based fragment (scFv) derived from an antibody, a nanoparticle, a growth factor sequence, a synthetic soluble factor-based sequence, a factor-based , which binds to the ectodomain receptor or extracellular domain of the receptor on the cell surface, which then conjugates to the transmembrane and co-stimulatory domains as described above.

Accordingly, the disease antigen may be a viral antigen.

The disease antigen may be a cell surface target or tumor antigen, an infected cell, a bacterially infected cell, a fungal or protozoan infected cell, or may be an active or inactivated viral fragment, peptide, protein, antigenic segment, or the like of such a virus. The cell surface target may include a tumor-specific antigen and/or a tumor-associated antigen.

Accordingly, the extracellular antigen-binding domain can recognize and bind to a tumor-specific or disease antigen that is only present on tumor/disease cells and does not bind to any other cells and/or disease antigens that are present on some disease cells, as well as on some healthy cells. Such disease antigens may include, but are not limited to, CD19, EGFR, EGFRvRIII, ErbB2, GM3, GD2, GD3, CD20, CD22, CD30, CD37, CD38, CD70, CD75, CD79b, CD33, CD138, gp100, NYESO-1 , MICA, MICB, MARTI, AFP, ROR1, ROR2, PSMA, PSCA, mutated Ras, p53, B-Raf, c-met, VEGF, carbonic anhydrase IX, WT1, embryonic tumor antigen, CA-125, MUC-1, MUC -3, epithelial tumor antigen and MAGE type antigen, including MAGEA1, MAGEA3, MAGEA4, MAGEA12, MAGEC2, BAGE, GAGE, XAGE1B, CTAG2, CTAG1, SSX2 or LAGE1 or viral antigens or combinations thereof, or post-translationally modified proteins that can include, but are not limited to, carbamylated and citrunylated proteins.

The cell surface antigen may be an immune checkpoint ligand, such as PD-L1 or PD-L2.

The CAR transmembrane domain may comprise one or more CD3 or CD4 or CD8 or CD28 transmembrane domains, or portions thereof.

In a co-stimulatory signaling region, a CAR may contain, for example, one or more intracellular signal 2 domains from CD28, CD137 (4-1BB), ICOS, CD27, OX40, LFA1, PD-1, CD150, CD154, CD244, NKG2D, DNAX-activated protein (DAP)-10, DAP-12, LIGHT, Fc-refeptor of y-chain, y-chain of conventional IL-2, IL-12 receptor.

According to a second aspect of the invention, there is provided a method for treating cancer, or cancer, in a mammal, preferably a human, comprising administering an effective amount of a T cell of the first aspect of the invention.

According to a third aspect of the invention, an isolated nucleic acid encoding SLC1A5, an SLC1A5 isoform, or a tryptophan or glutamine transporter operably linked to control sequences adapted to express a T cell transformed with a nucleic acid encoded tryptophan or glutamine transporter, such as SLC1A5, is provided.

A nucleic acid sequence for expressing SLC1A5, an SLC1A5 isoform, or a tryptophan or glutamine transporter may contain the following elements;

a promoter, for example, but not limited to, CMV, EF1a, MSCV, PGK, CAG, IRES, or UBC an SLC1A5 nucleic acid sequence, an SLC1A5 isoform, or a tryptophan or glutamine transporter, respectively, comprising an N-terminal Kozak sequence;

a splicer/polyadenylation RNA sequence, such as but not limited to BGH or SV40.

In embodiments, if the T cell contains a CAR, then the sequence of SLC1A5, SLC1A5 isoforms, or the tryptophan or glutamine transporter can be operably linked to a separate promoter, rather than the CAR promoter, to produce two independent mRNAs. Accordingly, expression of a CAR and a carrier encoded by a nucleic acid sequence encoding a carrier can be carried out by transcription under the control of a common bidirectional promoter to produce two independent mRNAs. Alternatively, expression of a CAR and a transporter sequence can be accomplished by transcription under the control of a single promoter and insertion of an internal ribosome entry site (IRES) between the two coding sequences to produce a single mRNA capable of translating the two proteins. Accordingly, the CAR and transporter sequence can be separated by a T2A self-cleaving sequence, resulting in a single mRNA under the control of a common promoter, translated into a single polypeptide, which will be co-translationally cleaved to produce two proteins.

According to a fourth aspect of the present invention, a vector is provided that contains the nucleic acid of the third aspect of the invention.

Any vector suitable for introducing a nucleic acid that can overexpress an SLC1A5 nucleic acid sequence, an SLC1A5 isoform, or a tryptophan or glutamine transporter can be used. The vector backbone may contain an origin of replication of bacterial origin, such as pBR322, and a selectable antibiotic resistance marker, such as, but not limited to, a beta-lactamase gene conferring resistance to the antibiotic ampicillin, to provide sufficient plasmid DNA in the bacterial host. Optionally, the vector may contain bacterial and phage integrase attachment sites (attB and attP) such as phiC31 in combination with endonuclease recognition sites such as I-SceI to provide minicircles lacking a bacterial backbone. The vector may also contain a sequence that encodes SLC1A5 or an isoform thereof, or alternatively a tryptophan or glutamine transporter linked to a suitable promoter sequence for expression in a target cell of interest, most preferably a T cell. Optionally, the vector may contain an antibiotic resistance gene for positive selection in mammalian cells, and may also contain a reporter gene to identify the expression of, for example, but not limited to, green fluorescence protein (GFP). Expression of the additional reporter and/or selection gene may be under the control of separate promoters, a bidirectional promoter, or by an IRES or a T2A self-cleaving sequence.

According to a fifth aspect of the present invention, a host T cell transformed with a nucleic acid of the third aspect or a vector of the fourth aspect of the present invention is provided.

The method of genetically modifying a T cell to introduce a nucleic acid encoding SLC1A5 or an alternative tryptophan or glutamine transporter may include any technique known to those skilled in the art.

Suitable methods include, but are not limited to, viral transduction with viruses, e.g. lentiviruses/retroviruses/adenoviruses, cellular transfection of nucleic acids by electroporation, nucleofection, lipid transfection reagents, nanoparticles, calcium chloride based transfection methods or bacterial based transposons, DNA transposons and retrotransposons, the TALENS technique or the CRISPR/Cas9 system.

Accordingly, the genetic information introduced to modify the T cell may be in the form of DNA (cDNA, plasmids, linear, episomal, mikicircular), RNA, or in vitro extracorporeally transcribed (IVT) RNA. In addition to the genetic information encoding the carrier(s) and/or CAR sequences, the genetic information may also encode proteins/enzymes/sequences necessary to integrate the genetic information into the host genome.

If lentiviruses/retroviruses/adenoviruses are used for transduction, then the introduction of chemicals can be used to enhance this process, as is understood by specialists in this field. These agents include, but are not limited to, hexadimethrin bromide (polybrene), fibronectin, recombinant human fibronectin (eg, RetroNectin-Takare Clonthech), DEAEdextran, and TransPlus viral transduction enhancer (ALSTEM Cell Advancements).

Accordingly, the introduction of nucleic acids encoding the transporter and/or CAR can be carried out in T cells, peripheral blood mononuclear cells (PBMC), umbilical cord blood mononuclear cells (CBMC) or expanded tissue T cells at any time during the culture process.

According to a sixth aspect of the present invention, there is provided a method for culturing T cells such that they express a third aspect nucleic acid or a fourth aspect vector capable of expressing a transporter. Optionally, in one embodiment, the method for culturing the host cell further comprises recovering the T cell from the cell culture medium.

In accordance with yet another aspect of the present invention, a method is provided for delivering a T cell of the present invention to a tumor cell expressing SLC1A5, an SLC1A5 isoform, or a glutamine or tryptophan transporter, wherein the microenvironment around the tumor cell is tryptophan depleted. Accordingly, in embodiments, tryptophan depletion can result in at least one, at least two, at least three, and at least four, at least five times reduction in tryptophan compared to the normal cellular microenvironment surrounding the cell. in the host animal. To assess tryptophan depletion, expression of a suitable transporter can be monitored, for example, by flow cytometry, Western blotting, immunocytochemistry, qPCR, or the like, and combinations thereof.

According to a further aspect of the present invention, a composition is provided comprising a T cell of the present invention, together with a therapeutic agent, in particular an anti-cancer agent.

Accordingly, the therapeutic agent may be selected from the group consisting of a radionucleotide, boron, gadolinium, or uranium, an immunomodulator, an immunoconjugate, a cytokine, a hormone, a hormone agonist, an enzyme, an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxic drug, a toxin, an angiogenesis inhibitor, an immune response checkpoint inhibitor, a therapeutic antibody, an antibody-drug conjugate (ADC), or combinations thereof.

The therapeutic agent may contain an immunoconjugate/ADC containing a cytotoxic drug. A suitable cytotoxic agent may be a drug, a prodrug, an enzyme, or a toxin.

In embodiments, a method for treating cancer in an individual, respectively, a mammal, in particular a human, may include treating the individual with a therapeutically effective amount of a T cell of the present invention. In embodiments, the T cell may be present in a therapeutically effective T cell formulation at a dose of 1 x 10 ⁴ cells per kg of body weight, greater than 5 x 10 ⁸ cells per kg of the individual's body weight per dose.

In embodiments, the method may include repeated administration of a therapeutically effective formulation of T cells.

In embodiments, the cancer targeted for treatment can be selected from, but is not limited to, kidney, brain, ovarian, cervical, lung, bladder, esophageal, colorectal, skin, melanoma, leukemia, myeloma, lymphoma, bone cancer, hepatocellular cancer, cancer of the endometrium, pancreas, uterus, head and neck, salivary gland, breast, prostate, or colon.

As used herein, the term SLC1A5 may refer to a neutral amino acid transporter, preferably an amino acid with zwitterions. Accordingly, the transporter can bind a neutral amino acid as a substrate, including glutamine, asparagine and branched chain amino acids, and aromatic amino acids. The carrier can also bind methylated, anionic and/or cationic amino acids.

SLC1A5 may also be referred to as ASCT2 or ATBO and may function as a sodium dependent amino acid transporter.

In embodiments, SLC1A5 may be R16, AAAT, NZA1, RDRC, ASCT-T, and N7BS1. SLC1A5 can also be referred to as 5th member of soluble carrier family 1, 5th member of soluble carrier family 1 (neutral amino acid transporters), sodium dependent neutral amino acid transporter type 2, RD114/simian retrovirus type D receptor, baboon virus M7 receptor, ATB(0), ASCT2, M7V1, RDRC, neutral amino acid transporter B(0), neutral amino acid transporter B, virus receptor RD114, M7VS1, AAAT, ATBO, R16 and RDR.

The human SLC1A5 nucleic acid sequence is available from the NIHNCBI websites with accession number BC000062. In embodiments, the amino acid sequence may be accession number AAH00062.1.

The SLC1A5 variant can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the AAH00062.1 sequence. It is understood that such a variant would encode a protein that can act as a carrier, in particular to enhance the uptake of tryptophan into the modified T cell. As indicated, SLC1A5 exists in truncated isoforms. Accordingly, variants are claimed that are fragments of SLC1A5 that can appropriately encode a protein that can act as a transporter. Functionality screening can be used to identify suitable proteins with N-terminus or C-terminal deletions encoded by such fragment variants.

SLC1A5.

The corresponding human SLC1A5 gene variant can be obtained using a homologue from another animal, such as a mouse or a rat, or the like. Accordingly, such homologues may have sequence homology of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% .

Homology is defined as the percentage of residues in an amino acid or nucleic acid sequence that are identical between the variant and SLC1A5 as described herein, after sequence alignment and gaps, if necessary, to obtain the maximum percent homology.

Methods and computer programs for aligning and searching for homology and sequence identity are well known in the art.

As used herein, an SLC1A5 variant may include modifications to the amino acid sequences of SLC1A5, where the variants are generated by introducing appropriate nucleotide changes in the nucleic acid encoding the SLC1A5 transporter. Modifications may include deletions, insertions, substitutions, or the like. Accordingly, amino acid changes can be made, and these amino acid changes include deletions, insertions and/or substitutions, or changes that involve changes in post-translational modification processes of the SLC1A5 transporter, such as modifications to the number and/or positions of glycosylation sites.

Accordingly, methods such as screening for alanine mutations can be used to locate suitable amino acid substitutions. Such a screen can be used in conjunction with a functional screen to determine where the substitutions, deletions and insertions will provide the appropriate functional activity of the polypeptide variants.

Polypeptide variants may also include modifications at the C- or N-terminus of the polypeptide. As is known in the art, suitable amino acid-encoding nucleic acid substitutions, or amino acid substitutions resulting in conservative substitutions, can be made, where conservative substitutions are substitutions for similar amino acids, based on the general properties of the side chains, e.g., hydrophobic, neutral, hydrophilic. , acidic, basic, chain arrangement or aromatic residues.

Accordingly, nucleic acid molecules encoding transporter amino acid sequence variants can be obtained by various methods known in the art. These methods may include, but are not limited to, preparation by site-directed mutagenesis, PCR-assisted mutagenesis, cassette mutagenesis, or the like.

In embodiments, therapeutically effective means an amount of T cells effective to treat a disease or disorder in a mammal, in particular cancer. A therapeutically effective amount in the context of T cells and cancer may be the amount of T cells necessary to reduce the number of cancer cells, for example, reduce the size of a tumor, inhibit or slow down the division of cancer cells, or stop the infiltration of cancer cells into peripheral organs, inhibit, slow down, or stopping tumor metastasis, inhibiting, slowing or stopping the growth of a malignant neoplasm and/or inhibiting, slowing or disappearing one or more symptoms associated with a malignant neoplasm.

Accordingly, the administration of a therapeutically effective amount of T cells can interfere with the growth and/or kill existing malignant cells. For cancer therapy, a therapeutically effective amount can, for example, be measured by assessing the time to disease progression and/or by determining the rate of response to treatment. Accordingly, in addition to the introduction of T cells can be applied to other anti-cancer treatments.

The term malignancy, as used herein, refers to a physiological condition in mammals, in particular humans, characterized by unregulated cell growth.

In embodiments, the cells may include benign, precancerous, malignant, metastatic, non-metastatic cells. Examples of malignancy include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Accordingly, malignancies may include squamous cell carcinoma, lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung carcinoma, malignant neoplasm of the abdomen, hepatocellular carcinoma, gastric cancer, including cancer of the gastrointestinal tract, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, rectal cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, cancer prostate cancer, vaginal cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, penile cancer, and head and neck cancer.

Accordingly, tumor response can be assessed by a change in tumor morphology, such as tumor extension, tumor size, and the like, or by MRI scan, X-ray scan, CT scan, bone imaging, or biopsy.

As used herein, an isolated nucleic acid molecule may be a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated with a natural source of the nucleic acid. Isolated nucleic acids may also include nucleic acids that are in a different form or environment than in nature.

An isolated nucleic acid also includes a nucleic acid molecule contained in a cell that normally expresses the nucleic acid, but which is located at a different location in the cell, such as at a different chromosomal location.

Control sequences used herein refer to DNA sequences expressing an operably linked coding sequence in a host organism. Accordingly, control sequences are suitable for the expression of an operably linked coding sequence in a T cell.

A nucleic acid that is operably linked as used herein is a nucleic acid that is operably linked to another nucleic acid sequence. For example, the secretory leader DNA is operably linked to the DNA of a polypeptide if it is expressed as a proprotein that allows secretion of the polypeptide. A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Another aspect of the present invention may include a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an antiangiogenic agent and a T cell of the present invention, forming a composition in which the components of the composition are delivered simultaneously, sequentially or separately in combination with an amount effective to stated goal.

In embodiments, the composition of the present invention may be administered after testing the individual, or a tumor cell taken from the individual, to determine whether a tryptophan-poor microenvironment surrounds the tumor cell.

Accordingly, a method of treating tumors expressing IDO or TDO is claimed, comprising the steps of obtaining a T cell or composition of the present invention in an individual with tumor cells expressing an increased level of IDO or TDO, optionally, the method may include the step of detecting the presence of increased expression of IDO or TDO in the tumor cell.

According to yet another aspect, a method is claimed for expressing a chimeric antigen receptor, e.g., a chimeric antigen receptor selected from a classical or co-stimulatory CAR, simultaneously with a glutamine and/or tryptophan transporter, comprising the steps of introducing genetic information encoding a suitable CAR with binding specificity to a target antigen or disease antigen, and a glutamine and/or tryptophan transporter contained in independent vectors/constructs or in the same construct. The SLC1A5 sequence, SLC1A5 isoform, or tryptophan or glutamine transporter can be under the control of the CAR promoter to produce two independent mRNAs. Expression of the CAR sequence and the carrier can be accomplished by transcription from a common bidirectional promoter to produce two independent mRNAs. Expression of a CAR sequence and a transporter can be accomplished by transcription from a single promoter and insertion of an IRES between the two coding sequences to produce a single mRNA capable of translating the two proteins. The CAR and transporter sequence can be separated by a self-cleavable T2A sequence, giving one mRNA under the control of a common promoter, translated one polypeptide, which will be co-translationally cleaved to give two proteins.

Accordingly, a T cell expressing a chimeric antigen receptor and a glutamine and/or tryptophan transporter is provided.

Accordingly, a method is provided for selecting a T cell that is capable of proliferation under conditions low in tryptophan and/or glutamine. The selection method may include the steps of growing T cells overexpressing SLC1A5 (or alternatively overexpressing a carrier) in a growth culture medium that contains suboptimal levels of L-tryptophan, e.g., L-tryptophan at a concentration of less than 5 μM. Cells expressing the appropriate transporter can also be enriched by cell division in growth culture media containing suboptimal levels of L-glutamine, eg L-glutamine at a concentration of less than 3 μM. You can also use a growth culture medium that contains suboptimal levels of L-tryptophan and L-glutamine. Alternatively, cells expressing the transporter can be enriched by cell division in a growth culture medium that contains an SLC1A5 inhibitor such as O-benzyl-8 042755

L-serine, to mimic low tryptophan conditions. Such growth conditions provide a technique by which T cells expressing the genetically introduced carrier can divide and be selected from other populations in cell culture, thereby selecting to proliferate unmodified T cells.

In embodiments, a T cell overexpressing a chimeric antigen receptor and a glutamine or tryptophan transporter can be selected by culturing the cells in low tryptophan and/or low glutamine media or in the presence of an SLC1A5 inhibitor such as O-benzyl-E. -serine.

In embodiments, an antibody with binding specificity for the glutamine and/or tryptophan transporter can be used to select for T cells that are capable of proliferating under conditions of low tryptophan and/or glutamine. Accordingly, the antibody may be selected from anti-SLC1A5, anti-SLC7A5, anti-LATl, or anti-SLC3A2.

In embodiments, an antibody directed against a transporter overexpressed on modified T cells that are administered to an individual for therapeutic purposes can be administered to the individual separately, by a safe mechanism that can neutralize the introduced modified T cells in the event of an adverse response to treatment. Such antibodies can provoke antibody-dependent cellular cytotoxicity (ADCC) to neutralize the modified T cells. Such therapeutic antibodies may include, but are not limited to, anti-SLC1A5, anti-SLC7A5, anti-LATl, or anti-SLC3A2.

Any document, reference, patent application or patent cited herein is expressly incorporated herein in its entirety by reference, which means that it should be considered as part of this document. The fact that a document, reference, patent application or patent cited in this document is not repeated in this text is for reasons of brevity only.

Reference to cited material or information contained in the text should not be taken as an admission that this material or information is part of the prior art or is known in the art.

The singular refers to one or more (for example, at least one) grammatical elements.

Approximate usually means an acceptable degree of error for the quantity being measured, taking into account the nature or accuracy of the measurements.

In the description, unless the context otherwise requires, the terms contains or includes, or variants such as containing or including, contain or include, mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers .

Preferred features and embodiments of each aspect of the invention are mutatis mutandis for each other aspect, unless the context otherwise requires.

Embodiments of the present invention will now be described by way of example with reference to the accompanying figures.

Fig. 1 illustrates a claimed nucleic acid construct of the invention in which the SLC1A5 gene is expressed under the control of the EF1alpha promoter along with a BGH polyadenylation signal for mRNA stability. The pEF-DEST51 vector (Life Technologies) also contains PUC origins of replication and a beta-lactamase gene (named AmpR) conferring resistance to the antibiotic ampicillin in order to allow sufficient division of plasmid DNA in the bacterial host.

Fig. 2 illustrates A) an unmodified T cell in which SLC1A5 transports glutamine in a sodium-dependent manner that provides a substrate for the SLC7A5/SLC3A2 (LAT1) antiporter complex, which is highly dependent on intracellular glutamine output to import amino acids such as tryptophan. B) A modified T cell that is engineered to overexpress SLC1A5 by transfecting or transducing the T cell with a vector containing SLC1A5, causing expression of SLC1A5 and thus increasing glutamine uptake into the cell. This provides an additional substrate (glutamine) for the antiporter complex SLC7A5/SLC3A2 (LAT1), thus increasing tryptophan import/uptake.

In FIG. 3 illustrates an example of the claimed mode of action of T cells overexpressing SLC1A5 and shows (a) IDO+ tumor cells that create a tryptophan-poor microenvironment that induces cell cycle arrest, decreased activation, and apoptosis of cytotoxic T cells. Tumor cells compensate for low tryptophan conditions by activating SLC1A5 expression. (B) If the T cell is equipped with the same compensation mechanism as that of tumor cells by overexpressing SLC1A5, the T cell can realize its activity in the tumor microenvironment with a low tryptophan content.

In FIG. 4 illustrates an example of the claimed mode of action of T cells overexpressing SLC1A5 and co-stimulatory CAR gamma-delta, and shows (A) IDO+ tumor cells generate

- 9 042755 microenvironment with a low content of tryptophan, which causes arrest of the cell cycle, reduces the activity and apoptosis of T cells expressing the chimeric antigen receptor γδ. Tumor cells compensate for low tryptophan conditions by activating SLC1A5 expression, which increases tryptophan import. (B) If gamma-delta CAR T cells are equipped with the same compensation mechanism as the tumor cell by overexpressing SLC1A5, then the gamma-delta CAR T cell will be able to be active in the tumor microenvironment with low tryptophan content and cause complete cytotoxic effector function by recognition of phosphoantigens via γδ TCR and disease antigen via CAR (or costimulatory CAR); CAR/SLC1A5 γδ T cell is able to display its activity and carry out cellular cytotoxicity.

Fig. 5 illustrates a CAR co-stimulatory construct containing the GMCSF-R secretion signaling domain, anti-CD19 scFv, CD28 loop, transmembrane and activation domain, and CD137 activation domain (4-1BB). Co-expression of SLC1A5 with the same construct can be achieved either by a C-terminal T2A self-cleaving peptide or by an internal ribosome binding site (IRES) upstream of the SLC1A5 sequence.

Fig. 6 illustrates the transduction efficiency and expression levels of SLC1A5 in γδ Vdelta2 T cells. PBMCs were transduced with lentivirus vectors carrying SLC1A5-L (accession #NP_005619.1) or SLC1A5-S (accession #NP 001138616.1) and GFP sequences 48 hours after being challenged with zoledronic acid. For an additional 16 days, the number of transduced cells was increased and the percentage of GFP positive cells was measured by flow cytometry. Transduction efficiency, as measured by GFP positive cells (%), was 16.7% (SLC1A5-S) and 20% (SLC1A5-L) (A). Cells were also stained intracellularly for SLC1A5 by fixation/permeabilization and analyzed by flow cytometry. At least 97% of γδ T cells expressed SLC1A5, regardless of transduction (c). However, SLC1A5 expression levels (as measured by mean fluorescence intensity, MFI) were higher in γδ T cells transduced with SLC1A5-L (~10-fold) or SLC1A5-S (~1.5-fold) than in non-transduced γδ T cells (B ). These data show that γδ T cells transduced with SLC1A5-L or SLC1A5-S have higher levels of transporter expression.

Fig. 7 illustrates resistance to the SLC1A45 inhibitor O-benzyl-L-serine (BenSer) and the resulting positive selection of Vdelta2 γδ T cells transduced with a lentivirus containing the SLC1A5-L sequence. PBMCs were transduced 48 hours after stimulation with zoledronic acid. The number of cells was increased in ALys medium with or without BenSer for a maximum of 21 days.

Cell counts at week 2 or 3 were analyzed by flow cytometry measuring survival (using propidium iodide) or GFP positive γδ T cells to determine the survival advantage of transduced γδ T cells under selective pressure. After three weeks of cultivation in the presence of BenSer, a decrease in the viability of non-transduced γδ T cells was found compared to γδ T cells at earlier stages of cultivation (from 80% on day 12 to less than 60% on day 23) (A ). However, γδ T cells transduced with the SLC1A5-L isoform did not show a decrease in viability and acquired resistance to BenSer, (A). In addition, an increase in GFP-positive γδ T cells was recorded after two to three weeks of cultivation in the presence of BenSer compared to control (~17% and ~14% increase, respectively (B and C). Thus, these data showed that overexpression of SLC1A5-L makes γδ T cells resistant to BenSer in a culture medium that mimics low levels of L-tryptophan.

Examples

To compensate for the lack or depletion of tryptophan caused by the expression of IDO and TDO, when tumor cells regulate the expression of amino acid transporters, including SLC1A5 and its truncated isoforms, which in turn increase the uptake of glutamine and tryptophan into the tumor cell, and causing tryptophan depletion of the microenvironment around the tumor cell cells, the present invention provides T cells that have increased expression of such amino acid transporters, including SLC1A5 and its truncated isoforms, to allow T cells to proliferate at such low tryptophan concentrations.

In a specific embodiment, SLC1A5 can be co-expressed on the same vector as the chimeric antigen construct that is expressed and presented on the T cell.

Accordingly, SLC1A5 may be expressed under the control of a promoter or linked to chimeric antigen receptor expression by an internal ribosome entry site (IRES) or a T2A cleavage sequence providing a single mRNA under the control of a common promoter, translating a single polypeptide that will be cleaved when translated from obtaining two proteins (see Fig. 5). As described herein, the vector in which the SLC1A5 transporter resides can be a mammalian expression vector such as the Gateway 'DEST' series vector (Life Technologies), the lentiviral vector can be such as pCDH from System Biosciences, a transposon vector, or a vector suitable to get mini rings.

- 10 042755

Co-expression of SLC1A5 has several advantages, namely:

transfected T cells expressing the chimeric antigen receptor and SLC1A5 have a growth advantage under low tryptophan and/or glutamine conditions, and thus these conditions can be used to select for cells expressing both the chimeric antigen receptor and the transporter;

T cells expressing only SLC1A5 or SLC1A5 in combination with a chimeric antigen receptor are more resistant to proliferative blockade after exposure to low tryptophan conditions, tumors expressing IDO or TDO;

T cells expressing only SLC1A5 or SLC1A5 in combination with a chimeric antigen receptor can be selectively depleted after adoptive cell transfer using an antibody specific for the SLC1A5 transporter;

as described herein, T cells that have a growth advantage under low tryptophan and/or glutamine conditions can be selected after transfection using low glutamine and/or tryptophan conditions in cell culture media;

alternatively, suitable T cells can be positively selected using, for example, antibodies capable of binding to SLC1A5 or the like. For example, it can be used in magnetic activated cell sorting (MACS), fluorescent cell sorting (FACS), or similar methods known to those skilled in the art.

Example 1

Preparation of a transfection vector for T cells DNA encoding the long SLC1A5 isoform was obtained from GeneArt (Life Technologies) in the backbone of pDONR221 between the attL1 and attL2 recombination sites. The SLC1A5 can be recombined into various appropriate Gateway vectors using LR clonase II and a recombinase reaction (Life Technologies). In this example, SLC1A5 was recombined into pEFDEST51 containing the EF1alpha promoter and the BGH polyadenylation signal sequence (see FIG. 1).

Example 2

Transfection of the T cell to express SLC1A5 at a level that allows the T cell to overcome the proliferative blockade of T cells by lowering the tryptophan concentration, for example below 5 µm.

T cells were electroporated with the vector described in Example 1 using the Nucleofection (Lonxo) or Neon (ThermoFisher) electroporation system. After reconstitution in complete medium (eg, IMDM) for 24 to 48 h, T cells were cultured in IMDM medium containing less than 5 μM L-tryptophan.

Example 3

An example of obtaining a long isoform SLC1A5 gene using a lentiviral system.

The long isoform SLC1A5 gene of Example 1 was cloned into the pCDH vector backbone (Systems Bioscience). Lentiviral supernatants were generated by co-transfection of HEK293T cells with the pCDH vector and a mixture of lentiviral packaging vectors expressing the gag, pol, rev and env genes required for virus production using Purefection transfection reagent (System Bioscience). Viral supernatants were collected within 48 and 72 h after transfection and concentrated using PEG-it (System Bioscience). T cells were plated and infected with lentivirus.

The long SLC1A5 isoform (SLC1A5-L) and its truncated isoform (SLC1A5-S) were transduced into γδ T cells using a lentiviral system containing the SLC1A5-L or SLC1A5-S sequences followed by the T2A and GFP sequences. Functional transduction efficiency was 16.7 (SLC1A5-S) and 20% (SLC1A5-L), based on GFP-positive γδ T cells (see FIG. 6A). In addition, the vast majority of γδ T cells expressed SLC1A5 whether they were transduced or not (see Fig. 6B). However, SLC1A5 expression levels were 10-fold higher (SLC1A5-L) or 1.5-fold higher (SLC1A5-S) in transduced γδ T cells compared to non-transduced γδ T cells (see Fig. 6C).

Example 4

An example of obtaining a long isoform of the SLC1A5 gene using a transposon-based system.

The long isoform of the SLC1A5 gene of Example 1 was cloned into the PB51x vector (System Bioscience). Cells were co-transfected with the PB51x vector and the 'Super' PiggyBac transposase-based expression vector (System Bioscience) using a Nucleofection (Lonza) or Neon (Thermo Fisher) electroporation system.

Example 5

Use of SLC1A5 as a selection marker and discussion of a medium that could provide selective ambient pressure.

T cells were transfected (as in Examples 2 and 4) or transduced (as in Example 3) with the SLC1A5 vector construct (as in Example 1), resulting in SLC1A5 overexpression. After transfection/transduction of T cells with a vector capable of expressing SLC1A5, T cells

Claims (15)

were challenged to recover over a period of 24 to 48 hours in a complete medium such as ALyS or IMDM. After a recovery period, T cells were cultured in ALyS or IMDM medium containing less than 5 μM L-tryptophan or containing less than 4 mM L-glutamine, or both. Growth was controlled in comparison to unmodified T cells. Since the actual concentration of L-tryptophan in the cell culture systems was not controlled over time, this may affect the response of gamma delta T cells. Therefore, the SLC1A5 inhibitor O-benzyl-L-serine (BenSer) was added to the culture medium to obtain a controlled selection pressure mimicking a low L-tryptophan condition. PBMCs transduced with lentiviral vectors carrying SLC1A5-S or SLC1A5-S (48 h after stimulation with zoledronic acid) were cultured in ALyS medium with or without BenSer. During the second and third weeks of culture, γδ T cells were analyzed by flow cytometry to measure cell viability and GFP positive cells to determine the survival advantage of transduced γδ T cells under selection pressure. After three weeks of culture, the presence of BenSer reduced the viability of non-decomposed γδ T cells compared to cells at earlier stages of culture (FIG. 7A). However, γδ T cells transduced with the SLC1A5-L isoform did not show such a decrease in viability and therefore became resistant to BenSer (FIG. 7A). In addition, the percentage of GFP-positive γδ T cells increased after two and three weeks of culture in the presence of BenSer compared to the vehicle control (approximately 17% and 14% increase, respectively, Fig. 7B-C). Thus, overexpression of the SLC1A5-L isoform renders gamma delta T cells resistant to BenSer in a culture medium that mimics low levels of L-tryptophan. Although the invention has been specifically demonstrated and described with reference to specific examples, a person skilled in the art will understand that various changes in form and detail can be made without departing from the scope of the present invention. CLAIM 1. A method for selecting modified T cells adapted to overexpress SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter, wherein the method includes the steps of: a) culturing a T cell population containing modified T cells adapted to overexpress SLC1A5, an SLC1A5 isoform, or an alternative tryptophan and glutamine transporter under low tryptophan and/or glutamine conditions provided by a medium with at least one of: L-tryptophan at a concentration of less than 5 μM and L-glutamine at a concentration of less than 3 μM, or in the presence of an SLC1A5 inhibitor to mimic the low tryptophan conditions provided by O-benzylA-serine in this medium; b) selecting those modified T cells that proliferate when cultured according to step a. 2. A method for selecting modified T cells adapted to overexpress SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter, according to claim 1, wherein the method further comprises the steps of: c) producing an antibody with binding specificity for at least one of: SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter, to a T cell expressing at least one of: SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter, d) optionally, detecting antibody binding to T cells in step a, and e) selecting cells to which the antibody has bound. 3. A method for selecting modified T cells adapted to overexpress SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter, according to claim 2, wherein the method also includes the steps of: f) isolating the cells to which the antibody has bound. 4. The method for selecting modified T cells according to claim 1, wherein the SLC1A5 inhibitor is O-benzyl-L-serine. 5. The method according to any one of claims 1 to 4, wherein the modified T cell simultaneously expresses a chimeric antigen receptor and a glutamine and/or tryptophan transporter, which is provided by the same construct. 6. A modified T cell adapted to overexpress SLC1A5 or an SLC1A5 isoform, wherein the T cell is adapted to express SLC1A5 or an SLC1A5 isoform at a level that is at least twice the level of expression seen in an unmodified activated T cell, where T -cell expresses gamma-delta T-cell receptor and co-stimulatory chimeric antigen receptor (CAR), - 12 042755 where the co-stimulatory CAR contains an antigen-binding domain, a transmembrane domain and an intracellular signaling domain, where the transmembrane domain contains one or more CD3 transmembrane domains, or CD4, or CD8, or CD28, or parts thereof; and where the intracellular signaling domain contains one or more intracellular signaling domains: CD28, CD137 (4-1BB), ICOS, CD27, OX40, LFA1, PD-1, CD150, CD154, CD244, NKG2D, DNAX-activating protein (DAP) - 10, DAP-12, LIGHT, γ chain Fc receptor, conventional IL-2 γ chain, or IL-12 receptor, where the intracellular signaling domain provides a co-stimulatory signal (signal 2 only) to the T cell upon binding of the extracellular antigen-binding domain with an antigen. 7. The modified T cell of claim 6, wherein the T cell expresses a gamma delta T cell receptor and a chimeric antigen receptor (CAR) wherein, when used, signal 1 is generated at the first TCR binding event on the gamma delta T cell with a target for binding to a cell recognized by the TCR, and signal 2 is generated by the second antigen binding event to the antigen-binding domain of the costimulatory CAR, and when signal 1 and signal 2 of the first and second binding events are combined, respectively, T cell activation occurs. 8. A modified T cell according to claim 6 or 7, wherein the T cell expresses a gamma-delta T cell receptor, wherein the gamma-delta (γδ) T cell has the Vγ9Vδ2 subtype. 9. A modified T cell according to any one of claims 6 to 8, wherein SLC1A5 is overexpressed in combination with SLC7A5 and SLC3A2 to form the LAT1 transporter. 10. The method according to any one of claims 1 to 5, wherein the isolated nucleic acid encoding the carrier is selected from a member of the carrier family with high affinity for glutamate and neutral amino acids (SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7); heavy subunits of heterodimeric amino acid carriers (SLC3A1, SLC3A2); представителя семейства натрий- и хлорид-зависимых натрий:нейромедиатор симпортеров (SLC6A1, SLC6A2, SLC6A3, SLC6A4, SLC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, SLC6A10, SLC6A11, SLC6A12, SLC6A13, SLC6A14, SLC6A15, SLC6A16, SLC6A17, SLC6A18, SLC6A19 , SLC6A20) or a member of the glycoprotein-associated family of cationic amino acid transporters (SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A9, SLC7A10, SLC7A11, SLC7A13, SLC7A14). 11. The modified T cell according to any one of claims 6-9, wherein the isolated nucleic acid encoding the transporter is selected from a member of the family of transporters with high affinity for glutamate and neutral amino acids (SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7 ); heavy subunits of heterodimeric amino acid carriers (SLC3A1, SLC3A2); представителя семейства натрий- и хлорид-зависимых натрий:нейромедиатор симпортеров (SLC6A1, SLC6A2, SLC6A3, SLC6A4, SLC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, SLC6A10, SLC6A11, SLC6A12, SLC6A13, SLC6A14, SLC6A15, SLC6A16, SLC6A17, SLC6A18, SLC6A19 , SLC6A20) or a member of the glycoprotein-associated family of cationic amino acid transporters (SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A9, SLC7A10, SLC7A11, SLC7A13, SLC7A14). 12. The use of a modified T cell according to any one of claims 6 to 9 in medicine. 13. The use of a modified T cell according to any one of claims 6 to 9 in the treatment of a malignant neoplasm or a virus. 14. Use according to claim 12, wherein the T cell is isolated from an individual with the disease being treated. 15. Use of a modified T cell adapted to overexpress SLC1A5, an SLC1A5 isoform, or an alternative tryptophan or glutamine transporter, wherein the T cell is adapted to express SLC1A5, an SLC1A5 isoform, or a tryptophan or glutamine transporter at a level that is at least twice the level expression observed in an unmodified activated T cell, where the T cell expresses the gamma-delta T cell receptor and the co-stimulatory chimeric antigen receptor (CAR), where the co-stimulatory CAR contains an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, -