CN118234848A

CN118234848A - Combination of chimeric receptor polypeptides with trans-metabolic molecules that redirect glucose metabolites out of the glycolytic pathway and therapeutic uses thereof

Info

Publication number: CN118234848A
Application number: CN202280072217.6A
Authority: CN
Inventors: K·麦吉尼斯; S·埃滕贝格; L·巴伦; M·法雷; C·威尔逊; G·莫茨; S·巴杜里; A·詹森; E·柯伊伯
Original assignee: Shudian Biotechnology Co
Current assignee: Shudian Biotechnology Co
Priority date: 2021-09-27
Filing date: 2022-09-27
Publication date: 2024-06-21

Abstract

A genetically engineered hematopoietic cell expressing one or more factors that redirect glucose metabolites; and optionally a chimeric receptor polypeptide (e.g., an antibody-coupled T cell receptor (ACTR) polypeptide, a Chimeric Antigen Receptor (CAR) polypeptide, or a TCR polypeptide) capable of binding to a target antigen of interest. Also disclosed herein is the use of the genetically engineered hematopoietic cells for inhibiting cells expressing a target antigen in a subject in need thereof.

Description

Combination of chimeric receptor polypeptides with trans-metabolic molecules that redirect glucose metabolites out of the glycolytic pathway and therapeutic uses thereof

Cross Reference to Related Applications

The present application claims the benefit of the date of filing of U.S. provisional application No. 63/248,629 filed on month 27 of 2021 and U.S. provisional application No. 63/399,324 filed on month 8 of 2022, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to genetically modified immune cells that express chimeric receptor polypeptides (e.g., chimeric antigen receptors, CARs) and factors that redirect metabolites out of the metabolic pathway. The invention further relates to CAR-NK or CAR-T and uses thereof, in particular for the treatment of cancer.

Background

Cancer immunotherapy, including cell-based therapies, is used to elicit an immune response that attacks tumor cells while sparing normal tissues. The cancer immunotherapy is a promising option for the treatment of various types of cancers, as it is possible to evade genetic and cellular mechanisms of drug resistance and target tumor cells while not damaging normal tissues.

Cell-based therapies may involve cytotoxic T cells with reactivity that is inclined to cancer cells (Eshhar et al, proc NATL ACAD SCI U S A, 90 (2): 720-724 (1993), geiger et al, J.Immunol (The Journal of Immunology), 162 (10): 5931-5939 (1999), brentjens et al, nat Med, 9 (3): 279-286 (2003), cooper et al, blood (Blood), 101 (4): 1637-1644 (2003), imai et al, leukemia (Leukemia), 18 (4): 676-684 (2004)). While cell-based immunotherapy shows promising therapeutic effects, it faces challenges arising from the specific nature of the Tumor Microenvironment (TME), which is the cellular environment created by the interaction between malignant and non-transformed cells.

Therefore, in view of TME, it is very important to develop strategies to improve the efficacy of cell-based immunotherapy.

Disclosure of Invention

The present disclosure is based on the development of strategies for cell-based immunotherapy for transferring or redirecting glucose metabolites out of glycolytic pathways in hematopoietic cells, such as immune cells, comprising cells expressing chimeric receptor polypeptides, such as antibody-coupled T cell receptor (ACTR) polypeptides, chimeric Antigen Receptor (CAR) polypeptides, or T Cell Receptor (TCR) polypeptides. Redirecting glucose metabolites out of the glycolytic pathway may be achieved by expressing (e.g., over-expressing) one or more factors (e.g., proteins or nucleic acids) such as the factors described herein in Hematopoietic Stem Cells (HSCs), preferably immune cells (e.g., αβ or γδ T cells or NK cells). For example, such genetically engineered immune cells are expected to have enhanced metabolic activity relative to the same type of natural hematopoietic cells in low glucose, low amino acid, low pH, and/or low oxygen environments (e.g., in TME). As such, hematopoietic cells such as HSCs or immune cells that co-express one or more factors (e.g., polypeptides or nucleic acids) that redirect glucose metabolites out of the glycolytic pathway in the hematopoietic cells and chimeric receptor polypeptides will exhibit superior biological activity (e.g., under low glucose, low amino acid, low pH, and/or hypoxic conditions), e.g., cell survival, cell proliferation, maintenance of their activated phenotype (e.g., increased cytokine production, e.g., IL-2 or IFN-gamma production), cytotoxicity, and/or in vivo antitumor activity.

Thus, provided herein are modified (e.g., genetically engineered) hematopoietic cells (e.g., HSCs), preferably immune cells (e.g., αβ or γδ T cells or NK cells), that have altered intracellular regulation of glucose concentration relative to wild-type immune cells of the same type. In some cases, the modified immune cells can express or overexpress a factor that redirects glucose metabolites, such as a polypeptide that transfers or redirects glucose metabolites out of the glycolytic pathway. A modified immune cell expressing any of the factors redirecting glucose metabolites refers to a genetically engineered immune cell into which exogenous nucleic acid encoding the factors has been introduced such that the encoded factors are expressed in the resulting modified immune cell, while the unmodified parent cell does not express such factors. A modified immune cell that overexpresses any of the factors redirecting glucose metabolites refers to a genetically engineered immune cell that is engineered to enhance the expression level of the factor relative to the unmodified parent cell. In some cases, the modified immune cells may be engineered to enhance expression of the endogenous gene encoding the factor. Alternatively, the modified immune cell may be engineered to transfect an exogenous nucleic acid encoding the factor, thereby producing additional amounts of the factor in the modified immune cell.

Factors that redirect glucose metabolites may transfer or redirect substrates out of the glycolytic pathway directly (e.g., glutamine-fructose-6-phosphate aminotransferase 1 (GFPT 1)) or indirectly by, for example, reducing the rate of glucose degradation in the glycolytic pathway (e.g., TP 53-inducible glycolytic and apoptosis regulator (TIGAR), pyruvate kinase muscle isoenzyme M2 (PKM 2) of the PKM2 variant). Exemplary polypeptides that redirect glucose metabolites out of the glycolytic pathway include, but are not limited to, pyruvate kinase muscle isozymes M2 (PKM 2), glutamine-fructose-6-phosphate aminotransferase 1 (GFPT 1), TP 53-inducible glycolytic and apoptosis regulator (TIGAR), and functional variants thereof (e.g., PKM2Y105E, PKM Y105D, PKM K422R and PKM 2H 391Y). In a specific example, the polypeptide that translocates or directs the glucose metabolite is TIGAR.

The modified immune cell may further express a chimeric receptor polypeptide, which may include: (a) an extracellular target binding domain; (b) a transmembrane domain; and (c) a cytoplasmic signaling domain (e.g., a cytoplasmic domain comprising an immunoreceptor tyrosine-based activation motif (ITAM)). In one embodiment, a genetically engineered immune cell has altered glucose metabolism compared to a native immune cell of the same type, wherein the immune cell: (i) Expressing or over-expressing a polypeptide that transfers or redirects glucose metabolites out of the glycolytic pathway; and (ii) a chimeric receptor polypeptide; wherein the chimeric receptor polypeptide comprises: (a) an extracellular target binding domain; (b) a transmembrane domain; and (c) a cytoplasmic signaling domain. Any of the chimeric polypeptides disclosed herein can further comprise at least one costimulatory signaling domain. In other embodiments, the chimeric receptor polypeptide can be free of a costimulatory signaling domain.

In some embodiments, the chimeric receptor polypeptide is an antibody-coupled T cell receptor (ACTR) that includes an extracellular Fc binding domain (a). In other embodiments, the chimeric receptor is a Chimeric Antigen Receptor (CAR) comprising an extracellular antigen binding domain (a). Alternatively or additionally, the cytoplasmic signaling domain (C) is located at the C-terminus of the chimeric receptor polypeptide. In some embodiments, the chimeric receptor polypeptide is a T Cell Receptor (TCR) comprising the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, or a CD3zTCR subunit, or a portion thereof.

In some embodiments, a chimeric receptor polypeptide described herein (e.g., ACTR polypeptide, CAR, or TCR polypeptide) can further comprise a hinge domain at the C-terminus of (a) and at the N-terminus of (b). In other embodiments, the chimeric receptor polypeptide can be free of any hinge domain. In yet other embodiments, the chimeric receptor polypeptide, e.g., ACTR polypeptide, can be free of any hinge domain other than the CD16A receptor. Alternatively or additionally, the chimeric receptor polypeptide further comprises a signal peptide at its N-terminus. In yet another embodiment, the cytoplasmic signaling domain (c) comprises an immunoreceptor tyrosine based activation motif (ITAM).

In some embodiments, the chimeric receptor polypeptides disclosed herein can be ACTR polypeptides that include an Fc binding domain (a). In some examples, the Fc binding domain of (a) may be an extracellular ligand binding domain of an Fc receptor, such as an extracellular ligand binding domain of an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor. In particular examples, the Fc binding domain is an extracellular ligand binding domain of CD16A (e.g., F158 CD16A or V158 CD 16A), CD32A, or CD 64A. In other examples, the Fc binding domain of (a) may be an antibody fragment that binds to the Fc portion of an immunoglobulin. For example, the antibody fragment may be a single chain variable fragment (ScFv), a single domain antibody (e.g., a nanobody). Alternatively, the Fc binding domain of (a) may be a naturally occurring protein that binds to the Fc portion of an immunoglobulin or Fc binding fragment thereof. For example, the Fc binding domain may be protein a or protein G or an Fc binding fragment thereof. In further examples, the Fc binding domain of (a) may be a synthetic polypeptide that binds to the Fc portion of an immunoglobulin. Examples include, but are not limited to, kunitz peptide, SMIP, avimer, affibody, DARPin, or anti-carrier.

In some embodiments, the chimeric receptor polypeptides disclosed herein can be CAR polypeptides that include an extracellular antigen-binding domain (a). In some embodiments, the extracellular antigen-binding domain of (a) is a single chain antibody fragment that binds to a tumor antigen, a pathogenic antigen, or an immune cell specific for a self antigen. In certain embodiments, the tumor antigen is associated with a hematologic tumor. Examples include, but are not limited to, CD19, CD20, CD22, kappa chain, CD30, CD123, CD33, leY, CD138, CD5, BCMA, CD7, CD40 and IL-1RAP. In certain embodiments, the tumor antigen is associated with a solid tumor. Examples include, but are not limited to, GD2, GPC3, FOLR (e.g., FOLR1 or FOLR 2), HER2, ephA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, epCAM, CEA, mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD, MUC16, L1-CAM, B7H3, and CAIX. In certain embodiments, the pathogenic antigen is a bacterial antigen, a viral antigen, or a fungal antigen, such as the antigens described herein. In some embodiments, the cytoplasmic signaling domain of (c) in any of the chimeric receptor polypeptides described herein (e.g., ACTR or CAR polypeptides) can be the cytoplasmic domain of CD3z or fcplr1γ.

In some embodiments, the hinge domain of any of the chimeric polypeptides described herein (e.g., ACTR or CAR polypeptides) can, where applicable, belong to CD28, CD16A, CD a, igG, murine CD8a, or DAP12. In other examples, the hinge domain is a non-naturally occurring peptide. For example, the non-naturally occurring peptide can be an extended recombinant polypeptide (XTEN) or (Gly ₄Ser)_n polypeptide), where n is an integer from 3 to 12 inclusive.

In specific examples, ACTR polypeptides as described herein may include: (i) a CD28 co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. For example, ACTR polypeptides include components (a) - (e) as shown in table 4.

In a specific example, a CAR polypeptide described herein can include: (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. In further specific examples, a CAR polypeptide described herein can include: (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (ii) A CD 8a transmembrane domain, a CD 8a hinge domain, or a combination thereof.

The genetically engineered immune cells described herein that express a factor (e.g., a polypeptide or nucleic acid) that redirects glucose metabolites, and optionally chimeric receptor polypeptides, can be cell lines or hematopoietic stem cells or their progeny. In some embodiments, the genetically engineered immune cells may be Natural Killer (NK) cells, monocytes/macrophages, neutrophils, eosinophils, αβt or γδ T cells.

In some examples, the immune cell is a T cell, wherein expression of an endogenous T cell receptor, an endogenous major histocompatibility complex, an endogenous β -2-microglobulin, or a combination thereof has been inhibited or eliminated. In particular embodiments, genetically engineered immune cells described herein (e.g., NK, αβt, or γδ T cells) can include a nucleic acid or a set of nucleic acids that collectively include: (a) A first nucleotide sequence encoding a factor (e.g., a polypeptide or nucleic acid) that redirects glucose metabolites; and optionally (b) a second nucleotide sequence encoding the Chimeric Antigen Receptor (CAR) polypeptide. The nucleic acid or the set of nucleic acids is a DNA or RNA molecule or a set of DNA or RNA molecules. In some embodiments, the immune cell comprises the nucleic acid comprising both the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the coding sequence of the factor (e.g., polypeptide or nucleic acid) that redirects glucose metabolites is located upstream of the coding sequence of the CAR polypeptide. In some embodiments, the coding sequence of the CAR polypeptide is located upstream of the coding sequence of the factor that redirects glucose metabolites. In some embodiments, the genetically engineered immune cell may further comprise a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosome jump site (e.g., P2A peptide), an Internal Ribosome Entry Site (IRES), or a second promoter.

In some embodiments, the nucleic acid or the set of nucleic acids is included in a vector or set of vectors, which may be an expression vector or set of expression vectors (e.g., a viral vector such as a lentiviral vector or a retroviral vector). A nucleic acid set or vector set refers to a set of two or more nucleic acid molecules or two or more vectors, each encoding one of the polypeptides of interest (i.e., a polypeptide or nucleic acid that redirects glucose metabolites out of the glycolytic pathway and a CAR polypeptide). Any of the nucleic acids described herein are also within the scope of the present disclosure.

In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the immune cells described herein and a pharmaceutically acceptable carrier.

Further, provided herein is a method for inhibiting cells expressing a target antigen in a subject (e.g., reducing the number of such cells, blocking cell proliferation, and/or inhibiting cell activity), the method comprising administering to a subject in need thereof a population of immune cells described herein that can co-express a factor (e.g., a polypeptide or nucleic acid) that redirects glucose metabolites and a CAR polypeptide. The subject (e.g., a human patient, such as a human patient with cancer) may have received or be receiving treatment with an anti-cancer therapy (e.g., an anti-cancer agent). In some embodiments, at least some of the cells expressing the target antigen are located in a low glucose environment, a low amino acid (e.g., low glutamine) environment, a low pH environment, and/or a low oxygen environment, such as a tumor microenvironment.

In some examples, the immune cells are autologous. In other examples, the immune cells are allogeneic. In any of the methods described herein, the immune cells can be activated, expanded, or activated and expanded ex vivo. In some cases, the immune cells include T cells that are activated in the presence of one or more of an anti-CD 3 antibody, an anti-CD 28 antibody, IL-2, phytohemagglutinin, and engineered artificial stimulation cells or particles. In other cases, the immune cells include natural killer cells that are activated in the presence of one or more of 4-1BB ligand, anti-4-1 BB antibody, IL-15, anti-IL-15 receptor antibody, IL-2, IL-12, IL-21, and K562 cells, engineered artificial stimulatory cells, or particles.

In some embodiments, the subject treated by the methods described herein can be a human patient having a cancer, such as a carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Additional exemplary target cancers include, but are not limited to, B cell-derived cancers, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer. Exemplary B cell derived cancers are selected from the group consisting of: acute lymphoblastic leukemia of the B lineage, chronic lymphoblastic leukemia of the B cells, and non-Hodgkin's lymphoma of the B cells.

The present disclosure also provides a nucleic acid or set of nucleic acids that collectively comprise: (A) A first nucleotide sequence encoding a factor that transfers or redirects glucose metabolites; and (B) a second nucleotide sequence encoding the chimeric receptor polypeptide (e.g., ACTR, CAR, or TCR polypeptide). An exemplary embodiment is a method for producing a modified immune cell in vivo, the method comprising administering to a subject in need thereof a nucleic acid or set of nucleic acids described herein that can co-express a factor (e.g., a polypeptide or nucleic acid) that redirects glucose metabolites and a CAR polypeptide.

Further, within the scope of the present disclosure is the use of genetically engineered immune cells described herein that co-express a factor (e.g., polypeptide or nucleic acid) that redirects glucose metabolites out of the glycolytic pathway and a CAR polypeptide for the treatment of a disease or disorder of interest, such as cancer or an infectious disorder, and their use for the manufacture of a medicament for an intended medical treatment.

The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages will become apparent from the detailed description of several embodiments and from the claims that follow.

Drawings

The following drawings form a part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which aspects may be better understood by reference to one or more of the drawings in conjunction with the detailed description of the specific embodiments presented herein.

FIG. 1 is a schematic illustration of an exemplary polypeptide (open box) redirecting glucose metabolites out of the glycolytic pathway. TIGAR reduces PFK activity. The activity of PKM2 and its loss of function (LoF) variants is lower than that of PKM 1. In addition GFPT1 competes with the glycolytic enzyme for substrate and its products are redirected into the biosynthetic pathway. Glycolytic enzymes belonging to the glycolytic pathway (filled boxes) are ordered together with their substrates and products.

FIG. 2 is a graph showing the effect of low glucose concentration on proliferation of immune cells expressing a chimeric antigen receptor.

FIG. 3 is a graph depicting the increase in glucose uptake of T cells transduced with GLUT1, GOT2 and TIGAR as a measure of fold change in luminescence relative to a control transduced with a mimetic (null).

Fig. 4 is a graph depicting free lactate production by T cells transduced with GLUT1, GOT2 and TIGAR relative to a control transduced with a mimetic (null) in the presence or absence of PMA and ionomycin stimulation as a measure of fold change in luminescence.

Figure 5 is an immunoblot showing transgene expression in NK92 cells with CAR or CAR and transgene alone (GOT 2 and TIGAR) versus control of mimetic transduction (null).

Fig. 6A and 6B are flow cytometry graphs depicting CAR expression in NK92 cells with CAR or CAR alone (fig. 6A) and transgenes (GOT 2 and TIGAR) (fig. 6B) for retroviral transduction relative to control (null) of mimetic transduction.

Detailed Description

Immune cell therapies involving genetically engineered T cells have shown promising results in cancer therapies.

In some embodiments, the disclosure provides for expression of chimeric receptors having an antigen binding domain (e.g., CAR) fused to one or more T cell activation signaling domains. Binding to cancer antigens via the antigen binding domain results in T cell activation and triggers cytotoxicity. Recent clinical trial results with respect to the infusion of autologous T lymphocytes expressing chimeric receptors provide convincing evidence for their clinical potential. (Brentjens, latouche et al, (Nature medical science, 9 (3): 279-286 (2003); pule et al, (Nature medical science, 14 (11): 1264-1270 (2008)); Brentjens et al, blood, 118 (18): 4817-4818 (2011); porter et al, new England journal of medicine (NEW ENGLAND Journal of Medicine) 365 (8): 725-733 (2011); kochenderfer et al, blood, 119 (12): 2709-2720 (2012); till et al, blood, 119 (17): 3940-3950 (2012); brentjens et al, science of transformation (SCI TRANSL MED), 5 (177): 177ra138 (2013)). Initially, the field focused on αβ T cells. Recently, cell-based therapies have expanded to include Natural Killer (NK) cells because of the unique advantages of natural killing, such as low risk of on-target/off-tumor toxicity, cytokine release syndrome, and neurotoxicity in normal tissues. The natural killer also exhibits natural cytotoxicity to tumor cells. Finally, ready cell therapy products can be prepared due to the reduction of Graft Versus Host Disease (GVHD) (Schmidt et al, front immunology (Front Immunol), 11:611163 (2020); Wang et al, cancer flash (CANCER LETT), 472:175-180 (2020); xie et al, E biomedical (EBioMedicine), 59:102975 (2020); gong et al, journal of hematology and oncology (J Hematol Oncol), 14 (1): 73 (2021); wrona et al, int J Mol Sci, 22 (11): (2021)). Another subtype of T cells, γδ T cells, has great potential in cell therapy, sharing similar advantages as NK cells that otherwise display immunomodulatory functions (Sievers et al, international journal of molecular science, 21 (10): 2020); park and Lee, experimental and molecular medicine (Exp Mol Med), 53 (3): 318-327 (2021)). In addition, sustained activation of these αβ T cells due to antigen-dependent or independent CAR activation may lead to failure and reduced biological activity, which is also an advantage of innate cells like NK and γδ T cells. Finally, both NK and γδ T cells expressing the chimeric receptor polypeptides may have enhanced effector functions such as increased inflammatory cytokine production, antigen acquisition and presentation or the ability to activate an adaptive immune response.

In other embodiments, provided herein are methods of expressing an antibody-coupled T cell receptor (ACTR) protein in a hematopoietic cell (e.g., a hematopoietic stem cell, an immune cell, such as an NK cell, or a T cell), the ACTR protein comprising an extracellular Fc binding domain. Hematopoietic cells expressing ACTR (e.g., T cells expressing ACTR, also referred to as "ACTR T cells") when administered to a subject with an anti-cancer antibody can enhance toxicity to antibody-targeted cancer cells by binding to the Fc domain of the antibody (Kudo et al, cancer research (CANCER RES), 74 (1): 93-103 (2014)).

In yet other embodiments, provided herein are methods of expressing an exogenous T Cell Receptor (TCR) protein in a hematopoietic cell (e.g., a hematopoietic stem cell, immune cell, such as an NK cell or T cell or NKT cell), the TCR protein comprising a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 zeta TCR subunit, or an extracellular domain of a CD3 delta TCR subunit, or a portion thereof. In some cases, the TCR is further modified (e.g., expressing multiple TCRs against the same or different target antigens or the same or different epitopes on the same antigen, or increasing affinity for the target or amino acid substitution to increase preferential TCR chain pairing). Hematopoietic cells that express TCRs (e.g., T cells that express TCRs, also referred to as "TCR T cells") can enhance toxicity to cancer cells targeted by the TCR-CD3 complex by binding to the peptide-MHC complex when administered to a subject (Shafer et al, immunological fronts, 13:835762 (2022); wieczorek et al, immunological fronts, 8:292 (2017)).

Tumor microenvironments have specific properties such as low glucose, low amino acids, low pH and/or low oxygen conditions, some of which may inhibit the activity of effector immune cells such as effector T cells. The present disclosure is based, at least in part, on the development of strategies to enhance effector word immune cell activity in tumor microenvironments. In particular, the present disclosure features methods for enhancing the metabolic activity of effector immune cells by transferring or redirecting glucose metabolism (e.g., transferring or redirecting one or more glucose metabolites out of the glycolytic pathway) in the effector immune cells, thereby enhancing their growth and biological activity. Glucose metabolites refer to any molecule involved in glucose metabolism, including substrates used in any reaction of glucose metabolism and/or any product produced in such reaction. The redirection of glucose metabolites may be regulated by a variety of factors, including redirecting substrate metabolism or reducing the expression level of polypeptides of the glucose cleavage rate of the glycolytic pathway. The present disclosure provides various methods of redirecting glucose metabolites out of the glycolytic pathway. Some examples are shown in fig. 1, including: overexpressing enzymes that compete with enzymes of the glycolytic pathway for substrates and thereby redirect these substrates into other biosynthetic pathways (e.g., GFPT 1); overexpression of enzymes that compete with enzymes of the glycolytic pathway for substrates and are less active than the glycolytic pathway (e.g., PKM2 variants); and/or over-express a polypeptide (e.g., TIGAR) that reduces the function of an enzyme in the glycolytic pathway. Alternatively, the redirection of glucose metabolites may be increased by modulating the expression of endogenous genes encoding proteins involved in redirecting glucose metabolism and/or modulating cellular trafficking or activity of such proteins.

Thus, the present disclosure provides genetically engineered immune cells (e.g., NK, αβt or γδt or NKT cells) having altered glucose metabolism compared to the same type of native immune cells. Such genetically engineered immune cells express or overexpress polypeptides that transfer or redirect glucose metabolites out of the glycolytic pathway in the immune cells, and express chimeric receptor polypeptides, such as antibody-coupled T cell receptor (ACTR) polypeptides, chimeric Antigen Receptor (CAR), or T Cell Receptor (TCR) polypeptides, including extracellular target binding domains, transmembrane domains, and cytoplasmic signaling domains. In preferred embodiments, the factor (e.g., polypeptide) encoded by a transgene introduced into an immune cell (e.g., exogenous to the host cell) is over-expressed as compared to the native immune cell of the same type. Also provided herein are uses of the genetically engineered immune cells, optionally in combination with an Fc-containing agent, where desired (e.g., when the immune cells express ACTR polypeptides), for improving immune cell proliferation and/or inhibiting or reducing target cells (e.g., target cancer cells) in a subject (e.g., a human cancer patient), e.g., by ADCC. The disclosure also provides pharmaceutical compositions and kits comprising the described genetically engineered immune cells.

The genetically engineered immune cells described herein expressing (e.g., over-expressing) factors that redirect glucose metabolites out of the glycolytic pathway can confer at least the following advantages. The expression of a factor that redirects glucose metabolism will redirect glucose metabolites in immune cells relative to native immune cells of the same type. As such, genetically engineered immune cells can proliferate better in low glucose, low amino acids, low pH, and/or low oxygen environments (e.g., tumor microenvironment), produce more cytokines, exhibit greater anti-tumor cytotoxicity, and/or exhibit greater NK, NKT, αβt, or γδ T cell survival relative to immune cells that do not express (or do not overexpress) a factor that redirects glucose metabolites, resulting in increased cytokine production, survival, cytotoxicity, and/or anti-tumor activity.

I. Factors that redirect glucose metabolites out of the glycolytic pathway

As used herein, a factor that redirects glucose metabolites refers to any factor (e.g., polypeptide, protein, or nucleic acid) that redirects glucose out of the glycolytic pathway so that glucose can be used for other biological pathways. For example, factors may redirect glucose metabolites from the glycolytic pathway to the following pathways: alcohol metabolism; carbohydrate and sugar metabolism; lipid and fatty acid metabolism; hormone metabolism; protein and amino acid metabolism; steroid metabolism; and/or vitamin and coenzyme metabolism. Such biological pathways include, but are not limited to, glycine, serine, and threonine metabolism; alanine, aspartic acid and glutamine metabolism; lysine biosynthesis; arginine and proline metabolism; pentose phosphate pathway; galactose metabolism; fructose and mannose metabolism; propionic acid metabolism; butyrate metabolism; glyoxylic acid and dicarboxylic acid metabolism; citric acid cycle (TCA); amino sugar and nucleotide sugar metabolism; starch and sucrose metabolism; methane metabolism; oxidative phosphorylation; fatty acid metabolism; glutathione metabolism; 2-oxo carboxylic acid metabolism; glycosyl Phosphatidylinositol (GPi) anchor biosynthesis; n-glycan biosynthesis; pantothenate and CoA biosynthesis; biosynthesis of terpene skeletons; pyrimidine metabolism; and/or purine metabolism. Such factors may redirect glucose out of the glycolytic pathway by any mechanism.

In some cases, the factor that redirects glucose metabolites may be a polypeptide. As illustrated in fig. 1, the factor that redirects glucose metabolites may be the following polypeptides: (i) Competing with enzymes of the glycolytic pathway (e.g., GFPT 1), thereby redirecting these substrates into other biosynthetic pathways (e.g., nucleotides through the pentose phosphate pathway, amino acids through the serine synthesis pathway, fatty acids shuttled through glycerol-3P, or glycosylated proteins through the hexosamine synthesis pathway); (ii) An enzyme that competes with and is less active than an enzyme of the glycolytic pathway for a substrate (e.g., PKM 2); and/or (iii) reduce the function of an enzyme in the glycolytic pathway (e.g., TIGAR).

In some embodiments, the polypeptide that transfers or redirects the glucose metabolite is genetically engineered. In some embodiments, the polypeptide may be mutated to mimic a polypeptide that redirects activation of the glucose metabolite (e.g., a phosphorylation mimetic) or mutated to affect its intracellular transport (e.g., transport to the cell surface) such that the activity of the polypeptide is increased or decreased.

Any such polypeptide that may belong to any suitable species (e.g., mammalian such as human) is contemplated for use with the compositions and methods described herein.

Thus, in one example, the polypeptide that transfers or redirects glucose metabolites is PKM2. In another example, the polypeptide that transfers or redirects the glucose metabolite is GFPT. In yet another example, the polypeptide that transfers or redirects glucose metabolites is TIGAR.

GFPT1 is the first enzyme in the hexosamine pathway and controls the entry of glucose into the hexosamine pathway, i.e. the redirection of glucose metabolism. Specifically, GFPT a catalyzes a reaction between L-glutamine and D-fructose 6-phosphate (fructose-6P) to produce L-glutamic acid and D-glucosamine 6-phosphate. Alternatively GFPT1 relates to the regulation of precursors of N-and O-linked glycosylation of proteins. fructose-6P is a shared substrate between GFPT1 and the enzyme PFK in the glycolytic pathway. Thus, GFPT1 consuming the enzymatic activity of fructose-6P effectively redirects glucose metabolites out of the glycolytic pathway and into the hexosamine and protein glycosylation pathway. An increase in GFPT1 expression or activity levels increases the redirection of glucose metabolites away from the glycolytic pathway. The amino acid sequence of an exemplary human GFPT enzyme (SEQ ID NO: 68) is provided below.

TIGAR function is to block glycolysis and redirect glucose metabolites into the pentose phosphate shunt pathway. TIGAR is directly opposite PFKFB3 in terms of shared regulation of the molecular fructose-2, 6-bisphosphate that increases the activity of the glycolytic pathway enzyme PFK. TIGAR degrades fructose-2, 6-bisphosphate, effectively reducing the enzymatic rate of PFK. This allows more glucose metabolites to be redirected into nucleotide synthesis and glycosylation pathways, such as pentose phosphate shunt pathways. An increase in TIGAR expression or activity level increases the redirection of glucose metabolites away from the glycolytic pathway. The amino acid sequence of an exemplary human TIGAR enzyme is provided below (SEQ ID NO: 69).

In particular embodiments, the polypeptide that transfers or redirects glucose metabolites out of the glycolytic pathway for any modified hematopoietic cell, such as an immune cell, is a TIGAR, more particularly a human TIGAR (SEQ ID NO: 69). Lactate production, measured as a luminescence reading of free L-lactate, is directly related to glucose metabolism by glycolysis, supplementing the glucose uptake rate, measured as a luminescence reading of free 2-deoxyglucose-6-phosphate (2 DG 6P). T cells co-expressing TIGAR are reported herein to show superior glucose uptake and lactate production compared to co-expressed GLUT1 (see WO2020/010110, the relevant disclosure of which is incorporated herein by reference for the subject matter and purpose cited herein) or GOT2 (see WO2020/037066, the relevant disclosure of which is incorporated herein by reference for the subject matter and purpose cited herein), and such cells show improved survival under low glucose conditions found in tumor microenvironments. Thus, therapeutic NK, αβ T cells or γδ T cells that co-express a chimeric receptor polypeptide as disclosed herein (e.g., CAR or ACTR polypeptide) and TIGAR will better adapt to the tumor microenvironment (e.g., nutritional deficiency) and exhibit better therapeutic activity than corresponding NK, αβ T or γδ T cell cells that do not co-express an exogenous TIGAR gene.

The term "TIGAR" encompasses functional equivalents of TIGAR (SEQ ID NO: 69) that, when expressed in T cells, show the same or substantially the same levels of glucose uptake and/or lactate production as shown in example 10. In the case of an enzymatic activity or function assay, substantially the same level means that the corresponding reading of the assay is + -25%, preferably + -20%, more preferably + -10%.

PKM2 is an isozyme for a pyruvate kinase, a family of enzymes that convert phosphoenolpyruvate (PEP) to pyruvate, a final step in the glycolytic pathway. PKM2 catalyzes at a slower rate than other pyruvate kinase isozymes (e.g., PKM 1). Thus, increasing the relative amount of PKM2 increases the availability of PEP to other biosynthetic pathways (e.g., NFAT pathway and IL-2 production) and redirects glucose metabolites as compared to other isozymes. An increase in PSAT1 expression or activity level increases the redirection of glucose metabolites away from the glycolytic pathway. The amino acid sequence of an exemplary PKM2 enzyme is provided below (SEQ ID NO: 70). The polypeptide that redirects glucose metabolites may be a naturally occurring polypeptide from a suitable species, e.g. a mammalian polypeptide, such as a polypeptide from a human or non-human primate. Such naturally occurring polypeptides are known in the art and can be obtained by searching publicly available gene databases, such as GenBank, for example, using any of the amino acid sequences described above as a query. Polypeptides for redirecting glucose metabolites of the present disclosure may share at least 85% (e.g., 90%, 95%, 97%, 98%, 99% or more) sequence identity with any of the exemplary proteins described above.

The "percent identity" of two amino acid sequences is determined using an algorithm (Karlin and Altschul, proc. Natl. Acad. Sci. USA, 87 (6): 2264-2268 (1990)) modified in (Karlin and Altschul, proc. Natl. Acad. Sci. USA, 90 (12): 5873-5877 (1993)). Such algorithms are incorporated in the NBLAST and XBLAST programs (version 2.0) of Altschul et al, journal of molecular biology (J Mol Biol), 215 (3): 403-410 (1990)). BLAST protein searches can be performed using the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. In the case of gaps between the two sequences, use may be made of the gapped BLAST as described in Altschul et al (Nucleic Acids Res) nucleic acids research 25 (17): 3389-3402 (1997)). When utilizing BLAST programs and gapped BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Alternatively, the polypeptide that redirects the glucose metabolite may be a functional variant of the natural counterpart. Such functional variants may contain one or more mutations outside the functional domain of the natural counterpart. The functional domains of the native polypeptides that redirect glucose metabolites may be known in the art or may be predicted based on their amino acid sequences. Mutations outside the functional domain are not expected to have a substantial effect on the biological activity of the protein. In some cases, the functional variant may exhibit increased activity of glucose uptake relative to the native counterpart. Alternatively, the functional variant may exhibit reduced activity of glucose uptake relative to the native counterpart. Additionally, functional variants may increase transport to the cell surface. Alternatively, the functional variant may reduce transport to the cell surface.

For example, in some embodiments, the polypeptide that redirects the glucose metabolite pathway may be a functional variant of PKM 2. Mutants that inhibit PKM2 function have been described previously, for example by inhibiting binding to fructose-1, 6-bisphosphate or by inhibiting the catalytic structures of PKM2, such as Y105E, Y105D, K R and H391Y (see, for example, gupta et al, J Biol Chem 285 (22): 16864-16873 (2010); iqbal et al, J biochemi 289 (12): 8098-8105 (2014); wang et al, protein and Cell (Protein Cell), 6 (4): 275-287 (2015); zhou et al, cancer research 78 (9): 2248-2261 (2018)). These variants are expected to have reduced activity relative to PKM2 and thus may be used as a means of modulating activity or overexpressing PKM2 polypeptides. In some embodiments, the PKM2 functional variant comprises at least one mutation selected from the list consisting of: Y105E, Y105D, K R and H391Y. The amino acid sequences of exemplary human PKM2 variant enzymes are provided below: PKM 2Y 105E is SEQ ID NO:71, PKM 2Y 105D is SEQ ID NO:72, PKM2K422R is SEQ ID NO:73, PKM 2H 391Y is SEQ ID NO:74.

Alternatively or additionally, the functional variant may contain conservative mutations at one or more positions in the natural counterpart (e.g., up to 20 positions, up to 15 positions, up to 10 positions, up to 5, 4, 3, 2, 1 positions). As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which they are made. Variants can be prepared according to methods for altering polypeptide sequences known to those of ordinary skill in the art, as found in references that program such methods, e.g., molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), edited by J.Sambrook et al, second edition, cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press, cold Spring Harbor, new York), 1989 or "molecular biology laboratory Manual (Current Protocols in Molecular Biology), edited by F.M. Ausubel et al, john Willi father publishing company, N.Y. (John Wiley & Sons, inc., new York). Conservative substitutions of amino acids include substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the factor that redirects glucose metabolites may be a molecule that modulates expression of an endogenous polypeptide that redirects glucose metabolites out of the glycolytic pathway. Such factors may be transcription factors or micrornas. In some cases, the factor that redirects glucose metabolites may be a nucleic acid (e.g., DNA, microrna, interfering RNA such as siRNA or shRNA or antisense nucleic acid) that modulates expression of one or more enzymes involved in glucose metabolism (e.g., enzymes in the glycolytic pathway). In further embodiments, the factor that redirects glucose metabolites may be a transcription factor that modulates the expression of one or more enzymes involved in glucose metabolism. In other embodiments, the factor that redirects glucose metabolites may be a molecule that mediates degradation of endogenous factors, as disclosed herein, e.g., E3 ligase as part of the ubiquitin/proteasome pathway. Additionally, the transport of factors that redirect glucose metabolites may be modulated, for example, by expressing polypeptides that increase their transport to a particular organelle or surface.

Table 1 below provides amino acid sequences of exemplary polypeptides that redirect glucose metabolites out of the glycolytic pathway.

Table 1: exemplary Polypeptides for redirecting glucose metabolites out of the glycolytic pathway

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Chimeric receptor polypeptides

As used herein, a chimeric receptor polypeptide refers to a non-naturally occurring molecule that can be expressed on the surface of a host cell, preferably an immune cell. The extracellular target binding domain of the chimeric receptor polypeptide targets an antigen of interest (e.g., an antigen associated with a disease such as cancer or an antigen associated with a pathogen; see discussion herein). The extracellular target binding domain can bind directly to an antigen of interest (e.g., an extracellular antigen binding domain in a CAR polypeptide as disclosed herein). Alternatively, the extracellular target binding domain may bind to the antigen of interest via an intermediate, for example an Fc-containing agent such as an antibody. Further, extracellular target binding may occur through engagement between TCR-CD3 complexes that are specific for peptide-MHC complexes (e.g., through binding of genetically engineered T cells expressing TCR polypeptides as disclosed herein to antigen presenting cells such as tumor cells displaying peptide-MHC complexes). The chimeric receptor polypeptide can further comprise a hinge domain, one or more co-stimulatory domains, or a combination thereof. In some cases, the chimeric receptor polypeptide can be free of a co-stimulatory domain. The chimeric receptor polypeptide is configured such that when expressed in a host cell, the extracellular target binding domain is located extracellular for direct or indirect binding to a target antigen. The optional co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling.

In some embodiments, the chimeric receptor polypeptide includes one or more of the following features: (i) The chimeric receptor polypeptide further comprises at least one or no co-stimulatory signaling domain; (ii) Cytoplasmic signaling domain (c) includes an immunoreceptor tyrosine based activation motif (ITAM); (iii) Cytoplasmic signaling domain (C) is located at the C-terminus of the chimeric receptor polypeptide; (iv) The chimeric receptor polypeptide further comprises a hinge domain located at the C-terminus of (a) and at the N-terminus of (b); (v) the chimeric receptor polypeptide does not contain any hinge domain; and (vi) the chimeric receptor polypeptide further comprises a signal peptide at its N-terminus.

In some embodiments, the chimeric receptor polypeptides described herein can further include a hinge domain, which can be located at the C-terminus of the extracellular target binding domain and at the N-terminus of the transmembrane domain. The hinge may be of any suitable length. In other embodiments, the chimeric receptor polypeptides described herein may have no hinge domain at all. In yet other embodiments, the chimeric receptor polypeptides described herein can have a shortened hinge domain (e.g., comprising up to 25 amino acid residues).

In some embodiments, a chimeric receptor polypeptide as described herein can include, from N-terminus to C-terminus, an extracellular target binding domain, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, a chimeric receptor polypeptide as described herein includes, from N-terminus to C-terminus, an extracellular target binding domain, a transmembrane domain, at least one costimulatory signaling domain, and a cytoplasmic signaling domain. In other embodiments, a chimeric receptor polypeptide as described herein includes, from N-terminus to C-terminus, an extracellular target binding domain, a transmembrane domain, a cytoplasmic signaling domain, and at least one costimulatory signaling domain.

In some embodiments, the chimeric receptor polypeptide can be an antibody-coupled T cell receptor (ACTR) polypeptide. As used herein, ACTR polypeptides (also known as ACTR constructs) refer to non-naturally occurring molecules that can be expressed on the surface of a host cell and include extracellular domains ("Fc-binding bodies" or "Fc-binding domains"), transmembrane domains, and cytoplasmic signaling domains that have binding affinity and specificity for the Fc portion of an immunoglobulin. In some embodiments, ACTR polypeptides described herein may further comprise at least one costimulatory signaling domain.

In other embodiments, the chimeric receptor polypeptides disclosed herein can be Chimeric Antigen Receptor (CAR) polypeptides. As used herein, CAR polypeptides (also known as CAR constructs) refer to non-naturally occurring molecules that can be expressed on the surface of a host cell and include extracellular antigen binding domains, transmembrane domains, and cytoplasmic signaling domains. The CAR polypeptides described herein can further comprise at least one co-stimulatory signaling domain.

The extracellular antigen-binding domain may be any peptide or polypeptide that specifically binds to a target antigen, including naturally occurring antigens associated with a medical condition (e.g., a disease), or antigenic moieties conjugated to a therapeutic agent that targets a disease-associated antigen.

In some embodiments, a CAR polypeptide described herein can further comprise at least one co-stimulatory signaling domain. The CAR polypeptide is configured such that when expressed on a host cell, the extracellular antigen binding domain is located extracellular for binding to the target molecule and cytoplasmic signaling domain. The optional co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling.

As used herein, the phrase "protein X transmembrane domain" (e.g., CD8 transmembrane domain) refers to any portion of a given protein, i.e., protein X, that spans a membrane, which is thermodynamically stable in the membrane.

As used herein, the phrase "protein X cytoplasmic signaling domain", e.g., CD3z cytoplasmic signaling domain, refers to any portion of a protein (protein X) that interacts with the interior of a cell or organelle and is capable of delivering a primary signal known in the art that results in immune cell proliferation and/or activation. The cytoplasmic signaling domain as described herein is distinct from the costimulatory signaling domain, which transmits secondary signals to fully activate immune cells.

As used herein, the phrase "protein X costimulatory signaling domain", e.g., a CD28 costimulatory signaling domain, refers to a portion of a given costimulatory protein (protein X, such as CD28, 4-1BB, OX40, CD27, or ICOS) that can transduce a costimulatory signal (secondary signal) into an immune cell (such as a T cell) resulting in complete activation of the immune cell.

In some embodiments, the extracellular target binding domain, preferably the antigen binding domain, is a single chain variable fragment (scFv) or a single domain antibody that binds to a tumor antigen, a pathogenic antigen, or an immune cell specific for an autoantigen.

In some embodiments, the TCR may comprise a modified T Cell Receptor (TCR). In some embodiments, the modified TCR may comprise a heterodimer comprising an alpha and/or beta chain and one or more CD3 chain (e.g., gamma, delta, E, or zeta) chains, and optionally engineered to bind to an antigen-specific peptide that is complexed with an antigen-presenting molecule, such as MHC class I or MHC class II, on a target cell (e.g., a tumor cell expressing WT1 or NY-ESO-1, a pathogen-infected cell such as HPV 16E 6 protein).

A. Extracellular target binding domain

Chimeric receptor polypeptides disclosed herein include extracellular domains that target an antigen of interest (e.g., an antigen described herein) by direct binding or indirect binding (via an intermediate such as an antibody). The chimeric receptor polypeptide can be an ACTR polypeptide that includes an Fc binding domain. Alternatively, the chimeric receptor polypeptide can be a CAR polypeptide that includes an extracellular antigen-binding domain.

(I) Fc binding domain

ACTR polypeptides described herein include an extracellular domain that is an Fc binding domain, i.e., capable of binding to the Fc portion of an immunoglobulin (e.g., igG, igA, igM or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey). Suitable Fc binding domains may be derived from naturally occurring proteins, such as mammalian Fc receptors or certain bacterial proteins (e.g., protein a, protein G). Alternatively, the Fc binding domain may be a synthetic polypeptide specifically engineered to bind with high affinity and specificity to the Fc portion of any of the antibodies described herein. For example, such an Fc binding domain may be an antibody or antigen binding fragment thereof that specifically binds to the Fc portion of an immunoglobulin. Examples include, but are not limited to, single chain variable fragments (scFv), domain antibodies, or single domain antibodies (e.g., nanobodies). Alternatively, the Fc binding domain may be a synthetic peptide that specifically binds to the Fc portion, such as a Kunitz domain, small Modular Immunopharmaceuticals (SMIPs), mimetic antibody protein drugs (adnectins), avimers, affibodies, DARPin, or anti-carrier proteins, which may be identified by screening a combinatorial library of peptides having Fc binding activity.

In some embodiments, the Fc binding domain is an extracellular ligand binding domain of a mammalian Fc receptor. As used herein, an "Fc receptor" is a cell surface-bound receptor that is expressed on the surface of many immune cells (including B cells, dendritic cells, natural Killer (NK) cells, macrophages, neutrophils, mast cells, and eosinophils) and that exhibits binding specificity for the Fc domain of an antibody. Fc receptors typically consist of at least two immunoglobulin (Ig) -like domains with binding specificity for the Fc (crystallizable fragment) portion of an antibody. In some cases, binding of the Fc receptor to the Fc portion of an antibody may trigger an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. The Fc receptor used to construct an ACTR polypeptide as described herein can be a naturally occurring polymorphic variant (e.g., CD 16V 158 variant) that may have increased or decreased affinity for Fc as compared to the wild-type counterpart. Alternatively, the Fc receptor may be a functional variant of a wild-type counterpart carrying one or more mutations (e.g., up to 10 amino acid residue substitutions, including 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 mutations) that alter the binding affinity to the Fc portion of an Ig molecule. In some cases, the mutation may alter the glycosylation pattern of the Fc receptor and thus alter the binding affinity to Fc.

Table 2 lists various exemplary polymorphisms in the extracellular domain of Fc receptors, see, for example (Kim et al, journal of molecular evolution (Journal of Molecular Evolution), 53 (1): 1-9 (2001)), which can be used in any of the methods or constructs described herein:

table 2: exemplary polymorphisms of Fc receptors

Amino acid numbering

19

48

65

89

105

130

134

141

142

158

FCR10

R

S

D

I

D

G

F

Y

T

V

P08637

R

S

D

I

D

G

F

Y

I

F

S76824

R

S

D

I

D

G

F

Y

I

V

J04162

R

N

D

V

D

F

H

I

V

M31936

S

N

I

D

F

H

I

V

M24854

S

N

I

E

D

S

H

I

V

X07934

R

S

N

I

D

F

H

I

V

X14356(FcgRII)

N

S

E

S

I

M31932(FcgRI)

S

T

N

R

E

A

F

T

I

G

X06948(FcaeI)

R

S

E

S

Q

S

E

S

I

V

Fc receptors are classified based on the isotype of the antibody to which they are able to bind. For example, fc-gamma receptors (fcγr) typically bind IgG antibodies such as one or more subtypes thereof (i.e., igG1, igG2, igG3, igG 4); fc-alpha receptors (fcαr) typically bind to IgA antibodies; and the Fc-epsilon receptor (fcer) typically binds IgE antibodies. In some embodiments, the Fc receptor is fcγ R, fc αr or fcεr. Examples of fcγrs include, but are not limited to, CD64A, CD64B, CD64C, CD32A, CD32B, CD a and CD16B. An example of an Fc aR is Fc aR 1/CD89. Examples of fcer include, but are not limited to, fceri and fcrii/CD 23. Table 3 lists exemplary Fc receptors and their binding activity to corresponding Fc domains for use in constructing ACTR polypeptides described herein:

Table 3: exemplary Fc receptors

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The choice of the ligand binding domain of the Fc receptor for the ACTR polypeptides described herein will be apparent to those skilled in the art. For example, it may depend on a variety of factors, such as the isotype of antibody desired to bind to the Fc receptor and the desired affinity of the binding interaction.

In some examples, the Fc binding domain is an extracellular ligand binding domain of CD16 that can incorporate naturally occurring polymorphisms that can modulate affinity for Fc. In some examples, the Fc binding domain is an extracellular ligand binding domain of CD16 that incorporates a polymorphism (e.g., valine or phenylalanine) at position 158. In some embodiments, the Fc binding domain is produced under conditions that alter its glycosylation state and its affinity for Fc.

Provided herein are amino acid sequences of human CD16AF158 (SEQ ID NO: 75) and CD16AV158 (SEQ ID NO: 76) variants, wherein F158 and V158 residues are indicated in bold/underlined.

CD16A F158 (F158 bold/underlined)

CD16A V158 (V158 bold/underlined)

In some embodiments, the Fc binding domain is an extracellular ligand binding domain of CD16 that incorporates modifications that render ACTR polypeptides specific for a subpopulation of IgG antibodies. For example, mutations that increase or decrease affinity for an IgG subtype (e.g., igG 1) can be incorporated.

Any of the Fc binding domains described herein may have suitable binding affinity for the Fc portion of a therapeutic antibody. As used herein, "binding affinity" refers to the apparent association constant or K _A.K_A is the inverse of the dissociation constant K _D. The extracellular ligand binding domain of the Fc receptor domain of an ACTR polypeptide described herein can have a binding affinity K _d for the Fc portion of an antibody of at least 10 ^-5、10^-6、10^-7、10^-8、10^-9、10^-10 M or less. In some embodiments, the Fc binding domain has a high binding affinity for an antibody, an isotype of an antibody, or a subtype thereof, as compared to the binding affinity of the Fc binding domain for another antibody, an isotype of an antibody, or a subtype thereof. In some embodiments, the extracellular ligand binding domain of the Fc receptor is specific for an antibody, an isotype of an antibody, or a subtype thereof, as compared to binding of the extracellular ligand binding domain of the Fc receptor to another antibody, an isotype of an antibody, or a subtype thereof.

Other Fc binding domains known in the art may also be used in ACTR constructs described herein, including for example constructs described in WO2015/058018A1 and WO2018/140960, the relevant disclosures of which are incorporated herein by reference for the purposes and subject matter cited herein.

(Ii) Extracellular antigen binding domains

The CAR polypeptides described herein include an extracellular antigen-binding domain that redirects specificity of immune cells (e.g., NK, αβt, or γδ T cells) that express the CAR polypeptides. As used herein, an "extracellular antigen-binding domain" refers to a peptide or polypeptide that has binding specificity for a target antigen of interest, which may be a naturally occurring antigen associated with a medical condition (e.g., expressed in a disease, condition, or cell type). Non-limiting examples of extracellular antigen binding domains are tumor antigens, pathogenic antigens and immune cells specific for self-antigens (Gubin et al, J CLIN INVEST, 125 (9): 3413-3421 (2015); LINNEMANN et al, nature medicine, 21 (1): 81-85 (2015)). The respective diseases and/or conditions to be treated include tumors, inflammatory conditions and autoimmune disorders. In some embodiments, the antigen is selectively expressed or over-expressed on cells of a disease or condition, such as tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues.

In some embodiments, the extracellular antigen-binding domain binds to a tumor antigen associated with a hematologic tumor or a solid tumor.

Non-limiting examples of hematologic tumor extracellular binding domains are domains of CD19, CD20, CD22, kappa chain, CD30, CD123, CD33, leY, CD138, CD5, BCMA, CD7, CD40 and IL-1 RAP. Non-limiting examples of solid tumor extracellular binding domains are domains of GD2, GPC3, FOLR (e.g., FOLR1 or FOLR 2), HER2, ephA2, EFGRVIII, IL13RA2, VEGFR2, ROR1, NKG2D, epCAM, CEA, mesothelin, MUC1, CLDN18.2, CD171, CD133, PSCA, cMET, EGFR, PSMA, FAP, CD, MUC16, L1-CAM, B7H3, and CAIX. In other cases, the tumor antigen comprises a tumor antigen derived from a cancer characterized by tumor-associated antigen expression such as HER2 expression. The antigen may comprise an epitope region or epitope peptide derived from a gene mutated in a tumor cell or derived from a gene transcribed at a different level in a tumor cell than a normal cell (e.g., survivin, mutated Ras, bcr/abl rearrangement, HER2, mutated, or wild-type p 53).

Next, the antigenic moiety may be conjugated to a therapeutic agent that targets a disease-associated antigen. The extracellular antigen binding domain as described herein does not include the extracellular domain of an Fc receptor and may not bind to the Fc portion of an immunoglobulin. The extracellular domain that does not bind to the Fc fragment means that the binding activity between the two cannot be detected using conventional assays, or only background or biologically insignificant binding activity can be detected using conventional assays.

In some cases, the extracellular antigen-binding domain of any of the CAR polypeptides described herein is a peptide or polypeptide capable of binding to a cell surface antigen (e.g., natural and mutated tumor antigens), or an antigen (or fragment thereof) that forms a complex with a major histocompatibility complex and can be presented on the cell surface of an antigen presenting cell. Such extracellular antigen-binding domains may be single chain antibody fragments (scFv) that may be derived from antibodies that bind with high binding affinity to a target cell surface antigen. Table 4 lists exemplary cell surface target antigens and exemplary antibodies that bind thereto.

Table 4: exemplary cell surface target antigens and exemplary antibodies binding thereto

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Depending on the target antigen of interest, the extracellular antigen-binding domain may include an antigen-binding fragment (e.g., scFv) derived from any of the antibodies listed in table 4. In some embodiments, depending on the target antigen of interest, an antigen binding fragment (e.g., scFv) can include the same heavy and light chain Complementarity Determining Regions (CDRs) as the antibodies listed in table 4. In some embodiments, depending on the target antigen of interest, an antigen-binding fragment (e.g., scFv) can include the same heavy chain variable region (VH) and light chain variable region (VL) as the antibodies listed in table 4.

In other embodiments, the extracellular antigen-binding domain of any of the CAR polypeptides described herein can be specific for a pathogenic antigen, such as a bacterial antigen, a viral antigen, or a fungal antigen. Some examples are provided below: influenza virus neuraminidase, hemagglutinin or M2 protein, human Respiratory Syncytial Virus (RSV) F glycoprotein or G glycoprotein, herpes simplex virus glycoprotein gB, gC, gD or gE, chlamydia MOMP or PorB protein, dengue virus core protein, matrix protein or glycoprotein E, measles virus hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, HIV 1 envelope glycoprotein, hepatitis b core antigen or surface antigen, diphtheria toxin, streptococcal 24M epitope, gonococcal pilin protein, pseudorabies virus G50 (gpD), pseudorabies virus II (gpB), pseudorabies virus III (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, coronavirus polypeptide, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, human papillomavirus E6 or E7, or human hepatitis c virus glycoprotein E1 or E2.

In addition, the extracellular antigen-binding domains of the CAR polypeptides described herein can be specific for a tag conjugated to a therapeutic agent that targets an antigen associated with a disease or disorder (e.g., a tumor antigen or a pathogenic antigen as described herein). In some cases, the tag conjugated to the therapeutic agent can be antigenic, and the extracellular antigen-binding domain of the CAR polypeptide can be an antigen-binding fragment (e.g., scFv) of an antibody having high binding affinity and/or specificity for the antigenic tag. Exemplary antigen tags include, but are not limited to, biotin, avidin, fluorescent molecules (e.g., GFP, YRP, luciferase, or RFP), myc, flag, his (e.g., polyhis such as 6 xHis), HA (heme lectin), GST, MBP (maltose binding protein), KLH (keyhole limpet hemocyanin (keyhole limpet hemocyanins)), trx, T7, HSV, VSV (e.g., VSV-g), glu-Glu, V5, E-tag, S-tag, KT3, E2, au1, au5, and/or thioredoxin.

In other cases, the tag conjugated to the therapeutic agent is a member of a ligand-receptor pair and the extracellular antigen-binding domain includes the other member of the ligand-receptor pair or a fragment thereof that binds to the tag. For example, the tag conjugated to the therapeutic agent can be biotin and the extracellular antigen-binding domain of the CAR polypeptide can include a biotin-binding fragment of avidin. See, e.g., urbanska et al, cancer research 72 (7): 1844-1852 (2012), lohmueller et al, tumor immunology (Oncoimmunology), 7 (1): e1368604 (2017)). Other examples include anti-tag CARs, where the extracellular antigen binding domain is a scFv fragment specific for the protein tag, such as FITC (Tamada et al, clinical Cancer research (CLIN CANCER RES), 18 (23): 6436-6445 (2012), kim et al, journal of the American society of chemistry (J Am Chem Soc), 137 (8): 2832-2835 (2015), cao et al, german application chemistry (ANGEW CHEM INT ED ENGL), 55 (26): 7520-7524 (2016), ma et al, proc. Natl. Acad. Sci., 113 (4): E450-458 (2016)), PNE (Rodgers et al, proc. Natl. Acad. Sci., 113 (4): E459-468 (2016)), blss-B (CARTELLIERI et al, la Blood Cancer journal (Blood Cancer J), 6 (8E 458 (Lohmueller, ham et al, J), biological element (173-7520) and tumor (2016) and Leu.1426 (2016) Cell (2016) and so forth (2016). The choice of antigen binding domain for the CAR polypeptides described herein will be apparent to those skilled in the art. For example, it may depend on a variety of factors, such as the type of target antigen and the desired affinity of the binding interaction.

The extracellular antigen-binding domain of any of the CAR polypeptides described herein can have suitable binding affinity for a target antigen (e.g., any of the targets described herein) or an epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K _A or K _D.K_A is the inverse of the dissociation constant (K _D). The binding affinity (K _D) of the extracellular antigen-binding domain for the CAR polypeptides described herein to a target antigen or epitope can be at least 10 ^-5、10^-6、10^-7、10^-8、10^-9、10^-10 M or less. The increased binding affinity corresponds to a decreased K _D. Higher affinity binding of the extracellular antigen-binding domain to the first antigen relative to the second antigen may be indicated by higher K _A (or smaller value K _D) for binding to the first antigen than K _A (or value K _D) for binding to the second antigen. In such cases, the extracellular antigen-binding domain is specific for the first antigen (e.g., the first protein in the first conformation or mimetic thereof) relative to the second antigen (e.g., the same first protein in the second conformation or mimetic thereof; or the second protein). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 37.5-fold, 50-fold, 70-fold, 80-fold, 91-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or 100,000-fold.

Binding affinity (or binding specificity) can be determined by various methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorometry). Exemplary conditions for assessing binding affinity are in HBS-P buffer (10mM HEPES pH 7.4, 150mM NaCl,0.005% (v/v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([ bound ]) is generally related to the concentration of free target protein ([ free ]) by the following equation:

[ binding ] = [ free ]/(kd+ [ free ])

However, an accurate determination of K _A is not always required, since sometimes the activity in e.g. a functional assay, e.g. an in vitro or in vivo assay is sufficient to obtain a quantitative measurement of affinity, to obtain a qualitative measurement of affinity or to obtain an inference of affinity, e.g. determined using methods such as ELISA or FACS analysis, proportional to K _A and thus can be used for comparison, e.g. to determine if the higher affinity is e.g. 2 times higher.

B. Transmembrane domain

The transmembrane domain of a chimeric receptor polypeptide described herein (e.g., ACTR polypeptide or CAR polypeptide) can be in any form known in the art. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. Transmembrane domains suitable for use in chimeric receptor polypeptides used herein can be obtained from naturally occurring proteins. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment, such as a hydrophobin segment that is thermodynamically stable in a cell membrane.

The transmembrane domains are classified based on the three-dimensional structure of the transmembrane domain. For example, the transmembrane domain may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the phospholipid bilayer of a cell. In addition, the transmembrane domains may also or alternatively be categorized based on transmembrane domain topology, including the number of transmembrane domains crossing the membrane and the orientation of the protein. For example, a single pass membrane protein passes through the cell membrane once, and as many Cheng Mo proteins pass through the cell membrane at least twice (e.g., 2,3,4,5,6, 7, or more times).

Depending on the topology of the membrane protein ends and relative to the extracellular and intracellular membrane-penetrating segments, a membrane protein may be defined as type I, type II or type III. Type I membrane proteins have a single membrane spanning region and are oriented such that the N-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell and the C-terminus of the protein is located on the cytoplasmic side. Type II membrane proteins also have a single membrane spanning region but are oriented such that the C-terminus of the protein is located on the extracellular side of the lipid bilayer of the cell and the N-terminus of the protein is located on the cytoplasmic side. Type III membrane proteins have multiple segments across the membrane and can be further sub-classified based on the number of transmembrane segments and the position of the N and C termini.

In some embodiments, the transmembrane domain of the chimeric receptor polypeptides described herein is derived from a single pass membrane protein type I. Preferably, the transmembrane domain belongs to a membrane protein ：CD8α、CD8β、4-1BB/CD137、CD27、CD28、CD34、CD4、FcεRIγ、CD16A、OX40/CD134、CD3ζ、CD3ε、CD3γ、CD3δ、TCRα、TCRβ、TCRζ、CD32、CD64、CD45、CD5、CD9、CD22、CD37、CD80、CD86、CD40、CD40L/CD154、VEGFR2、FAS、FGFR2B、CD2、IL15、IL15R、IL21、DNAM-1、2B4、NKG2D、NKp44 and NKp46 selected from the group consisting of. In some embodiments, the transmembrane domain is from a membrane protein ：CD8a、CD8b、4-1BB、CD28、CD34、CD4、FcεRIγ、CD16A、OX40、CD3z、CD3e、CD3g、CD3d、TCRα、CD32、CD64、VEGFR2、FAS、FGFR2B、DNAM-1、2B4、NKG2D、NKp44 and NKp46 selected from the group consisting of. In some examples, the transmembrane domain belongs to CD8 (e.g., the transmembrane domain belongs to CD8 a). In some examples, the transmembrane domain belongs to 4-1BB/CD137. In other embodiments, the transmembrane domain belongs to CD28. In other embodiments, the transmembrane domain belongs to NKG2D, NKp, 44 or NKp46. In other examples, the transmembrane domain belongs to CD34. In yet other examples, the transmembrane domain is not derived from human CD8a. In some embodiments, the transmembrane domain of the chimeric receptor polypeptide is a single-pass alpha helix.

The amino acid sequences of exemplary transmembrane domains are provided in table 5:

Table 5: exemplary transmembrane domains

Transmembrane domains from multi-pass membrane proteins may also be suitable for use in the chimeric receptor polypeptides described herein. The multi-pass membrane protein may include complex alpha helical structures (e.g., at least 2, 3, 4, 5, 6, 7, or more alpha helices) or beta sheet structures. Preferably, the N-and C-termini of the multi-pass membrane protein are located on opposite sides of the lipid bilayer, e.g., the N-terminus of the protein is located on the cytoplasmic side of the lipid bilayer and the C-terminus of the protein is located on the extracellular side. In some cases, the reverse orientation of such native transmembrane proteins can be constructed to effectively orient the chimeric receptor polypeptide (e.g., CAR) within the immune cell membrane. One or more helices from a multi-pass membrane protein may be used to construct the chimeric receptor polypeptides described herein.

The transmembrane domain for the chimeric receptor polypeptides described herein can also include at least a portion of a synthetic non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic non-naturally occurring alpha helix or beta sheet. In some embodiments, a protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in US 7,052,906B1 and WO 2000/032776A2, the relevant disclosures of each of which are incorporated herein by reference.

In some embodiments, the amino acid sequence of the transmembrane domain does not include a cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises a cysteine residue. In some embodiments, the amino acid sequence of the transmembrane domain comprises two cysteine residues. In some embodiments, the amino acid sequence of the transmembrane domain includes more than two cysteine residues (e.g., 3, 4, 5, or more).

The transmembrane domain may include a transmembrane region and a cytoplasmic region located at the C-terminal side of the transmembrane domain. The cytoplasmic region of the transmembrane domain may include three or more amino acids and, in some embodiments, helps orient the transmembrane domain in the lipid bilayer. In some embodiments, one or more cysteine residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine residues are present in the cytoplasmic region of the transmembrane domain. In some embodiments, the cytoplasmic region of the transmembrane domain comprises a positively charged amino acid. In some embodiments, the cytoplasmic region of the transmembrane domain includes the amino acids arginine, serine, and lysine.

In some embodiments, the transmembrane region of the transmembrane domain comprises a hydrophobic amino acid residue. In some embodiments, the transmembrane region consists essentially of hydrophobic amino acid residues such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a poly-leucine-alanine sequence.

The hydrophilicity, hydrophobicity, or hydrophilicity characteristics of a protein or protein segment can be assessed by any method known in the art, including, for example, kyte and Doolittle hydrophilicity assays.

C. costimulatory signaling domains

In addition to stimulating antigen-specific signals, many immune cells (e.g., NK or T cells) also require co-stimulation to promote cell proliferation, differentiation and survival, as well as activating effector functions of the cells. As used herein, the term "costimulatory signaling domain" refers to at least one fragment of a costimulatory signaling protein that mediates signal transduction within a cell to induce an immune response such as effector function (secondary signal). As is known in the art, activation of immune cells such as T cells typically requires two signals: (1) An antigen-specific signal (primary signal) triggered by the engagement of an antigen peptide/MHC complex presented by a T Cell Receptor (TCR) and an antigen presenting cell, typically driven by cd3ζ as a component of the TCR complex; and (ii) a co-stimulatory signal (secondary signal) triggered by the interaction between the co-stimulatory receptor and its ligand. Costimulatory receptors transduce costimulatory signals (secondary signals) as a complement to TCR trigger signaling and modulate immune cell-mediated responses such as T cells, NK cells, macrophages, neutrophils or eosinophils.

Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity. The costimulatory signaling domains of any costimulatory molecule may be compatible for use in the chimeric receptor polypeptides described herein. The type of co-stimulatory signaling domain is selected based on the following factors: such as the type of immune cell in which the chimeric receptor polypeptide is to be expressed (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) and the desired immune effector function (e.g., ADCC). Thus, one embodiment is a chimeric receptor polypeptide of a genetically engineered immune cell comprising at least one costimulatory signaling domain. Examples of costimulatory signaling domains for chimeric receptor polypeptides can be cytoplasmic signaling domains of costimulatory proteins, including, but not limited to: members of the B7/CD28 family (e.g., ,B7-1/CD80、B7-2/CD86、B7-H1/PD-L1、B7-H2、B7-H3、B7-H4、B7-H6、B7-H7、BTLA/CD272、CD28、CTLA-4、Gi24/VISTA/B7-H5、ICOS/CD278、PD-1、PD-L2/B7-DC and PDCD 6); Members of the TNF superfamily (e.g., 4-1BB/TNFRSF9/CD137, 4-1BB ligand/TNFSF 9, BAFF/BLyS/TNFSF13B, BAFF R/TNFRSF13C, CD/TNFRSF 7, CD27 ligand/TNFSF 7, CD30/TNFRSF8, CD30 ligand/TNFSF 8, CD40/TNFRSF5, CD40/TNFSF5, CD40 ligand/TNFSF 5, DR3/TNFRSF25, GITR/TNFRSF18, and, GITR ligand/TNFSF 18, HVEM/TNFSF 14, LIGHT/TNFSF14, lymphotoxin- α/tumor necrosis factor- β, OX 40/TNFSF 4, OX40 ligand/TNFSF 4/CD2525, RELT/TNFSF 19L, TACI/TNFSF 13B, TL a/TNFSF15, TNF- α, and TNF RII/TNFRSF 1B); Members of the SLAM family (e.g., ,2B4/CD244/SLAMF4、BLAME/SLAMF8、CD2、CD2F-10/SLAMF9、CD48/SLAMF2、CD58/LFA-3、CD84/SLAMF5、CD229/SLAMF3、CRACC/SLAMF7、NTB-A/SLAMF6 and SLAM/CD 150); And any other costimulatory molecule, such as CD2, CD7, CD53, CD82/Kai-1, CD90/Thy1, CD96, CD160, CD200, CD300a/LMIR1, HLA class I, HLA-DR, ikaros, integrin alpha 4/CD49d, integrin alpha 4 beta 1, integrin α4β7/LPAM-1、LAG-3、TCL1A、TCL1B、CRTAM、DAP10、DAP12、Dectin-1/CLEC7A、DPPIV/CD26、EphB6、TIM-1/KIM-1/HAVCR、TIM-4、TSLP、TSLP R、 lymphocyte function-associated antigen-1 (LFA-1), NKG2D, NKG2C, NKp30, NKp44, NKp46 and JAMAL. In certain embodiments, the chimeric receptor polypeptide can contain a CD28 costimulatory signaling domain or a 4-1BB (CD 137) costimulatory signaling domain. In some embodiments, the at least one costimulatory signaling domain is selected from the group consisting of ：4-1BB、CD28、CD8α、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL, or any variant thereof.

Functional variants of any of the costimulatory signaling domains described herein are also within the scope of the disclosure such that the costimulatory signaling domain is capable of modulating an immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises a mutation (e.g., 1,2, 3, 4, 5, or 8) of up to 10 amino acid residues, such as an amino acid substitution, deletion, or addition, as compared to the wild-type counterpart. Such co-stimulatory signaling domains including one or more amino acid variants (e.g., amino acid substitutions, deletions, or additions) may be referred to as variants.

Mutations in the amino acid residues of the costimulatory signaling domain may result in increased signaling transduction and stimulation of the immune response relative to costimulatory signaling domains that do not include mutations. Mutations in the amino acid residues of the costimulatory signaling domain may result in reduced signaling transduction and reduced stimulation of the immune response relative to a costimulatory signaling domain that does not include the mutation. For example, mutations at residues 186 and 187 of the native CD28 amino acid sequence may result in an increase in costimulatory activity and induction of an immune response by the costimulatory domain of the chimeric receptor polypeptide. In some embodiments, the mutation is a substitution of a glycine residue at each of positions 186 and 187 with a glycine residue of the CD28 co-stimulatory domain known as the CD28 _LL→GG variant. Thus, a suitable variant of CD28 is the CD28 _LLàGG variant.

Additional mutations may be made in the costimulatory signaling domain that may increase or decrease the costimulatory activity of the domain, as will be apparent to one of ordinary skill in the art. In some embodiments, the costimulatory signaling domain is selected from the group consisting of: 4-1BB, CD28, OX40, and CD28 _LL→GG variants. In one embodiment, the at least one costimulatory signaling domain is a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain.

In some embodiments, the chimeric receptor polypeptide can contain a single costimulatory domain, such as a CD27 costimulatory domain, a CD28 costimulatory domain, a 4-1BB costimulatory domain, an ICOS costimulatory domain, an OX40L costimulatory domain, a 2B4 costimulatory domain, a GITR costimulatory domain, a NKG2D costimulatory domain, a NKp30 costimulatory domain, a NKp44 costimulatory domain, a NKp46 costimulatory domain, a DAP10 costimulatory domain, a DAP12 costimulatory domain, a DNAM1 costimulatory domain, a LFA-1 costimulatory domain, an HVEM costimulatory domain, or a JAMAL costimulatory domain.

The type of co-stimulatory signaling domain may be selected based on the following factors: such as the type of host cell (e.g., αβt, γδt or NK cells) and the desired immune effector function used with the chimeric receptor polypeptide.

In some embodiments, the chimeric receptor polypeptide can include more than one costimulatory signaling domain (e.g., 2,3, or more). In one embodiment, the chimeric receptor polypeptide comprises at least two co-stimulatory signaling domains. In a preferred embodiment, the chimeric receptor polypeptide comprises two costimulatory signaling domains. In some embodiments, the chimeric receptor polypeptide comprises two or more of the same costimulatory signaling domains, e.g., two copies of the costimulatory signaling domain of CD 28. In some embodiments, the chimeric receptor polypeptide comprises two or more costimulatory signaling domains from different costimulatory proteins, such as any two or more costimulatory proteins described herein. In some embodiments, the chimeric receptor polypeptide can include two or more co-stimulatory signaling domains from different co-stimulatory receptors, such as any two or more co-stimulatory receptors described herein, e.g., CD28 and 4-1BB, CD28 and CD27, CD28 and ICOS, CD28 _LL→GG variants and 4-1BB, CD28 and OX40, or CD28 _LL→GG variants and OX40. In some embodiments, the two costimulatory signaling domains are CD28 and 4-1BB. In some embodiments, the two costimulatory signaling domains are the CD28 _LL→GG variant and 4-1BB. In some embodiments, the two costimulatory signaling domains are CD28 and OX40. In some embodiments, the two costimulatory signaling domains are a CD28 _LL→GG variant and OX40. In some embodiments, the chimeric receptor polypeptides described herein can contain a combination of CD28 and ICOSL. In some embodiments, the chimeric receptor polypeptides described herein can contain a combination of CD28 and CD 27. In certain embodiments, the 4-1BB costimulatory domain is located at the N-terminus of the CD28 or CD28 _LL→GG variant costimulatory signaling domain.

In some embodiments, one of the co-stimulatory signaling domains is a CD28 co-stimulatory signaling domain and the other co-stimulatory domain is selected from the group consisting of ：CD8α、4-1BB、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL co-stimulatory signaling domains. In some embodiments, one of the co-stimulatory signaling domains is a CD 8a co-stimulatory signaling domain and the other co-stimulatory domain is selected from the group consisting of ：CD28、4-1BB、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL co-stimulatory signaling domains. In some embodiments, one of the costimulatory signaling domains is a 4-1BB costimulatory signaling domain and the other costimulatory domain is selected from the group consisting of ：CD8α、CD28、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL costimulatory signaling domains.

In some embodiments, the chimeric receptor polypeptides described herein do not include a costimulatory signaling domain.

The amino acid sequences of exemplary co-stimulatory domains are provided in table 6.

Table 6: exemplary costimulatory domains

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Alternatively, any of the chimeric receptor polypeptides can be free of any costimulatory signaling domain.

D. Cytoplasmic signaling domains

Any cytoplasmic signaling domain can be used to produce the chimeric receptor polypeptides described herein (e.g., ACTR polypeptides or CAR polypeptides). Such cytoplasmic domains can be any signaling domain involved in triggering cell signaling (primary signaling) that leads to immune cell proliferation and/or activation. The cytoplasmic signaling domain as described herein is not a costimulatory signaling domain, as is known in the art, which delivers a costimulatory or secondary signal to fully activate immune cells (e.g., CAR-T).

The cytoplasmic signaling domains described herein can include an immune receptor tyrosine based activation motif (ITAM) domain (e.g., at least one ITAM domain, at least two ITAM domains, or at least three ITAM domains) or can be free of ITAM. As used herein, "ITAM" is a conserved protein motif that is typically present at the tail of signaling molecules expressed in many immune cells. The ITAM motif may comprise two repeats of amino acid sequence YxxL/I, each x being independently any amino acid, separated by 6-8 amino acids, resulting in the conserved motif YxxL/Ix _(6-8) YxxL/I. The ITAM within a signaling molecule is important for intracellular signal transduction, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. ITAM can also act as a docking site for other proteins involved in signaling pathways. Examples of ITAMs for use in chimeric receptor polypeptides included within cytoplasmic signaling domains can be, but are not limited to: cd3γ, cd3ε, cd3δ each contain a single ITAM motif, whereas each zeta chain contains 3 different ITAM domains (ζa, ζb and ζc). The number and ITAM sequences are also important in the design of CARs (Bettini et al J Immunol 199 (5): 1555-1560 (2017); jayaraman et al E biomedical 58:102931 (2020)).

In some embodiments, the cytoplasmic signaling domain belongs to cd3ζ or fcepsilonr 1 γ. The amino acid sequence of one exemplary cytoplasmic signaling domain of human CD3z is provided below:

In other examples, the cytoplasmic signaling domain is not derived from human cd3ζ. In yet other examples, when the extracellular Fc binding domain of the same chimeric receptor polypeptide is derived from CD16A, the cytoplasmic signaling domain is not derived from an Fc receptor.

In a specific embodiment, several signaling domains may be fused together to produce an additive or synergistic effect. Non-limiting examples of useful additional signaling domains include part or all of one or more of TCR zeta chain, CD28, OX40/CD134, 4-1BB/CD137, fcεRIgamma, ICOS/CD278, IL2 Ralpha/CD 122, IL-2 Rgamma/CD 132, and CD 40.

In other embodiments, the cytoplasmic signaling domains described herein are free of ITAM motifs. Examples include, but are not limited to, the cytoplasmic signaling domain of Jak/STAT, toll-Interleukin receptor (TIR), and tyrosine kinase.

E. Hinge domain

In some embodiments, a chimeric receptor polypeptide described herein, such as an ACTR polypeptide or a CAR polypeptide, further comprises a hinge domain located between the extracellular ligand binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is typically found between two domains of a protein and can allow flexibility in the protein and movement of one or both domains relative to each other. Any amino acid sequence that provides such flexibility and mobility of the extracellular ligand binding domain relative to the transmembrane domain of the chimeric receptor polypeptide can be used.

The hinge domain of any protein known in the art, including hinge domains, is suitable for use in the chimeric receptor polypeptides described herein. In some embodiments, the hinge domain is at least a portion of the hinge domain of a naturally occurring protein and imparts flexibility to the chimeric receptor polypeptide. In one embodiment, the chimeric receptor polypeptide comprises a hinge domain that is a hinge domain selected from the list of: CD28, CD16A, CD, igG, murine CD8a and DAP12. In some embodiments, the hinge domain belongs to CD8 (e.g., the hinge domain belongs to CD8 a). In some embodiments, the hinge domain is part of a hinge domain of CD8, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD 8. In some embodiments, the hinge domain belongs to CD28. In some embodiments, the hinge domain is part of CD28, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD28. The hinge domain and/or the transmembrane domain may be linked to additional amino acids (e.g., 15aa, 10-aa, 8-aa, 6-aa, or 4-aa) at the N-terminal portion, the C-terminal portion, or both. Examples can be found, for example, in YING et al, nature medicine 25 (6): 947-953 (2019).

In some embodiments, the hinge domain belongs to the CD16A receptor, e.g., the entire hinge domain of the CD16A receptor or a portion thereof, which may consist of up to 40 consecutive amino acid residues (e.g., 20, 25, 30, 35, or 40) of the CD16A receptor. Such chimeric receptor polypeptides (e.g., ACTR polypeptides) may not contain hinge domains from different receptors (not CD16A receptors). In some cases, the chimeric receptor polypeptides described herein can be free of hinge domains from any non-CD 16A receptor. In some cases, such chimeric receptor polypeptides may not contain any hinge domain.

Hinge domains of IgG antibodies, such as IgG, igA, igM, igE or IgD antibodies, are also suitable for use in the chimeric receptor polypeptides described herein. In some embodiments, the hinge domain is a hinge domain that is linked to constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain belongs to an antibody and includes the hinge domain of an antibody and one or more constant regions of an antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of an antibody. In some embodiments, the hinge domain includes the hinge domain of an antibody and the CH2 and CH3 constant regions of an antibody. In some embodiments, the antibody is IgG, igA, igM, igE or an IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, igG2, igG3, or IgG4 antibody, preferably IgG1 and IgG4. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides can also be used as hinge domains of the chimeric receptor polypeptides described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular target binding domain and the N-terminus of the transmembrane domain is a peptide linker, such as (Gly _xSer)_n linker, wherein x and N independently may be integers between 3 and 12, including 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more.

In other embodiments, the hinge domain is an extended recombinant polypeptide (XTEN), which is an unstructured polypeptide consisting of hydrophilic residues of different lengths (e.g., 10-80 amino acid residues). The amino acid sequence of XTEN peptides will be apparent to those skilled in the art and can be found, for example, in US 8,673,860, the relevant disclosure of which is incorporated herein by reference. In some embodiments, the hinge domain is an XTEN peptide and comprises 60 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 30 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 45 amino acids. In some embodiments, the hinge domain is an XTEN peptide and comprises 15 amino acids.

Any of the hinge domains used in preparing the chimeric receptor polypeptides as described herein can contain up to 250 amino acid residues. In some cases, the chimeric receptor polypeptide can contain a relatively long hinge domain, e.g., containing 150-250 amino acid residues (e.g., 150-180 amino acid residues, 180-200 amino acid residues, or 200-250 amino acid residues). In other cases, the chimeric receptor polypeptide can contain a mid-sized hinge domain that can contain 60-150 amino acid residues (e.g., 60-80, 80-100, 100-120, or 120-150 amino acid residues). In some cases, the hinge domain may be a flexible linker consisting of glycine and serine amino acids between 15 and 60 amino acids in length, preferably consisting of Gly ₄ Ser units, in particular one of the linkers of SEQ ID No. 15 to SEQ ID No. 17. Alternatively, the chimeric receptor polypeptide can contain a short hinge domain, which can contain less than 60 amino acid residues (e.g., 1-30 amino acids or 31-60 amino acids). In some embodiments, the chimeric receptor polypeptides described herein (e.g., ACTR polypeptides) do not contain a hinge domain or do not contain a hinge domain from a non-CD 16A receptor. The amino acid sequences of exemplary hinge domains are provided in table 7.

Table 7: exemplary hinge Domains

F. Signal peptides

In some embodiments, a chimeric receptor polypeptide (e.g., ACTR polypeptide or CAR polypeptide) can also include a signal peptide (also referred to as a signal sequence) located at the N-terminus of the polypeptide. Typically, the signal sequence is a peptide sequence that targets the polypeptide to a desired site in the cell. In some embodiments, the signal sequence targets the chimeric receptor polypeptide to the secretory pathway of the cell and will allow the chimeric receptor polypeptide to integrate and anchor into the lipid bilayer. The compatibility of signal sequences comprising naturally occurring proteins or synthetic non-naturally occurring signal sequences for use in the chimeric receptor polypeptides described herein will be apparent to those of skill in the art. In some embodiments, the signal sequence is derived from CD8 alpha (SEQ ID NO: 1). In some embodiments, the signal sequence is from CD28 (SEQ ID NO: 2). In other embodiments, the signal sequence is from a murine kappa chain. In yet other embodiments, the signal sequence is from CD16. See table 8 below.

Table 8: exemplary Signal peptides

Signal peptides	Sequence(s)	SEQ ID NO.
			CD8a	MALPVTALLLPLALLLHAARP	SEQ ID NO:1
CD28	MLRLLLALNLFPSIQVTG	SEQ ID NO:2
			Murine k chain	METDTLLLWVLLLWVPGSTG	SEQ ID NO:3

In some cases, any of the chimeric receptor polypeptides disclosed herein can further comprise a protein tag, examples of which are provided in table 9 below.

Table 9: exemplary protein tags

Protein tag	Sequence(s)	SEQ ID NO.
			2XV5 tag	GKPIPNPLLGLDSTGKPIPNPLLGLDST	SEQ ID NO:62
6XHis tag	HHHHHH	SEQ ID NO:63
			2XFlag tag	DYKDDDDKDYKDDDDK	SEQ ID NO:64
3XHA tag	YPYDVPDYAYPYDVPDYAYPYDVPDYA	SEQ ID NO:65

Examples of ACTR polypeptides

Exemplary ACTR constructs for use in the methods and compositions described herein may be found, for example, in the present specification and figures, or may be found in WO2016/040441A1, WO2017/161333, and WO2018/140960, each of which is incorporated herein by reference for its purposes. ACTR polypeptides described herein may include a CD16A extracellular domain, a transmembrane domain, and a cd3ζ cytoplasmic signaling domain that have binding affinity and specificity for the Fc portion of IgG molecules. In some embodiments, the ACTR polypeptide may further comprise one or more costimulatory signaling domains, one of which may be a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain. The ACTR polypeptide is configured such that when expressed on a host cell, the extracellular ligand binding domain is located extracellular for binding to the target molecule and the cd3ζ cytoplasmic signaling domain. The co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling.

In some embodiments, ACTR polypeptides as described herein may include, from N-terminus to C-terminus, an Fc binding domain such as a CD16A extracellular domain, a transmembrane domain, optionally one or more costimulatory domains (e.g., a CD28 costimulatory domain, a 4-1BB costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, or an ICOS costimulatory signaling domain), and a CD3 zeta cytoplasmic signaling domain.

Alternatively or additionally, ACTR polypeptides described herein may contain two or more costimulatory signaling domains, which may be linked to each other or separated by a cytoplasmic signaling domain. The extracellular Fc-binding, transmembrane domain, optional costimulatory signaling domain, and cytoplasmic signaling domain in the ACTR polypeptide may be linked to each other directly or through a peptide linker. In some embodiments, any of the ACTR polypeptides described herein may include a signal sequence at the N-terminus.

Table 10 provides exemplary ACTR polypeptides described herein. These exemplary constructs have, in order from N-terminal to C-terminal, a signal sequence, an Fc binding domain (e.g., the extracellular domain of an Fc receptor), a hinge domain, and a transmembrane, while the positions of the optional co-stimulatory domain and cytoplasmic signaling domain may be switched.

Table 10: exemplary Components of ACTR Polypeptides

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Examples of h.car polypeptides

Exemplary CAR polypeptides for use in the methods and compositions described herein can be found, for example, in the present specification and figures, or are known in the art. The CAR polypeptides described herein can include an extracellular domain comprising a single chain antibody fragment (scFv) having binding affinity and specificity for an antigen of interest (e.g., an antigen of interest listed in table 4), a transmembrane domain (e.g., a transmembrane domain listed in table 5), preferably a CD8a transmembrane domain, a costimulatory domain (e.g., a costimulatory domain listed in table 6), and a cd3ζ cytoplasmic signaling domain. In some embodiments, the CAR polypeptide can further comprise a hinge domain (e.g., a hinge domain listed in table 7).

In a specific example, a CAR polypeptide described herein can include: (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; and (ii) a CD28 transmembrane domain, a CD28 hinge domain, or a combination thereof. In further specific examples, a CAR polypeptide described herein can include: (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (ii) A CD8 a transmembrane domain, a CD8 a hinge domain, or a combination thereof. In some embodiments, the CAR polypeptide may further comprise one or more costimulatory signaling domains, one of which may be a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain. In other examples, a CAR polypeptide described herein can include: (i) a CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (ii) A CD28 transmembrane domain, a CD8 a hinge domain, or a combination thereof.

In an exemplary embodiment, the CAR polypeptide comprises: (i) a CD8 a hinge domain; (ii) a CD8 a transmembrane domain; (iii) A CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (iv) a cd3ζ cytoplasmic signaling domain or combination thereof. In other embodiments, the CAR polypeptide comprising two co-stimulatory domains further comprises: (i) a CD8 a or CD28 hinge domain; (ii) a CD8 a or CD28 transmembrane domain; (iii) A CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (iv) OX40L co-stimulatory domain, or 2B4 co-stimulatory domain, or DAP10 co-stimulatory domain, or DNAM-1 co-stimulatory domain, or NKG2D co-stimulatory domain, or NKp30 co-stimulatory domain, or NKp44 co-stimulatory domain, or NKp46 co-stimulatory domain, or JAMAL co-stimulatory domain; (v) a cd3ζ cytoplasmic signaling domain or combination thereof. In another exemplary embodiment, the CAR polypeptide comprising two co-stimulatory domains further comprises: (i) a CD8 a hinge domain; (ii) a CD28 transmembrane domain; (iii) A CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (iv) An OX40L co-stimulatory domain, or a 2B4 co-stimulatory domain, or a DAP10 co-stimulatory domain, or a DNAM-1 co-stimulatory domain, or JAMAL co-stimulatory domain; (v) a cd3ζ cytoplasmic signaling domain or combination thereof.

In another exemplary embodiment, the CAR polypeptide comprising two co-stimulatory domains further comprises: (i) a CD8 a hinge domain; (ii) A CD28 transmembrane domain, or an NKp44 transmembrane domain, or an NKG2D transmembrane domain, or an NKp46 transmembrane domain; (iii) A CD28 co-stimulatory domain, or a 4-1BB co-stimulatory domain, or a 2B4 co-stimulatory domain, or a DAP10 co-stimulatory domain; (iv) An OX40L co-stimulatory domain, or a 2B4 co-stimulatory domain, or a DAP10 co-stimulatory domain, or a DAP12 or DNAM-1 co-stimulatory or JAMAL co-stimulatory domain; (v) A cd3ζ cytoplasmic signaling domain, or a DAP12 cytoplasmic signaling domain, or a 2B4 cytoplasmic signaling domain, or a combination thereof. In an exemplary embodiment, the CAR polypeptide comprises: (i) a CD8 a hinge domain; (ii) a CD28 transmembrane domain; (iii) A CD28 co-stimulatory domain or a 4-1BB co-stimulatory domain; (iv) an OX40L co-stimulatory domain or an OX40 co-stimulatory domain; (v) a cd3ζ cytoplasmic signaling domain or combination thereof.

For example, the CAR polypeptide can include an amino acid sequence selected from SEQ ID NO:78 or SEQ ID NO:79 provided below.

The CAR polypeptide is configured such that when expressed on a host cell (e.g., a T or NK cell), the extracellular antigen binding domain is located extracellular for binding to a target molecule (e.g., a tumor antigen) and the cd3ζ cytoplasmic signaling domain. The co-stimulatory signaling domain may be located in the cytoplasm for triggering activation and/or effector signaling.

In some embodiments, a CAR polypeptide as described herein can include, from N-terminus to C-terminus, an extracellular antigen binding domain, a transmembrane domain, optionally one or more costimulatory domains (e.g., a CD28 costimulatory domain, a 4-1BB costimulatory signaling domain, an OX40L costimulatory signaling domain, an OX40 costimulatory signaling domain, a CD27 costimulatory signaling domain, a 2B4 costimulatory signaling domain, or an ICOS costimulatory signaling domain), and a CD3 ζ cytoplasmic signaling domain.

Alternatively or additionally, the CAR polypeptides described herein can contain two or more co-stimulatory signaling domains that can be linked to each other or separated by a cytoplasmic signaling domain. The extracellular antigen binding domain, transmembrane domain, optional costimulatory signaling domain, and cytoplasmic signaling domain in the CAR polypeptide can be linked to each other directly or through a peptide linker. In some embodiments, any of the CAR polypeptides described herein can include a signal sequence at the N-terminus.

Tables 11-13 provide exemplary CAR polypeptides for CAR- αβ T cells, CAR-NK cells, and CAR- γδ T cells described herein. These exemplary constructs have, in order from the N-terminus to the C-terminus, a signal sequence, an antigen binding domain (e.g., an scFv fragment that targets an antigen such as a tumor antigen or a pathogenic antigen), a hinge domain, and a transmembrane, while the positions of the optional co-stimulatory domain and the cytoplasmic signaling domain can be switched.

Table 11: exemplary CAR constructs for expression in αβ T cells

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Table 12: exemplary CAR constructs for expression in NK cells

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Table 13: exemplary CAR constructs for expression in γδ T cells

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Table 14: exemplary GPC 3-targeting CAR constructs

An amino acid sequence of an exemplary anti-GPC 3 scFv for construction of an anti-GPC CAR construct, and an exemplary anti-GPC 3 CAR construct comprising such an amino acid sequence, is provided below:

anti-GPC 3 scFv

GPC3-CAR1 against

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GPC3-CAR 2 against

TCR polypeptides

The present disclosure provides a modified immune cell (e.g., an αβ T cell, γδ T cell, or NKT cell) that includes an exogenous T Cell Receptor (TCR) on a target cell (e.g., recognizing a peptide antigen presented on a Major Histocompatibility (MHC) molecule associated with a disease such as cancer; see discussion herein). As used herein, a TCR complex is expressed on T or NKT cells and associates with a CD3 complex. Engagement of the TCR complex results in activation of T cells in the form of proliferation, cytokine secretion and cytolytic activity. Provided herein are T Cell Receptor (TCR) -engineered immune cells (e.g., T and NKT cells) that can be used to improve cellular immunotherapy. For example, the present disclosure provides modified immune cells comprising a vector encoding a chimeric polypeptide comprising: (i) An extracellular single-chain variant fragment that specifically binds to an epitope (e.g., a tumor (neo) epitope or a tumor-associated antigen); (ii) an intracellular activation domain; and (iii) coupling the extracellular single-stranded variant fragment to a transmembrane linker of an intracellular activation domain. Most typically, the first, second and third segments are arranged such that the extracellular single-chain variant fragment, the intracellular activation domain and the linker form a single chimeric polypeptide.

(I) peptide-MHC complexes

Major Histocompatibility Complex (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. Human MHC is known as Human Leukocyte Antigen (HLA). MHC class I molecules are heterodimers with a membrane spanning the alpha chain (with three alpha domains) and non-covalently associated beta 2 microglobulin. MHC I consists of HLAA, B and C and B2 microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both spanning the membrane. Each chain has two domains. MHC II is a heterodimer of several HLA (HLA-DP, DQ, DR). MHC class I molecules deliver cytosolic-derived peptides to the cell surface where peptide-MHC complexes are recognized by CD8 ⁺ T cells. MHC class II molecules deliver peptides derived from the vesicle system to the cell surface where they are recognized by CD4 ⁺ T cells. TCRs can recognize a range of peptide antigens including tumor-associated antigens (TAAs), cancer germ line antigens (CGAs), and tumor-specific antigens (TSAs) including viral antigens and neoantigens (listed in table 1 of the following document: shafer et al, immunological front, 13:835762 (2022)).

(Ii) CD3 complexes

As used herein, CD3 refers to a six-chain polyprotein complex associated with an antigen signal in T cells. In mammals, the complex includes a homodimer of one CD3 gamma chain, one CD3 delta chain, two CD3 epsilon chains, and one CD3 zeta chain. The CD3 gamma, CD3 beta and CD3 epsilon chains are highly related cell surface proteins of the immunoglobulin superfamily containing single immunoglobulin domains. The transmembrane regions of the cd3γ, cd3β and cd3ε chains are negatively charged, a property that allows these chains to associate with positively charged TCR chains. The intracellular tails of the cd3γ, cd3β and cd3ε chains each contain a single conserved motif, known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each cd3ζ chain has three conserved motifs. Without wishing to be bound by theory, it is believed that ITAM is important for the signaling ability of the TCR complex. CD3 as used in the present disclosure is preferably an endogenous complex of T or NKT cells for cell therapy. Second, T or NKT cells may be from a variety of animal species, including humans, mice, rats, or other mammals.

(Iii) TCR complex

As used herein, a TCR complex refers to a complex formed by association of CD3 with a TCR. TCRs consist of two heterodimeric TCR chains tcrα, tcrβ (in αβt cells) or tcrγ, tcrδ (in γδ T cells) and six CD3 chains that form a multiprotein complex that recognizes peptide antigens presented on MHC. The TCR complex forms a ligand binding site, and the CD3 complex protein mediates signaling and subsequent T cell activation. TCR alpha is encoded by the TRA gene, beta chain is encoded by TRB, gamma chain is encoded by TRG and delta chain is encoded by TRD.

The component of a physiological TCR complex refers to a TCR chain (i.e., tcrα, tcrβ, tcrγ, or tcrδ), a CD3 chain (i.e., cd3γ, cd3δ, cd3ε, or cd3ζ), or a complex formed from two or more TCR chains or CD3 chains (e.g., a complex of tcrα and tcrβ, a complex of tcrγ and tcrδ, a complex of cd3ε and cd3δ, a complex of cd3γ and cd3ε, or a sub-TCR complex of tcrα, tcrβ, cd3γ, cd3δ, and two cd3ε chains). In some cases, a complex may be formed from two or more TCR chains or CD3 chains (e.g., a complex of tcrα and tcrβ, a complex of tcrγ and tcrδ, a complex of cd3ε and cd3δ, a complex of cd3γ and cd3ε, or a sub-TCR complex of tcrα, tcrβ, cd3γ, cd3δ, and two cd3ε chains).

TCRs consist of heterodimeric TCRs linked by disulfide bonds (α/β or γ/δ TCRs) and form a non-covalent polyprotein complex with the CD3 chain. TCR chains are type I proteins consisting of an extracellular region, a transmembrane region, and a short cytoplasmic tail. The extracellular domain contains a hypervariable V region responsible for antigen recognition and a constant C region near the membrane. The transmembrane and cytoplasmic domains form non-covalent interactions with the CD3 chain to stabilize the TCR complex and mediate downstream signaling (Kuhns et al, immunoreview (Immunol Rev), 250 (2012); wucherpfennig et al, cold spring harbor medical view (Cold Spring Harb Perspect Biol), 2:a005140 (2010)). A review of TCRs and their designs is provided in the following documents: blankenstein et al, contemporary immunology perspective (Curr Opin Immunol), 33:112-9 (2015) (incorporated herein by reference for the subject matter and purposes cited herein). Methods for producing an engineered TCR are described, for example, in the following documents: bowerman et al, molecular immunology (Mol Immunol), 46:3000-8 (2009), the technology of which is incorporated herein by reference. In addition, antibodies that bind to a particular antigen can be isolated from TCRs that bind to the antigen to screen libraries of complementary vα or vβ domains, respectively, using vα or vβ domains. In certain embodiments, the TCR is found on the surface of a T cell and associates with a CD3 complex. TCR sources as used in the present disclosure may be from a variety of animal species, such as human, mouse, rat, rabbit, or other mammals.

The TCR complex further comprises: (a) an extracellular domain; a transmembrane domain; and (c) a cytoplasmic domain. The extracellular domain may be derived from natural or recombinant sources. In the case where the source is natural, the domain may be derived from any protein, except membrane-bound or transmembrane proteins. In one aspect, the extracellular domain is capable of associating with the transmembrane domain. Non-limiting examples of extracellular domains particularly useful in the present disclosure may comprise the α, β, γ, or δ chain of a TCR, or the extracellular domain of CD3 epsilon, CD3 γ, or CD3 δ, or in alternative embodiments, the extracellular domain of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154.

In some embodiments, the antigen binding domain may comprise one member of an interaction pair, e.g., the antigen binding domain may be one member of an interaction pair comprising a receptor and a ligand, or a fragment thereof. The receptor or ligand or fragment thereof may be referred to as an antigen binding domain. Another member not referred to as an antigen binding domain may include an epitope that specifically binds to the antigen binding domain.

The second antigen binding domain may be linked to any member of the TCR complex, and the TCR may be an alpha/beta or gamma/delta TCR. The second antigen binding domain may be linked to at least one of a TCR chain, cluster of differentiation 3 (CD 3) chain, or CD3 z chain. The second antigen binding domain may be linked to a transmembrane receptor of a TCR, such as TCR delta, TCR gamma, TCR alpha or TCR beta. The second antigen binding domain may be linked to a CD3 chain, such as CD3 epsilon, CD3 delta, or CD3 gamma. The second antigen binding domain may be linked to a CD3 z chain.

In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a CD3 epsilon chain and a second antigen-binding domain fused to a CD3 delta chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a cd3δ chain and a second antigen-binding domain fused to a cd3γ chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a CD 3a chain or a CD3 β chain and a second antigen-binding domain fused to a CD3 epsilon chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a CD3 β chain or a CD3 δ chain and a second antigen-binding domain fused to a CD3 epsilon chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to an alpha chain or a TCR gamma chain and a second antigen-binding domain fused to a CD3 gamma chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a TCR β chain or a TCR δ chain and a second antigen-binding domain fused to a CD3 γ chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a TCR alpha chain or a TCR gamma chain and a second antigen-binding domain fused to a CD3 delta chain. In some embodiments, the modified TCR complex comprises a first antigen-binding domain fused to a TCR β chain or a TCR δ chain and a second antigen-binding domain fused to a δ chain.

The transmembrane domain may in turn be derived from natural or recombinant sources. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the TCR complex binds to a target (i.e., peptide-MHC). Non-limiting examples of transmembrane domains particularly useful in the present disclosure may comprise an alpha, beta, gamma or delta chain of TCR、CD28、CD3ε、CD3γ、CD3δ、CD3 z、CD45、CD4、CD5、CD8、CD9、CD 16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154. The transmembrane domain may comprise one or more additional amino acids adjacent to the transmembrane region, for example one or more amino acids associated with an extracellular region of a protein from which the transmembrane is derived. In one aspect, a transmembrane domain is a domain that associates with one of the other domains of the TCR used. In some examples, the transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

In general, the extracellular domain and the transmembrane domain may be encoded by a single genomic sequence. In alternative embodiments, the sequence may be designed to include a transmembrane domain that is heterologous to the extracellular domain.

Optionally, a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length may form a link between the transmembrane domain and the cytoplasmic region of the TCR polypeptide. Glycine-serine duplex provides a particularly suitable linker (e.g., SEQ ID NOS: 15-17).

The cytoplasmic domain of the TCR may comprise an intracellular domain. In some embodiments, the intracellular domain is from cd3γ, cd3δ, cd3ε, tcra, tcrβ, tcrγ, or tcrδ. In some embodiments, if the TCR complex comprises a cd3γ, δ, epsilon polypeptide; TCR α, TCR β, TCR γ, and TCR δ subunits typically have a short (e.g., 1-19 amino acids in length) intracellular domain and typically lack a signaling domain, then the intracellular domain comprises the signaling domain. The intracellular signaling domain is generally responsible for activating at least one of the normal effector functions of the immune cells into which the TCR has been introduced. Although the intracellular domains of tcrα, tcrβ, tcrγ and tcrδ do not have a signaling domain, they are able to recruit proteins having the primary intracellular signaling domains described herein, such as CD3 z, which serves as the intracellular signaling domain.

In some cases, the TCR subunits include: (i) at least a portion of a TCR extracellular domain; (ii) a TCR transmembrane domain; and (iii) a TCR intracellular domain, wherein at least two of (i), (ii) and (iii) are from the same TCR subunit. In some cases, the TCR extracellular domain comprises an extracellular domain of a TCR α chain, a TCR β chain, a TCR γ chain, a TCR δ chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 δ TCR subunit, and a functional fragment thereof, or a portion thereof.

In some examples, the TCR subunits comprise transmembrane domains comprising the transmembrane domains of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 z TCR subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof.

In some cases, the TCR subunit comprises a TCR intracellular domain of CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR gamma, or TCR delta. In some embodiments, the intracellular domain comprises a stimulatory domain of a protein comprising an intracellular signaling domain of CD3 epsilon, CD3 gamma, CD3 delta, and functional fragments thereof.

In various embodiments of aspects herein, the modified TCR complex comprises a previously identified TCR. In some cases, whole exome sequencing can be used to identify TCRs. For example, TCRs may target neoantigens or neoepitopes identified by whole exome sequencing of target cells. Alternatively, TCRs may be identified from autologous, allogeneic or xenogeneic libraries.

The modified T Cell Receptor (TCR) complex may comprise a second antigen-binding domain that exhibits binding to a second epitope. The second antigen binding domain may comprise any protein or molecule capable of binding to an epitope. In some embodiments, the second antigen binding domain comprises a heterologous sequence that exhibits binding to a second epitope. Non-limiting examples of the second antigen binding domain of the TCR complex include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, or functional derivatives, variants, or fragments thereof, including, but not limited to, fab ', F (ab') 2, fv, single chain Fv (scFv), minibodies, diabodies, and single domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VH H) of camelid nanobodies. In some embodiments, the second antigen binding domain comprises a single domain antibody (sdAb).

Since thymus is selected in the range of about 1-100. Mu.M, TCRs naturally have relatively low affinity. Amino acid substitutions in the variable region can increase affinity to the low μΜ and nM range. Yeast and phage display methods have been used to generate very high affinity TCRs in the pM range. Increasing TCR affinity may also lead to loss of specificity, however finer structure-directed design approaches may be used to increase affinity without decreasing specificity. TCRs can also be engineered to contain single chain proteins from the variable regions of TCR alpha and TCR beta chains fused by peptide linkers of the antigen recognition domain and CD3z domain to confer signaling. Additional co-stimulatory domains may also be included to improve signaling (WILLEMSEN et al, gene Ther, 7:1369-77 (2000); zhang et al, cancer Gene therapy (CANCER GENE THER), 11:487-96 (2004); plaksin et al, journal of immunology, 158:2218-27 (1997); chung et al, proc. Natl. Acad. Sci. USA, 91:12654-8 (1994)).

Hematopoietic cells expressing factors that redirect glucose metabolites and optionally chimeric receptor polypeptides

Provided herein are genetically engineered host cells (e.g., hematopoietic cells such as HSCs and immune cells, e.g., T cells or NK cells) that express one or more of the factors that redirect glucose metabolites (e.g., redirect glucose metabolites out of the glycolytic pathway) as described herein. In some embodiments, the factor (e.g., polypeptide) is encoded by a transgene introduced into the host cell (e.g., exogenous to the host cell). The genetically engineered host cell may further express a chimeric receptor polypeptide (e.g., an ACTR-expressing cell, e.g., an ACTR T cell, a CAR-expressing cell, e.g., a CAR-T cell, or a TCR-expressing cell, e.g., a TCR-T cell), as also described herein. In some embodiments, the host cell is a hematopoietic cell or progeny thereof. In some embodiments, the hematopoietic cells may be hematopoietic stem cells. In some embodiments, the genetically engineered immune cells may be Natural Killer (NK) cells, natural Killer T (NKT) cells, monocytes/macrophages, neutrophils, eosinophils, αβ T or γδ T cells. In other embodiments, the host cell is an immune cell, such as an αβ T cell, γδ T cell (e.g., naive T cell, effector memory T cell, central memory T cell, double negative T cell, effector T cell, thI cell, thII cell, th17 cell, th22 cell) or NK cell. In some embodiments, the immune cells are αβ T cells. In some embodiments, the immune cell is a γδ T cell. In some embodiments, the γδ T cells are vγ9δ2t cells. In some embodiments, the γδ T cells are vδ1T cells. In some embodiments, γδ T cells comprise vγ9δ2tcr, vγ10/vδ2tcr, and/or vγ2/vδ2tcr. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cell is a NKT cell.

In a preferred embodiment, the immune cell is an αβ T cell, and wherein the chimeric receptor polypeptide is a CAR polypeptide comprising the components as shown in table 11. In another preferred embodiment, the immune cell is an NK cell, and wherein the chimeric receptor polypeptide is a CAR polypeptide comprising a component as shown in table 12. In yet another preferred embodiment, the immune cell is a γδ T cell and wherein the chimeric receptor polypeptide is a CAR polypeptide comprising the components as set forth in table 13.

In some other embodiments, the genetically engineered immune cells described herein may be derived from a cell line, for example selected from NK-92, NK-92MI, YTS and KHYG-1, preferably NK-92 cells. In other embodiments, the genetically engineered immune cells described herein may be derived from Peripheral Blood Mononuclear Cells (PBMCs), hematopoietic Stem Cells (HSCs), umbilical Cord Blood Stem Cells (CBSCs), or induced pluripotent stem cells (ipscs).

In some embodiments, genetically engineered hematopoietic cells or immune cells (e.g., T cells or NK cells) such as HSCs can co-express any of the CAR constructs with any of the factors that redirect glucose metabolites, such as polypeptides that divert or redirect glucose metabolites (e.g., PKM2, GFPT1, or TIGAR), as disclosed herein. In some embodiments, the CAR construct may include a costimulatory domain from 4-1BB or CD28, and the polypeptide that transfers or redirects the glucose metabolite is PKM2, GFPT1, or TIGAR. The CAR construct can further include a hinge and transmembrane domain from CD8 (e.g., CD8 a) or CD 28.

In some examples, genetically engineered hematopoietic cells or immune cells (e.g., T cells or NK cells) such as HSCs can be engineered to co-express any of the CAR constructs (e.g., anti-GPC 3 CARs disclosed herein) and TIGARs. In particular embodiments, the genetically engineered cells include T cells that co-express a CAR and a TIGAR. In other embodiments, the genetically engineered cells include NK cells that co-express the CAR and TIGAR.

In other embodiments, genetically engineered hematopoietic cells or immune cells (e.g., T cells or NK cells) such as HSCs can co-express any of the ACTR constructs, such as the constructs disclosed herein, with any of the factors that redirect glucose metabolites, such as polypeptides that divert or redirect glucose metabolites (e.g., PKM2, GFPT1, or TIGAR). In some embodiments, ACTR constructs may include a costimulatory domain from 4-1BB or CD28, and the polypeptide that transfers or redirects glucose metabolites is PKM2, GFPT1, or TIGAR. ACTR constructs may further include hinge and transmembrane domains from CD8 or CD 28.

In some examples, genetically engineered hematopoietic cells or immune cells (e.g., T cells or NK cells), such as HSCs, can be engineered to co-express any of the ACTR constructs (e.g., CD16A-V158ACTR disclosed herein) and TIGAR. In particular embodiments, the genetically engineered cells include T cells that co-express ACTR and TIGAR. In other embodiments, the genetically engineered cells include NK cells that co-express ACTR and TIGAR.

Alternatively, the genetically engineered host cells disclosed herein may not express any chimeric receptor polypeptides.

In some embodiments, genetically engineered immune cells that may overexpress one or more factors that redirect glucose metabolites as disclosed herein may be derived from tumor-infiltrating lymphocytes (TILs). Overexpression of factors that redirect glucose metabolites may enhance antitumor activity or TIL in the tumor microenvironment. In particular embodiments, TILs are selected that are reactive/targeted to specific peptides presented by MHC complexes.

TIL and/or T cells expressing the genetically modified TCR may target peptide-MHC complexes, where the peptide may be derived from a pathogen, a tumor antigen, or an autoantigen. Some examples are provided in table 15 below.

Any of the CAR constructs disclosed herein or antibodies used with ACTR T cells can also target any of the peptides in such peptide/MHC complexes.

In other embodiments, genetically engineered hematopoietic cells or immune cells (e.g., T cells or NKT cells) such as HSCs may co-express TCR polypeptides with any of the factors that redirect glucose metabolites, such as polypeptides that divert or redirect glucose metabolites (e.g., PKM2, GFPT, or TIGAR).

Table 15: exemplary peptide-MHC targets

Target(s)	Indication of disease
		NY-ESO-1	Sarcoma, MM
MAGE-A10	NSCLC, bladder, HNSCC
		MAGE-A4	Sarcomas, other
PMEL	Melanoma (HEI)
		WT-1	Ovary
AFP	HCC
		HPV-16E6	Cervical of the uterus
HPV-16E7	Cervical of the uterus

In some embodiments, the host cell is an immune cell such as a T cell or NK cell. In some embodiments, the immune cell is a T cell. For example, the T cells may be cd4+ helper cells or CD8 ⁺ cytotoxic cells or a combination thereof. Alternatively or additionally, the T cells may be suppressor T cells such as T _reg cells. In some embodiments, the immune cells are NK cells. In other embodiments, the immune cells may be derived cell lines, such as NK-92 cells. The population of immune cells may be obtained from any source, such as Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, or tissues such as spleen, lymph nodes, thymus, or tumor tissues. In one embodiment, the lymphocytes are obtained from tumor tissue, i.e., tumor-infiltrating lymphocytes (TILs). Alternatively, the population of immune cells may be derived from stem cells, such as Peripheral Blood Mononuclear Cells (PBMCs), hematopoietic Stem Cells (HSCs), umbilical cord blood stem cells, and induced pluripotent stem cells (ipscs). Sources suitable for obtaining the desired host cell type will be apparent to those skilled in the art. In some examples, the immune cells may be a mixture of different types of T cells and/or NK cells known in the art. For example, the immune cells can be a population of immune cells isolated from a suitable donor (e.g., a human patient). In a preferred embodiment, the population of immune cells is derived from PBMCs that can be obtained from a patient (e.g., a human patient) in need of the treatment described herein. The desired host cell type (e.g., T cell or NK cell) can be expanded in a population of cells obtained by co-incubating the cells with a stimulatory molecule. As a non-limiting example, anti-CD 3 and anti-CD 28 antibodies may be used to expand T cells.

In further embodiments of the invention, the genetically engineered immune cells comprise a nucleic acid or a set of nucleic acids, which together comprise; a first nucleotide sequence encoding a factor that transfers or redirects glucose metabolites; and a second nucleotide sequence encoding the chimeric receptor polypeptide. In some cases, the factor that redirects the glucose metabolite for introduction into the host cell is the same as the endogenous protein of the host cell. The introduction of additional copies of the coding sequence for the factor redirecting the glucose metabolite into the host cell will increase the expression level of the polypeptide (i.e. overexpression) relative to the native counterpart. In some cases, the factor that redirects the glucose metabolite for introduction into the host cell is heterologous to the host cell, i.e., is not present or expressed in the host cell. Such heterologous factors that redirect glucose metabolites may be naturally occurring proteins that are not expressed in the native host cell (e.g., from a different species, or from a different cell type of the same species). Alternatively, the heterologous factor that redirects glucose metabolites may be a variant of a native protein, as described herein. In some examples, the exogenous (i.e., non-host cell natural) copy of the encoding nucleic acid may exist extrachromosomally. In other examples, the exogenous copy of the coding sequence may be integrated into the chromosome of the host cell and may be located at a site different from the native locus of the endogenous gene.

Such genetically engineered host cells have the ability to increase the rate of glycolysis and may, for example, have the ability to increase the extraction of glucose from the environment. Thus, such genetically engineered host cells may exhibit better growth and/or biological activity under low glucose, low amino acid, low pH, and/or low oxygen conditions, e.g., in a tumor microenvironment.

When expressing a chimeric receptor polypeptide as disclosed herein, the genetically engineered cell can recognize and inhibit the target cell directly (e.g., by an immune cell expressing the CAR or a T cell expressing the TCR) or by an Fc-containing therapeutic agent such as an anti-tumor antibody (e.g., by an immune cell expressing ACTR). Given the high proliferation rates, biological activities and/or survival rates expected of genetically engineered cells such as T cells, NKT and NK cells in low glucose, low amino acids, low pH and/or low oxygen environments (e.g., tumor microenvironment), genetically engineered cells are expected to have higher therapeutic efficacy relative to chimeric receptor polypeptides, T, NKT or NK cells that do not express or express low levels or low active forms of factors that redirect glucose metabolites.

To construct immune cells expressing any of the factors described herein that redirect glucose metabolites, and optionally chimeric receptor polypeptides, stable or transiently expressed expression vectors of the factors that redirect glucose metabolites and/or chimeric receptor polypeptides can be created and introduced into immune host cells by conventional methods as described herein. For example, nucleic acids encoding factors that redirect glucose metabolites and/or chimeric receptor polypeptides can be cloned into one or two suitable expression vectors, such as a viral vector or a non-viral vector operably linked to a suitable promoter. In some cases, each of the coding sequences for the chimeric receptor polypeptide and the factor that redirects the glucose metabolite are located on two separate nucleic acid molecules and can be cloned into two separate vectors that can be introduced into a suitable host cell simultaneously or sequentially. In other embodiments, the coding sequences for the chimeric receptor polypeptide and the factor that redirects the glucose metabolite are on one nucleic acid molecule and can be cloned into one vector. Thus, one embodiment is an immune cell comprising a nucleic acid comprising a first nucleotide sequence and a second nucleotide sequence. The coding sequences for the chimeric receptor polypeptide and the factor that redirects glucose metabolites may be operably linked to two different promoters such that expression of the two polypeptides is controlled by the different promoters. Alternatively, the coding sequences for the chimeric receptor polypeptide and the factor that redirects the glucose metabolite may be operably linked to one promoter, such that expression of both polypeptides is controlled by a single promoter. Suitable sequences may be inserted between the coding sequences of the two polypeptides so that the two isolated polypeptides may be translated from a single mRNA molecule. Such sequences, e.g., IRES or ribosome jump sites, are well known in the art. Thus, one embodiment is that the nucleic acid further comprises a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosome jump site, an Internal Ribosome Entry Site (IRES) or a promoter. Additional description is provided below.

In further embodiments, the nucleic acid or set of nucleic acids is included in one or more viral vectors. The nucleic acid and vector may be contacted with the restriction enzyme under suitable conditions to produce complementary ends on each molecule that can be paired with each other and ligated with the ligase. Alternatively, the synthetic nucleic acid linker may be linked to the end of the nucleic acid encoding the factor that redirects glucose metabolites and/or the chimeric receptor polypeptide. Synthetic linkers may contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector/plasmid/viral vector will depend on the host cell type used to express the factor redirecting the glucose metabolite and/or chimeric receptor polypeptide, but should be suitable for integration and replication in eukaryotic cells.

Various promoters may be used to express factors that redirect the glucose metabolites and/or chimeric receptor polypeptides described herein, including, but not limited to, a Cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as Rous sarcoma (Rous sacoma) virus LTR, HIV-LTR, HTLV-1LTR, a simian virus 40 (SV 40) early promoter, a human EF 1-alpha promoter, or a herpes simplex tk virus promoter. Additional promoters for expressing factors that redirect glucose metabolites and/or chimeric receptor polypeptides include any constitutively active promoter in the immune cell. Alternatively, any regulatable/inducible promoter may be used so that its expression can be regulated within immune cells. Suitable induction systems are known in the art, see for example Kallunki et al (Kallunki et al, cells 8 (8): 796 (2019)).

Alternatively, the carrier may comprise, for example, some or all of the following: selectable marker genes such as neomycin gene or kanamycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences for immediate early genes from human CMV for high level transcription; an intron sequence from the human EF 1-alpha gene; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 or polyomavirus origin of replication and ColE1 for appropriate episomal replication; internal ribosome binding sites (IRES), universal multiple cloning sites; t7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNAs; a "suicide switch" or "suicide gene" that when triggered results in vector-carrying cell death (e.g., HSV thymidine kinase or an inducible caspase, such as iCasp 9); and a reporter gene for assessing expression of a factor that redirects glucose metabolites and/or chimeric receptor polypeptides.

In a specific embodiment, such a vector further comprises a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes death of cells expressing the suicide gene. Suicide genes may be genes that confer sensitivity to an agent, e.g., a drug, on a cell expressing the gene and cause cell death when the cell is contacted or exposed to the agent. Suicide genes are known in the art (see, e.g., springer, C.J., suicide gene therapy: methods and reviews (Suicide GENE THERAPY: methods AND REVIEWS), hu Mana Press (Humana Press) (2004)) and comprise, e.g., the Herpes Simplex Virus (HSV) Thymidine Kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8.

Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of preparing vectors for expressing factors that redirect glucose metabolites and/or chimeric receptor polypeptides can be found, for example, in US 2014/0106449, which is incorporated herein by reference in its entirety.

Any vector including a vector encoding a nucleic acid sequence of a factor and/or chimeric receptor polypeptide described herein that redirects glucose metabolites is also within the scope of the present disclosure. Such vectors or sequences encoding factors that redirect glucose metabolites and/or chimeric receptor polypeptides contained therein may be delivered to host cells, such as host immune cells, by any suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection with agents such as liposomes, or viral transduction (e.g., retroviral transduction, such as lentiviral transduction).

In some embodiments, vectors for expressing factors that redirect glucose metabolites and/or chimeric receptor polypeptides are delivered to host cells by viral transduction (e.g., retroviral transduction, such as lentiviral or gamma-retroviral transduction). Exemplary viral delivery methods include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; and WO 91/02805;US 5,219,740 and US 4,777,127;GB 2,200,651; and EP 0345242), alphavirus-based vectors and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984; and WO 95/00655). In some embodiments, the vector for expressing the factor that redirects glucose metabolites and/or chimeric receptor polypeptide is a retrovirus. In some embodiments, the vector for expressing the factor that redirects glucose metabolites and/or chimeric receptor polypeptide is a lentivirus.

Examples of references describing retroviral transduction include: anderson et al, U.S. Pat. No. 5,399,346; (Mann et al, cells, 33 (1): 153-159 (1983)); US 4,650,764; US 4,980,289; (Markowitz et al J Virol, 62 (4): 1120-1124 (1988)); US 5,124,263; WO 95/07358 and (Kuo et al, blood 82 (3): 845-852 (1993)). WO 95/07358 describes efficient transduction of primary B lymphocytes. See also WO 2016/040441A1, all of which are incorporated herein by reference for the purposes and subject matter of the references cited herein.

In examples where a vector encoding a factor that redirects glucose metabolites and/or a chimeric receptor polypeptide is introduced into a host cell using a viral vector, viral particles capable of infecting immune cells and carrying the vector may be produced by any method known in the art and can be found, for example, in WO 91/02805A2, WO 98/09271A1 and US 6,194,191. Viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles with immune cells.

In some embodiments, RNA molecules encoding factors that redirect glucose metabolites and/or any of the chimeric receptor polypeptides and/or chimeric receptor polypeptides as described herein can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into a suitable host cell, such as the host cells described herein, by known methods, such as Rabinovich, komarovskaya et al (Human gene therapy (Human GENE THERAPY), 17 (10): 1027-1035 (2006)).

In some cases, the nucleic acid encoding the factor that redirects glucose metabolites and the nucleic acid encoding the appropriate chimeric receptor polypeptide may be cloned into separate expression vectors, which may be introduced into an appropriate host cell simultaneously or sequentially. For example, an expression vector (or RNA molecule) for expressing a factor that redirects glucose metabolites may first be introduced into a host cell, and transfected host cells expressing a factor that redirects glucose metabolites may be isolated and cultured in vitro. An expression vector (or RNA molecule) for expression of a suitable chimeric receptor polypeptide can then be introduced into a host cell expressing a factor that redirects glucose metabolites, and transfected cells expressing both polypeptides can be isolated. In another example, an expression vector (or RNA molecule) each for expressing a factor redirecting glucose metabolites and a chimeric receptor polypeptide may be introduced simultaneously into a host cell, and transfected host cells expressing both polypeptides may be isolated by conventional methods.

In other cases, the nucleic acid encoding the factor that redirects glucose metabolites and the nucleic acid encoding the chimeric receptor polypeptide may be cloned into the same expression vector. Polynucleotides for expressing chimeric receptor polypeptides and factors that redirect glucose metabolites (including vectors in which such polynucleotides are operably linked to at least one regulatory element) are also within the scope of the present disclosure. Non-limiting examples of useful vectors of the present disclosure include viral vectors, such as retroviral vectors including gamma retroviral vectors and lentiviral vectors, and adeno-associated viral vectors (AAV vectors).

In some cases, nucleic acids encoding factors that redirect glucose metabolites and/or chimeric receptor polypeptides can be delivered into host cells via a transposon (e.g., piggybac). In some cases, the encoding nucleic acid may be delivered into the host cell by gene editing, e.g., by CRISPR, TALEN, zinc Finger Nuclease (ZFN), or meganuclease.

In some cases, a nucleic acid described herein can include two coding sequences, one encoding a chimeric receptor polypeptide as described herein, and the other encoding a polypeptide capable of redirecting glucose out of the glycolytic pathway (i.e., a factor that redirects glucose metabolites). In some cases, recombinant TCRs comprising TCR chains (e.g., α and β TCR chains) are isolated by self-cleaving 2A peptides, such as P2A or T2A. A nucleic acid comprising two coding sequences described herein may be configured such that the polypeptides encoded by the two coding sequences may be expressed as independent (and physically separated) polypeptides. To achieve this, the nucleic acids described herein may contain a third nucleotide sequence located between the first coding sequence and the second coding sequence. The third nucleotide sequence may, for example, encode a ribosome jump site. Ribosome jump sites are sequences that impair normal peptide bond formation. This mechanism results in translation of additional open reading frames from one messenger RNA. The third nucleotide sequence may, for example, encode a P2A, T A or F2A peptide (see, for example, kim, lee et al, public science library complex (PLoS One), 6 (4): e18556 (2011)). See table 16 below.

Table 16: exemplary ribosome-jumping peptides

Ribosome jump site	Sequence(s)	SEQ ID NO
			P2A	ATNFSLLKQAGDVEENPGP	SEQ ID NO:67
T2A	EGRGSLLTCGDVEENPGP	SEQ ID NO:82
			E2A	QCTNYALLKLAGDVESNPGP	SEQ ID NO:83
F2A	AVKQTLNFDLLKLAGDVESNPGP	SEQ ID NO:84

In another embodiment, the third nucleotide sequence may encode an Internal Ribosome Entry Site (IRES). IRES is an RNA element that allows translation to be initiated in a terminal independent manner, as well as allowing translation of additional open reading frames from one messenger RNA. Alternatively, the third nucleotide sequence may encode a promoter that controls expression of the second polypeptide. The third nucleotide sequence may also encode more than one ribosome jump sequence, IRES sequence, additional promoter sequence or a combination thereof.

The nucleic acid may also comprise additional coding sequences (including but not limited to fourth and fifth coding sequences) and may be configured such that the polypeptides encoded by the additional coding sequences are expressed as further independent and physically separated polypeptides. To this end, the further coding sequence may be separated from the further coding sequence by one or more nucleotide sequences encoding one or more ribosome jump sequences, IRES sequences or a further promoter sequence.

An exemplary IRES sequence is provided as follows (SEQ ID NO: 85):

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In some examples, a nucleic acid (e.g., an expression vector or RNA molecule as described herein) can include coding sequences for both a factor that redirects glucose metabolites (e.g., a factor described herein) and a suitable chimeric receptor polypeptide, separated in any order by a third nucleotide sequence (e.g., SEQ ID NO: 67) encoding a P2A peptide. Thus, two isolated polypeptides, i.e., a factor that redirects glucose metabolites and a chimeric receptor, can be produced from such nucleic acids, wherein the P2A portion SEQ ID NO 67 is linked to an upstream polypeptide (encoded by an upstream coding sequence) and residue P from the P2A peptide is linked to a downstream polypeptide (encoded by a downstream coding sequence). In some examples, the chimeric receptor polypeptide is an upstream polypeptide and the factor that redirects glucose metabolites is a downstream factor. In other examples, the factor that redirects glucose metabolites is an upstream factor and the chimeric receptor polypeptide is a downstream polypeptide. In some embodiments, a nucleic acid (e.g., an expression vector or RNA molecule as described herein) can include a factor that redirects glucose metabolites (e.g., a factor described herein) and a coding sequence for both a suitable TCR, ACTR, or CAR polypeptide, separated in any order by a third nucleotide sequence encoding a P2A peptide (e.g., SEQ ID NO: 67). Thus, two isolated polypeptides, a factor that redirects glucose metabolites and a TCR, ACTR or CAR, can be produced from such nucleic acids, wherein the P2A portion SEQ ID NO:67 is linked to an upstream polypeptide (encoded by an upstream coding sequence) and residue P from the P2A peptide is linked to a downstream polypeptide (encoded by a downstream coding sequence). In some embodiments, the TCR, ACTR, or CAR polypeptide is an upstream polypeptide, and the factor that redirects glucose metabolites is a downstream factor. In other embodiments, the factor that redirects glucose metabolites is an upstream factor and the TCR, ACTR, or CAR polypeptide is a downstream polypeptide.

In some examples, the nucleic acids described above may further encode a linker (e.g., a GSG linker) between two segments of the encoded sequence, such as between the upstream polypeptide and the P2A peptide.

In a specific example, the nucleic acids described herein are configured such that two isolated polypeptides are expressed in a nucleic acid transfected host cell: (i) A first polypeptide comprising, from N-terminus to C-terminus, a suitable CAR (e.g., as set forth in tables 11-14 or SEQ ID NO:78-SEQ ID NO: 79), a peptide linker (e.g., a GSG linker), and a ATNFSLLKQAGDVEENPG (SEQ ID NO: 67) segment derived from a P2A peptide; and (ii) a second polypeptide comprising, from N-terminus to C-terminus, a P residue derived from a P2A peptide and a factor that redirects glucose metabolites (e.g., any of the following SEQ ID NOs: 68-74).

In a specific example, the nucleic acids described herein are configured such that two isolated polypeptides are expressed in a nucleic acid transfected host cell: (i) A first polypeptide comprising, from N-terminus to C-terminus, a suitable ACTR (see table 7), a peptide linker (e.g., GSG linker), and a ATNFSLLKQAGDVEENPG (SEQ ID NO: 67) segment derived from a P2A peptide; and (ii) a second polypeptide comprising, from N-terminus to C-terminus, a P residue derived from a P2A peptide and a factor that redirects glucose metabolites (e.g., any of the following SEQ ID NOs: 68-SEQ ID NO: 74). In some cases, additional polypeptides of interest may also be introduced into the host immune cell.

After introducing into a host cell a nucleic acid (e.g., an RNA molecule) encoding any of the factors redirecting glucose metabolites and/or chimeric receptor polypeptides and/or vectors encoding chimeric receptor polypeptides provided herein or encoding chimeric receptor polypeptides and/or factors redirecting glucose metabolites, the cell can be cultured under conditions that allow for expression of the factors redirecting glucose metabolites and/or chimeric receptor polypeptides. In embodiments in which the nucleic acid encoding the factor that redirects glucose metabolites and/or chimeric receptor polypeptide is regulated by a regulatable promoter, the host cell may be cultured under conditions in which the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter and the immune cells are cultured in the presence of the inducing molecule or under conditions that produce the inducing molecule. It will be apparent to those skilled in the art that determining whether a factor that redirects glucose metabolites and/or a chimeric receptor polypeptide is expressed or not may be assessed by any known method, for example, by quantitative reverse transcriptase PCR (qRT-PCR) detection of the factor that redirects glucose metabolites and/or mRNA encoding the chimeric receptor polypeptide or by methods including western blotting, fluorescence microscopy and flow cytometry detection of the factor that redirects glucose metabolites and/or chimeric receptor polypeptide protein.

Alternatively, expression of the chimeric receptor polypeptide can be performed in vivo after administration of the immune cells to a subject. As used herein, the term "subject" refers to any mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. For example, the subject may be a primate. In a preferred embodiment, the subject is a human.

Alternatively, redirecting expression of a factor and/or chimeric receptor polypeptide of a glucose metabolite in any of the immune cells disclosed herein can be achieved by introducing an RNA molecule encoding the factor and/or chimeric receptor polypeptide that redirects the glucose metabolite. Such RNA molecules may be prepared by in vitro transcription or chemical synthesis. The RNA molecule can then be introduced into a suitable host cell, such as an immune cell (e.g., T or NK cell), by, for example, electroporation. For example, RNA molecules can be synthesized and introduced into host immune cells according to the methods described in the following documents: rabinovich, komarovskaya et al (human Gene therapy 17 (10): 1027-1035 (2006)) and WO 2013/040557.

In certain embodiments, vectors or RNA molecules comprising factors that redirect glucose metabolites and/or chimeric receptor polypeptides may be introduced into host cells or immune cells in vivo. As a non-limiting example, this can be accomplished by directly administering to a subject (e.g., by intravenous administration) a vector or RNA molecule encoding one or more factors that redirect glucose metabolites and/or one or more chimeric receptor polypeptides described herein, thereby producing host cells comprising the factor that redirect glucose metabolites and/or chimeric receptor polypeptides in vivo.

The method for preparing a host cell expressing any of the factors and/or chimeric receptor polypeptides described herein that redirect glucose metabolites may further comprise activating the host cell ex vivo. Activating a host cell means stimulating the host cell into an activated state in which the cell may be able to perform effector functions. The method of activating the host cell will depend on the type of host cell used to express the factor that redirects the glucose metabolite and/or the chimeric receptor polypeptide. For example, T cells may be activated ex vivo in the presence of one or more molecules, including but not limited to: anti-CD 3 antibodies, anti-CD 28 antibodies, IL-2, phytohemagglutinin, engineered artificially stimulated cells or particles, or a combination thereof. The engineered artificial stimulatory cells may be artificial antigen presenting cells known in the art. See, for example, neal, bailey et al (journal of immunology research (J Immunol Res Ther) 2 (1): 68-79 (2017)), and Turtle and Riddell (journal of Cancer (Sudbury, mass.), 16 (4): 374-381 (2010)), the relevant disclosures of each of which are incorporated herein by reference for the purposes and subject matter recited herein.

In other examples, NK cells may be activated ex vivo in the presence of one or more molecules such as 4-1BB ligand, anti-4-1 BB antibody, IL-2, IL-15, anti-IL-15 receptor antibody, IL12, IL-21, K562 cells and/or engineered artificially stimulated cells or particles. In some embodiments, the host cell expressing any of the factors that redirect glucose metabolites and/or chimeric receptor polypeptides described herein (ACTR-/CAR-/TCR-and/or factors that redirect cells expressing glucose metabolites) is activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be apparent to those skilled in the art and may comprise assessing the expression of one or more cell surface markers associated with cell activation, expression or secretion of cytokines, and cell morphology.

Methods for preparing host cells expressing any of the factors and/or chimeric receptor polypeptides described herein that redirect glucose metabolites may include ex vivo expansion of the host cells. Expansion of the host cell may involve any method that causes an increase in the number of cells expressing the factor that redirects the glucose metabolite and/or the chimeric receptor polypeptide, e.g., allowing the host cell to proliferate or stimulating the host cell to proliferate. The method used to stimulate host cell expansion will depend on the type of host cell used to express the factor that redirects the glucose metabolite and/or the chimeric receptor polypeptide, and will be apparent to those skilled in the art. In some embodiments, the host cells expressing the factor that redirects glucose metabolites and/or any factor in the chimeric receptor polypeptides and/or chimeric receptor polypeptides described herein are amplified ex vivo prior to administration to a subject.

In some embodiments, the host cells expressing the factor that redirects glucose metabolites and/or chimeric receptor polypeptide are expanded and activated ex vivo prior to administration of the cells to a subject. Host cell activation and amplification may be used to allow integration of the viral vector into the genome and expression of genes encoding factors that redirect glucose metabolites and/or chimeric receptor polypeptides as described herein. If mRNA electroporation is used, activation and/or expansion may not be required, although electroporation on activated cells may be more efficient. In some cases, the factor that redirects glucose metabolites and/or chimeric receptor polypeptide is transiently expressed in a suitable host cell (e.g., for 3-5 days). Transient expression may be advantageous if there is potential toxicity, and should aid in the initial stages of clinical testing for possible side effects. Any of the host cells expressing the factor redirecting the glucose metabolite and/or chimeric receptor polypeptide may be admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure. Thus, a pharmaceutical composition comprising the genetically engineered immune cells of the invention is another embodiment. The genetically engineered immune cells are preferably admixed with a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable" as used in connection with the compositions of the present disclosure refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not normally produce adverse reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. By "acceptable" is meant that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acid, vector, cell, or therapeutic antibody) and does not adversely affect the subject to whom the composition is administered. Any of the pharmaceutical compositions used in the methods of the invention may include a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized form or an aqueous solution.

Pharmaceutically acceptable carriers comprising buffers are well known in the art and may include phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; a preservative; a low molecular weight polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; amino acids; a hydrophobic polymer; a monosaccharide; disaccharides; and other carbohydrates; a metal complex; and/or nonionic surfactants. See, for example, ramington: pharmaceutical science and practice (Remington: THE SCIENCE AND PRACTICE of Pharmacy), 20 th edition (2000) LiPink, williams and Wilkins Inc. (Lippincott WILLIAMS AND WILKINS), K.E.Hoover editions.

The pharmaceutical compositions of the present disclosure may also contain one or more active compounds as desired for the particular indication being treated, preferably active compounds having complementary activities that do not adversely affect each other. Non-limiting examples of possible additional active compounds include, for example, IL-2 and various agents known in the art and listed in the discussion of combination therapies below.

Immunotherapy using genetically engineered hematopoietic cells described herein

The genetically engineered hematopoietic cells (e.g., hematopoietic stem cells, immune cells, such as NK cells or T cells) disclosed herein can be used in immunotherapy against a variety of conditions, such as cancer, infectious diseases, and autoimmune diseases. Thus, another embodiment of the invention is a method for inhibiting a cell expressing a target antigen in a subject, the method comprising administering to a subject in need thereof a population of genetically engineered immune cells as set forth herein or a pharmaceutical composition as set forth herein comprising the population of genetically engineered immune cells.

A. Combination immunotherapy of genetically engineered hematopoietic cells expressing ACTR polypeptides and Fc-containing therapeutics

Exemplary ACTR polypeptides of the disclosure confer T lymphocyte antibody-dependent cellular cytotoxicity (ADCC) capability and enhance ADCC in NK cells. The receptor, when bound by an antibody that binds to a cell, triggers T cell activation, sustained proliferation, and specific cytotoxicity to the bound cell.

The extent of affinity of CD16 for the Fc portion of Ig is a key determinant of ADCC and, thus, of the clinical response to antibody immunotherapy. CD16 with the V158 polymorphism, which has higher binding affinity for Ig and mediates superior ADCC, was selected as an example relative to CD16 with the F158 polymorphism. Although the F158 receptor is less potent at inducing T cell proliferation and ADCC than the V158 receptor, the F158 receptor may have lower in vivo toxicity than the V158 receptor, which makes it useful in some clinical situations.

Redirecting glucose metabolites to co-express factors with ACTR polypeptides in immune cells will facilitate cell-based immunotherapy, such as T cell therapy or NK cell therapy, by allowing cells to effectively grow and/or function in low glucose, low amino acid, low pH, and/or low oxygen environments. Antibody-directed cytotoxicity can be stopped as long as needed, simply by stopping antibody administration. Clinical safety may be further enhanced by using mRNA electroporation to express factors that transiently redirect glucose metabolites and/or ACTR polypeptides to limit any potential autoimmune reactivity.

Thus, in one embodiment, the present disclosure provides a method for enhancing the efficacy of antibody-based immunotherapy of cancer in a subject in need thereof, the subject being treated with an Fc-containing therapeutic agent, such as a therapeutic antibody, that can bind to cells expressing an antigen. The Fc-containing therapeutic agent contains an Fc moiety, such as a human or humanized Fc moiety, that can be recognized and bound by an Fc binding portion of ACTR expressed on an engineered immune cell (e.g., the extracellular domain of human CD 16A). Exemplary ACTR constructs are provided in table 10 above.

The methods described herein can include introducing into a subject a therapeutically effective amount of an antibody and a therapeutically effective amount of a genetically engineered host cell, such as a hematopoietic cell, e.g., an immune cell (e.g., T or NK cell) that co-expresses a factor of the disclosure that redirects glucose metabolites and an ACTR polypeptide. A subject (e.g., a human patient, such as a human cancer patient) has been or is being treated with an Fc-containing therapeutic agent specific for a target antigen. The target antigen may be any molecule associated with a disease or condition, including but not limited to a tumor antigen, a pathogenic antigen (e.g., a bacterium, fungus, or virus), or an antigen present on a diseased cell, as described herein.

In the context of the present disclosure, the terms "treatment", "treatment" and the like, in relation to any other disease condition described herein, mean alleviating or alleviating at least one symptom associated with such condition, or slowing or reversing the progression of such condition. Within the meaning of the present disclosure, the term "treatment" also means preventing, delaying the onset of the disease (i.e. the period of time prior to the clinical manifestation of the disease) and/or reducing the risk of disease progression or disease exacerbation. For example, the term "treatment" in connection with cancer may mean elimination or alleviation of the tumor burden of a patient, or prevention, delay or inhibition of metastasis, etc.

As used herein, the term "therapeutically effective" as used in a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients (e.g., a first pharmaceutical composition comprising an antibody and a second pharmaceutical composition comprising a population of T or NK cells expressing factors that redirect glucose metabolites and/or antibody-coupled T cell receptor (ACTR) constructs) is administered, an effective amount of the combination may or may not comprise an amount of each ingredient that is effective when administered alone. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a compound or pharmaceutical composition sufficient to delay the manifestation of, prevent the progression of, slow or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

Host cells (e.g., hematopoietic cells, e.g., immune cells such as T and NK cells) expressing the factors that redirect glucose metabolites and ACTR or CAR polypeptides described herein can be used to enhance ADCC and/or enhance the efficacy of antibody-based immunotherapy and/or enhance growth and/or proliferation of immune cells in a low glucose environment in a subject. In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has been treated or is being treated with any of the therapeutic antibodies described herein.

To practice the methods described herein, an effective amount of host cells, e.g., immune cells (e.g., NK cells and/or T lymphocytes) expressing any of the factors that redirect glucose metabolites and ACTR polypeptides described herein and ACTR polypeptides, and an effective amount of antibodies or a composition thereof, may be administered to a subject in need of treatment by a suitable route, e.g., intravenous administration. As used herein, an effective amount refers to the amount of the corresponding agent (e.g., NK cells and/or T lymphocytes expressing factors that redirect glucose metabolites, ACTR polypeptides, antibodies, or a combination thereof) that imparts a therapeutic effect to a subject upon administration. It will be apparent to those skilled in the art whether the amount of cells or compositions described herein achieves a therapeutic effect. As will be appreciated by those of skill in the art, the effective amount will vary depending upon the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, sex (gender/sex) and weight, the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of a health practitioner. In some embodiments, the effective amount alleviates, slows, alleviates, ameliorates, reduces symptoms of or delays progression of any disease or disorder in the subject. In some embodiments, the subject in need of treatment is a human. In some embodiments, the subject in need of treatment is a human cancer patient. In some embodiments, a subject in need of treatment has one or more pathogenic infections (e.g., viral, bacterial, and/or fungal infections).

The methods of the present disclosure may be used to treat any cancer or any pathogen. Specific non-limiting examples of cancers that can be treated by the methods of the present disclosure include, for example, lymphomas, breast cancer, gastric cancer, neuroblastomas, osteosarcomas, lung cancer, skin cancer, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastomas, gliomas, glioblastomas, thyroid cancer, hepatocellular carcinoma, esophageal cancer, and cervical cancer. In certain embodiments, the cancer may be a solid tumor.

The methods of the present disclosure may also be used to treat infectious diseases that may be caused by bacterial, viral, or fungal infections. In such cases, the genetically engineered immune cells can be used with an Fc-containing therapeutic agent (e.g., an antibody) that targets a pathogenic antigen (e.g., an antigen associated with the bacteria, virus, or fungus that caused the infection). Specific non-limiting examples of pathogenic antigens include, but are not limited to, bacterial, viral, and/or fungal antigens. Some examples are provided below: influenza virus neuraminidase, hemagglutinin or M2 protein, human Respiratory Syncytial Virus (RSV) F glycoprotein or G glycoprotein, herpes simplex virus glycoprotein gB, gC, gD or gE, chlamydia MOMP or PorB protein, dengue virus core protein, matrix protein or glycoprotein E, measles virus hemagglutinin, herpes simplex virus type 2 glycoprotein gB, poliovirus I VP1, HIV 1 envelope glycoprotein, hepatitis b core antigen or surface antigen, diphtheria toxin, streptococcus 24M epitope, gonococcal pilin protein, pseudorabies virus G50 (gpD), pseudorabies virus II (gpB), pseudorabies virus III (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein or human hepatitis c virus glycoprotein E1 or E2.

In some embodiments, the immune cells are administered to the subject in an amount effective to enhance ADCC activity by at least 20% and/or at least 2-fold, e.g., 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more.

The immune cells are co-administered with an Fc-containing therapeutic agent, such as a therapeutic antibody, in order to target cells expressing an antigen to which the Fc-containing therapeutic agent binds. In some embodiments, more than one Fc-containing therapeutic agent, such as more than one antibody, may be used in conjunction with immune cells. Antibody-based immunotherapy may be used to treat, alleviate or mitigate the symptoms of any disease or disorder for which immunotherapy is deemed useful in a subject.

Antibodies (used interchangeably in plural form) are immunoglobulin molecules capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses not only intact (i.e., full length) polyclonal or monoclonal antibodies, but also antigen binding fragments thereof including the Fc region, fusion proteins including antibody portions, humanized antibodies, chimeric antibodies, diabodies, single domain antibodies (e.g., nanobodies), linear antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule that includes the antigen-recognition site of the desired specificity and the Fc region, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. Antibodies comprise antibodies of any class, such as IgD, igE, igG, igA or IgM (or subclasses thereof), and the antibodies need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the amino acid sequence of the antibody in its heavy chain constant domain. There are five main classes of immunoglobulins: igA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), for example, igG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Antibodies for use in the present disclosure contain an Fc region that can be recognized by commonly used ACTR-and/or factors that redirect immune cells expressing glucose metabolites. The Fc region may be a human or humanized Fc region.

Any of the antibodies described herein may be monoclonal or polyclonal. "monoclonal antibody" refers to a homogeneous antibody population, and "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the source of the antibody or the manner in which the antibody is made.

In one example, the antibody used in the methods described herein is a humanized antibody. Humanized antibodies are forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains or antigen binding fragments thereof containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not found in either the recipient antibody or the introduced CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have a modified Fc region as described in WO 99/58372. Antibodies as used herein may be glycosylated (e.g., fucosylated) or defucosylated. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) altered with respect to the original antibody, also referred to as "derived from" one or more CDRs from the original antibody. Humanized antibodies may also be involved in affinity maturation.

In another example, the antibodies described herein are chimeric antibodies, which may comprise heavy constant regions and light constant regions from a human antibody. Chimeric antibodies refer to antibodies having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies derived from one species of mammal (e.g., non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications may be made in the variable and/or constant regions.

Hematopoietic cells, e.g., immune cells (e.g., T and/or NK cells) or HSCs expressing any of the factors that redirect glucose metabolites and/or ACTR polypeptides disclosed herein, and/or ACTR polypeptides, can be administered to a subject that has been treated or is being treated with an Fc-containing antibody. For example, immune cells may be administered to a human subject concurrently with antibodies. Alternatively, immune cells may be administered to a human subject during antibody-based immunotherapy. In some embodiments, immune cells and antibodies can be administered to a human subject at least 4 hours apart, e.g., at least 12 hours apart, at least 1 day apart, at least 3 days apart, at least one week apart, at least two weeks apart, or at least one month apart.

In some embodiments, an antibody described herein specifically binds to a corresponding target antigen or epitope thereof. Antibodies that "specifically bind" to an antigen or epitope are terms well known in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, longer in duration, and/or with greater affinity to a particular target antigen than it reacts to an alternative target. An antibody "specifically binds" to a target antigen or epitope if it binds more strongly, more avidly, more easily, and/or for a longer period of time than it does with other substances. For example, an antibody that specifically (or preferentially) binds to an antigen or an epitope therein is an antibody that: the antibodies bind to such target antigens with greater affinity, higher avidity, easier and/or longer duration than other antigens or other epitopes in the same antigen. It will also be appreciated by this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically bind to or preferentially bind to a second target antigen. As such, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. In some examples, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes in the same antigen.

In some embodiments, an antibody as described herein has suitable binding affinity for a target antigen (e.g., any of the targets described herein) or an epitope thereof. Antibodies for use in the methods of immunotherapy described herein can bind (e.g., specifically bind) to a target antigen of interest or a specific region or epitope therein. Table 4 above lists exemplary target antigens of interest and exemplary antibodies specific thereto.

B. Immunotherapy of genetically engineered hematopoietic cells expressing CAR polypeptides

The genetically engineered hematopoietic cells described herein (e.g., hematopoietic stem cells, immune cells, such as T or NK cells) that co-express a factor that redirects glucose metabolites and a CAR polypeptide can be used in immunotherapy, such as T cell therapy (both αβ and γδ T cells) or NK cell therapy, to inhibit diseased cells expressing an antigen targeted by the CAR polypeptide directly or indirectly (e.g., by a tag-conjugated therapeutic agent that binds to the CAR polypeptide). By allowing the cells to effectively grow and/or function in low glucose, low amino acid, low pH, and/or low oxygen environments, such as tumor microenvironments, factors that redirect glucose metabolites co-expressed with CAR polypeptides in immune cells will facilitate cell-based immunotherapy. Clinical safety may be further enhanced by using mRNA electroporation to express factors and/or CAR polypeptides that transiently redirect glucose metabolites to limit any potential non-tumor specific reactivity.

The methods described herein can include introducing into a subject a therapeutically effective amount of a genetically engineered host cell, such as a hematopoietic cell, e.g., an immune cell (e.g., an αβt, γδt, or NK cell) that co-expresses a factor of the disclosure that redirects glucose metabolites and a CAR polypeptide (see illustrative examples in tables 11-14). A subject (e.g., a human patient, such as a human cancer patient) may additionally have been treated or be treated with an anti-cancer or anti-infective therapy, including but not limited to an anti-cancer therapeutic or an anti-infective agent. The CAR has an antigen binding domain that can bind to any target antigen. Such target antigens may be any molecule associated with a disease or condition, including but not limited to tumor antigens, pathogenic antigens (e.g., bacteria, fungi, or viruses), or antigens present on diseased cells, as described herein. In some embodiments, the target antigen binding domain targets a native tumor antigen protein. In other embodiments, the target antigen binding domain targets a variant (e.g., a mutation) of the tumor antigen protein. Some examples include EGFRVIII SCFV recognizing tumor-specific variants of EGFR (Wang, jiang et al, report on cancer 472:175-180 (2020)).

Host cells (e.g., hematopoietic cells, e.g., immune cells such as T and NK cells) expressing the factors and CAR polypeptides of the redirected glucose metabolites described herein can be used to inhibit cells expressing a target antigen and/or to enhance growth and/or proliferation of immune cells in a low glucose environment, a low amino acid environment, a low pH environment, and/or a low oxygen environment, e.g., a tumor microenvironment. In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has additionally been treated or is being treated with any of the therapeutic antibodies described herein.

To practice the methods described herein, an effective amount of hematopoietic cells, e.g., immune cells (NK and/or T cells) or a composition thereof expressing any of the factors that redirect glucose metabolites and CAR polypeptides described herein and CAR polypeptides, can be administered to a subject in need of treatment by a suitable route, such as intravenous, subcutaneous, and intradermal administration. As used herein, an effective amount refers to the amount of the corresponding agent (e.g., NK and/or T cells expressing a factor that redirects glucose metabolites, CAR polypeptide, or a composition thereof) that confers a therapeutic effect on a subject upon administration. It will be apparent to those skilled in the art whether the amount of cells or compositions described herein achieves a therapeutic effect. As will be appreciated by those of skill in the art, the effective amount will vary depending upon the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, sex (gender/sex) and weight, the duration of the treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of a health practitioner. In some embodiments, the effective amount alleviates, slows, alleviates, ameliorates, reduces symptoms of or delays progression of any disease or disorder in the subject. In some embodiments, the subject in need of treatment is a human. In some embodiments, the subject in need of treatment is a human cancer patient. In some embodiments, a subject in need of treatment has one or more pathogenic infections (e.g., viral, bacterial, and/or fungal infections).

The methods of the present disclosure may be used to treat any cancer or any pathogen. Specific non-limiting examples of cancers that can be treated by the methods of the present disclosure include, for example, lymphomas, breast cancer, gastric cancer, neuroblastomas, osteosarcomas, lung cancer, skin cancer, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastomas, gliomas, glioblastomas, thyroid cancer, hepatocellular carcinoma, esophageal cancer, and cervical cancer. In certain embodiments, the cancer may be a solid tumor. In certain embodiments, the cancer may be a liquid tumor. Accordingly, a preferred embodiment is a method for inhibiting cells expressing a target antigen in a subject, wherein the subject is a human patient suffering from cancer and the target antigen is a tumor antigen; wherein the cancer is selected from the group consisting of: carcinoma, lymphoma, sarcoma, blastoma, and leukemia, preferably wherein said cancer is selected from the group consisting of: cancers of B cell origin, breast cancer, stomach cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer; or the B cell derived cancer is selected from the group consisting of: acute lymphoblastic leukemia of B lineage, chronic lymphoblastic leukemia of B cells, and non-hodgkin's lymphoma of B cells.

The methods of the present disclosure may also be used to treat infectious diseases that may be caused by bacterial, viral, or fungal infections. In such cases, genetically engineered immune cells expressing a CAR polypeptide specific for a pathogenic antigen (e.g., an antigen associated with the bacteria, virus, or fungus that caused the infection) can be used to eliminate the infected cell. Specific non-limiting examples of pathogenic antigens include, but are not limited to, bacterial, viral, and/or fungal antigens.

In some embodiments, the immune cells are administered to the subject in an amount effective to inhibit at least 20% and/or at least 2-fold of the cells expressing the target antigen, e.g., 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more of the cells expressing the target antigen.

Additional therapeutic agents (e.g., antibody-based immunotherapeutic agents) may be used to treat, alleviate or mitigate the symptoms of any disease or disorder for which the therapeutic agent is deemed useful to the subject.

The efficacy of cell-based immunotherapy as described herein may be assessed by any method known in the art and will be apparent to the skilled medical practitioner. For example, the efficacy of a cell-based immunotherapy may be assessed by the survival rate of the subject or the tumor or cancer burden in the subject or a tissue or sample thereof. In some embodiments, the immune cells are administered to a subject in need of treatment in an amount effective to enhance the efficacy of the cell-based immunotherapy by at least 20% and/or at least 2-fold, e.g., enhancing the efficacy of the antibody-based immunotherapy by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, as compared to the efficacy in the absence of immune cells that express the factor that redirects glucose metabolites and/or the CAR polypeptide.

In any of the compositions or methods described herein, the immune cells (e.g., NK and/or T cells) can be autologous to the subject, i.e., the immune cells can be obtained from a subject in need of treatment, genetically engineered to express a factor that redirects glucose metabolites and/or a CAR polypeptide, and then administered to the same subject. In a specific embodiment, the autoimmune cells (e.g., T or NK cells) are activated and/or expanded ex vivo prior to reintroduction into the subject. Administration of autologous cells to a subject may result in reduced rejection of host cells compared to administration of non-autologous cells.

Alternatively, the host cell is an allogeneic cell, i.e., the cell is obtained from a first subject, genetically engineered to express a factor that redirects glucose metabolites and/or a CAR polypeptide, and administered to a second subject that is different from the first subject but that belongs to the same species. For example, the allogeneic immune cells may be derived from a human donor and administered to a human recipient different from the donor. In particular embodiments, the T cell is an allogeneic T cell in which expression of the endogenous T cell receptor is inhibited or eliminated. In a specific embodiment, the allogeneic T cells are activated and/or expanded ex vivo prior to introduction into the subject. T lymphocytes may be activated by any method known in the art, for example, in the presence of anti-CD 3/CD28, IL-2, IL-15, phytohemagglutinin, engineered artificially stimulated cells or particles, or a combination thereof. In certain aspects, the starting population of NK or T cells is obtained from isolated monocytes using a ficoll-paque density gradient. In some aspects, the method further comprises depleting monocytes of CD3, CD14 and/or CD19 cells to obtain the starting population of NK cells. In some aspects, the method further comprises depleting monocytes CD3, CD14 and CD19 cells to obtain the starting population of NK cells. In a particular aspect, the depleting includes performing magnetic sorting. In other aspects, NK cells can be positively selected using sorting, magnetic bead selection, or other methods to obtain an initial population of NK cells.

Additionally, immune cells such as NK cells are derived from cord blood stem cells or Induced Pluripotent Stem Cells (iPSCs), and immunotherapy is provided from "off-the-shelf" sources (Li et al, cell Stem cells (CELL STEM CELL), 23 (2): 181-192.e185 (2018); liu et al, leukemia (32) (2): 520-531 (2018); morgan et al, front of immunology, 11:1965 (2020), wrona, borowiec et al, international molecular sciences, 22 (11): 2021)). In particular embodiments, the starting population of NK cells is obtained from cord blood. In other embodiments, the cord blood has been previously frozen. In some embodiments, the cells are derived from a cell line (e.g., NK-92 and Vγ9Vδ2T cells).

NK and T cells (αβt or γδ T cells) can be activated by any method known in the art, for example in the presence of one or more agents selected from the group consisting of: CD137 ligand proteins, CD137 antibodies, IL-15 receptor antibodies, IL-2, IL-12, IL-21, and cells from the K562 cell line and/or engineered artificially stimulated cells or particles. For a description of useful methods for expanding NK cells see, e.g., US 7,435,596 and US 8,026,097. For example, NK cells used in the compositions or methods disclosed herein can be preferentially expanded by exposure to cells lacking or poorly expressing major histocompatibility complex I and/or II molecules, and which have been genetically modified to express membrane-bound IL-15 and 4-1BB ligand (CD 137L). Such cell lines include, but are not necessarily limited to, K562[ ATCC, CCL 243; (Lozzio and Lozzio), blood (3): 321-334 (1975), klein et al, (Int J Cancer) 18 (4): 421-431 (1976)), and Wilms tumor cell lines HFWT (Fehniger and Caligiouri), international immunology comments (Int Rev Immunol) 20 (3-4): 503-534 (2001), harada et al, (Exp Hematol) experimental blood (32 (7): 614-621 (2004)), endometrial tumor cell line HHUA, melanoma cell line HMV-II, hepatoma cell line HuH-6, small lung Cancer cell lines Lu-130 and Lu-134-A, neuroblastoma cell lines NB 19 and N1369, embryonal carcinoma cell lines NEC 14 from testis, cervical Cancer cell line TNB-2 and bone marrow metastasis cell line TNB 1 (Harada et al, (J319) J Cancer (2002) J Cancer (CANCER RES) J2002). Preferably, the cell lines used lack or poorly express both MHC I and II molecules, such as K562 and HFWT cell lines. Instead of a cell line, a solid support may be used. Such a support should preferably be attached to its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and/or proliferation response, or capable of binding to a molecule having such an effect, thereby acting as a scaffold. The support may have attached to its surface a CD137 ligand protein, a CD137 antibody, an IL-15 protein or an IL-15 receptor antibody. Preferably, the support will have IL-15 receptor antibodies and CD137 antibodies bound to its surface.

In one embodiment of the described compositions or methods, the subject is administered a therapeutically effective amount of IL-2 after introduction (or reintroduction) of the T lymphocytes, NK cells, or both T lymphocytes and NK cells into the subject.

In a further aspect, the method further comprises cryopreserving the population of engineered NK or T cells. In some cases, the engineered NK or γδ T cells are cryopreserved. Further provided herein are genetically engineered populations of cryopreserved NK or T cells.

According to the present disclosure, a patient may be treated by infusing a therapeutically effective dose of immune cells, such as T or NK cells, comprising the factor redirecting glucose metabolites and/or CAR polypeptide of the present disclosure, ranging from about 10 ⁵ to 10 ¹⁰ or more cells (cells/Kg) per kilogram body weight. Infusion may be repeated at a frequency and number of times that the patient can tolerate until the desired response is reached. The appropriate infusion dosage and schedule will vary from patient to patient but can be determined by the treating physician for a particular patient. Typically, an initial dose of about 10 ⁶ cells/kg is infused, gradually increasing to 10 ⁸ cells/kg or more. IL-2 may be co-administered to expand infused cells. The amount of IL-2 per square meter of body surface is about 1-5X 10 ⁶ International units.

The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within an acceptable standard deviation in accordance with the practice in the art. Alternatively, "about" may mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5% and still more preferably up to ±1% of a given value. Alternatively, especially for biological systems and methods, the term may mean within an order of magnitude, preferably within a factor of 2. When a particular value is described in the present disclosure and claims, unless otherwise indicated, the term "about" is implicit and in this context means within the acceptable error range for the particular value.

The efficacy of the compositions or methods described herein can be assessed by any method known in the art and will be apparent to the skilled medical practitioner. For example, the efficacy of a composition or method described herein can be assessed by survival of the subject or cancer or pathogen load in the subject or a tissue or sample thereof. In some embodiments, the compositions and methods described herein can be based on the safety or toxicity of a subject's therapy (e.g., administering immune cells expressing factors that redirect glucose metabolites and CAR polypeptides), e.g., by the overall health of the subject and/or in the presence of an adverse event or serious adverse event.

C. Other immunotherapy

In some embodiments, genetically engineered immune cells expressing one or more of the factors that redirect glucose metabolites (e.g., PKM2, GFPT1, or TIGAR) may be derived from natural immune cells specific for diseased cells (e.g., cancer cells or pathogen-infected cells). Such genetically engineered immune cells (e.g., tumor-infiltrating lymphocytes or TILs) may not co-express any chimeric receptor polypeptides and may be used to destroy target disease cells, such as cancer cells. Genetically engineered TILs that express one or more factors that redirect glucose metabolites but do not express chimeric receptors may be used with bispecific antibodies (BiTE) that are capable of binding to target tumor cells and TILs.

In some embodiments, the genetically engineered immune cells expressing one or more of the factors that redirect glucose metabolites (e.g., PKM2, GFPT1, or TIGAR) may be T _reg cells. Such T _reg cells can co-express a chimeric receptor polypeptide as disclosed herein. Alternatively, T _reg cells may not co-express any chimeric receptor polypeptides and may be used for the intended therapy.

Some embodiments provide a composition comprising an effective amount of the engineered NK or T cells of the embodiments for treating a disease or disorder in a subject. Also provided herein is a use of a composition comprising an effective amount of the engineered NK or T cells of the examples for treating an immune-related disorder in a subject. Further embodiments provide a method of treating an immune-related disorder in a subject, the method comprising administering to the subject an effective amount of the engineered NK or γδ T cells of the embodiments. In an exemplary embodiment, the method does not include performing HLA matching. In particular embodiments, NK or γδ T cells are KIR-ligands that are mismatched between the subject and the donor. In further specific embodiments, the method does not include performing HLA matching. In certain embodiments, the lack of HLA matching does not result in graft versus host disease or toxicity.

D. immunotherapy of genetically engineered hematopoietic cells expressing TCR polypeptides

Genetically engineered hematopoietic cells described herein (e.g., hematopoietic stem cells, immune cells, such as T or NKT cells, ipscs) that co-express factors that redirect glucose metabolites and TCR polypeptides can be used in immunotherapy, such as T cell therapy (both αβ and γδ T cells) or NKT cell therapy, to inhibit diseased cells expressing antigens targeted directly by the TCR polypeptides (e.g., by recognizing specific peptide-MHC). By allowing the cells to effectively grow and/or function in low glucose, low amino acid, low pH and/or low oxygen environments, such as tumor microenvironments, factors that redirect glucose metabolites co-expressed with TCR polypeptides in immune cells will facilitate cell-based immunotherapy. Clinical safety may be further enhanced by using mRNA electroporation to express factors and/or TCR polypeptides that transiently redirect glucose metabolites to limit any potential non-tumor specific reactivity.

The methods described herein can include introducing into a subject a therapeutically effective amount of a genetically engineered host cell, such as a hematopoietic cell, e.g., an immune cell (e.g., an αβt, γδt, or NKT cell) that co-expresses a factor of the disclosure that redirects glucose metabolites and a TCR polypeptide. A subject (e.g., a human patient, such as a human cancer patient) may additionally have been treated or be treated with an anti-cancer or anti-infective therapy, including but not limited to an anti-cancer therapeutic or an anti-infective agent.

TCRs have antigen binding domains that can bind any target antigen through processed peptide-MHC complexes. Such target antigens may be any molecule associated with a disease or condition, including but not limited to tumor antigens, pathogenic antigens (e.g., bacteria, fungi, or viruses), or antigens present on diseased cells, as described herein. In some embodiments, the target antigen binding domain targets a native tumor antigen protein. In other embodiments, the target antigen binding domain targets a variant (e.g., a mutation) of the tumor antigen protein.

Host cells (e.g., hematopoietic cells, e.g., immune cells such as T and NKT cells) expressing the factors and TCR polypeptides described herein that redirect glucose metabolites can be used to inhibit cells expressing a target antigen and/or to enhance growth and/or proliferation of immune cells in a low glucose environment, a low amino acid environment, a low pH environment, and/or a low oxygen environment, e.g., a tumor microenvironment. In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient. In some embodiments, the subject has additionally been treated or is being treated with any of the therapeutic antibodies described herein.

To practice the methods described herein, an effective amount of hematopoietic cells, e.g., immune cells (NKT and/or T cells) or a composition thereof expressing any of the factors that redirect glucose metabolites and TCR polypeptides described herein and TCR polypeptides, may be administered to a subject in need of treatment by a suitable route, such as intravenous or subcutaneous administration. As used herein, an effective amount refers to the amount of the corresponding agent (e.g., NKT and/or T cells expressing a factor that redirects glucose metabolites, TCR polypeptide, or a composition thereof) that confers a therapeutic effect on a subject upon administration. It will be apparent to those skilled in the art and disclosed herein to determine whether the amount of a cell or composition described herein achieves a therapeutic effect.

The methods of the present disclosure may be used to treat any cancer or any pathogen. Specific non-limiting examples of cancers that can be treated by the methods of the present disclosure include, for example, lymphomas, breast cancer, gastric cancer, neuroblastomas, osteosarcomas, lung cancer, skin cancer, prostate cancer, colorectal cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastomas, gliomas, glioblastomas, thyroid cancer, hepatocellular carcinoma, esophageal cancer, and cervical cancer. In certain embodiments, the cancer may be a solid tumor. The methods of the present disclosure may also be used to treat infectious diseases, which may be viral infections.

In some embodiments, the immune cells are administered to the subject in an amount effective to inhibit at least 20% and/or at least 2-fold of the cells expressing the target peptide antigen, e.g., 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more of the cells expressing the target antigen. Additional therapeutic agents (e.g., antibody-based immunotherapeutic agents) may be used to treat, alleviate or mitigate the symptoms of any disease or disorder for which the therapeutic agent is deemed useful to the subject.

The efficacy of cell-based immunotherapy as described herein may be assessed by any method known in the art and will be apparent to the skilled medical practitioner. In some embodiments, the immune cells are administered to a subject in need of treatment in an amount effective to enhance the efficacy of the cell-based immunotherapy by at least 20% and/or at least 2-fold, e.g., enhancing the efficacy of the antibody-based immunotherapy by 50%, 80%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more, as compared to the efficacy in the absence of immune cells expressing factors that redirect glucose metabolites and/or TCR polypeptides.

In any of the compositions or methods described herein, the immune cells (e.g., NKT and/or T cells) may be autologous to the subject, i.e., the immune cells may be obtained from a subject in need of treatment, genetically engineered to express factors that redirect glucose metabolites and/or TCR polypeptides, and then administered to the same subject. In a specific embodiment, the autoimmune cells (e.g., T or NKT cells) are activated and/or expanded ex vivo prior to reintroduction into the subject. Administration of autologous cells to a subject may result in reduced rejection of host cells compared to administration of non-autologous cells. NKT and T cells (αβt or γδ T cells) may be activated by any method known in the art.

In one embodiment of the described compositions or methods, after introducing (or reintroducing) T cells or NKT cells into a subject, a therapeutically effective amount of IL-2 is administered to the subject. In a further aspect, the method further comprises cryopreserving the engineered iPSC, NKT, or T cell population.

According to the present disclosure, a patient may be treated by infusing a therapeutically effective dose of immune cells, such as T or NKT cells, including the factor redirecting glucose metabolites and/or TCR polypeptides of the present disclosure, ranging from about 10 ⁵ to 10 ¹⁰ or more cells (cells/Kg) per kilogram body weight. Infusion may be repeated at a frequency and number of times that the patient can tolerate until the desired response is reached. The appropriate infusion dosage and schedule will vary from patient to patient but can be determined by the treating physician for a particular patient. Typically, an initial dose of about 10 ⁶ cells/kg is infused, gradually increasing to 10 ⁸ cells/kg or more.

The efficacy of the compositions or methods described herein can be assessed by any method known in the art and will be apparent to the skilled medical practitioner. For example, the efficacy of a composition or method described herein can be assessed by survival of the subject or cancer or pathogen load in the subject or a tissue or sample thereof. In some embodiments, the compositions and methods described herein can be based on the safety or toxicity of a subject's therapy (e.g., administering immune cells expressing factors that redirect glucose metabolites and TCR polypeptides), e.g., by the overall health of the subject and/or in the presence of adverse events or serious adverse events.

V. combination therapy

The compositions and methods described in this disclosure may be used in conjunction with therapies for other types of cancers, such as chemotherapy, surgery, radiation therapy, gene therapy, etc., or anti-infective therapy. Such therapies may be administered simultaneously or sequentially (in any order) with immunotherapy according to the present disclosure. When co-administered with additional therapeutic agents, the appropriate therapeutically effective dose of each agent may be reduced due to additive or synergistic effects.

In some embodiments, immune cells (e.g., T and/or NK cells) expressing any of the factors and/or chimeric receptor polypeptides disclosed herein that redirect glucose metabolites may be administered to a subject that has been treated or is being treated with an additional therapeutic agent (e.g., an additional anti-cancer therapeutic agent). For example, immune cells may be administered to a human subject concurrently with an additional therapeutic agent. In some embodiments, the immune cells may be administered to a human subject prior to the additional therapeutic agent. In some embodiments, the immune cells may be administered to the human subject after the additional therapeutic agent.

The co-expression of factors that redirect glucose metabolites and genetically engineered immune cells (e.g., T cells or NK cells) of the CAR polypeptide specific for the tag can be used in conjunction with a therapeutic agent conjugated to the tag. Such genetically engineered immune cells can bind to and inhibit growth of diseased cells by a therapeutic agent capable of binding to an antigen associated with the diseased cells, such as tumor cells. Any of the antibodies listed in table 4 above or other antibodies also listed in table 4 that are specific for the same target antigen may be conjugated to a suitable tag (e.g., a tag described herein) and used in conjunction with immune cells that co-express a factor that redirects glucose metabolites and a CAR polypeptide that is specific for the tag.

The treatment of the present disclosure may be combined with other immunomodulatory treatments such as therapeutic vaccines (including but not limited to GVAX, dendritic Cell (DC) based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to agents that enhance 41BB, OX40, etc.). In some embodiments, genetically engineered immune cells (e.g., T cells or NK cells) that co-express a factor that redirects glucose metabolites and a CAR polypeptide are combined with an immunomodulatory treatment.

Non-limiting examples of other therapeutic agents that can be used in combination with the immunotherapy of the present disclosure include: (i) Anti-angiogenic agents (e.g., TNP-470, platelet factor 4, thrombospondin-1, metalloproteinase tissue inhibitors (TIMP 1 and TIMP 2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF-soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptor, placenta-associated protein, and those listed by (Carmeliet and Jain, nature), 407 (6801): 249-257 (2000)); (ii) VEGF antagonists or VEGF receptor antagonists, such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase, and any combination thereof; and (iii) chemotherapeutic compounds such as pyrimidine analogs (5-fluorouracil, fluorouridine, capecitabine (capecitabine), gemcitabine (gemcitabine), and cytarabine), purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine (cladribine))); Antiproliferative/antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine and vinorelbine), microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole (nocodazole), epothilone (epothilone) and noveboard (novelline), epipodophyllotoxins (etoposide) and teniposide (teniposide)), DNA damaging agents (actinomycin, amsacrine (amsacrine), anthracycline (anthracycline), Bleomycin (bleomycin), busulfan (busulfan), camptothecin (camptothecin), carboplatin, chlorambucil (chlorambucil), cisplatin, cyclophosphamide (cyclophosphamide), cyclophosphamide (cytoxan), dactinomycin (dactinomycin), daunorubicin (daunorubicin), doxorubicin (doxorubicin), epirubicin (epirubicin), hexamethylmelamine (hexamethyhnelamine), and pharmaceutical compositions, Oxaliplatin (oxaliplatin), ifosfamide, melphalan (melphalan), dichloromethyldiethylamine (merchlorehtamine), mitomycin (mitomycin), mitoxantrone (mitoxantrone), nitrosourea, plicamycin (plicamycin), procarbazine (procarbazine), taxol (taxol), taxotere (taxotere), teniposide, triethylthiophosphamide (triethylenethiophosphoramide) and etoposide (VP 16)); Antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin)), idarubicin (idarubicin), anthracycline, mitoxantrone, bleomycin, plicamycin (mithramycin (mithramycin)), and mitomycin; enzymes (L-asparaginase that systematically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents, such as nitrogen mustard (dichloromethyl diethylamine, cyclophosphamide and analogues, melphalan, chlorambucil), ethyleneimine and methyl melamine (hexamethylmelamine and thiotepa), alkyl sulfonate-busulfan, nitrosoureas (carmustine (carmustine) (BCNU) and analogues, streptozotocin (streptozocin)), triazene-dacarbazine (trazenes-Dacarbazinine) (DTIC); Antiproliferative/antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide (aminoglutethimide); hormones, hormone analogs (estrogens, tamoxifen, goserelin (goserelin), bicalutamide (bicalutamide), nilutamide (nilutamide)) and aromatase inhibitors (letrozole, anastrozole); Anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin (aspirin), dipyridamole (dipyridamole), ticlopidine (ticlopidine), clopidogrel (clopidogrel), abciximab (abciximab); an anti-migration agent; antisecretory agents (brefeldin); immunosuppressants (cyclosporine (cyclosporine), tacrolimus (FK-506), sirolimus (sirolimus) (rapamycin)), azathioprine, mycophenolate; Anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast Growth Factor (FGF) inhibitors); angiotensin receptor blockers; a nitric oxide donor; an antisense oligonucleotide; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); AKT inhibitors (e.g., MK-2206 2HCl, piperifacin (Perifosine) (KRX-0401), GSK690693, patadine (Ipatasertib) (GDC-0068), AZD5363, prednisolone (uprosertib), arhat (afuresertib), or troxiribine (triciribine)); mTOR inhibitors, topoisomerase inhibitors (doxorubicin), amsacrine, camptothecine, daunorubicin, dactinomycin, geniposide (eniposide), epirubicin, etoposide, idarubicin and mitoxantrone, topotecan (topotecan) and irinotecan (irinotecan)), corticosteroids (cortisone (cortisone), dexamethasone (dexamethasone), hydrocortisone (hydrocortisone), methylprednisolone (methylpednisolone), prednisone (prednisone) and prednisolone (prednisolone)); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; chromatin breaking agents.

For examples of additional useful agents, see also: physician's desk reference (THE PHYSICIAN' S DESK REFERENCE), 59 th edition, (2005), thomson P D R, montvale n.j.); gennaro et al, eds. "Remington' S THE SCIENCE AND PRACTICE of Pharmacy", 20 th edition, (2000) Lippincott Williams and Wilkins, inc. (Lippincott WILLIAMS AND WILKINS, baltimore Md.) of Ballmol, balld; braunwald et al, harrison' S PRINCIPLES of INTERNAL MEDICINE, 15 th edition, (2001), maglauca Hill, N.Y. (MCGRAW HILL, NY); berkow et al, edited the merck diagnosis and therapy handbook (The Merck Manual of Diagnosis AND THERAPY) (1992), merck research laboratory of Rahway n.j., MERCK RESEARCH Laboratories).

Administration of the additional therapeutic agent may be by any suitable route, including systemic administration as well as direct administration to the disease site (e.g., to a tumor).

In some embodiments, the methods involve administering an additional therapeutic agent (e.g., an antibody) to the subject at one dose. In some embodiments, the methods involve administering an additional therapeutic agent (e.g., an antibody) to the subject in multiple doses (e.g., at least 2, 3, 4, 5, 6, 7, or 8 doses). In some embodiments, the additional therapeutic agent (e.g., antibody) is administered to the subject in a plurality of doses, and the first dose of the additional therapeutic agent (e.g., antibody) is administered to the subject about 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the immune cells that express the factor that redirects the glucose metabolite and/or the CAR polypeptide. In some embodiments, the first dose of the additional therapeutic agent (e.g., antibody) is administered to the subject between about 24-48 hours prior to administration of the immune cells that express the factor that redirects glucose metabolites and/or the CAR polypeptide. In some cases, the additional therapeutic agent may be an antibody specific for the target antigen of interest, such as the antibodies listed in table 4 and other antibodies specific for the same target antigen.

In some embodiments, a first dose of an additional therapeutic agent (e.g., an antibody) is administered to the subject prior to administration of the immune cells expressing the factor that redirects glucose metabolites and/or the CAR polypeptide. In some embodiments, the subject is administered an additional therapeutic agent (e.g., an antibody) prior to administration of the immune cells expressing the factor that redirects glucose metabolites and/or the CAR polypeptide, and then administered about once every two weeks. In some embodiments, the first two doses of the additional therapeutic agent (e.g., antibody) are administered about one week apart (e.g., about 6, 7,8, or 9 days). In certain embodiments, the third and subsequent doses are administered about once every two weeks.

In any of the embodiments described herein, the timing of administration of the additional therapeutic agent (e.g., antibody) is approximate and includes three days before and three days after the specified date (e.g., administration every three weeks encompasses administration on day 18, day 19, day 20, day 21, day 22, day 23, or day 24).

The immune system-induced efficacy for disease therapy may be enhanced by combination with other agents, e.g., agents that reduce tumor burden prior to administration of CAR-T or CAR-NK. Antibody-drug conjugates (ADCs) can be effective in reducing tumor burden for multiple types of cancer. Many exemplary ADCs (Mullard), reviewed naturally: drug discovery (Nat Rev Drug Discov), 12 (5): 329-332 (2013), coatings et al, clinical cancer research (CLINICAL CANCER RESEARCH), 25 (18): 5441-5448 (2019), zhao et al, pharmaceutical journal (Acta Pharmaceutica Sinica B), 10 (9): 1589-1600 (2020), fu et al, signal transduction and Targeted therapy (Signal Transduction AND TARGETED THERAPY), 7 (1): 93 (2022)) are known in the art. Any such known ADC may be used in combination with a CAR-T or CAR-NK construct as described herein. Thus, in some embodiments, when the ADC is used in combination with CAR-T or CAR-NK, the ADC is administered prior to CAR-T or CAR-NK. In some embodiments, the ADC is used in combination with a CAR-T or CAR-NK as disclosed herein. In some embodiments, the first dose of ADC is administered to the subject prior to administration of the immune cells expressing the factor that redirects glucose metabolites and/or the CAR polypeptide.

In another embodiment, immune system-induced efficacy for disease therapy may be enhanced by combination with other immunotherapeutic agents, such as cytokines (e.g., agonists of IL-2/IL-15R, βγ such as IL-2, IL-15 (IL-2/IL-15 superagonists), IL-7 or IL-12 or derivatives thereof) or immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG 3 antibodies, anti-CTLA 4 antibodies or anti-TIM 3 antibodies) that stimulate CAR-T or CAR-NK cells in vivo.

In some embodiments, the method further comprises administering a lymphopenia treatment, preferably selected from cyclophosphamide and fludarabine. Such lymphocyte removal treatment is preferably performed prior to infusion of the CAR-expressing hematopoietic cells to allow greater T cell expansion of the infused cells (Shank et al, pharmacotherapy, 37 (3): 334-345 (2017)).

The efficacy of the methods described herein can be assessed by any method known in the art and will be apparent to the skilled medical practitioner and/or to the practitioner described herein. For example, the efficacy of antibody-based immunotherapy can be assessed by the survival rate of the subject or the cancer burden in the subject or a tissue or sample thereof. In some embodiments, antibody-based immunotherapy is based on the safety or toxicity of the therapy in the subject, e.g., assessed by the overall health status of the subject and/or in the presence of adverse events or serious adverse events.

Kit for therapeutic use

The present disclosure also provides kits for use in the compositions described herein. For example, the disclosure also provides kits comprising a population of immune cells (e.g., T or NK cells constructed in vitro or in vivo) expressing factors that redirect glucose metabolites and optionally chimeric receptor polypeptides for inhibiting growth of diseased cells, e.g., tumor cells and/or enhancing immune cell growth and/or proliferation in a low glucose environment, a low amino acid environment, a low pH environment, and/or a low oxygen environment, e.g., in a tumor microenvironment. The kit may further comprise a therapeutic agent or a therapeutic agent conjugated to a tag (e.g., a tag described herein) to which the chimeric receptor polypeptide expressed on the immune cells binds. Such kits may comprise one or more containers comprising a population of genetically engineered immune cells (e.g., T and/or NK cells) as described herein that co-express a factor that redirects glucose metabolites and a chimeric receptor polypeptide as described herein, and optionally a therapeutic agent or a therapeutic agent conjugated to a tag.

In some embodiments, the kit includes a factor that redirects in vitro amplified immune cells expressing the glucose metabolite and expressing the chimeric receptor polypeptide. In another embodiment, the kit includes factors that redirect immune cells expressing glucose metabolites and expressing chimeric receptor polypeptides and antibodies specific for cell surface antibodies present on activated T cells, such as anti-CD 5 antibodies, anti-CD 38 antibodies, or anti-CD 7 antibodies. In an exemplary embodiment, the kit includes a factor that redirects NK or T cells expressing the glucose metabolite and expressing the CAR. Factors that redirect immune cells expressing glucose metabolites and expressing chimeric receptor polypeptides may express any of the chimeric receptor polypeptide constructs known in the art or disclosed herein.

Alternatively, the kits disclosed herein may include a nucleic acid or set of nucleic acids described herein that collectively encode any of the chimeric receptor polypeptides as described herein and any of the factors that redirect glucose metabolites.

In some embodiments, the kit may additionally include instructions for any of the methods described herein. The included instructions can include descriptions of administering the first and second pharmaceutical compositions to a subject to achieve a desired activity, e.g., inhibiting growth of target cells and/or enhancing growth and/or proliferation of immune cells in a low glucose environment, a low amino acid (e.g., a low glutamine environment) environment, a low pH environment, and/or a low oxygen environment (e.g., a low glucose, low amino acid, low pH, or low oxygen tumor microenvironment) in the subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of treatment. Non-limiting examples of such identification methods may comprise expression of a target in blood, DNA, or tissue (e.g., immunohistochemistry). Further, in some cases, the cut-off range may be used to adjust the therapeutic dose.

In some embodiments, the instructions include a description of administering a population of genetically engineered immune cells (e.g., T or NK cells), and optionally a description of administering a tag-conjugated therapeutic agent. Instructions related to the use of immune cells (e.g., T or NK cells) as described herein, optionally to label conjugated therapeutic agents, typically comprise information about the dosage, dosing regimen, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is for treating a disease or disorder in a subject, delaying the onset of a disease or disorder in a subject, and/or alleviating a disease or disorder in a subject.

The kits provided herein employ suitable packaging. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packaging, and the like. Packages for use in combination with specific devices such as inhalers, nasal administration devices or infusion devices are also contemplated. The kit may have a sterile inlet port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile inlet port. The at least one active agent in the second pharmaceutical composition is an antibody as described herein. The at least one active agent in the first pharmaceutical composition is a population of immune cells (e.g., T lymphocytes or NK cells) expressing the chimeric receptor polypeptides and factors that redirect glucose metabolites as described herein.

The kit optionally may provide additional components such as buffers and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the kit described above.

Further embodiments of the invention are nucleic acids or groups of nucleic acids, which together comprise; a first nucleotide sequence encoding the above-described factor that transfers or redirects glucose metabolites; and a second nucleotide sequence encoding the chimeric receptor polypeptide described above. The isolated nucleic acid or nucleic acid set may be used to make genetically engineered immune cells of the invention. Thus, a further embodiment is a method for in vitro manufacturing of modified immune cells by transfecting immune cells with a nucleic acid or a set of nucleic acids and selecting/enriching the transfected immune cells. Selection of transfected cells can be performed by methods known in the art (e.g., flow cytometry) by selecting expression of chimeric receptor polypeptides on the surface of immune cells. The immune cells may optionally be activated in vitro by any method known in the art, as described above for NK and T cells.

Such isolated nucleic acids or groups of nucleic acids may be in the form of a vector or group of vectors. Thus, a method for producing a modified immune cell in vivo, the method comprising administering a nucleic acid or a set of nucleic acids, preferably a vector or a set of vectors, according to the invention to a subject in need thereof. The corresponding immune cells of the subject will be transfected with a nucleic acid or nucleic acid set (e.g., gamma-retrovirus or lentivirus) and result in the expression or overexpression of a polypeptide that transfers or redirects the glucose metabolite out of the glycolytic pathway and chimeric receptor polypeptide.

General technique

Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as: molecular cloning: laboratory Manual, second edition (Sambrook et al, 1989), cold spring harbor Press (Cold Spring Harbor Press); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); Molecular biology methods (Methods in Molecular Biology), hu Mana press; cell biology: laboratory Manual (Cell Biology: A Laboratory Notebook) (J.E.Cellis editions, 1989), academic Press (ACADEMIC PRESS); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney edit, 1987); cell and tissue culture treatises (Introduction to Cell and Tissue Culture) (J.P.Mather and P.E.Roberts, 1998), proleman Press (Plenum Press); cell and tissue culture: laboratory procedures (Cell and Tissue Culture: laboratory Procedures) (A.Doyle, J.B.Griffiths and D.G.Newell editions, 1993-8) John Willi father-son publishing company (J.Wiley and Sons); enzymology method (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)); manual of laboratory immunology (Handbook of experimental immunology) (D.M. Weir and C.C. Blackwell editions); gene transfer Vectors for mammalian cells (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos. Eds., 1987); molecular biology laboratory guidelines (F.M. Ausubel et al editions 1987); PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, eds., 1994); immunological experimental guidelines (Current Protocols in Immunology) (J.E. Coligan et al, editions, 1991); Instructions on the fine-compiled molecular biology laboratory Manual (Short Protocols in Molecular Biology) (John Willi's father-son publishing company, 1999); immunobiology (Immunobiology) (c.a. janeway and p.transitions, 1997); antibodies (P.Finch, 1997); antibody: practical methods (Antibodies: A PRACTICE applications) (D.Catty. Edit, IRL Press, 1988-1989); Monoclonal antibody: practical methods (Monoclonal antibodies: A PRACTICAL appreach) (p.shepherd and c.dean editions, oxford university press (Oxford University Press), 2000); use of antibodies: laboratory manuals (Using anti-ibodies: a laboratory manual) (E.Harlow and D.Lane (Cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press), 1999); Antibodies (The Antibodies) (M.Zanetti and J.D.Capra editors Hawude academy of sciences (Harwood Academic Publishers), 1995); DNA cloning: practical methods (DNA Cloning: A PRACTICAL application), volumes I and II (D.N.Glover edit 1985); nucleic acid hybridization (Nucleic Acid Hybridization) (B.D.Hames and S.J.Higgins, editions (1985); Transcription and translation (Transcription and Translation) (B.D.Hames and S.J.Higgins editions (1984), animal cell culture (R.I.Freshney editions (1986), immobilized cells and enzymes (Immobilized Cells and Enzymes) (IRL Press (1986)), B.Perbal, molecular cloning Utility guide (A PRACTICAL Guide To Molecular Cloning) (1984); ausubel et al (editions).

Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present disclosure to its fullest extent. Accordingly, the following specific examples should be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subjects mentioned herein.

Examples

The following examples are intended only to illustrate methods and embodiments according to the present invention and, therefore, should not be construed as imposing limitations upon the claims.

Example 1: effect of expressing factors that redirect glucose metabolites out of the glycolytic pathway in a lower glucose environment on immune cell function expressing ACTR polypeptides

The transgene encoding a factor (e.g., polypeptide) that redirects glucose metabolites is co-expressed with ACTR polypeptides in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding an ACTR polypeptide and a factor that redirects glucose metabolites, e.g., isolated by P2A ribosome jump sequences. T and/or NK cells are mixed with tumor target cells such as IGROV-1 cells and tumor targeting antibodies such as anti-FOLRalpha antibodies at a given effector to target (E: T) ratio. The reaction was then incubated at 37℃in a 5% CO ₂ incubator for a period of time (e.g., 6-8 days) at various starting concentrations of glucose (e.g., 0-20 mM). T and/or NK cell function is then assessed, for example, using cytokine production or proliferation assays or for resistance to chronic stimuli. Cytokine production (e.g., IL-2 and/or IFN-gamma) is measured from the reaction supernatant. For proliferation experiments, co-cultures were harvested and stained with α -CD3, α -CD14, α -CD33, α -CD45, α -CD56 antibodies and live-dead cell staining. As a measure of T cell proliferation, live T cells were enumerated in CD45 ⁺CD33^-CD3⁺CD14^-CD56^- cells and live-dead cell staining was assessed by flow cytometry. And in the case of NK cells, CD45 ⁺CD33^-CD3^-CD14^-CD56⁺ was enumerated and the live-dead cell staining was assessed by flow cytometry.

T and/or NK cells expressing factors that redirect glucose metabolites other than ACTR polypeptides exhibit enhanced T and/or NK cell function, including, for example, enhanced cytokine production or enhanced proliferation, relative to T cells expressing ACTR alone. This enhanced function may be more pronounced at lower glucose concentrations. These experiments show that expressing factors that redirect glucose metabolites in immunity (e.g., T and/or NK) has a positive impact on immune cell activity.

Example 2: effect of expressing factors that redirect glucose metabolites out of the glycolytic pathway on immune cell function of expressed ACTR polypeptides in environments with higher concentrations of soluble inhibitors

The transgene encoding a factor (e.g., polypeptide) that redirects glucose metabolites is co-expressed with ACTR polypeptides in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding an ACTR polypeptide and a factor that redirects glucose metabolites, e.g., isolated by P2A ribosome jump sequences. T and/or NK cells are mixed at a given effector to target (E: T) ratio with tumor target cells such as IGROV-1 cells and tumor targeting antibodies such as anti-folra antibodies in media containing varying concentrations of soluble inhibitors (e.g., tgfβ, PGE ₂, kynurenine, and/or adenosine) present in the tumor microenvironment. The reaction was then incubated in a 5% CO ₂ incubator at 37℃for a period of time (e.g., 6-8 days). NK and/or T cell function is then assessed, for example, using cytokine production or proliferation assays or for resistance to chronic stimulation. Cytokine production (e.g., IL-2 and/or IFN-gamma) is measured from the reaction supernatant. For proliferation experiments, NK and/or T cell co-cultures were harvested, stained and evaluated by flow cytometry (see example 1).

T and/or NK cells expressing factors other than polypeptides that redirect glucose metabolites exhibit enhanced cellular functions, including, for example, enhanced cytokine production, relative to NK and/or T cells expressing ACTR alone. This enhanced function can be achieved at higher concentrations of the soluble inhibitor. These experiments show that expressing factors that redirect glucose metabolites in immune (e.g., T or NK) cells has a positive impact on immune cell activity.

Example 3: effect of expressing factors that redirect glucose metabolites out of the glycolytic pathway in environments where immunosuppressive cells are present in excess on immune cell function expressing ACTR polypeptides

The transgene encoding a factor (e.g., polypeptide) that redirects glucose metabolites is co-expressed with ACTR polypeptides in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding an ACTR polypeptide and a factor that redirects glucose metabolites, e.g., isolated by P2A ribosome jump sequences. T and/or NK cells are mixed with tumor target cells, such as IGROV-1 cells, and tumor-targeting antibodies, such as anti-folra antibodies, in the presence of immunosuppressive cells (e.g., bone marrow-derived suppressor cells and/or regulatory T cells) at a given effector to target (E: T) ratio. The reaction was then incubated in a 5% CO ₂ incubator at 37℃for a period of time (e.g., 3-10 days). Immune cell (NK and/or T cell) function is then assessed, for example, using cytokine production or cell proliferation assays or for resistance to chronic stimulation. Cytokine production (e.g., IL-2 and/or IFN-gamma) is measured from the reaction supernatant. Proliferation experiments were performed and evaluated as described in example 1.

T and/or NK cells expressing factors that redirect glucose metabolites other than ACTR or CAR polypeptides exhibit enhanced T and/or NK cell function, including, for example, enhanced cytokine production or enhanced proliferation, relative to T and/or NK cells expressing ACTR or CAR alone. This enhanced function may be achieved in the presence of an increased number (e.g., a greater number or percentage) of immunosuppressive cells. These experiments show that expressing factors that redirect glucose metabolites in immune (e.g., T and/or NK) cells has a positive impact on immune cell activity.

Example 4: expression of factors that redirect glucose metabolites out of the glycolytic pathway on tumor models

The transgene encoding a factor (e.g., polypeptide) that redirects glucose metabolites is co-expressed with ACTR polypeptides in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding an ACTR polypeptide and a factor that redirects glucose metabolites, e.g., isolated by P2A ribosome jump sequences. Antitumor activity of transduced T and/or NK cells was assessed in a mouse tumor model. For these experiments, tumor cell lines, such as IGROV-1, were inoculated into NSG ^TM(NOD scidγ,NOD.Cg-Prkdc^scidIL2rg^tm1Wjl/SzJ, strain 005557) mice. Tumor-targeting antibodies, such as anti-folra antibodies and T and/or NK cells expressing ACTR or ACTR alone and factors that redirect glucose metabolites, are then administered to tumor-bearing mice. Tumor growth was monitored throughout the experiment.

In combination with tumor-targeting antibodies, T and/or NK cells expressing factors that redirect glucose metabolites other than ACTR polypeptides show enhanced anti-tumor activity relative to T and/or NK cells expressing ACTR polypeptides alone. Additionally, in combination with tumor-targeting antibodies, T and/or NK cells expressing factors that redirect glucose metabolites other than ACTR polypeptides may exhibit enhanced T and/or NK cell activity, including, for example, enhanced proliferation, enhanced T and/or NK cell persistence, and/or enhanced cytokine production, relative to T and/or NK cells expressing ACTR polypeptides alone. These experiments demonstrate that expression of factors that redirect glucose metabolites in ACTR-expressing immune (e.g., T and/or NK) cells has a positive impact on immune cell function in vivo.

Example 5: effect of reduced glucose concentration on T cell function

The gamma-retrovirus encoding the exemplary GPC 3-targeting CAR expression construct of SEQ ID No. 78 is produced by recombinant techniques and is used to infect primary human T cells to produce cells expressing a GPC 3-targeting CAR polypeptide on their cell surface. The function of CAR-expressing cells targeting GPC3 was then tested using a six-day flow-based proliferation assay. Specifically, 200,000 untransduced mimetic T cells or T cells expressing a CAR construct targeting GPC3 were incubated with 50,000 GPC3 ⁺ hepatocellular carcinoma JHH7 or Hep3B tumor cells at a ratio of 4:1 (effector cells/CAR-expressing T cells to target cells). The CO-cultures were incubated in a 5% CO ₂ incubator at 37℃for six days in the presence of different concentrations of glucose. At the end of six days, the co-cultures were harvested and stained with anti-CD 3 antibody. The number of CD3 positive cells was assessed by flow cytometry as a measure of T cell proliferation. At lower glucose concentrations, less CAR-T proliferation was observed (figure 2). These experiments indicate that the low glucose environment may have a negative impact on CAR-T cell proliferation activity.

Example 6: effect of factors that redirect glucose metabolites out of the glycolytic pathway on immune cell function using GPC 3-targeted CAR-T or CAR-NK expression constructs expression

The gamma-retrovirus encoding the exemplary GPC 3-targeting CAR polypeptide expression construct (SEQ ID NO:78 or SEQ ID NO: 79) is produced by recombinant techniques and is used to infect primary human T and/or NK cells to produce cells expressing the GPC 3-targeting CAR polypeptide on their cell surfaces. Additionally, a gamma retrovirus encoding an exemplary GPC 3-targeting CAR polypeptide and a factor that redirects glucose metabolites out of the glycolytic pathway (PKM 2, PKM2Y 105E variant, PKM2Y105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT H391Y variant, or TIGAR) (SEQ ID NO:68-SEQ ID NO: 74) is produced by recombinant techniques and used to infect primary human T and/or NK cells to produce cells expressing the GPC 3-targeting polypeptide and the factor that redirects glucose metabolites. In constructs encoding CAR polypeptides and factors that redirect glucose metabolites, the two polypeptides are separated, for example, by a P2A ribosome jump sequence. The variant to be expressed is SEQ ID NO:68-SEQ ID NO:74 (e.g., CAR+PKM2, CAR+PKM2Y E, CAR +PKM2Y D, CAR +PKM2K422R, CAR +PKM2H2H391Y, CAR + GFPT1 or CAR+TIGAR). The function of CAR-expressing cells targeting GPC3 was then tested using a six-day flow-based proliferation assay. Specifically, 200,000 untransduced mimetic T or NK cells, T or NK cells expressing a CAR polypeptide that targets GPC3, or T or NK cells expressing a CAR polypeptide that targets GPC3 and a factor that redirects glucose metabolites were incubated with 50,000 GPC3 ⁺ hepatocellular carcinoma JHH7 tumor cells at a ratio of 4:1 (effector cells/CAR-expressing T or NK cells to target cells). The CO-cultures were incubated at 37℃for six days in the presence of 1.25mM glucose (tumor associated) and 10mM glucose (approximately peripheral blood levels) at 5% CO ₂. At the end of six days, the co-cultures were harvested and stained with α -CD3, α -CD14, α -CD33, α -CD45, α -CD56 antibodies and live-dead cell staining. As a measure of T cell proliferation, live T cells were enumerated in CD45 ⁺CD33^-CD3⁺CD14^-CD56^- cells and live-dead cell staining was assessed by flow cytometry. And in the case of NK cells, CD45 ⁺CD33^-CD3^-CD14^-CD56⁺ was enumerated and the live-dead cell staining was assessed by flow cytometry.

Immune cells expressing factors that redirect glucose metabolites other than CAR polypeptides exhibit enhanced T and/or NK cell proliferation relative to T and/or NK cells expressing the CAR construct alone. This enhanced proliferation also occurs at tumor-associated low glucose concentrations. These experiments demonstrate that expressing factors that redirect glucose metabolites in immune (e.g., T and/or NK) cells has a positive effect on CAR-T and/or CAR-NK cell proliferation activity.

Example 7: effect of expressing factors that redirect glucose metabolites out of the glycolytic pathway on immune cell function of expressed CAR polypeptides in environments with higher soluble inhibitor concentrations

The transgene encoding a factor (e.g., polypeptide) that redirects glucose metabolites is co-expressed with the CAR polypeptide in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding a CAR polypeptide and a factor that redirects glucose metabolites isolated, for example, by a P2A ribosome jump sequence. Transduced T and/or NK cells are mixed at a given effector to target (E: T) ratio with tumor target cells, such as HepG2 cells, in medium containing varying concentrations of soluble inhibitors (e.g., TGF beta, PGE ₂, kynurenine, and/or adenosine) present in the tumor microenvironment. The reaction was then incubated in a 5% CO ₂ incubator at 37℃for a period of time (e.g., 6-8 days). NK and/or T cell function is then assessed, for example, using cytokine production or proliferation assays or for resistance to chronic stimulation. Cytokine production (e.g., IL-2 and/or IFN-gamma) is measured from the reaction supernatant. For proliferation experiments, NK and/or T cell co-cultures were harvested, stained and evaluated by flow cytometry (see example 1).

T and/or NK cells expressing factors that redirect glucose metabolites other than CAR polypeptides exhibit enhanced T and/or NK cell function, including, for example, enhanced cytokine production or enhanced proliferation, relative to T and/or NK cells expressing the CAR alone. This enhanced function can be achieved at higher concentrations of the soluble inhibitor. These experiments show that expressing factors that redirect glucose metabolites in immune (e.g., T and/or NK) cells has a positive impact on immune cell activity.

Example 8: effect of expressing factors that redirect glucose metabolites out of the glycolytic pathway on immune cell function of expressed CAR polypeptides in environments where immunosuppressive cells are present in excess

The transgene encoding the factor redirecting the glucose metabolite is co-expressed with the CAR polypeptide in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding a CAR polypeptide and a factor that redirects glucose metabolites isolated, for example, by a P2A ribosome jump sequence. Transduced T and/or NK cells are mixed with tumor target cells, such as HepG2 cells, at a given effector to target (E: T) ratio in the presence of immunosuppressive cells (e.g., bone marrow derived suppressor cells and/or regulatory T cells). The reaction was then incubated in a 5% CO ₂ incubator at 37℃for a period of time (e.g., 3-10 days). T and/or NK cell function is then assessed, for example, using cytokine production or cell proliferation assays or for resistance to chronic stimulation. Cytokine production (e.g., IL-2 and/or IFN-gamma) is measured from the reaction supernatant. Proliferation experiments were performed and evaluated as described in example 1.

T and/or NK cells expressing factors that redirect glucose metabolites other than CAR polypeptides exhibit enhanced T and/or NK cell function, including, for example, enhanced cytokine production or enhanced proliferation, relative to T and/or NK cells expressing the CAR alone. This enhanced function may be achieved in the presence of an increased number (e.g., a greater number or percentage) of immunosuppressive cells. These experiments show that expressing factors that redirect glucose metabolites in immune (e.g., T and/or NK) cells has a positive impact on immune cell activity.

Example 9: expression of an agent that redirects glucose metabolites out of the glycolytic pathway in a tumor model for T cell function

The transgene encoding a factor that redirects glucose metabolites is co-expressed with a Chimeric Antigen Receptor (CAR) polypeptide in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding a CAR polypeptide and a factor that redirects glucose metabolites isolated, for example, by a P2A ribosome jump sequence. Antitumor activity of transduced T and/or NK cells was assessed in a mouse tumor model. For these experiments, tumor cell lines, e.g., hepG2, were inoculated into NSG ^TM(NOD scidγ,NOD.Cg-Prkdc^scidIL2rg^tmWj1/SzJ, strain 005557) mice. T and/or NK cells expressing the CAR alone or the CAR and a factor that redirects glucose metabolites are then administered to tumor-bearing mice. Tumor growth was monitored throughout the experiment.

T and/or NK cells expressing factors that redirect glucose metabolites in addition to CAR polypeptides show enhanced anti-tumor activity relative to T or NK cells expressing CAR polypeptides alone. Additionally, T and/or NK cells expressing factors that redirect glucose metabolites other than CAR polypeptides may exhibit enhanced T and/or NK cell activity, including, for example, enhanced proliferation, persistence, and/or cytokine production, relative to T and/or NK cells expressing CAR polypeptides alone. These experiments demonstrate that expression of factors that redirect glucose metabolites in CAR-expressing T and/or NK cells has a positive impact on T or NK cell function in vivo.

Example 10: expression of GLUT1, GOT2 and TIGAR increases glucose uptake and lactate production

Healthy donor PBMCs were stimulated with anti-CD 3 and anti-CD 28 until day 2, followed by transduction with V5-tagged transgenes packaged into lentiviral vectors. The transgene encodes GLUT1 (SEQ ID NO: 80), GOT2 (SEQ ID NO: 81) or TIGAR (SEQ ID NO: 69). Transduced cells were replenished with fresh IL-2 daily until day 10. 10,000 cells/well (384 well plate) were resuspended in PBS and glucose uptake was determined. The fold change for each transgene was compared to the fold change for null (non-transduced control; baseline fold change 1) T cells under the same conditions to evaluate luminescence readings. Cells transduced with GLUT1, GOT2 or TIGAR showed elevated levels of glucose uptake, indicating higher metabolic activity. Data represent three donors. See fig. 3.

Further, healthy donor PBMCs were stimulated with anti-CD 3 and anti-CD 28 until day 2, followed by transduction with V5-tagged transgenes packaged into lentiviral vectors (described above). Transduced cells were replenished with fresh IL-2 daily until day 9. On day 9, T cell subsets were stimulated with PMA and ionomycin for 24 hours. 10,000 harvested cells/well (384 well plates) were resuspended in RPMI without FBS and incubated at 37 ℃ for 2 hours to remove residual lactate from the medium and to determine lactate production. The fold change for each transgene was compared to the fold change for null (non-transduced control; baseline fold change 1) T cells under the same conditions to evaluate luminescence readings. Stimulated cells transduced with GLUT1, GOT2 and TIGAR showed elevated lactate production levels, indicating a higher metabolic adaptation in nutrient-deficient environments. See fig. 4.

Taken together, the results of this example demonstrate that T cells transduced with GLUT1, GOT2 or TIGAR show enhanced metabolic activity and adaptability in nutrient deficient environments, and TIGAR shows the best effect among the three. This indicates that therapeutic T cells co-expressing GLUT1, GOT2, or TIGAR (specifically TIGAR) (e.g., T cells expressing ACTR or CAR polypeptides as disclosed herein) will better adapt to the tumor microenvironment (which may lack nutrition) and exhibit better therapeutic activity than corresponding T cells not transduced with GLUT1, GOT2, or TIGAR genes. See also WO2020/010110 and WO2020/037066, the relevant disclosures of each of which are incorporated herein by reference for the subject matter and purposes cited herein.

Example 11: influence of expression of factors that redirect glucose metabolism in immune cells expressing ACTR polypeptides

Transgenes encoding factors (e.g., polypeptides) that redirect glucose metabolites are co-expressed in the same T and/or NK cells that express ACTR polypeptides. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are stimulated with anti-CD 3 and anti-CD 28 for a period of time (e.g., 1-4 days) and subsequently transduced with a virus (e.g., lentivirus or γ -retrovirus) encoding ACTR polypeptide and factors that redirect glucose metabolites that may be isolated, for example, by P2A ribosome jump sequences. Transduced cells are supplemented with cytokines (e.g., IL-2) for 3-10 days. All reactants were incubated at 37℃in a 5% CO ₂ incubator. For glucose uptake measurement, cells were harvested and glucose uptake was determined using the glucose uptake Glo kit. This luminescence-based assay was evaluated and the data expressed as fold change. Supplemental cellular metabolic flux assays were performed using a hippocampal extracellular flux analyzer to capture changes in basal Oxygen Consumption Rate (OCR).

T and/or NK cells expressing factors that redirect glucose metabolites other than ACTR polypeptides are expected to show enhanced glucose uptake. This enhanced function suggests enhanced metabolic adaptation and has a positive impact on immune cell activity.

Example 12: influence of expression of factors that redirect glucose metabolism in immune cells expressing CAR polypeptides

Transgenes encoding factors (e.g., polypeptides) that redirect glucose metabolites are co-expressed in the same T and/or NK cells that express the CAR polypeptide. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are stimulated with anti-CD 3 and anti-CD 28 for a period of time (e.g., 1-4 days) and subsequently transduced with a virus (e.g., lentivirus or γ -retrovirus) encoding a CAR polypeptide and a factor that redirects the glucose metabolites, which can be isolated, for example, by a P2A ribosome jump sequence. Transduced cells are supplemented with cytokines (e.g., IL-2) for 3-10 days. All reactants were incubated at 37℃in a 5% CO ₂ incubator. For glucose uptake measurement, cells were harvested and glucose uptake was determined using the glucose uptake Glo kit. This luminescence-based assay was evaluated and the data expressed as fold change. Supplemental cellular metabolic flux assays were performed using a hippocampal extracellular flux analyzer to capture changes in basal Oxygen Consumption Rate (OCR).

T and/or NK cells expressing factors that redirect glucose metabolites other than CAR polypeptides are expected to show enhanced glucose uptake. This enhanced function suggests enhanced metabolic adaptation and has a positive impact on immune cell activity.

Example 13: effect of expressing factors that redirect lactate production in immune cells expressing ACTR polypeptides

Transgenes encoding factors (e.g., polypeptides) that redirect lactate production are co-expressed with ACTR polypeptides in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are stimulated with anti-CD 3 and anti-CD 28 for a period of time (e.g., 1-4 days) and subsequently transduced with a virus (e.g., lentivirus or γ -retrovirus) encoding ACTR polypeptide and factors that redirect glucose metabolites, e.g., isolated by P2A ribosomal jump sequences. Transduced cells are supplemented with cytokines (e.g., IL-2) and additionally with stimulators (e.g., PMA and/or ionomycin) for 3-10 days. All reactants were incubated at 37℃in a 5% CO ₂ incubator. Cells were harvested and lactate production was determined using Lactate Glow assay. This luminescence-based assay was evaluated and the data expressed as fold change.

T and/or NK cells expressing factors other than ACTR polypeptides that redirect lactate production are expected to show enhanced lactate production. This enhanced function suggests enhanced metabolic adaptation and has a positive impact on immune cell activity.

Example 14: effect of expressing factors that redirect lactate production in immune cells expressing CAR polypeptides

The transgene encoding a factor (e.g., polypeptide) that redirects lactate production is co-expressed with the CAR polypeptide in the same T and/or NK cells. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM2K422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are stimulated with anti-CD 3 and anti-CD 28 for a period of time (e.g., 1-4 days) and subsequently transduced with a virus (e.g., lentivirus or γ -retrovirus) encoding a CAR polypeptide and a factor that redirects the glucose metabolites, e.g., isolated by the P2A ribosomal jump sequence. Transduced cells are supplemented with cytokines (e.g., IL-2) and additionally with stimulators (e.g., PMA and/or ionomycin) for 3-10 days. All reactants were incubated at 37℃in a 5% CO ₂ incubator. Cells were harvested and lactate production was determined using Lactate Glow assay. This luminescence-based assay was evaluated and the data expressed as fold change.

T and/or NK cells expressing factors other than CAR polypeptides that redirect lactate production are expected to show enhanced lactate production. This enhanced function suggests enhanced metabolic adaptation and has a positive impact on immune cell activity.

Example 15: production of retroviral particles

On day 1, 12×10 ⁶ low-passage HEK293T cells were plated on 15cm coated tissue culture plates in DMEM medium containing 10% FBS. The next day, day 2, the cells were 80% confluent. On day 3, cells were transfected. 3ml of a transfection mixture containing 10. Mu.g GAG/Pol, 6.6. Mu.g GALV helper cells, 20. Mu.g transfer plasmid and 74. Mu.l PEIPro transfection reagent (catalog No. 115-010, bo Li Pusi Co. (PolyPlus)) was prepared and added to the cell culture plate. 6 hours after transfection, the transfected cells were supplemented with fresh DMEM medium containing 10% FBS medium. Viral supernatants were harvested 24 hours and 36 hours post-transfection and concentrated by 0.45 μm filter and stored at-80 ℃ until further use.

Example 16: initiation and transduction of immune cells

Immune cells (e.g., NK and/or T cells) are isolated from fresh blood samples or derived from cell lines.

Peripheral Blood Mononuclear Cells (PBMCs) containing immune cells were isolated by a density gradient method using Ficoll-paque. Briefly, equal volumes of whole blood and PBS were carefully mixed by inversion, overlaid on Ficoll-paque, and subsequently centrifuged at 400g for 30min at room temperature. PBMC were removed from the buffy coat (see Low and Wan Abas, international biomedical research (Biomed Res Int), 2015:239362 (2015)). PBMCs were stimulated with anti-CD 3 and anti-CD 28 until day 2 prior to transduction.

NK-92 cell lines were used to assess NK cell function. 1X 10 ⁶ NK-92 cells in T75 flask and in the 10% FBS containing RPMI medium with IL-2 (100 UI/ml) stimulation. Cells were maintained for one week by supplementing IL-2 (100 UI/ml) every 48 hours.

The transgene encoding a factor (e.g., polypeptide) that redirects glucose metabolites is co-expressed in the same immune (NK and/or T) cells as ACTR (see table 7) or CAR (see tables 8-11) polypeptides. The transgene is, for example, PKM 2Y 105E variant, PKM 2Y 105D variant, PKM 2K 422R variant, PKM 2H 391Y variant, GFPT1 or TIGAR (e.g., SEQ ID NO:68-SEQ ID NO: 74). T and/or NK cells are transduced with a virus encoding an ACTR or CAR polypeptide and a factor that redirects glucose metabolites isolated, for example, by P2A ribosome jump sequences. Briefly, 1X 10 ⁶ cells were mixed with 1ml of virus supernatant (see example 1), in a total volume of 2ml, centrifuged at 1200g for 45 min, and subsequently plated into 24 well plates. The cells were then incubated at 37℃in a 5% CO ₂ incubator. In the case of NK-92 transduced cells, the growth of the cultures was monitored every 48 hours and split to a final concentration of 0.5X10 ⁶ cells/ml by supplementing IL-2 (100 UI/ml) every 48 hours.

Transgenic expression of transduced immune cells was assessed by immunoblotting. Transduced cells (e.g., NK-92) were harvested by centrifugation at 1500rpm for 5 minutes at room temperature. The supernatant was removed and the cell pellet was washed twice in 1XPBS, then flash frozen in liquid nitrogen and stored at-80℃until further use. The cell pellet was then lysed in 200. Mu.l of SDS lysis buffer (catalog NP0008; novex) containing a1 XHALT protease inhibitor cocktail (catalog number 78430; thermoFisher, inc.) followed by sonication. The suspension was centrifuged at 15,000rpm for 15 minutes at room temperature, and the supernatant containing the total protein was collected. Total protein concentration was measured using Pierce 660nm protein assay (catalog number 1861426; semer Feier technologies Co. (Thermo Scientific)) followed by immunoblotting. 10 μg total protein was loaded into each lane of Novex ^TM to 12% Tris-Glycine Plus, 1.0mm, 20 well Midi protein gel (Invitrogen), transferred onto PVDF membranes using Transblot Turbo (burle company (Biorad)) and blocked for 1 hour at room temperature using LICOR blocking buffer. Transgenic (e.g., GOT2, TIGAR) in membranes were probed with mouse α -actin (3700S, CST; dilution 1:2000), rabbit α -TIGAR (14751S CST; dilution 1:1000) and rabbit α -Got2 (NBP 232241, novus; dilution 1:2000) antibodies overnight at 4 ℃ (in 0.1% Tween 20+LIcor blocking buffer). The next day, the membranes were washed three times for 5 minutes with 1 XTBS containing 0.1% Tween20 detergent (w/v). The membranes were then incubated with standard rabbit or mouse secondary antibodies (LICOR; dilution 1:10,000) for 1 hour. Membranes were washed three times for 5 minutes with 1 XTBS containing 0.1% Tween20 detergent (w/v). Immunoblots were imaged using a CLX imager (LICOR) and processed in Image Studio software (v 5.2; LICOR).

Example 17: analysis of CAR expression in transduced immune cells

Immune cells (e.g., NK and/or T cells) were isolated from fresh blood samples or derived from cell lines and transduced as described in example 16. On day 7 post transduction, cells were harvested by centrifugation at 1500rpm for 5 minutes at room temperature. The supernatant was removed and the cell pellet was washed twice in 1 XPBS, followed by staining with Live read Aqua (catalog number L34966; siemens Feisher company) for 10 minutes at room temperature. Cells were washed twice in 1X PBS followed by staining with primary and secondary antibodies in 1X PBS containing 2% FBS to assess CAR expression. Live single cells were selected and compared to non-transduced controls (null) to determine CAR expression. The data was analyzed using FlowJo 10.7.1 version of software (tree star (TREE STAR INC)).

Co-expression of the CAR construct alone or together with TIGAR or GOT2 has been demonstrated in NK92 cells, an IL-2 dependent NK cell line derived from lymphoma patients. Transgenic overexpression of cells harvested on day 7 was analyzed by immunoblotting (see figure 5). Due to endogenous expression of GOT2, GOT2 expression was observed in all constructs and controls, whereas a stronger band was observed in case of co-expression of GOT2 with CAR. No endogenous TIGAR expression was observed under these conditions. Only when TIGAR is co-expressed with CAR can strong expression be seen. In addition, harvested cells were stained with recombinant antibodies directed against the Fc portion of the CAR and the presence of CAR on the surface of NK cells was assessed using flow cytometry. Figure 6 shows that all constructs expressed CARs on the surface of NK92 cells.

OTHER EMBODIMENTS

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the foregoing description, one skilled in the art can readily ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the following claims.

Equivalent(s)

Although a number of embodiments of the invention have been described and illustrated herein, various other devices and/or structures for performing functions and/or obtaining results and/or one or more of the advantages described herein will be apparent to those of ordinary skill in the art, and each such variation and/or modification is deemed to be within the scope of embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, embodiments of the invention may be practiced otherwise than as specifically described and claimed. Embodiments of the invention of the present disclosure relate to each individual feature, system, article, material, kit, and/or method described herein. In addition, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, any combination of two or more such features, systems, articles, materials, kits, and/or methods is included within the scope of the present disclosure.

All definitions and uses herein are to be understood as controlling dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated herein by reference to the subject matter of each reference, which in some cases may encompass the entire document.

The indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one" unless explicitly indicated to the contrary.

As used herein in the specification, the phrase "and/or" should be understood to refer to "either or both" of such combined elements, i.e., the elements co-exist in some cases and in other cases separately. The various elements listed with "and/or" should be interpreted in the same manner, i.e. "one or more" of the elements so combined. In addition to elements specifically identified with the "and/or" clause, other elements may optionally be present, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "a and/or B" when used in conjunction with an open language such as "comprising" may refer in one embodiment to a alone (optionally including elements other than B); in another embodiment, only B (optionally including elements other than a); in yet another embodiment, both a and B (optionally including other elements); etc.

As used herein in the specification and claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" and/or "should be construed as inclusive, i.e., including at least one element of a plurality of elements or list of elements, but also including more than one element thereof, and optionally other unlisted items. Only conversely clearly indicated terms, such as "only one of …" or "exactly one of …" or "consisting of …" when used in the claims, will refer to exactly one element of a list comprising a plurality of elements or elements. In general, when there are exclusive terms in advance, such as "either," "one of …," "only one of …," or "exactly one of …," the term "or" as used herein should be interpreted to indicate only exclusive alternatives (i.e., "one or the other, not both"). "consisting essentially of …" when used in the claims should have the ordinary meaning as used in the patent statutes.

As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements is to be understood as meaning at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element specifically listed within the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements may optionally be present other than those specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, in one embodiment, "at least one of a and B" (or equivalently, "at least one of a or B," or equivalently "at least one of a and/or B") may refer to at least one optionally comprising more than one a, absent B (and optionally comprising elements other than B); in another embodiment, it may refer to at least one optionally comprising more than one B, absent a (and optionally comprising elements other than a); in yet another embodiment, it may refer to at least one optionally comprising more than one a, and optionally comprising at least one of more than one B (and optionally comprising other elements); etc.

It should also be understood that in any method claimed herein that comprises more than one step or operation, the order of the steps or operations of the method is not necessarily limited to the order of the steps or steps of the method recited unless explicitly stated to the contrary.

Claims

1. A genetically engineered immune cell having altered glucose metabolism as compared to a native immune cell of the same type, wherein the immune cell:

(i) Expressing or over-expressing a polypeptide that transfers or redirects glucose metabolites out of the glycolytic pathway; and

(Ii) A chimeric receptor polypeptide; wherein the chimeric receptor polypeptide comprises:

(a) An extracellular target binding domain;

(b) A transmembrane domain; and

(C) Cytoplasmic signaling domains.

2. The genetically engineered immune cell of claim 1, wherein the polypeptide (i) that transfers or redirects glucose metabolites is selected from the group consisting of: pyruvate kinase muscle isozymes M2 (PKM 2), glutamine-fructose-6-phosphate aminotransferase 1 (GFPT 1), and TP 53-inducible glycolysis and apoptosis regulator (TIGAR).

3. The genetically engineered immune cell of claim 2, wherein the polypeptide that transfers or redirects glucose metabolites is TIGAR.

4. The immune cell of any one of claims 1 to 3, wherein the chimeric receptor polypeptide comprises one or more of the following features:

(i) The chimeric receptor polypeptide further comprises at least one or no co-stimulatory signaling domain;

(ii) The cytoplasmic signaling domain (c) includes an immune receptor tyrosine based activation motif (ITAM);

(iii) The cytoplasmic signaling domain (C) is located at the C-terminus of the chimeric receptor polypeptide;

(iv) The chimeric receptor polypeptide further comprises a hinge domain located at the C-terminus of (a) and at the N-terminus of (b);

(v) The chimeric receptor polypeptide does not contain any hinge domain; and

(Vi) The chimeric receptor polypeptide further includes a signal peptide at its N-terminus.

5. The genetically engineered immune cell of any one of claims 3 to 4, wherein the chimeric receptor polypeptide is preferably a chimeric receptor antigen (CAR) polypeptide, wherein (ii) (a) is an extracellular antigen binding domain.

6. The genetically engineered immune cell of claim 5, wherein the extracellular antigen binding domain of (ii) (a) is a single chain variable fragment (scFv) or single domain antibody that binds to a tumor antigen, a pathogenic antigen, or an immune cell specific for a self antigen.

7. The genetically engineered immune cell of claim 6, wherein the extracellular antigen binding domain of (ii) (a) binds to the tumor antigen associated with a hematologic tumor or a solid tumor.

8. The genetically engineered immune cell of claim 6, wherein the extracellular antigen binding domain of (ii) (a) binds to the pathogenic antigen, which is a bacterial antigen, a viral antigen, or a fungal antigen.

9. The genetically engineered immune cell of any one of claims 1 to 8, wherein the transmembrane domain belongs to a membrane protein ：CD8α、CD8β、4-1BB、CD27、CD28、CD34、CD4、FcεRIγ、CD16A、OX40、CD3ζ、CD3ε、CD3γ、CD3δ、TCRα、TCRβ、TCRζ、CD32、CD64、CD45、CD5、CD9、CD22、CD37、CD80、CD86、CD40、CD40L/CD154、VEGFR2、FAS、FGFR2B、CD2、IL15、IL15R、IL21、DNAM-1、2B4、NKG2D、NKp44 and NKp46 selected from the group consisting of.

10. The genetically engineered immune cell of any one of claims 1 to 9, wherein the chimeric receptor polypeptide comprises the at least one costimulatory signaling domain that belongs to costimulatory molecules ：4-1BB、CD28、CD8α、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL, or a functional variant thereof, selected from the group consisting of.

11. The genetically engineered immune cell of any one of claims 1 to 10, wherein the at least one costimulatory signaling domain is a CD28 costimulatory signaling domain or a 4-1BB costimulatory signaling domain.

12. The genetically engineered immune cell of any one of claims 1 to 10, wherein the chimeric receptor polypeptide comprises two costimulatory signaling domains.

13. The genetically engineered immune cell of claim 12, wherein

(I) One of the co-stimulatory signaling domains is a CD28 co-stimulatory signaling domain; and wherein the other costimulatory domain is selected from the group consisting of ：CD8α、4-1BB、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL costimulatory signaling domains;

(ii) One of the co-stimulatory signaling domains is a CD 8a co-stimulatory signaling domain; and wherein the other costimulatory domain is selected from the group consisting of ：CD28、4-1BB、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL costimulatory signaling domains; or alternatively

(Iii) One of the costimulatory signaling domains is a 4-1BB costimulatory signaling domain; and wherein the other co-stimulatory domain is selected from the group ：CD8α、CD28、2B4、OX40、OX40L、ICOS、CD27、GITR、HVEM、TIM1、LFA1、CD2、DAP10、DAP12、DNAM-1、NKG2D、NKp30、NKp44、NKp46 and JAMAL co-stimulatory signaling domains consisting of.

14. The genetically engineered immune cell of any one of claims 1 to 13, wherein the cytoplasmic signaling domain of (c) is a cd3ζ or fcsr1γ cytoplasmic domain.

15. The genetically engineered immune cell of any one of claims 1 to 14, wherein the chimeric receptor polypeptide comprises the hinge domain (iv) which is a hinge domain selected from the list of: CD28, CD16A, CD a, igG, murine CD 8a and DAP12.

16. The genetically engineered immune cell of any one of claims 1 to 15, wherein the immune cell is an αβt, γδt or Natural Killer (NK) cell.

17. The genetically engineered immune cell of claim 16, wherein the immune cell is an αβ T cell, and wherein the chimeric receptor polypeptide is a CAR polypeptide comprising a component as shown in table 8.

18. The genetically engineered immune cell of claim 16, wherein the immune cell is an NK cell, and wherein the chimeric receptor polypeptide is a CAR polypeptide comprising the components as shown in table 9.

19. The genetically engineered immune cell of claim 16, wherein the immune cell is a γδ T cell, and wherein the chimeric receptor polypeptide is a CAR polypeptide comprising the components as set forth in table 10.

20. The genetically engineered immune cell of any one of claims 1 to 19, wherein

(I) The immune cells are derived from a cell line; or alternatively

(Ii) The immune cells are derived from Peripheral Blood Mononuclear Cells (PBMCs), hematopoietic Stem Cells (HSCs), umbilical cord blood stem cells, or induced pluripotent stem cells (ipscs).

21. The genetically engineered immune cell of any one of claims 1 to 20, wherein the immune cell comprises a nucleic acid or a set of nucleic acids that together comprise:

(A) A first nucleotide sequence encoding a factor that transfers or redirects glucose metabolites; and

(B) A second nucleotide sequence encoding the chimeric receptor polypeptide.

22. The genetically engineered immune cell of claim 21, wherein the immune cell comprises the nucleic acid comprising both the first nucleotide sequence and the second nucleotide sequence.

23. The genetically engineered immune cell of claims 21 to 22, wherein the nucleic acid further comprises a third nucleotide sequence located between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a ribosome jump site, an Internal Ribosome Entry Site (IRES) or a promoter.

24. The genetically engineered immune cell of claim 23, wherein the nucleic acid or group of nucleic acids is included within one or more viral vectors.

25. A pharmaceutical composition comprising the genetically engineered immune cell of any one of claims 1 to 24.

26. A method for inhibiting cells of a subject that express a target antigen, the method comprising administering to a subject in need thereof the population of genetically engineered immune cells of any one of claims 1-24 or the pharmaceutical composition of claim 25 comprising the population of genetically engineered immune cells.

27. The method for inhibiting cells expressing a target antigen in a subject according to claim 26, wherein the subject is a human patient having cancer and the target antigen is a tumor antigen; wherein the cancer is selected from the group consisting of: carcinomas, lymphomas, sarcomas, lymphomas and leukemias, preferably wherein

(I) The cancer is selected from the group consisting of: cancers of B cell origin, breast cancer, stomach cancer, neuroblastoma, osteosarcoma, lung cancer, skin cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, mesothelioma, pancreatic cancer, head and neck cancer, retinoblastoma, glioma, glioblastoma, liver cancer, and thyroid cancer; or alternatively

(Ii) The B cell derived cancer is selected from the group consisting of: acute lymphoblastic leukemia of the B lineage, chronic lymphoblastic leukemia of the B cells, and non-Hodgkin' slymphoma B-cell lymphoma.

28. A nucleic acid or set of nucleic acids, collectively comprising:

(A) A first nucleotide sequence encoding a factor that transfers or redirects glucose metabolites as described in any one of claims 1 to 3; and

(B) A second nucleotide sequence encoding the chimeric receptor polypeptide of any one of claims 4 to 24.

29. A method for producing a modified immune cell in vivo, the method comprising administering the nucleic acid or set of nucleic acids of claim 28 to a subject in need thereof.