Methods for increasing immune cell function and persistence

Abstract

Disclosed are methods for enhancing the memory-like state of immune cells by treating the cells with itaconate. Enhancing the memory-like state improves immune cell function and persistence. Also disclosed are methods of using the enhanced immune cells for treating cancer and autoimmune diseases.

Description

METHODS FOR INCREASING IMMUNE CELL FUNCTION AND PERSISTENCECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 668,733 filed on July 8, 2024. The content of which is incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (70258102689. xml; Size: (38,197 bytes; and Date of Creation: July 7, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Adoptive cell therapies with CAR-T cells and tumor infiltrating lymphocytes (TILs) are at the forefront of discovery as anti-cancer treatments. While adoptive T cell therapies have shown a lot of promise, these therapies do not generate durable responses. One of the primary reasons for the inefficiencies in response to adoptive T cell therapies is that T cells enter into a state of functional exhaustion after initial effector activity in vivo. To counteract these challenges, several new approaches are currently under development to prevent or reverse the dysfunctional states in adoptively transferred T cells. Several studies have shown that the state of exhaustion in T cells is epigenetically encoded and is influenced by metabolic pathways. Studies have shown that genetic targeting of epigenetic enzymes such as ten-eleven-translocation (TET) family of DNA demethylases, can promote similar enhancement of T cell function, however, TET enzymes are known tumor suppressors and the safety of this approach, remains in question. Therefore, alternative approaches of metabolically enhancing T cell function are of interest.SUMMARY

[0004] In an aspect, provided herein is a method for enhancing a memory-like state of an immune cell, the method comprising contacting the immune cell with an itaconate ester. The itaconate ester may be a cell permeant itaconate ester. The itaconate ester may be selected from 4-octyl-itaconate, dimethyl itaconate, and 4-ethyl itaconate. In embodiments, the itaconate ester is 4-octyl-itaconate.

[0005] The immune cell may be contacted for between about 1 and about 30 days. The immune cell may be contacted for at least about 10 days. The immune cell may be contacted with betweenabout 25 pmol / ml and about 400 pmol / ml 4-octyl-itaconate. The immune cell may be contacted with about 75 pmol / ml 4-octyl-itaconate.

[0006] The method may further comprise contacting the immune cell with IL-2. The IL-2 may be at about 20 ng / ml. The immune cell may be a T cell, a tumor infiltrating lymphocyte (TIL), a B cell, a natural killer (NK) cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell. The immune cell may express a chimeric antigen receptor (CAR) that targets at least one antigen expressed on a cancer cell. In embodiments, the immune cell is a CAR-T cell.

[0007] The method may further comprise, prior to contacting the CAR-T cell with 4-octyl- itaconate, engineering the T cell to express the CAR. The CAR may comprise an antigen binding region that targets CD 19 or a hapten. In embodiments, the antigen binding region targets CD 19. The antigen binding region may comprise a sequence having at least 95% identity to SEQ ID NO: 32. In embodiments, the antigen binding region targets the hapten. The hapten may be 4-hydroxy- 3-nitrophenylacetyl. The antigen binding region may comprise a sequence having at least 95% identity to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0008] The CAR may comprise an immune cell activation domain comprising at least two of a CD28 signaling domain, a 4-1BB domain, and a CD3(j signaling domain. The CAR may comprise a sequence having at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 31.

[0009] In another aspect, provided herein is an immune cell prepared by any of the methods described herein.

[0010] In another aspect, provided herein is a kit comprising: the immune cell described herein, wherein the immune cell is a CAR-T cell, wherein the CAR antigen binding region binds to a hapten; and a modified antibody conjugated to the hapten.

[0011] In another aspect, provided herein is a method for treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first pharmaceutical composition comprising the immune cell described herein. If the immune cell expresses a CAR that targets a hapten, the method may further comprise administering to the subject a therapeutically effective amount of a second pharmaceutical composition comprising a modified antibody conjugated to the hapten. The subject may have a cancer or an autoimmune disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0013] FIG. 1. Itaconate enhances the generation of T stem cell memory population in human CAR T cells. Human peripheral blood mononuclear cells were transduced with lentivirus for hCD19-CAR and hMASS-CAR. Cells were cultured with 4-octyl-itaconate 75 pmol / ml for 10 days. Flow plot shows the frequencies of T stem cell memory (TSCM). CD45RA+CD95+CCR7+CD62L+ population. Bar graph shows the frequency of TSCM. Oneway Anova.

[0014] FIG. 2. Itaconate enhances the T central memory population in human CAR T cells. Human peripheral blood mononuclear cells were transduced with lentivirus for hCD19-CAR and hMASS-CAR. Cells were cultured with 4-octyl-itaconate 75 pmol / ml for 10 days. Flow plot shows the frequencies of T stem cell memory (TSCM). CD45RA+CD95+CCR7+CD62L+ population. Bar graph shows the frequency of TSCM. One-way Anova.

[0015] FIG. 3. Use of itaconate during human CAR T cell generation enhances the cytotoxic CD8 T cells population. Human peripheral blood mononuclear cells were transduced with lentivirus for hCD19-CAR and hMASS-CAR. Cells were cultured with 4-octyl-itaconate 75 pmol / ml for 10 days. Bar graph shows the ratio of CD4 / CD8. 2-way ANOVA.

[0016] FIG. 4. Itaconate enhances the cytotoxicity of human CAR T cells in vitro. Human CAR T cells transduced with hMASS-CAR and hCD19 CAR were co-cultured with human lymphoma cell line BJAB. hMASS-CAR was combined with NP-labelled anti-human CD19 antibody. The cytotoxicity was measured by comparison to untreated BJAB cells. 2-way Anova.

[0017] FIG. 5. Itaconate decreases the level of exhaustion markers on CD8 T cells. Bar graph shows the percentage of PD+ and TIGIT+ cells. 2-way ANOVA.

[0018] FIGS. 6A-6B. Itaconate promotes a T cell memory phenotype in T cells. A. Flow plots showing T cells from Tet2 wildtype (WT) and Tet2 knockout (KO) cultured in mIL-2 (20ng / ml) with DMSO or Itaconate (lOOuM). B. Bar graph showing the frequency of T central memory (TCM) cells on Day 13.

[0019] FIG. 7A-7B. Itaconate promotes enhances the levels of TCF-1 in T cells. Itaconate treatment enhances the level of TCF-1 transcription factor. A. Histograms showing the percentage of TCF-1 high cells in DMSO and Itaconate (lOOuM) for ten days. B. The overlay histogram with median fluorescence intensity of TCF-1 in cells treated with DMSO and Itaconate.

[0020] FIG. 8. Itaconate treated CAR-T cells are cytotoxic and can persist in peripheral blood. Anti-human CD-19 CAR expressing mouse T cells were cultured in mIL-2 (20ng / ml) with DMSO or Itaconate (75uM) for ten days. CAR-T cells were then co-cultured with hCD19 expressing B16F10 melanoma cells at 1 :5 and 1 :25, tumor cell to CAR T cell ratio. Bar graph shows the percentage of live B16F10 cells after 18 hrs of co-culture.

[0021] FIGS. 9A-9C. CAR-T cells treated with itaconate establish a greater population of TSCM cells in the lymph nodes and spleen. Anti-human CD-19 CAR expressing mouse T cells were cultured in mIL-2 (20ng / ml) with DMSO or Itaconate (75uM) for ten days. DMSO and Itaconate (75uM) cells were transplanted into CD45.2 congenic mice. A. Mice were bled every week for 3 weeks. Displayed bar graph of CD45.1 transferred cells in peripheral blood. B. After 4 weeks post transplantation mice were sacrificed, spleen and lymph nodes were analyzed. B and C. Bar graphs showing the frequencies of Central memory, T stem cell memory and effector cells. Multiple t-test on graph pad prism.

[0022] FIG. 10. Metabolomics Data: CD19 CAR T cells were cultured with 75uM of Itaconate for 10 days. Cells were harvested and metabolomics was performed. Heatmap of top 50 metabolites significantly different among DMSO and Itaconate.

[0023] FIG. 11. 5hmc decreases in CD4 T cells treated with itaconate. Treatment with Itaconate 10 days at 75uM

[0024] FIG. 12. 5hmc decreases in CD8 T cells treated with itaconate. Treatment with Itaconate 10 days at 75uM

[0025] FIG. 13. Itaconate doesn’t alter the rate of expansion until day 10.

[0026] FIG. 14. Schematic of MASS-CAR strategy. The illustrated MASS-CAR consists of the leader sequence, signal sequence, anti-NP single chain variable fragment, CD28 and / or 41-BB, CD3 zeta, P2A, and Thy 1.1 domains. MASS-CAR recognizes NP conjugates to antibodies, nanobodies, ligands-receptor pairs, bi-specific or tri-specific T cell engagers, Natural Killer cell engagers, etc.

[0027] FIGS. 15A-15E. Itaconate enhances functional T cell memory in vivo. CD45.1 + and CD45.2+ P14 T cells were treated with itaconate or DMSO, respectively, for 10 days. After treatment, equal numbers of cells from each group were mixed at a 1 : 1 ratio, and a total of 50,000 cells per mouse were adoptively transferred. The following day, recipient mice were rechallenged with LCMV. Seven days post-rechallenge, spleens were harvested and analyzed for P 14 T cell expansion. (A) Representative flow cytometry plot showing the ratio of pre-labeled CD45.1+ (itaconate-treated) to CD45.2+ (DMSO-treated) P14 cells. (B) Quantification of the frequency of itaconate-treated versus untreated P14 cells. (C) Absolute numbers of total P14 cells recovered from the spleen. (D) Frequency of memory precursor effector cells (CD8+CD27+CD127+KLRG1-). (E) Frequency of terminal effector T cells (CD8+KLRG1+CD127-). Statistical significance is calculated by paired T-test (B-E). *p <0.05, ** p <0.01.

[0028] FIG. 16. Downregulated genes in Itaconate treated CAR T cells

[0029] FIG. 17. Upregulated genes in Itaconate treated CAR T cells

[0030] FIG. 18. Examples of differentially expressed genesDETAILED DESCRIPTION

[0031] In a first aspect, the present disclosure provides a method for enhancing the memory-like state of an immune cell, the method comprising contacting the immune cell with an itaconate ester.

[0032] The terms “memory-like state”, “stem-like state”, and “stem-like memory state” are used interchangeably herein to describe a condition of an immune cell in which an acquired immune response to an invading antigen is maintained long-term after the antigen has been eliminated. The memory -like state may be characterized by reduced levels of exhaustion markers (PD1 and TIGIT), reduced ten-eleven-translocation (TET) enzymatic activity, and / or increased inosine. Enhancing the memory-like state may prevent immune cells from entering a dysfunctional state, and it may yield better and more durable immune cell function.

[0033] The inventors found that 4-octyl-itaconate enhances the memory-like state of T cells, preventing the cells from entering a dysfunctional state at which they are no longer responsive to a specific antigen.

[0034] Itaconate is a fatty acid produced in the tricarboxylic acid (TCA) cycle. Studies suggest that itaconate and itaconate-like compounds act on the cell to regulate potentially deleteriousinflammation response caused by microorganisms, viruses, autoimmune disease, oxidative stress, and other tissue injuries. The itaconate ester may be a cell permeant itaconate ester. Itaconate-like compound 4-octyl itaconate has been used to mimic the biological effects of itaconate. This itaconate analog has a greater ability to pass through the cell membrane. Other analogs and metabolites of itaconate may be used, e.g. dimethyl itaconate, 4-ethyl itaconate, etc.

[0035] The immune cell may be contacted with the itaconate ester for between about 1 and about 30 days, or any duration or range in between. In exemplary embodiments, the immune cell is contacted with the itaconate ester for between about 10 and about 30 days. The immune cell may be contacted with the itaconate ester for at least about 10 days.

[0036] The immune cell may be contacted with between about 25 pmol / ml and about 400 pmol / ml 4-octyl-itaconate. The immune cell may be contacted with about 75 pmol / ml 4-octyl-itaconate.

[0037] The immune cell may be further contacted with IL-2. The IL-2 may be at about 20 ng / ml.

[0038] The term “contacting” refers to bringing the disclosed 4-octyl itaconate and the cell together in such a manner that the 4-octyl itaconate can cross the cell membrane. The contacting may be done by adding the 4-octyl itaconate to a medium or buffer containing the cell or a culture of the cell.

[0039] The immune cell may be a mammalian cell. In embodiments, the immune cell is a human cell. The immune cell may be a T cell, a natural killer (NK) cell, a B cell, an invariant natural killer T (iNKT) cell, a macrophage, an innate lymphoid cell, or any tumor-infiltrating immune cell. The T cell may be any adaptive T cell. The immune cell may be engineered to express a chimeric antigen receptor (CAR). Accordingly, the immune cell may be a be a tumor a chimeric antigen receptor T cell (CAR-T). The immune cell may also be a CAR-NK cell, CAR-B cell, a CAR- macrophage, or a CAR-iNKT cell. The immune cell may be derived from a subject, engineered to express the CAR, treated with the itaconate ester, then used to treat the subject. Any suitable means for delivering the CAR constructs and vectors to the host cell may be used including, but not limited to, transfection, transduction, transformation, and nanoparticle delivery.

[0040] The terms “chimeric antigen receptor”, “chimeric receptor”, and “CAR” refer to a polypeptide having a binding specificity to a desired target and operably connected to e.g., as a fusion or as separate chains linked by one or more disulfide bonds, etc.) the intracellular part of an immune cell activation domain. When expressed, a CAR is present at the plasma membrane of an immune cell. More particularly, CARs are engineered receptors which, when expressed, graftan antigen specificity onto a cytotoxic cell, such as a T cell, a natural killer (NK) cell, a macrophage, etc. For example, CARs are engineered to give T cells the new ability to target a specific protein. CARs comprise an extracellular domain having at least one antigen specific targeting region / antigen binding domain / antigen binding region that binds to an antigen, a transmembrane domain (TM), and an intracellular domain (ID) including a T cell activation domain, the T cell activation domain comprising one or more co- stimulatory domains (CSD), in a combination that is not naturally found together on a single protein. The antigen specific targeting region may be a single chain antibody (scFv). CARs known in the art typically bind to antigens expressed on tumor cells. In exemplary embodiments, the CAR expressed by the enhanced immune cells binds to CD 19. In other exemplary embodiments, the CAR bind to a hapten.

[0041] The CAR may comprise all elements necessary for the desired expression and function of the CAR. For example, the CAR may include a leader sequence and / or signal peptide for directing expression of the CAR at the cell membrane. The CAR may comprise a transmembrane domain. The CAR may comprise a hinge region linking the extracellular domain to the transmembrane domain. The intracellular domain may comprise one or two cytoplasmic costimulatory domains that enhance T cell activation. Costimulatory domains that may be included in the CARs described herein include, but are not limited, to CD28, CD3(^, CD27, CD134, and 4-1BB / CD137 domains. CARs may further include factors that enhance T cell expansion, persistence, and anti-tumoral activity, such as cytokine or cytokine signaling domains including, but not limited to, IL-2, IL- 12, JAK, and STAT3 / 5. In exemplary embodiments, the CAR comprises at least two of a CD28 costimulatory domain, a 4-1BB costimulatory domain, and a CD3(^ costimulatory domain. The CD28 costimulatory domain may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. The CD3(^ costimulatory domain may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. The 4-1BB costimulatory domain may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 18.

[0042] The CAR may be any generation of CAR. For Example, the CAR may be a first generationCAR having a single intracellular signaling domain, or a second generation CAR having a costimulatory domain (e.g. CD28, CD134 (OX-40), or CD137 (4-1BB)) to the intracellular signaling domain, a third generation CAR having a second intracellular costimulatory domain, a fourth generation CAR based on a second generation CAR but including a protein (e.g. a nuclear factor of the activated T-cell (NF AT)) that is constitutively or inducible expressed upon CAR activation, a fifth generation CAR based on a second generation CAR but including an intracellular domain of a cytokine receptor, etc.

[0043] In embodiments, the CAR comprises an antigen binding region that targets CD 19. The antigen binding region may comprise a sequence having at least 95% identity to SEQ ID NO: 32.

[0044] In embodiments, the CAR comprises an antigen binding region that targets a hapten. In such embodiments, the CAR-immune cell also expresses a modified antibody labeled with the hapten. The hapten may be a nitrophenol (NP). The nitrophenol may be a di -nitrophenol, a trinitrophenol, or any other nitrophenol. The hapten may be 4-hydroxy-3 -nitrophenylacetyl group. The modified antibody may be labeled with multiple molecules of the hapten. The modified antibody may be labeled with at least two different haptens. The antibody may be modified with the hapten by lysine conjugation, cysteine conjugation or any other suitable labeling techniques. (See, e.g., Stephen J. Walsh et al. Chem Soc Rev 2021, 50, 1305-1353). An immune cell expressing a CAR comprising an antigen binding region targeting a hapten, and a modified antibody labeled with the hapten employs a strategy “MASS-CAR (Modified Antibody Specific Switchable CAR)”, illustrated in FIG. 14, which uses chemical labeling of antibodies to direct CAR-T cell activity to specific tumors while also leveraging the therapeutic benefits of antibody biologies, leading to more holistic anti-tumor immune responses. The MASS-CAR strategy and methods for expressing a MASS-CAR system in an immune cell are further described in International Application No. PCT / US2024 / 022358, which is incorporated herein by reference in its entirety.

[0045] The hapten-modified antibody may be of any specificity, and antibodies of more than one specificity, such as bispecific antibodies, may be modified with haptens for use as described herein. The hapten-modified antibody may be specific to a tumor specific antigen. Tumor specific antigens include, but are not limited to, CD19, HER2, BCMA, CD33, CD123, PD1, PDL1, Tim3, Timl, LAG3, TIGIT, TNFRSF17, SDC1, MS4A1, CD22, TNFRSF8, CD33, CD38, CDS, NCAM1, CD70, ULBP1, ULBP2, IL 1 RAP, CEACAMS, MET, EPHA2, ERBB2, GPC3, Mucl, PDCD1,CD274, KDR, IL13RA2, F0LH1, FAP, CA9, FOLR1, LI CAM, R0R1, CD23, CD44, CD174, SLAMF7, GD2, PSCA, GPNMB, CD276, CSPG4, CD133, TEM1PSA, TRP-2, EpCAM, GPC3, MSLN, EGFR, EGFRVIII, p21,RAS, p53, CDK-4, -catenin, Notch receptors, and MAGE. The hapten-modified antibody may be specific to an antigen associated with an autoimmune disease. Antigens associated with autoimmune diseases include, but are not limited to aminoacyl tRNA synthetase, Mi-2, signal recognition particle (SRP), transcriptional intermediary factor 1 -gamma (TIFl-y; TRIM 33), nuclear matrix protein-2 (NXP2; M0RC3), 3-hydroxy-3-methylgultaryl-coA reductase (HMGCR), melanoma-associated differentiation gene-5 (MDA5), small ubiquitin-like modifier activating enzymes SAE-1 and SAE-2, topoisomerase- 1, centromere proteins A, B & C (“CENPs”), fibrillarin, nucleophosmin (NPM; B23), RNA polymerases I, II & III, EXOSC9, EXOSCIO, Ku 70 / 80 components of DNA-dependent protein kinase, Ro52 (TRIM 21), Ro60, La (SS-B), gamma interferon-inducible protein- 16 (IFI16), proteinase-3 (PR3), myeloperoxidase (MPO), a citrullinated protein, peptidyl arginine-deiminase-4 (PAD4), double stranded DNA (dsDNA), a component of the Sm splicing ribonucleoprotein, Ul-RNP, ribosomal protein P, cardiolipin, an anionic phospholipid / protein complex, N-methyl-D-aspartate (NMDA) receptor, and tissue transglutaminase.

[0046] A “hapten” is a small molecule that elicits an immune response only when attached to a large carrier, such as a protein. Nitrophenols (NPs) are a known class of small molecule haptens that are immunogenic when conjugated to a carrier. The nitrophenol may be a di -nitrophenol, a trinitrophenol, or any other nitrophenol. In exemplary embodiments, nitrophenol is 4-hydroxy-3- nitrophenylacetyl. The hapten may be digoxigenin. The engineered polynucleotide antigen binding region may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 2 (NP binding domain), SEQ ID NO: 3 (NP binding domain with a W33L mutation), or SEQ ID NO: 4 (NP binding domain with a K59R mutation).

[0047] In exemplary embodiments, the CAR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1 (NP CAR with CD3ij and CD28 signaling domains), SEQ ID NO: 5 (NP CAR with CD3(^ and CD28 signaling domains; W33L NP domain mutant), SEQ ID NO: 6 (NP CAR with CD3ij and CD28 signaling domains; K59R NP domain mutant), SEQ ID NO: 7 (NP CAR with CD3(j and 4-1BB signaling domains),SEQ ID NO: 8 (NP CAR with CD3(j and 4-1BB signaling domains; W33L NP domain mutant), SEQ ID NO: 9 (NP CAR with CD3(^ and 4-1BB signaling domains; K59R NP domain mutant), SEQ ID NO: 10 (NP CAR with CD3i 4-1BB and CD28 signaling domains), SEQ ID NO: 11 (NP CAR with CD3(^, 4-1BB and CD28 signaling domains; W33L NP domain mutant), SEQ ID NO: 12 (NP CAR with CD3(^, 4-1BB and CD28 signaling domains; K59R NP domain mutant), or SEQ ID NO: 22 (NP CAR with CD3 , 4- IBB and CD28 signaling domains; K59R NP domain mutant).

[0048] The term “antibody” as used herein refers to immunoglobulin molecules or other molecules which comprise an antigen binding domain, and is intended to include whole antibodies (e.g., IgG, IgA, IgE, IgM, or IgD), monoclonal antibodies, chimeric antibodies, humanized antibodies, and antibody fragments, including single chain variable fragments (ScFv), single domain antibodies (nanobodies), and antigen-binding fragments, genetically engineered antibodies, among others, as long as the characteristic properties (e.g., ability to bind CD30) are retained. The term "antibody fragment" as used herein is intended to include any appropriate antibody fragment that displays antigen binding function, for example, Fab, Fab', F(ab')2, scFv, Fv, dsFv, ds-scFv, Fd, mini bodies, monobodies, and multimers thereof and bispecific antibody fragments.

[0049] As stated above, antibody fragments / antigen binding fragments comprise an antigen binding domain. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (e g. single chain antibodies or single chain Fv (scFv), (see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)).

[0050] The term "fragment" as used herein refers to fragments of biological relevance (functional fragment), e.g., fragments which can contribute to or enable antigen binding, e.g., form part or all of the antigen binding site or can contribute to the prevention of the antigen interacting with its natural ligands. Fragments may comprise a heavy chain variable region (VH domain) and light chain variable region (VL). Fragments may comprise one or more of the heavy chain complementarity determining regions (CDRHs) of the antibodies or of the VH domains, and one or more of the light chain complementarity determining regions (CDRLs), or VL domains to form the antigen binding site.

[0051] Antibodies can be genetically engineered from CDRs and monoclonal antibody sequences by using conventional techniques such as, for example, synthesis by recombinant techniques or chemical synthesis. Techniques for producing antibody fragments are well known and described in the art.

[0052] One may wish to engraft one or more CDRs from monoclonal antibodies into alternate scaffolds. For example, standard molecular biological techniques can be used to transfer the DNA sequences encoding the antibody's CDR(s) to (1) full IgG scaffold of human or other species; (2) a scFv scaffold of human or other species, or (3) other specialty vectors. If the CDR(s) have been transferred to a new scaffold all of the previous modifications described can also be performed. For example, one could consult Biotechnol Genet Eng Rev, 2013, 29:175-86 for a review of useful methods.

[0053] The antibodies can be wholly or partially synthetically produced. Thus, the antibody may be from any appropriate source, for example recombinant sources and / or produced in transgenic animals or transgenic plants. Thus, the antibodies can be produced in vitro or in vivo. The antibodies may comprise all or a portion of a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgE, IgM or IgD constant region.

[0054] The antibodies may comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions may be produced wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art.

[0055] The term "complementarity determining regions" or "CDRs," as used herein, refers to part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B-cells and T-cells respectively, where these molecules bind to their specific antigen. As the most variable parts of the molecules, CDRs are crucial to the diversity of antigen specificities generated by lymphocytes. There are three CDRs (CDR1, CDR2 and CDR3), arranged non-consecutively, on the amino acid sequence of a variable domain of an antigen binding site. Since the antigen binding sites are typically composed of two variable domains (on two different polypeptide chains, heavy and light chain), there are six CDRs for each antigen binding site that can collectively come into contact with the antigen. A single whole antibody has two antigen binding sites and therefore contains twelve CDRs. Sixty CDRs can be found on a pentameric IgM molecule.

[0056] Within the variable domain, CDR1 and CDR2 may be found in the variable (V) region of a polypeptide chain, and CDR3 includes some of V, and all of diversity (D, heavy chains only) and joining (J) regions. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Among these, CDR3 shows the greatest variability as it is encoded by a recombination of VJ in the case of a light chain region and VDJ in the case of heavy chain regions. The tertiary structure of an antibody is important to analyze and design new antibodies. The human VH complex is composed of approximately 100 gene segments per haploid genome, including at least 51 functional genes, as judged by successful rearrangement in cloned cDNA. On the basis of nucleic acid sequence homology, the VH genes have been grouped into 6-7 families (VH 1-7). Among the seven families, the VH3 family is the largest. The antibodies disclosed herein may be derived from the VH3-74 family or the VH3-33 family or its paralog.

[0057] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibodies of a single amino acid composition that specifically binds to a single epitope of the antigen.

[0058] The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Other forms of "chimeric antibodies" are those in which the class or subclass has been modified or changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies." Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. In some embodiments, the antibodies are chimeric antibodies including heavy chain constant domains from non-human mammals (e.g., mouse, rat, rabbit, or non-human primate). In some embodiments, the antibodies disclosed in the present invention are chimeric antibodies including constant regions from rabbit heavy chain immunoglobulin sequences. Suitable heavy chain constant region sequences from non-human mammals, including mouse, rat, rabbit, and non-human primate are known in the art.

[0059] The antibodies may be human antibodies, as they may include the constant region from human germline immunoglobulin sequences. The term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from a host cell such as an SP2-0, NS0 or CHO cell (like CHO KI) or froman animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and, in some embodiments, constant regions derived from human germline immunoglobulin sequences in a rearranged form.

[0060] An antibody may also be T cell engager or a natural killer (NK) cell engager. A “T cell engager” refers to an antibody engineered to bind a target antigen expressed on a cancer cell and at least one molecule on a T cell, such as CD3. As such, T cell engagers are multispecific. The terms “bispecific T cell engager”, “BiTE”, and “BTE” are used interchangeable herein and refer to the antibody that are capable of binding two distinct antigens at the same time. Particularly, the bispecific antibodies are capable of binding to the surface of tumor cells and T cells simultaneously, allowing for activation of the T cells and the targeting killing of tumor cells bound to the bispecific antibody.

[0061] The T cell engager may be tri-specific. The term "tri-specific" means that the binding protein is able to specifically bind to at least three distinct moieties (e.g., antigen binding sites). Typically, a tri-specific molecule comprises three different binding sites, each of which is specific for a different moiety (e.g., antigen). The tri-specific binding proteins described herein may be capable of simultaneously binding three moieties, particularly three moieties expressed on two distinct cells (e.g., a tumor cell and a T cell). The tri-specific T cell engagers may be capable of binding two cancer cell antigens and a T cell antigen. The terms “trispecific T cell engager”, “TriTE”, and “TrTE” are used interchangeable herein and refer to the antibodies that are capable of binding three distinct antigens at the same time. Particularly, the tri-specific antibodies are capable of binding to the surface of tumor cells and T cells simultaneously, allowing for activation of the T cells and the targeting killing of tumor cells bound to the tri-specific antibodies.

[0062] "Percentage of sequence identity" or "percent similarity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield thepercentage of sequence identity.

[0063] The term "substantial identity" or "substantial similarity" of polynucleotide or peptide sequences means that a polynucleotide or peptide comprises a sequence that has at least 75% sequence identity. Alternatively, percent identity can be any integer from 75% to 100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0064] As used herein, the terms “protein” or “polypeptide” or “peptide” may be used interchangeable to refer to a polymer of amino acids. Typically, a “polypeptide” or “protein” is defined as a longer polymer of amino acids, of a length typically of greater than 50, 60, 70, 80, 90, or 100 amino acids. A “peptide” is defined as a short polymer of amino acids, of a length typically of 50, 40, 30, 20 or less amino acids. A protein typically comprises a polymer of naturally or non- naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0065] In a second aspect, provided herein is an immune cell prepared by the methods described herein. The immune cell may exhibit an enhanced memory-like state.

[0066] In a third aspect, provided herein is a pharmaceutical composition comprising the immune cell prepared by the methods described herein and a pharmaceutically acceptable delivery vehicle.

[0067] As used herein, the term “pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammal. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Examples of compositions appropriate for such therapeutic applications include preparations for parenteral, subcutaneous, transdermal, intradermal, intramuscular, intracoronarial, intramyocardial, intraperitoneal, intravenous or intraarterial (e.g., injectable), orintratracheal administration, such as sterile suspensions, emulsions, and aerosols. In some cases, pharmaceutical compositions appropriate for therapeutic applications may be in admixture with one or more pharmaceutically acceptable excipients, diluents, or carriers such as sterile water, physiological saline, glucose or the like.

[0068] In a fourth aspect, provided herein is a kit comprising the immune cell described herein, wherein the immune cell is a CAR-T cell that targets a hapten, and a modified antibody conjugated to the hapten. The engineered immune cell and hapten-modified antibody are able to functionally interact with each other in such a way that when the modified antibody binds to the host cell and a target cell, e.g. a cancer cell, the immune cell exerts an immune response upon the target cell. Activation of the immune response may include T cell activation and response, including cytotoxicity, as well as a B cell or antibody mediated response. The immune cell and the hapten- modified antibody may be provided in separate containers.

[0069] In a fifth aspect, provided herein is a method of treating a subject in need thereof by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the memory-like enhanced immune cell described herein. The subject may have cancer or an autoimmune disease. The subject may be in need of an induced or enhanced immune response.

[0070] Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Treating a cancer can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; (d) reducing or ameliorating at least one symptom of cancer; and (e) extending the survival of the subject. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. When the engineered host cell is an immune cell, the term “treatment” may further include activating an immune response in the subject, wherein the immune response targets the cancer cells. The cancer may be a solid tumor or a hematological malignancy. Cancers that may be treated using the compositions and methods described herein include, but are not limited to B cell cancers, myeloid leukemias, T cell cancers, breast cancers, pancreatic cancers, colorectal cancers, brain cancers, head and neck cancers, melanomas, etc.

[0071] Autoimmune diseases are a group of diseases arising from when the body’ s immune systemmistakenly attacks healthy tissues, organs, and cells. In methods for treating autoimmune diseases, the hapten-modified antibody may be specific to an antigen associated with an autoimmune disease. Treating an autoimmune disease can be characterized by reducing are preventing one or more symptoms caused by an autoimmune disease. Symptoms include, but are not limited to fatigue, frequent fevers, joint pain and swelling, stomach pain or digestion issues, and swollen glands. Autoimmune diseases that may be treated using the compositions and methods described herein include, but are not limited to from scleroderma, Sjogren’s syndrome, vasculitis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and celiac disease. For further information, see Rosen and Casciola-Rosen “Autoantigens as Partners in Initiation and Propagation of Autoimmune Rheumatic Diseases” Ann. Rev. Immunol. 2016 May 20;34:395-420, which is incorporated herein by reference.

[0072] The method may further comprise administering to the subject a therapeutically effective amount of the hapten labeled antibody described herein when the immune cell is a CAR-T cell targeting the hapten described herein. In embodiments, a pharmaceutical composition comprising the immune cell is administered first, followed by a pharmaceutical composition comprising the modified antibody. The immune response generated by the engineered host cell and hapten- modified antibody can be increased or decreased if changes to the administration of either composition is altered. For example, an immune response will be generated if the engineered cell and the hapten-modified antibody interact, however if administration of the hapten-modified antibody is stopped, the immune response will be less activated. Alternatively, if administration of the engineered host cell is stopped, the host cell-specific immune response will decrease.

[0073] An “effective amount” or a “therapeutically effective amount” means the amount of the immune cell or modified antibody sufficient to treat the cancer. The amount of the pharmaceutical compositions to be administered is dependent on a variety of factors, including the severity of the condition, the age, sex, and weight of the subject, the frequency of administration, the duration of treatment, and the like. The pharmaceutical compositions may be administered at any suitable dosage, frequency, and for any suitable duration necessary to achieve the desired therapeutic effect. Either or both of the pharmaceutical composition may be administered once per day or multiple times per day, e.g. twice per day or three or more times per day. Either or both of the composition may be administered daily, every other day, every three days, every four days, every five days, every six days, once per week, once every two weeks, or less than once every two weeks.The compositions may be administered for any suitable duration to achieve the desired therapeutic effect, i.e., enhanced transplant tolerance. For example, either or both of the compositions may be administered to the subject for one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, one month, two months, three months, six months, 1 year, or more than 1 year. Any suitable dose of the pharmaceutical compositions may be used. Suitable doses will depend on the therapeutic agent, intended therapeutic effect, body weight of the individual, age of the individual, and the like. In general, suitable dosages of the disclosed nanocarriers or pharmaceutical compositions comprising the same may range from about 0.025 mg nanocarrier / kg body weight to 200 mg nanocarrier / kg body weight. For example, suitable dosages may be about 0.025 mg / kg, about 0.03 mg / kg, about 0.05mg / kg, about 0.10 mg / kg, about 0.15mg / kg, about 0.30mg / kg, about 0.5mg / kg, about 0.75 mg / kg, about l.Omg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 1.75 mg / kg, about 2.0 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, or about 200 mg / kg.

[0074] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations, but nonetheless, may be ascertained by the skilled artisan from this disclosure, the documents cited herein, and the knowledge in the art.

[0075] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any agent of the present technology used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0076] As used herein, the term “administering” refers to dispensing or delivering the therapeutic to the subject by any suitable route for delivery of the therapeutic agent to the desired location inthe subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, transdermal delivery, and administration by the intranasal or respiratory tract route. In exemplary embodiments, the compositions are administered by intravenous injection.

[0077] Formulations may be designed or intended for parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intraperitoneal, intrathecal, intraocular and epidural) administration. In general, aqueous and non-aqueous liquid or cream formulations are delivered by a parenteral, oral or topical route. In other embodiments, the compositions may be present as an aqueous or a non-aqueous liquid formulation or a solid formulation suitable for administration by any route, e.g., oral, topical, buccal, sublingual, parenteral, aerosol, a depot such as a subcutaneous depot or an intraperitoneal or intramuscular depot. In some cases, pharmaceutical compositions are lyophilized. In other cases, pharmaceutical compositions as provided herein contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 20thedition, 2000, ed. A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J., USA) or phosphate buffered saline (PBS).

[0078] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The preparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vialsof diluent, syringes and needles) for a course of treatment. Tn all cases, a composition for parenteral administration must be sterile and should be formulated for ease of injectability. The composition should be stable under the conditions of manufacture and storage, and must be shielded from contamination by microorganisms such as bacteria and fungi.

[0079] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fdtered solution thereof.

[0080] The preferred route may vary with, for example, the subject’s pathological condition or age or the subject’s response to therapy or that is appropriate to the circumstances. The formulations can also be administered by two or more routes, where the delivery methods are essentially simultaneous or they may be essentially sequential with little or no temporal overlap in the times at which the composition is administered to the subject.

[0081] The term “subject” or “patient” are used herein interchangeably to refer to a mammal, such as a human, to be treated by the methods and compositions described herein. “Mammals” means any member of the class Mammalia including, but not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. The subject may be a human.

[0082] In embodiments, the method of treatment may further comprise administering another anticancer treatment. Cancer treatment includes, but is not limited to chemotherapy, radiation, bone marrow transplant, surgery and immunotherapy.

[0083] Miscellaneous

[0084] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0085] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context inwhich they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0086] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0087] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0088] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally wouldbe accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0089] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0090] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0091] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0092] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0093] EXAMPLES

[0094] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0095] Example 1

[0096] To identify the epigenetic and metabolic regulators of stem-like states in T cells, we performed a screen to test known naturally occurring metabolites that transiently inhibit TET enzymatic activity to induce a memory-like state in T cells. In order to measure the activity of TET enzymes in the CAR-T cells, 5-hydroxy-methylcytosine (5hmc) was immunofluorescence stained and quantified using flow cytometry. 5hmc is a product of TET catalytic activity converting 5- methylcytosine to 5hmc. Itaconate has been reported to be a competitive inhibitor of dioxygenases, such as TET enzymes, by binding to the same site on the TET enzymes as the crucial cofactor a- ketoglutarate which is essential for TET activity. In CAR-T cells treated with itaconate, there is a two-fold decrease in 5hmc mean fluorescence intensity indicating inhibition of TET activity as expected (FIGS. 11-12).

[0097] Through these studies we identified itaconate as potential candidate which is shown to dampen TET enzymatic activity through either direct allosteric inhibition and indirect effects on other cellular metabolites, such as succinate and alpha-ketoglutarate. Itaconate is a naturally occurring metabolite generated by the conversion of the TCA cycle metabolite cis-aconitate by the Aconitase Decarboxylase 1. Transient treatment of T cells with itaconate (the cell permeant form, 4-octyl-itaconate) led to generation of both human and mouse T cells with a strong memory-like signatures and reduced expression of markers associated with T cell exhaustion (FIGS. 1-2). These effects were apparent in both activated T cells and T cells expressing Chimeric antigen receptors (FIG. 2). Transfer of itaconate treated T cells, also led to enhanced persistence in vivo in a memory state (FIG. 9). Moreover, itaconate treatment further enhanced memory-like phenotypes in TET2 KO cells (FIG. 6), suggesting that the effects of itaconate go beyond just TET inhibition.

[0098] Metabolic characterization of T cells treated with itaconate revealed upregulation ofinosine (FTG. 11), indicating that itaconate treatment triggers a metabolic circuit that may further enhance T cell function. In a metabolic study, CAR-T cells treated with itaconate show higher expression of several metabolites associated with increased antitumor efficacy such as proline, inosine, taurine, and pantothenic acid. The metabolism of proline and presence of taurine have been associated with increased T cell effector function by virtue of decreased apoptosis and greater cytokine secretion (Ye, L. et al, 2022, Ping, Y. et al, 2022). Additionally, the presence of inosine in T cells has been linked to increased sternness of T cells and, consequently, better CAR-T efficacy (Klysz, D. D. et al, 2024). Itaconate-treated cells also show downregulation of alanine, a metabolite crucial for T cell activation whose absence promotes T cell memory differentiation (Wang, W. and Zou, W., 2020). Furthermore, the downregulation of L-arginino-succinate, an intermediate in the urea cycle responsible for ammonia detoxification, in treated cells may suggest an increased rate of detoxification which has been linked to greater CD8 memory differentiation (Tang, K. et al, 2023). Interestingly, the metabolite cystathionine is shown to be downregulated in treated cells. Cystathionine is a crucial component of the transsulfuration pathway that regulates synthesis of SAM, the ubiquitous methyl donor, and sulfur containing amino acids such as methionine and cysteine. A byproduct of the transsulfuration pathway is hydrogen sulfide. It has been previously reported that disruptions in the pathway leading to hydrogen sulfide deficiencies have resulted in lower expression of TET1 and TET2 in T cells (Yang, R. et al, 2015). Altogether our work identifies itaconate as an epigenetic and metabolic modifier (Epimod) which is a promising candidate for boosting T cell function in the context of adoptive T cell therapies.

[0099] Several studies have shown that the patients with less CD4 / CD8 have better response to CAR T cell therapy. Use of itaconate further reduces CD4 / CD8 expression. (FIG. 3).

[0100] Cytotoxicity of MASS-CAR cells and CAR-T cells is enhanced when they’re cultured with itaconate (FIGS. 4 and 8).

[0101] Itaconate decreases the level of exhaustion markers (PD1 and TIGIT) on CD8 T cells (FIG. 5).

[0102] Itaconate also enhances the levels of TCF-1, a transcription factor that promotes the differentiation of memory cells, in T cells. (FIG. 7).

[0103] Itaconate does not alter the rate of expansion of CAR T and MASS-CAR cells (FIG. 13).

[0104] Example 2

[0105] Experimental design for testing the effect of itaconate on T cell memory. To evaluate whether itaconate enhances T cell memory responses in vivo, we utilized the LCMV Armstrong infection model. CD8+P14 T cells — derived from transgenic mice expressing a TCR specific for the LCMV glycoprotein epitope GP33-41 — were cultured for 10 days in the presence or absence of 75 uM itaconate, along with IL-2 to support expansion. Following treatment, equal numbers of itaconate-treated and untreated P14 cells were mixed at a 1 : 1 ratio, and a total of 50,000 cells were adoptively transferred into each recipient mouse. The results show that itaconate treatment increased the expansion of the T cells (FIG. 15).

[0106] Bulk RNA sequencing of CAR-T cells treated with or without Itaconate. MASS-CAR and CD19-CAR T cells were transduced and cultured in the presence or absence of 75 qM itaconate for 10 days. Following treatment, cells were harvested and processed for bulk RNA sequencing. Differentially expressed genes (DEGs) were identified by comparing itaconate-treated versus untreated CAR-T cells within both CAR construct groups. FIGS. 16-18, and Tables 1 and 2 show differentially regulated genes in itaconate treated CAR T cells.

[0107] Table 1. Downregulated genes

[0108] Table 2. Upregulated genes

[0109] Table3. Informal Sequence Listing

Claims

CLAIMSWhat is claimed is:

1. A method for enhancing a memory-like state of an immune cell, the method comprising contacting the immune cell with an itaconate ester.

2. The method of claim 1, wherein the itaconate ester is a cell permeant itaconate ester.

3. The method of claim 2, wherein the itaconate ester is selected from 4-octyl-itaconate, dimethyl itaconate, and 4-ethyl itaconate.

4. The method of claim 3, wherein the itaconate ester is 4-octyl-itaconate.

5. The method of any one of claims 1-4, wherein the immune cell is contacted for between about 1 and about 30 days.

6. The method of claim 5, wherein the immune cell is contacted for at least about 10 days.

7. The method of any one of claims 4-6, wherein the immune cell is contacted with between about 25 pmol / ml and about 400 pmol / ml 4-octyl-itaconate.

8. The method of claim 7, wherein the immune cell is contacted with about 75 pmol / ml 4- octyl -itaconate.

9. The method of any one of claims 1-8, further comprising contacting the immune cell with IL-2.

10. The method of claim 9, wherein the IL-2 is at about 20 ng / ml.11 . The method of any one of claims 1-10, wherein the immune cell is a T cell, a tumor infiltrating lymphocyte (TIL), a B cell, a natural killer (NK) cell, an invariant natural killer T (iNKT) cell, a macrophage, or an innate lymphoid cell.

12. The method of claim 11, wherein the immune cell expresses a chimeric antigen receptor (CAR) that targets at least one antigen expressed on a cancer cell.

13. The method of claim 12, wherein the immune cell is a CAR-T cell.

14. The method of claim 13, wherein prior to contacting the CAR-T cell with 4-octyl- itaconate, the T cell is engineered to express the CAR.

15. The method of any one of claims 12-14, wherein the CAR comprises an antigen binding region that targets CD 19 or a hapten.

16. The method of claim 15, wherein the antigen binding region targets CD 19.

17. The method of claim 16, wherein the antigen binding region comprises a sequence having at least 95% identity to SEQ ID NO: 32.

18. The method of claim 15, wherein the antigen binding region targets the hapten.

19. The method of claim 18, wherein the hapten is 4-hydroxy-3-nitrophenylacetyl.

20. The method of claim 18 or 19, wherein the antigen binding region comprises a sequence having at least 95% identity to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

21. The method of any one of claims 12-20, wherein the CAR comprises an immune cell activation domain comprising at least two of a CD28 signaling domain, a 4- IBB domain, and a CD3^ signaling domain.

22. The method of claim 21, wherein the CAR comprises a sequence having at least 95% identity to SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 22, or SEQ ID NO: 31.

23. An immune cell prepared by the method of any one of claims 1-19.

24. A kit comprising: the immune cell of claim 23, wherein the immune cell is a CAR-T cell, wherein the CAR antigen binding region binds to a hapten; and a modified antibody conjugated to the hapten.

25. A method for treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a first pharmaceutical composition comprising the immune cell of claim 23.

26. The method of claim 25, wherein when the immune cell expresses a CAR that targets a hapten, the method further comprises administering to the subject a therapeutically effective amount of a second pharmaceutical composition comprising a modified antibody conjugated to the hapten.

27. The method of claim 25 or 26, wherein the subject has a cancer or an autoimmune disease.

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