Tumor infiltrating lymphocytes

Abstract

A method for reprogramming native CD4+ T-cells is provided. The method comprises incubating the CD4+ T-cells with a Class 1 HDAC inhibitor for a period of time sufficient to increase cytotoxicity of the CD4+ T-cells in comparison to the cytotoxicity of native CD4+ T-cells. The method may be utilized to prepare cytotoxic tumor infiltrating CD4+ T-cells for use to treat cancer, including MHC-I deficient cancers.

Description

TUMOR INFILTRATING LYMPHOCYTESFIELD OF THE INVENTION

[0001] The present invention generally relates to the reprogramming of immune cells, and in particular, to the methods of preparing tumor-infiltrating lymphocytes useful in the treatment of cancers.BACKGROUND

[0002] T-cells are critical contributors to cancer immunity. CD8+T-cells, in particular, play a pivotal role in mediating anti-tumor immune responses by recognizing and eliminating cancerous cells through the detection of cancer peptides presented on major histocompatibility complex-I (MHC-I). However, tumors have evolved various strategies to evade CD8+T-cell responses, including the downregulation of MHC-I expression. This adaptation is crucial for avoiding T-cell recognition and is observed in 40-90% of human tumors. The downregulation of MHC-I can be either reversible or irreversible. Notably, irreversible MHC-I deficiency is a characteristic feature in several cancer types, including melanoma, head and neck squamous cell carcinoma (HNSCC), breast, lung, bladder, and colorectal cancer. These irreversible cases present a significant challenge to the effective application of current immunotherapies.

[0003] Adoptive cell therapy (ACT) is an emerging cancer treatment strategy that has demonstrated promising complete and long-term responses in clinical trials. ACT involves the ex- vivo modification of patient-derived T-cells through artificial or genetic engineering to enhance their tumoricidal capabilities, followed by their reinfusion into the patient to specifically target cancer cells. These therapies primarily rely on the cytolytic capabilities of CD8+T-cells, which identify and destroy cancer cells through the recognition of peptide-MHC-I complexes. However, the effectiveness of ACT is limited in tumors that lack MHC-I complexes. This limitation has prompted the exploration of alternative methods to effectively target and combat MHC-I deficient tumors.

[0004] Recent discoveries discussed a subset of CD4+tumor-infiltrating lymphocytes (TILs) with cytolytic properties similar to CD8+T-cells. These CD4+TILs show an upregulation in the expression of cytotoxic molecules including Perforin (Prf) and Granzyme B (GzmB) across6366488511various cancer types. These cells have also been identified in bladder cancer, utilizing Prf and GzmB for effective tumor elimination in an MHC-II-dependent manner. In a similar study, CD4+TILs efficiently eliminate melanoma cancer cells through direct and indirect recognition of the MHC-II complex in a murine model. In addition, these cytotoxic CD4+TILs in melanoma, head and neck squamous cell carcinomas (HNSCC), breast cancer, and hepatocellular carcinoma, with increased GzmB levels, were reported previously. While these cell subsets have potent tumoricidal activities, their origins and the key factors contributing to their cytotoxic transition remain elusive.

[0005] Recent discoveries uncover the potential role of human gut microbiota in modifying the therapeutic responses in cancer patients. Clinical investigations have demonstrated that fecal microbiota transplantation from melanoma patients who respond well to immune checkpoint inhibitors (ICIs) can induce similar responses in patients resistant to these treatments. Additionally, studies have shown that metabolites derived from gut microbiome may activate effector T-cells and enhance anti-tumor activity.

[0006] Short-chain fatty acids (SCFAs), a class of microbial metabolites produced by anaerobic bacteria through the fermenting of soluble dietary fibre, have potential immunomodulatory effects. SCFAs, such as butyrate, have been shown to induce the differentiation of colonic T regulatory cells in murine models, subsequently reducing the development of colitis. Furthermore, pentanoate, produced by the gut commensal Megasphaera massiliensis, can enhance the tumoricidal activity of CD8+T-cells. These metabolites induce the production of pro-inflammatory cytokines and cytotoxic molecules, thereby increasing the efficacy of cancer cell therapy in murine models.

[0007] On the other hand, branched-chain fatty acids (BCFAs), another class of microbial metabolites characterized by their branched structure, are produced in lower quantities compared to SCFAs by bacteria. Despite their presence in the gut and oral cavity, the immunomodulatory role of BCFAs remains unclear.

[0008] The oral cavity represents a complex ecosystem, second to the gut in microbial diversity, harboring around 700 bacterial species. In addition to the gut microbiome, certain oral pathogens including Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) can produce a range of SCFAs and BCFAs in severe oral inflammatory conditions including propanoic63664885\1acid, butyric acid, isovaleric acid and isobutyric acid. While extensive research has focused on gastrointestinal SCFAs and their immunomodulatory effects, the potential of metabolites derived from the oral microbiome and their immunomodulatory effects remains unexplored. Challenges in CD4+TIL therapy, notably the scarcity of these cells and the unknown mechanisms of their cytotoxic enhancement, have rendered this approach impractical for treating MHC-I deficient tumors.

[0009] Thus, in view of the foregoing, it would be desirable to develop a method of preparing novel tumor-infiltrating immune cells which are useful to treat cancers.SUMMARY OF THE INVENTION

[0010] A method has now been developed to reprogram CD4+T-cells via epigenetic engineering to enhance the cytotoxic capabilities and / or upregulate migration-related pathways of these cells, priming the cells for effective targeting of tumors, including MHC-I deficient cancers.

[0011] Thus, in one aspect of the invention, a method for reprogramming native CD4+T- cells is provided comprising incubating the CD4+T-cells with a Class I histone deacetylase (HDAC) inhibitor for a period of time sufficient to increase cytotoxicity of the CD4+T-cells in comparison to the cytotoxicity of the native CD4+T-cells.

[0012] In embodiments, the reprogrammed CD4+T-cells exhibit increased expression of at least one cytotoxic protein in comparison to the expression of the cytotoxic protein in the native CD4+T-cells. In one embodiment, the cytotoxic protein is perforin. In another embodiment, the cytotoxic protein is a granzyme.

[0013] In other embodiments, the reprogrammed CD4+T-cells exhibit increased expression of a migration pathway protein in comparison to the expression of the migration pathway protein in the native CD4+cells. In one embodiment, the migration pathway protein is the CX3C motif chemokine receptor 1 (CX3CR1). In another embodiment, the migration pathway protein is Lymphocyte Function- Associated Antigen (LFA-1).

[0014] In one embodiment, the reprogrammed CD4+T-cells exhibit downregulated expression of a pro-apoptotic protein. In one embodiment, the pro-apoptotic protein is a protein of the Fas pathway such as the Fas receptor (CD95) or a Fas ligand (CD95).6366488511

[0015] In one embodiment, the HD AC inhibitor is selected from the group consisting of hydroxamic acids, cyclic tetrapeptides, depsipeptides, benzamides, electrophilic ketones and aliphatic acids.

[0016] In one embodiment, the HD AC inhibitor is an aliphatic acid. The aliphatic acid may be an SCFA such as butyric acid, propanoic acid, or a pharmaceutically acceptable derivative or salt thereof, or the aliphatic acid may be a BCFA such as isovaleric acid, or the aliphatic acid may include a mixture of one or more SCFAs and / or one or more BCFAs.

[0017] In one embodiment, the HD AC inhibitor is present in a bacterial cell-free supernatant. The supernatant may be obtained from an oral bacterium, such as Porphyromonas bacteria or Fusobacterium bacteria. In one embodiment, the supernatant is obtained from the bacteria P. gingivalis. In another embodiment, the supernatant is obtained from the bacteria F. nucleatum.

[0018] In one embodiment, the CD4+T-cells are naive. In another embodiment, the CD4+T-cells are antigen-experienced cells.

[0019] In another aspect of the invention, a method for reprogramming native CD4+T- cells is provided comprising incubating the CD4+T-cells with one or more SCFAs and / or BCFAs for a period of time sufficient to increase cytotoxicity of the CD4+T-cells in comparison to the cytotoxicity of the native CD4+T-cells.

[0020] In one embodiment, the CD4+T-cells are incubated with one or more SCFAs such as butyric acid, propanoic acid, or a pharmaceutically acceptable derivative or salts thereof. In another embodiment, the CD4+T-cells are incubated with a BCFA such as isovaleric acid. In one embodiment, the CD4+T-cells are incubated with a mixture of SCFA(s) and BCFA(s).

[0021] In another aspect of the invention, reprogrammed CD4+cells prepared by the foregoing methods are provided.

[0022] In other aspects of the invention, reprogrammed CD4+T-cells having enhanced cytotoxicity and compositions comprising the cells are provided.6366488511

[0023] In other aspects, reprogrammed CD4+T-cells which exhibit increased cytotoxicity in comparison to naive CD4+T-cells are provided wherein said CD4+T-cells additionally exhibit: i) upregulation of a migration marker such as CX3CR1 or LFA-1, and / or ii) downregulation of pro-apoptotic proteins such as a protein of the Fas pathway, e.g. a Fas receptor (CD95) or a Fas ligand (CD95), as compared to native CD4+T-cells.

[0024] In a further aspect of the invention, a method for preparing CD4+TILs with enhanced cytotoxicity against tumor cells is provided. The method comprises incubating CD4+T- cells isolated from a patient tumor with dendritic cells (DCs) preloaded with tumor-specific peptides in a medium enriched with an HD AC inhibitor for a period of time sufficient to produce the tumor infiltrating CD4+TILs.

[0025] In another aspect of the invention, tumor-infiltrating CD4+T-cells prepared as described herein are provided and compositions comprising these cells.

[0026] In another aspect, a method of treating a patient with cancer is provided comprising administering reprogrammed CD4+T-cells or tumor-infiltrating CD4+TILs as described herein which have been reprogrammed with an HD AC inhibitor to exhibit enhanced cytotoxicity and / or increased tumor-cell killing capacity against cancer.

[0027] In a further aspect, a method of treating a patient with cancer is provided comprising administering to the patient HD AC inhibitor encapsulated within a delivery system modified to target CD4+T-cells. In embodiments, the delivery system is modified to incorporate CD4- targeting antibodies on the surface thereof. In other embodiments, the delivery system comprises lipid nanoparticles (LNPs).

[0028] In embodiments, the present methods of treating a patient with cancer are effective to treat an MHC-I deficient cancer, such as melanoma, head and neck squamous cell carcinoma (HNSCC), breast cancer, lung cancer, bladder cancer, prostate cancer, neuroblastoma, hematological malignancy and colorectal cancer.

[0029] These and other aspects of the invention are described in the following detailed description by reference to the following figures.6366488511BRIEF DESCRIPTION OF THE FIGURES

[0030] Figure 1. Pg- and Fn-derived supernatant induces cytotoxicity in naive CD4+T-cells. (A) Schematic representation of in-vitro model to reprogram CD4+T-cells and evaluate bacterial metabolites on modulating CD4+T-cell responses. (B) Flow cytometry analysis of cytotoxic markers (Perforin and Granzyme B) after treating naive CD4+T-cells with different oral bacterial supernatants in two different dilutions (1 :20 and 1 :40 of supernatant to total media volume) including S. salivarius (n = 5), S. oralis (n = 5), A. naeslundii (n = 5), S. sanguinis (n = 5), S. mutans (n = 5), V. dispar (n = 5), A. actinomycetemcomitans (n = 5), / '. forsythia (n = 5), F. nucleatum (n = 5) and P. gingivalis (n = 5). Data are presented as means ± SEM by the Kruskal- Wallis test. (C) Graphic demonstration of spin column separation method for fractionating Pg supernatant. (D) Flow cytometry measurement of the Prf and GzmB marker on naive CD4+T-cells treated with smaller than 3kD fraction (n = 9), bigger than 3kD fraction (n = 9), and carrier (n = 9). In both Perforin and Granzyme B,0.0001.

[0031] Figure 2. Isovaleric acid, butyric acid and propanoic acid selectively modulate CD4+T-cells cytotoxicity by upregulating the expression of the pore-forming protein perforin (Prf) and the serine protease granzyme B (GzmB). (A) The production of SCFAs and BCFAs by 10 human oral bacteria was measured by Gas chromatography. All bacteria were grown in-vitro until the stationary growth phase before the measurement of fatty acids in supernatants. (B) Representative flow cytometry contour plots showing the expression of Prf and GzmB in CD4+T-cells treated with different SCFAs and BCFAs, including acetic acid, lactic acid, isobutyric acid, and isovaleric acid. (C) Flow cytometry analysis of Prf and GzmB expression in CD4+T-cells treated with different concentrations (from 0.1 to 2.5 mM) of seven abundant SCFAs and BCFAs secreted by Porphyromonas gingivalis and Fusobacterium nucleatum. Significant differences are indicated as follows: ***P < 0.001, ****P < 0.0001. (D) Flow cytometry analysis of Perforin and Granzyme B after treating naive CD4+T-cells with an optimum dose of different SCFAs and BCFAs. Data are presented as means ± SEM by the Mann-Whitney test. (E) Viability of CD4+T- cells treated with varying concentrations of each SCFA or BCFA, assessed by live / dead staining with FACS.

[0032] Figure 3. Impact of Isovaleric Acid on CD4+T-cell Proliferation. (A)Representative histograms showing the proliferation of CD4+T-cells in the presence of PBS6366488511(control) and varying concentrations of isovaleric acid (0.1, 0.5, 1, and 2.5 mM). The percentages indicate the proportion of proliferating CD4+T-cells. (B) Flow cytometry analysis of CD4+T-cell proliferation at different concentrations of isovaleric acid. The graph shows the percentage of proliferating CD4+T-cells at each concentration. Significant differences are indicated as follows: ***P < 0.001, ****P < 0.0001.

[0033] Figure 4. Upregulation of LFA-1 in CD4+T-cells treated with isovaleric acid or P. gingivalis supernatant. (A) Representative flow cytometry contour plots showing LFA-1 expression on vehicle-treated versus isovaleric acid-treated CD4+T-cells. Percentages indicate the proportion of LFA-1+cells within the CD4+population. (B) Quantification of LFA-1+CD4+T- cells after isovaleric acid treatment compared to vehicle control (n = 4). (C) Representative flow cytometry contour plots showing LFA-1 expression on vehicle-treated versus P. gingivalis supernatant-treated CD4+T-cells. (D) Quantification of LFA-1+CD4+T-cells after P. gingivalis supernatant treatment compared to vehicle control (n = 4). Data represent mean ± SEM. Statistical analysis was performed using the Mann-Whitney U test, p < 0.05 was considered significant.

[0034] Figure 5. Isovaleric acid enhances cytotoxic and migratory gene programs while reducing pro-apoptotic Fas expression in CD4+T-cells. (A) Gene expression analysis shows a marked increase in GZMB, Prfl, and CX3CR1 mRNA levels in CD4+T-cells following isovaleric acid treatment, confirming molecular reprogramming towards a cytotoxic phenotype. Significant differences are indicated as follows: **P < 0.01; and (B) Downregulation of Fas expression in CD4+T-cells following isovaleric acid treatment. Mean fluorescence intensity (MFI) of Fas expression was measured in vehicle-treated versus isovaleric acid-treated CD4+T-cells. Isovaleric acid treatment significantly reduced Fas levels compared to vehicle control (n = 5). Data represent mean ± SEM; Mann-Whitney U test, / ? = 0.0079.

[0035] Figure 6. Effect of bacterial supernatant on class I HD AC activity. (A) Kinetic HDAC activity assay of bacterial supernatants shows that P. gingivalis and F. nucleatum supernatants markedly suppressed HDAC activity compared to vehicle control, with inhibition comparable to the positive control TSA. (B) Bacterial supernatant effect on HDAC isoforms (HDAC1, HDAC2, HDAC3) activity. Data represent three independent experiments. Statistical analysis was performed using the Mann-Whitney U test; *p < 0.05, **p < 0.01.6366488511

[0036] Figure 7. Cytotoxicity of P. gingivalis-reprogrammed TRP-1 CD4+T-cells against B16F10 melanoma cells. (A) IFN-y pretreatment significantly increased MHC-II expression on B16F10 melanoma cells, enabling recognition by TRP-1 CD4+T-cells (200 U / mL IFN-y, 48 h). statistical analysis by Mann-Whitney U test. ****p < 0.0001. (B) At an effector-to- target (E:T) ratio of 1 : 1, P. gingivalis (Pg) supernatant-treated TRP-1 CD4+T-cells induced a robust increase in B16F10 apoptosis compared with vehicle-treated CD4+T-cells. Apoptosis was defined as Annexin V+melanoma cells (including Annexin V+ / PI early apoptotic and Annexin V+ / PI+late apoptotic populations). Cytotoxic activity was completely abrogated when B16F10 cells were pretreated with an anti-MHC-II blocking antibody. (C) Similar results were observed at an E:T ratio of 3: 1, where Pg-reprogrammed CD4+T-cells exhibited strong cytotoxicity that was dependent on MHC-II recognition. Data represent mean ± SEM from independent experiments; statistical analysis by Kruskal-Wallis. ***p < 0.001.

[0037] Figure 8. Cytotoxicity of Fusobacterium nucleatum-reprogrammed TRP-1 CD4+T-cells against B16F10 melanoma cells. (A) At an effector-to-target (E:T) ratio of 1 : 1, Fn supernatant-treated TRP-1 CD4+T-cells induced significantly higher apoptosis of IFN-y- pretreated B16F10 melanoma cells compared with vehicle-treated controls. Apoptosis was quantified by flow cytometry as Annexin V+melanoma cells (early + late apoptosis). Cytotoxicity was abolished when B16F10 cells were pretreated with an anti-MHC-II blocking antibody, confirming MHC-II dependence. (B) Similar results were observed at an E:T ratio of 3 : 1, where Fn-reprogrammed CD4+T-cells demonstrated robust cytotoxicity that was strictly dependent on MHC-II recognition. Data represent mean ± SEM from independent experiments; statistical analysis by Kruskal-Wallis. ***p < 0.001.

[0038] Figure 9. Cytotoxicity of isovaleric acid-reprogrammed TRP-1 CD4+T-cells against B16F10 melanoma cells. (A) At an effector-to-target (E:T) ratio of 1 : 1, TRP-1 CD4+T- cells treated with ImM isovaleric acid induced significantly higher apoptosis of IFN-y-pretreated B16F10 melanoma cells compared with vehicle-treated controls. Apoptosis was quantified by flow cytometry as Annexin V+melanoma cells (early + late apoptosis). Cytotoxicity was abolished when B16F10 cells were pretreated with an anti-MHC-II blocking antibody, confirming MHC-II dependence. (B) Similar results were observed at an E:T ratio of 3: 1, where isovaleric acid- reprogrammed CD4+T-cells demonstrated robust cytotoxicity that was strictly dependent on6366488511MHC-II recognition. Data represent mean ± SEM from independent experiments; statistical analysis by Kruskal-Wallis. ***p < 0.001.

[0039] Figure 10. A schematic of a TIL therapy method in accordance with an aspect of the invention, using HD AC inhibitor to reprogram CD4+T-cells isolated from a cancer patient’s blood and tumor biopsies, expand the reprogrammed cytotoxic CD4+TILs by exposing them to dendritic cells (DCs) loaded with tumor-specific peptides, and subsequent reinfusion of the CD4+TILs into the patient for tumour targeting.DETAILED DESCRIPTION

[0040] In one aspect of the invention, a method for engineering mammalian CD4+T-cells to produce cells having enhanced CD4+cytotoxicity is provided comprising incubating the CD4+T-cells with a Class I histone deacetylase (HDAC) inhibitor for a period of time sufficient to reprogram the CD4+T-cells.

[0041] CD4+T-cells, also referred to as CD4+cells, CD4 cells, T helper cells or T4 cells, are lymphocytes that play a role in immune cell signalling. For use in the present method, CD4+cells from mammals are used, including human and non-human mammals. The CD4+cells may be obtained from any suitable biological sample including blood, plasma, serum and tissue, from either healthy individuals or non-healthy individuals, such as individuals with cancer. Biological samples associated with cancer may also be used including tumor tissue, bone marrow aspirate, ascites fluid, pleural effusion, tumor-draining lymph nodes, and cerebrospinal fluid samples. The CD4+cells are isolated from the biological sample using well-established techniques, including leukapheresis to isolate mononuclear white blood cells, followed by cell sorting using methods such as fluorescence-activated cell sorting (FACS), density gradient centrifugation, magnetic- activated cell sorting (MACS) or activation-induced markers (AIM) method. In one embodiment, the CD4+cells are naive, i.e. not previously exposed to tumor antigen. In another embodiment, the CD4+cells are tumor antigen-experienced CD4+T-cells or memory T4 cells.

[0042] The CD4+cells are then incubated with a Class I histone deacetylase (HDAC) inhibitor which inhibits the activity of Class I histone deacetylase (HDAC) enzymes including HDAC1, HDAC2, HDAC3 and HDAC8 enzymes. HDAC enzymes remove acetyl groups from histones and other nuclear proteins, which condenses chromatin and affects gene expression.63664885\1HD AC inhibitors in accordance with the invention block this enzyme activity. Examples of HD AC inhibitors for use in the present method include, but are not limited to, hydroxamic acids such as trichostatin A (TSA), vorinostat, belinostat, resminostat, abexinostat, givinostat, LAQ824, ivaltinostat, nanatinostat and panobinostat; cyclic tetrapeptides such as trapoxin B, depsipeptides such as nomidepsin and bocodepsin hydrochloride; benzamides such as entinostat, tacedinaline, zabadinostat and mecotinostat; electrophilic ketones such as trifluoromethyl ketones, and aliphatic acids including short and branched chain fatty acids (SCFA / BCFA).

[0043] In one embodiment, the HDAC inhibitor is an aliphatic acid such as an SCFA, i.e. a saturated fatty acid comprising two to six carbon atoms. Examples include acetic acid, propanoic acid, butyric acid, and pentanoic (valeric) acid. In other embodiments, the HDAC inhibitor is a branched chain fatty acid, i.e. a saturated fatty acid with one or more branches, for example, a methyl, ethyl or propyl branch. In one embodiment, BCFAs for use in the present method may include 3-10 carbon atoms (3-6 base carbon atoms with additional carbon branches). Examples include isobutyric acid, anteisobutyric (or 2-methylbutryic) acid, isovaleric acid, anteisovaleric acid, antecaproic acid, antecaprylic acid, antecapric acid, valproic acid, and pharmaceutically acceptable derivatives or salts thereof.

[0044] A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M. et a (1977) J. Pharm. Set. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0045] In one embodiment, the CD4+cells may be exposed to the SCFA and / or BCFA in the form of a cell-free bacterial supernatant obtained from an SCFA / BCFA-producing bacteria. In this regard, the supernatant may be processed to specifically include a small molecule fraction, i.e.6366488511small molecules of up to 3kD, while excluding larger, potentially interfering molecules such as bacterial peptides and toxins. In one embodiment, the SCFA / BCFA-producing bacteria is of the genus, Porphyromonas, which may include but is not limited to, P. asaccharolytica, P. endodontalis, P. gingivalis, P. catoniae, P. pasteri, P. somerae, P. uenonis, P. cangingivalis, P. canoris, P. cansulsi, P. circumdentaria, P. crevioricanis, P. gingivicanis, P. salivosa, P. macacae, P. gulae, P. levii or combinations thereof. In one embodiment, the Porphyromonas sp is an oral bacterium such as P. endodontalis or P. gingivalis. In another embodiment, the Porphyromonas sp is an isovaleric acid-producing bacterium such as P. gingivalis. In a further embodiment, the SCFA-producing bacteria is of the genus, Fusobacterium, and preferably, the Fusobacterium sp. is a butyric acid- and propanoic acid-producing bacterium such as Fusobacterium nucleatum.

[0046] Alternatively, the CD4+cells may be exposed to one or more SCFA / BCFAs isolated from the bacterial supernatant. SCFA / BCFAs may also be isolated from other sources including foods such as dairy and beef products, plants and essential oils. Methods to isolate SCFA / BCFAs from a given source are known in the art, and may vary with the source from which the SCFA / BCFA is isolated. Synthetic SCFA / BCFAs may also be used. Chemical synthetic methods for preparing a given SCFA / BCFA are also known in the art. Many SCFA / BCFAs are commercially available from Sigma-Aldrich and other chemical manufacturers. Preferred SCFAs for use in the present method are butyric acid and propanoic acid, and a preferred BCFA is isovaleric acid.

[0047] In accordance with the present method, the CD4+cells are exposed to the selected HD AC inhibitor in an amount which results in an enhanced expression of cytotoxic markers in the CD4+cells, namely, expression of a cytotoxic marker in an amount that is greater than the baseline expression of the cytotoxic marker in native (untreated) CD4+cells, i.e. CD4+cells which have not been exposed to an HDAC inhibitor. By “enhanced expression” is meant an increase in the expression of a cytotoxic marker by at least about 25%, 50%, 75%, 100% or greater than the baseline expression of the cytotoxic marker. The amount of HDAC inhibitor used will be an amount that results in an increase in cytotoxicity of the CD4+cells as compared to their cytotoxicity prior to HDAC inhibitor exposure, while not having a significant adverse effect on other properties of the cells such as proliferation capacity and viability. In this regard, an amount of HDAC inhibitor is used which retains at least about 50% of the proliferation capacity of the CD4+cells,6366488511and preferably at least about 75% or more of CD4+proliferation capacity. It will be appreciated that the amount of HD AC inhibitor may vary with the HD AC inhibitor used.

[0048] In one embodiment, the amount of SCFA, such as butyric acid or propanoic acid, useful to reprogram CD4+cells is an amount in the range of 0.1 - 1 mM butyric acid, or 0.5 - 2.5 mM propanoic acid. In another embodiment, the amount of a BCFA, such as isovaleric acid, useful to reprogram the CD4+cells is an amount in the range of 0.1- 2.5 mM, preferably an amount in the range of 0.1-1 mM. As one of skill in the art will appreciate, reduced amounts of an SCFA or BCFA may be useful when used in combination with another SCFA / BCFA or another molecule that induces cytotoxicity in CD4+cells.

[0049] The cytotoxic marker may be any marker that is toxic to cells, and in particular, to tumor cells. In one embodiment, the cytotoxic marker is a pore-forming protein perforin (Prf). In another embodiment, the cytotoxic marker is a serine protease granzyme B (GzmB) and other forms of granzymes, including Granzyme A, Granzyme M, and Granzyme H. In another embodiment, the cytotoxic marker is a pro-inflammatory cytokine such as interferon-gamma (IFN- y) or tumor necrosis factor-alpha (TNF-a). In another embodiment, the cytotoxic marker is the antimicrobial and cytotoxic protein is Granulysin (GNLY).

[0050] Incubation and expansion of the CD4+cells with an HD AC inhibitor may also result in the upregulation of a migration marker, i.e. a marker which enhances the ability of the CD4+cells to infiltrate a tumor microenvironment (TME). The migration marker exhibits an increase in expression in comparison to the baseline expression of this marker in native CD4+cells which have not been incubated or treated with the HD AC inhibitor, for example, an increase in expression of at least about 10%, 20%, 30%, 40%, 50% or more in comparison to expression levels in untreated CD4+cells. In one embodiment, the migration marker is CX3CR1, also known as CX3C motif chemokine receptor 1 or G-protein coupled receptor 13 (GPR13).

[0051] In another embodiment, the migration marker is an emigration marker such as LF A-1 or Lymphocyte Function-Associated Antigen, an adhesion molecule that facilitates firm binding of T-cells to endothelial ICAM-1, thereby enabling efficient transmigration into the tumor microenvironment. Its upregulation augments the migratory capacity of reprogrammed CD4+T- cells, complementing the chemokine-driven trafficking mediated by CX3CR1. Importantly, increased LFA-1 expression enhances the stability of the immunological synapse, improving the6366488511affinity and sensitivity of T-cell receptor interactions with cancer antigen-MHC complexes. Upregulation of migration markers such as CX3CR1 and LFA-1 equips the cytotoxic CD4+T-cells with superior migratory and antigen-recognition capabilities that provides efficient tumor infiltration and heightened antitumor activity and recognition.

[0052] In addition to the increased cytotoxicity and migration properties of the reprogrammed CD4+T-cells, incubation of the cells with an HDAC inhibitor may also result in downregulation of one or more pro-apoptotic proteins such as proteins of the Fas pathway (the Fas receptor (APO-2 or CD95) and Fas ligand (FasL or CD95L)), the BAX protein (bcl-2-like protein 4) and the BID protein (bcl-2-like protein 11). The downregulation of proteins involved in activation-induced cell death, such as Fas, which are frequently engaged by regulatory T cells (Tregs) to suppress effector T-cell function, their downregulation provides a survival advantage to the reprogrammed CD4+T-cells. By lowering pro-apoptotic protein expression, these cells are less susceptible to Treg-mediated apoptosis, thereby preserving their effector capacity and enabling sustained activity within the tumor microenvironment. This dual mechanism ensures that the reprogrammed CD4+T-cells are not only more cytotoxic and migratory, but also more resistant to immunosuppressive pressures that would otherwise limit their therapeutic potential.

[0053] In another aspect of the invention, a method for preparing tumour infiltrating CD4+T-cells or CD4+TILs is provided. The method comprises incubating CD4+T-cells with tumorspecific peptides in a medium enriched with an HDAC inhibitor, e.g. an SCFA or BCFA, for a period of time sufficient to produce CD4+TILs.

[0054] The term “tumor-infiltrating” refers to CD4+cells modified to target specific tumor cells. In this regard, the CD4+cells are modified to recognize specific tumor-related antigens, for example, by incubation of CD4+cells with tumor-related peptides which may include: tumorspecific antigens, such as TRP-1, MAGE- A3 and NY-ESO-1 antigens; tumor-associated antigens such as carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP); or neoantigens including proteomic variants such as mutated TP53, or viral-derived neoantigens such as HPV E6 / E7 oncoprotein.

[0055] To prepare tumor-infiltrating CD4+cells, CD4+cells obtained from a patient, as previously described, are incubated with tumor-specific peptides derived from the patient to6366488511expand tumor-specific CD4+cells for targeting the patient’s tumor. The term “patient” is used herein to refer to mammalian patients, including human and non-human mammals (such as cats, dogs, rodents, goats, sheep, horses, pigs, cattle and others). In one embodiment, tumor suspension comprising a cocktail of tumor-specific proteins and peptides is prepared from a patient’s tumor biopsy, and the tumor suspension is incubated with the patient’s dendritic cells (DCs) to encourage the uptake of tumor-specific proteins and / or peptides by the DCs. The tumor suspension will comprise a sufficient concentration of tumor-specific proteins and peptides to promote adequate uptake of the tumor proteins / peptides by the DCs. In one embodiment, the DCs are incubated with a tumor suspension comprising at least about 1-lOpg / mL of tumor protein / peptides. The incubation is conducted in an appropriate medium and for a period of time sufficient to achieve expression of tumor-specific proteins / peptides by the DCs, e.g. about 2-10 hours. DCs loaded with tumor-specific proteins / peptides are then co-cultured with CD4+cells, for example, at a ratio of DCs to CD4+cells of about 1 : 10 to 1 : 100, in the presence of a selected HD AC inhibitor, as illustrated in Fig. 10. The incubation is conducted for a period of time sufficient for cells to proliferate and yield activated tumor-infiltrating CD4+cells that exhibit upregulated expression of cytotoxic proteins such as Prf and GzmB, and / or upregulated expression of a migration marker such as CX3CR1 or LFA-1 by the CD4+cells. The term “activated” refers to the ability of the CD4+cells to specifically target the tumor-specific proteins. peptides, e.g. by expressing tumorspecific T-cell receptors which are detectable using established techniques. It will be appreciated by one of skill in the art that the CD4+cells may be incubated with the HDAC inhibitor concurrently with the tumor-specific DCs, or may alternatively be incubated with the HDAC inhibitor prior to co-culturing with the DCs, or subsequent to co-culturing with the DCs.

[0056] In another embodiment, tumor-specific CD4+T-cells may be detected in and isolated from a patient tumor sample using an appropriate method, for example, using tumor peptide-MHC-II tetramers. Tumor-specific CD4+cells are then expanded using artificial expansion reagents, such as anti-CD3 / CD28 beads, to generate a sufficient number of CD4+cells for use in the present method. The CD4+cells are then cultured in media enriched with HDAC inhibitor to yield activated tumor-infiltrating CD4+cells with enhanced cytotoxicity.

[0057] Reprogrammed CD4+cells, including tumor-infiltrating CD4+cells, are useful in a method of treating a patient with a tumor, e.g. a patient with cancer. The terms “treat”, “treating”6366488511or “treatment” are used herein to refer to methods that favorably alter a cancer, including those that moderate, reverse, reduce the severity of, or protect against, the progression of the cancer. The present reprogrammed CD4+cells, including tumor-infiltrating programmed to target the specific tumor in a patient, are administered to the patient in a therapeutically effective amount. The term "therapeutically effective amount" is an amount of the CD4+cells required to treat the cancer but which does not exceed an amount that may cause significant adverse effects to the patient in need of treatment. Dosages of CD4+cells that are therapeutically effective will vary on many factors including the nature of the condition to be treated, the patient being treated (e.g. age, weight, general health) and the dosage form utilized for administration. Appropriate dosages for use in such a treatment include dosages sufficient to result in infiltration of CD4+TILs into the tumor microenvironment (TME) of at least about 10%, and preferably, infiltration of the TME by greater than 10%, for example, at least 20%, 30%, 40%, 50% or greater. In one embodiment, the dosage of in vzfro-derived tumor-infiltrating CD4+cells is a dosage in the range of about 0.1-5 billion cells depending on the weight of the patient, for a sufficient period of time, to achieve treatment. The treatment regimen may include daily administration of the CD4+cells, or administration more or less frequently, e.g. on alternate days, weekly, or multiple dosages per day. The term “about” is used herein to mean an amount that may differ somewhat from the given value, by an amount that would not be expected to significantly affect activity or outcome as appreciated by one of skill in the art, for example, a variance of from 1-10% from the given value.

[0058] The method is useful to treat a variety of cancers, including both MHC-I-expressing tumors and MHC-I deficient tumors. Of particular note is the use of the present reprogrammed cells to treat MHC-I deficient cancer cells since CD4+cells, unlike CD8 cells, do not rely on the recognition of peptide-MHC-I complexes. Examples of cancers that may be treated using the presently reprogrammed CD4+cells, include but are not limited to, melanoma, Head and Neck Squamous Cell Carcinoma (HNSCC), breast cancer, lung cancer, bladder cancer, prostate cancer, neuroblastoma, hematological malignancies and colorectal cancer.

[0059] Reprogrammed CD4+cells, including CD4+TILs, in accordance with the invention may be formulated for therapeutic use by combination with a pharmaceutically acceptable carrier to form a composition. The expression "pharmaceutically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable.63664885\1As one of skill in the art will appreciate, the selected carrier may vary with the intended mode of administration. In one embodiment, reprogrammed CD4+cells may be formulated for administration by infusion or injection, and thus, are formulated as a suspension in a medicalgrade, physiologically acceptable carrier, such as an aqueous solution in sterile and pyrogen-free form, optionally buffered or made isotonic. The carrier may be a carbohydrate-containing solution (e.g. dextrose) or a saline solution comprising sodium chloride and optionally buffered. Suitable saline solutions may include varying concentrations of sodium chloride, for example, normal saline (0.9%), half-normal saline (0.45%), quarter-normal saline (0.22%), and solutions comprising greater amounts of sodium chloride (e.g. 3%-7%, or greater). Saline solutions may optionally include additional components, e.g. carbohydrates such as dextrose and the like. Examples of saline solutions including additional components, include Ringer’s solution, e.g. lactated or acetated Ringer’s solution, phosphate buffered saline (PBS), TRIS (hydroxymethyl) aminomethane hydroxymethyl) aminomethane)-buffered saline (TBS), Hank’s balanced salt solution (HBSS), Earle’s balanced solution (EBSS), standard saline citrate (SSC), HEPES- buffered saline (HBS) and Gey’s balanced salt solution (GBSS). The cells may also be formulated for administration by routes including, but not limited to, inhalation. In this regard, aerosol formulations may be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, stabilizers, anti-oxidants and anti-microbial agents, to increase shelf-life and prevent microbial growth over prolonged storage periods.

[0060] The present reprogrammed CD4+cells may be formulated for targeted delivery in a cell delivery system. Suitable cell delivery systems include biopolymers such as alginate, chitosan, hyaluronan, dextran, collagen, gelatin and albumin, hydrogels and films such as nitinol films, as a scaffold to load cells for delivery. Liposome technology may also be used to encapsulate the cells for administration.

[0061] The reprogrammed cytotoxic CD4+cells may be administered alone or in combination with one or more other cytotoxic treatments, which may have the same or different cytotoxic properties. Administration “in combination” refers to either an admixture together, or administration separately, at the same or different times. In one embodiment, the present CD4+cells may be administered together with a different immune cell, such as a CD8 cell, natural killer63664885\1cell, and other types of lymphocytes, which may or may not also be reprogrammed by incubation with an SCFA and / or BCFA, or otherwise genetically modified such as by chimeric antigen receptor (CAR) modification or antigen-loading.

[0062] In other embodiments, the present reprogrammed CD4+cells may be administered in combination with cancer chemotherapies such as alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors and others.

[0063] In addition to ex-vivo modification, the present invention also encompasses the direct in vivo reprogramming of CD4+T-cells in a further embodiment. In this regard, HDAC inhibitors may be administered to a subject for targeted delivery to CD4+cells in vivo. Thus, HDAC inhibitors may be modified to target CD4+cells, for example by conjugation with a CD4- targeting entity such as conjugation with anti-CD4 antibody. Alternatively, the HDAC inhibitor may be encapsulated within a delivery system such as lipid nanoparticles (LNPs) or the like which are modified to target CD4+cells, e.g. by conjugation of anti-CD4 antibody to the surface of LNPs, to enable highly specific delivery of the encapsulated HDAC inhibitor into CD4+T-cells in vivo following systemic administration. Anti-CD4 antibodies for this use are commercially available, for example, from Thermo Fisher Scientific, Abeam and Novus Bio, or may be prepared using techniques well-established in the art. Lipid nanoparticles for this purpose may comprise one or more phospholipids such as, but not limited to, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, l,2-distearoyl-sn-glycero-3 -phosphoethanolamine, cholesterol, DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine) and DSPC (l,2-distearoyl-sn-glycero-3- phosphocholine), which may be modified by PEGylation or with charge modifiers to optimize the nanoparticles for in vivo delivery to CD4+cells.

[0064] Thus, HDAC inhibitors, including SCFAs and BCFAs such as propanoic acid, butyric acid, and isovaleric acid, may be encapsulated within CD4-targeted LNPs and administered intravenously, orally, or intratumorally. This approach allows selective delivery of reprogramming agents directly to endogenous CD4+T-cells in vivo to reprogram the CD4+cells, thereby inducing within the CD4+cells cytotoxic activity, enhanced migration (via upregulation of CX3CR1 and / or LFA-1), and reduced susceptibility to regulatory T-cell suppression (via downregulation of FAS). Such in vivo reprogramming advantageously bypasses the need for leukapheresis, ex vivo culture,63664885\1and reinfusion of engineered cells, offering a simplified and scalable therapeutic strategy for adoptive cell therapy against both MHC-I-expressing and MHC-I-deficient tumors.

[0065] Aspects of the present invention provide a transformative approach in TIL therapy, leveraging HDAC inhibitors to reprogram CD4+T-cells either ex vivo wherein the CD4+T-cells are isolated from cancer patients or in vivo by administration of HDAC inhibitors for targeted delivery to CD4+T-cells. The novel methodology utilizes HDAC inhibitors to induce cytotoxic characteristics in CD4+TILs. This approach offers an alternative for targeting MHC-I deficient tumors, which are typically resistant to conventional CD8+T-cell-based therapies. By harnessing the cytotoxic potential of CD4+T-cells and enhancing their migratory and survival capabilities, this method addresses the limitations posed by treating MHC-I deficient variants.

[0066] Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.Example 1 - Reprogramming CD4+ T-cell Cytotoxicity

[0067] A method to reprogram CD4+T-cell cytotoxicity was developed as described in the following.

[0068] The capacity of oral bacteria including commensals and pathogens that can possibly modulate T-cell immune responses and generate cytotoxic CD4+TILs was evaluated (Fig. 1A).

[0069] To recapitulate the effect of oral bacteria on the proliferation and generation of CD4+TILs in-vitro, naive CD4+cells from healthy individuals were isolated to proliferate in T- cell media enriched with different oral bacteria cell-free supernatants including six commensals and four pathogens. The supernatant of P. gingivalis (Pg) and F. nucleatum were determined to be capable of inducing cytotoxicity by upregulating perforin (Prf) and Granzyme B (GzmB) expression on the CD4+ T-cells (Fig IB).

[0070] To rule out the effect of Pg-related peptides and toxins, a spin column separation method was used with a threshold of 3kD to segregate peptides larger than 3kD in the cell-free supernatant from small molecules containing SCFAs and BCFAs (smaller than 3kD) (Fig. 1C). The effect of these fractions to induce cytotoxicity on naive CD4+T-cells was further evaluated.63664885\1The results confirmed that only the smaller than 3kD fraction containing SCFAs and BCFAs were able to upregulate Prf and GzmB (Fig. ID).

[0071] Pg and Fn secrete various forms of short-chain and branched fatty acids. Metabolomics analysis uncovered the ability of Pg to secrete various forms of SCFAs and BCFAs including acetic acid, propanoic acid, butyric acid, isobutyric acid, and isovaleric acid, and the ability of Fn to secrete propanoic acid and butyric acid (Fig. 2A). To determine the specific role of certain SCFAs, and exclude the effects of other SCFAs and BCFAs, synthetic forms of these fatty acids separately were administered to isolated CD4+T-cells at different concentrations. The results demonstrated that isovaleric acid, propanoic acid and butyric acid significantly upregulated the expression of Prf and GzmB (Fig. 2B, 2C, 2D). The optimum concentrations for isovaleric acid, butyric acid, and propanoic acid had no toxic effect on the viability of CD4+T-cells (Fig. 2E)

[0072] Impact of Isovaleric Acid on CD4 T-cell Proliferation. To analyze the impact of isovaleric acid on the proliferation of CD4+T-cells, a proliferation assay was performed on treated CD4+T-cells. The analysis confirmed that isovaleric acid at low concentrations (0.1 and 0.5 mM) does not affect the proliferation of CD4+T-cells while it does induce cytotoxicity of the cells efficiently through upregulating Prf and GzmB (Fig. 3A, 3B). Therefore, the data suggest that isovaleric acid is a promising candidate for expanding CD4+T-cells effectively against cancer cells within a short time frame and with greater efficacy.

[0073] Upregulation of cytotoxic molecules and migration-related pathways by isovaleric acid Next, a qPCR assay to evaluate the gene expression levels of Prfl and GZMB in CD4+T-cells was employed. The qPCR results demonstrated a significant upregulation of Prfl and GZMB expression following treatment with isovaleric acid (Fig. 5A).

[0074] To investigate the impact of isovaleric acid on CX3CR1 expression, further assays were conducted. The findings showed that isovaleric acid not only upregulates cytotoxic gene expression such as Prf and GzmB but also increases the gene expression level of the chemokine receptor, CX3CR1. This enhancement improves the ability of CD4+T-cells to migrate to and infiltrate within the TME (Fig. 5A).6366488511

[0075] Downregulation of Fas expression - Treatment of CD4+T-cells with SCFA / BCFAs results in a reduction of Fas (CD95) expression. Treatment with isovaleric acid significantly reduced Fas expression compared to vehicle-treated CD4+T-cells (Fig. 5B).

[0076] Upregulation of LFA-1 - Increased expression of lymphocyte function-associated antigen 1 (LFA-1, integrin aL[32) in reprogrammed cytotoxic CD4+T-cells following treatment with P. gingivalis supernatant and isovaleric acid was also identified (Fig. 4). Upregulation of LFA-1 expression is shown in flow cytometry contour plots showing LFA-1 expression in isovaleric acid-treated CD4+T-cells versus vehicle treated T-cells (Fig. 4A). The proportion of LFA-U cells within the CD4+population is shown in Fig. 4B. Similarly, flow cytometry was used to determine LFA-1 expression on vehicle-treated versus P. gingivalis supernatant-treated CD4+T-cells (Fig. 4C), the latter of which was also shown to be upregulated to a similar degree (Fig. 4D)Discussion

[0077] The potential role of oral microbial-derived SCFA / BCFAs in cancer therapy has not yet been studied. In this work, it has surprisingly been shown that SCFA / BCFAs such as isovaleric acid, butyric acid and propanoic acid exert a remarkable immunomodulatory effect on CD4+T-cells observed by the significant upregulation of the expression of Prf and GzmB, which are nearly undetectable in naive CD4+T-cells. On the other hand, other SCFA / BCFAs at a range of different concentrations showed no such impact on CD4+T-cells. Therefore, the current findings support the use of selective SCFAs or BCFAs, obtainable from the oral microbiome, including isovaleric acid, butyric acid and propanoic acid to induce desired cytotoxic characteristics in CD4+T-cells, for subsequent use in ACT therapies to target cancers, including MHC-I and MHC-L deficient cancers.

[0078] In addition, isovaleric acid-induced CD4+T-cells generated in the current study showed an increase in the expression of CX3CR1 in CD4+T-cells, which indicates enhanced migration characteristics of the induced cells and the ability to infiltrate the tumor microenvironment.

[0079] Thus, collectively, the present CD4+T-cells induced to express cytotoxic Prf and GzmB and having enhanced infiltration capacity represent an effective treatment to target6366488511heterogeneous cancer cell communities and to achieve broader and more favourable responses in patients.Example 2 - Inhibition of Class I histone deacetylase (HD AC) enzymes

[0080] It was identified that supernatants derived from Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) potently inhibit the activity of Class I histone deacetylase (HDAC) enzymes, including HDAC1, HDAC2, and HDAC3, in CD4+ T-cells (Fig. 6A, 6B). Since Class I HDACs normally act to maintain a repressive chromatin state in cells, their inhibition relieves transcriptional constraints and allows for the upregulation of effector and cytotoxic molecules within the cells.

[0081] Experimental - Enzymatic activity assays demonstrated that P. gingivalis and nucleatum supernatants significantly reduced total HDAC as compared with supernatants from other bacteria and vehicle controls (Fig. 6A). The inhibitory effect was sustained over time and closely mirrored that of trichostatin A (TSA), a known pan-HDAC inhibitor. When isoform- specific assays were performed, a clear pattern emerged: both bacterial supernatants selectively suppressed HDAC1, HDAC2, and HDAC3, which are the central members of the Class I HDAC family (Fig. 6B). Other oral bacteria tested, including S. mutans, S. sanguinis, and A. actinomycetemcomitans, did not produce the same degree of inhibition, highlighting the specificity of this effect to Pg and Fn.

[0082] This data is in line with the robust increase of cytotoxic markers such as perforin (PRF1) and granzyme B (GZMB) in CD4+T-cells treated with such bacterial supernatants.Example 3 - Cytotoxicity Assay with P. gingiva / is-Reprogrammed CD4+T-cells

[0083] To evaluate the cytotoxic potential of reprogrammed CD4+T-cells, we utilized TRP-1 -specific CD4+T-cells isolated from the spleen of TRP-1 transgenic mice. These cells recognize the melanoma antigen TRP-1 expressed by MHC-II of the B16F10 melanoma cell line but, in their naive state, lack intrinsic cytotoxic function and are unable to induce apoptosis of target cells.

[0084] B16F10 melanoma cells were first pretreated with 200U / mL interferon-gamma(IFN-y) for 48 hours to upregulate surface MHC-II expression (Fig. 7A), thereby enabling CD4+T-cell recognition of TRP-1. Following this, naive TRP-1 CD4+T-cells were treated ex vivo with6366488511either vehicle control or P. gingivalis (Pg) culture supernatant, and subsequently co-cultured for 24 hours with IFN-y-pretreated B16F10 cells at effector-to-target (E:T) ratios of 1 : 1 and 3: 1. These relatively low E:T ratios were deliberately chosen to rigorously test the killing capacity of reprogrammed CD4+T-cells under limiting conditions, avoiding artificially inflated cytotoxicity at higher effector doses.

[0085] Apoptosis of melanoma cells was quantified by flow cytometry (FACS) using Annexin V and propidium iodide (PI) staining. For the analysis, Annexin V+B16F10 cells (including both Annexin V / PI early apoptotic and Annexin V+ / PI+late apoptotic populations) were designated as apoptotic. Pg-treated CD4+T-cells induced a significant increase in Annexin V+apoptotic B16F10 cells compared to vehicle-treated controls, demonstrating that reprogramming with Pg confers robust cytotoxic potential to CD4+T-cells at both 1 : 1 and 3: 1 ratios (Fig. 7B, 7C).

[0086] To confirm the specificity of this killing mechanism, B16F10 cells were pretreated with a blocking antibody against MHC-II prior to co-culture. Under these conditions, the cytotoxic activity of Pg-reprogrammed CD4+T-cells was completely abrogated, indicating that apoptosis was mediated strictly through MHC-II-restricted antigen recognition rather than nonspecific killing (Fig.7B, 7C).

[0087] Together, these results establish that Pg supernatant reprograms TRP-1 CD4+T- cells into potent cytotoxic effectors that infiltrate and kill tumor targets in an antigen-specific and MHC-II-restricted manner.Example 4 - Cytotoxicity Assay with Fusobacterium n u cleat uin CD4+T-cells

[0088] We next investigated whether Fusobacterium nucleatum (Fn) culture supernatant could similarly reprogram TRP-1 CD4+T-cells into cytotoxic effectors. TRP-1 CD4+T-cells from transgenic mice were treated ex vivo with either vehicle control or Fn supernatant and co-cultured for 24 hours with IFN-y-pretreated B16F10 melanoma cells at effector-to-target (E:T) ratios of 1 : 1 and 3 : 1. As with the Pg assays, relatively low E:T ratios were used to stringently evaluate the cytotoxic potential of reprogrammed CD4+T-cells.

[0089] Apoptosis of B16F10 cells was quantified by flow cytometry (FACS) using Annexin V / PI staining, with total Annexin V+melanoma cells (early + late apoptosis) designated6366488511as apoptotic. Fn-treated CD4+T-cells induced a significant increase in melanoma cell apoptosis compared with vehicle-treated controls at both 1 : 1 and 3: 1 ratios, demonstrating that Fn-derived factors are also capable of conferring cytotoxic function to CD4+T-cells (Fig. 8A, B).

[0090] To confirm antigen specificity and rule out nonspecific cytotoxicity, B16F10 cells were pretreated with an anti-MHC-II blocking antibody before co-culture. Under these conditions, the killing activity of Fn-reprogrammed CD4+T-cells was completely abolished, confirming that cytotoxicity was MHC-II restricted (Fig. 8A, B).

[0091] These results demonstrate that, similar to Pg, Fn supernatant reprograms otherwise non-cytotoxic TRP-1 CD4+T-cells into potent cytotoxic effectors, further supporting the general principle that oral microbial-derived products can reprogram CD4+T-cells to recognize and kill tumor targets through an MHC-II-dependent mechanism.Example 5 - Cytotoxicity Assay with Isovaleric Acid-Reprogrammed CD4+T-cells

[0092] We treated naive TRP-1 CD4+T-cells isolated from TRP-1 transgenic mice with ImM isovaleric acid or vehicle control. As in the previous assays, B16F10 melanoma cells were pretreated with IFN-y (200 U / mL, 48 hours) to upregulate surface MHC-II expression, thereby permitting TRP-1 CD4+T-cell recognition of their cognate antigen (Trp-1). Following treatment, isovaleric acid- or vehicle-exposed CD4+T-cells were co-cultured with IFN-y-pretreated B 16F 10 cells at effector-to-target (E:T) ratios of 1 : 1 and 3: 1 for 24 hours.

[0093] Apoptosis of melanoma cells was measured by FACS using Annexin V and PI staining, with Annexin V+B16F10 cells (early + late apoptosis) designated as apoptotic. Isovaleric acid-treated CD4+T-cells induced a significant increase in apoptosis of B16F10 melanoma cells compared with vehicle-treated controls at both 1 : 1 and 3: 1 E:T ratios (Fig. 9A, 9B). Importantly, when B 16F 10 cells were pretreated with an anti-MHC-II blocking antibody, the cytotoxic activity of isovaleric acid-reprogrammed CD4+T-cells was completely abolished, confirming that the killing was mediated strictly through MHC-II-restricted recognition of TRP-1 antigen rather than nonspecific effects (Fig. 9A, 9B).

[0094] Together, these findings demonstrate that isovaleric acid is sufficient to reprogram naive CD4+T-cells into functional cytotoxic lymphocytes capable of inducing antigen-specific, MHC-II-restricted tumor cell apoptosis.6366488511Example 6 - Cytotoxic Enhancement of CD4+T-Cells Against MHC-I Deficient Tumors

[0095] A method to prepare reprogrammed CD4+ T-cells for use as an adoptive cell therapy in a patient has been developed as follows and as illustrated in Fig. 10.

[0096] Peripheral Blood Collection and Isolation of CD4+T-Cells -Peripheral blood is collected from a patient, and white blood cells (WBCs) are isolated via leukapheresis. Naive CD4+T-cells are enriched using magnetic bead separation targeting CD4+surface markers. Enriched CD4+T-cells are rested in T-cell culture media (such as RPMI 1640) to achieve a steady state. Suitable growth conditions include incubating at 37°C and a 5% CO2 level.

[0097] Tumor Biopsy and Isolation of Tumor-Infiltrating CD4+T-Cells - A tumor biopsy is then obtained from the patient, followed by mechanical homogenization of the biopsy to prepare a single-cell suspension. CD4+T-cells are then isolated from the tumor biopsy using magnetic beads and rested in T-cell culture media to stabilize the cells.

[0098] Monocyte Isolation and Differentiation into Dendritic Cells (DCs) - Monocytes are isolated from the patient’s peripheral blood using magnetic bead sorting. Isolated monocytes are cultured with a cytokine cocktail containing GM-CSF (Granulocyte-Macrophage Colony- Stimulating Factor) and IL-4 (Interleukin-4) for 5-7 days to differentiate the monocytes into immature dendritic cells (iDCs).

[0099] Tumor Peptide Uptake by Dendritic Cells - The differentiated DCs are then cocultured with the single-cell suspension of the tumor biopsy for 2-10 hours to allow for the uptake and processing of tumor-specific peptides. Following peptide uptake, DCs express tumor peptides on their surface through the MHC complex.

[0100] Expansion of CD4+T-Cells Against Tumor Peptides - The rested CD4+T-cells (from both peripheral blood and tumor biopsy) are co-cultured with the peptide-loaded DCs to stimulate antigen-specific expansion. Initial expansion is conducted over 5-7 days at 37°C and a 5% CO2 level in T-cell media, allowing for the proliferation of CD4+T-cells with specificity against tumor antigens.

[0101] Polyclonal and Rapid Expansion of CD4+T-Cells - The expanded tumor-specific CD4+T-cells undergo further polyclonal and rapid expansion using anti-CD3 / CD28 beads in a media enriched with an HDACi such as isovaleric acid or Porphyromonas gingivalis supernatant.6366488511This expansion continues until the CD4+T-cells reach a target concentration of 1 to 10 million cells per kilogram of the patient’s body weight.

[0102] Preparation for Transfusion - The expanded cytotoxic CD4+T-cells are harvested, washed, and prepared for transfusion back into the patient. Quality control measures, including sterility testing and functional assays, are performed to ensure the safety and efficacy of the cell product.6366488511

Claims

CLAIMS1. A method for reprogramming native CD4+T-cells comprising incubating the CD4+T-cells with a Class I histone deacetylase (HDAC) inhibitor for a period of time sufficient to increase cytotoxicity of the CD4+T-cells in comparison to the cytotoxicity of the native CD4+T-cells.

2. The method of claim 1, wherein the reprogrammed CD4+T-cells exhibit increased expression of at least one cytotoxic protein in comparison to the expression of the cytotoxic protein in the native CD4+T-cells.

3. The method of claim 2, wherein the cytotoxic protein is perforin.

4. The method of claim 2, wherein the cytotoxic protein is a granzyme.

5. The method of claim 1, wherein the reprogrammed CD4+T-cells exhibit increased expression of a migration pathway protein in comparison to the expression of the migration pathway protein in the native CD4+cells.

6. The method of claim 5, wherein the migration pathway protein is the CX3C motif chemokine receptor 1 (CX3CR1) or Lymphocyte Function-Associated Antigen (LFA-1).

7. The method of claim 1, wherein the reprogrammed CD4+T-cells exhibit downregulated expression of a pro-apoptotic protein.

8. The method of claim 7, wherein the pro-apoptotic protein is a protein of the Fas pathway selected from the Fas receptor (CD95) and Fas ligand (CD95).

9. The method of claim 1, wherein the HDAC inhibitor is selected from the group consisting of hydroxamic acids, cyclic tetrapeptides, depsipeptides, benzamides, electrophilic ketones and aliphatic acids.

10. The method of claim 9, wherein the aliphatic acid is an SCFA selected from butyric acid, propanoic acid, or a pharmaceutically acceptable derivative or salt thereof.

11. The method of claim 1, wherein the HDAC inhibitor is a BCFA.63664885\112. The method of claim 11, wherein the BCFA is isovaleric acid.

13. The method of claim 1, wherein the HD AC inhibitor is present in a bacterial cell-free supernatant.

14. The method of claim 13, wherein the supernatant is obtained from an oral bacterium.

15. The method of claim 13, wherein the supernatant is obtained from Porphyromonas bacteria.

16. The method of claim 15, wherein the bacteria is P. gingivalis.

17. The method of claim 13, wherein the supernatant is obtained from F. nucleatum.

18. The method of claim 1, wherein the CD4+T-cells are naive.

19. The method of claim 1, wherein the CD4+T-cells are antigen-experienced cells.

20. Reprogrammed CD4+T-cells prepared by the method of any one of claims 1 to 19.

21. A method for preparing tumour infiltrating CD4+T-cells comprising incubating CD4+T- cells isolated from a patient tumor with dendritic cells (DCs) preloaded with tumor-specific peptides in medium enriched with an HD AC inhibitor for a period of time sufficient to produce the tumour infiltrating CD4+T-cells.

22. The method of claim 21, wherein the HD AC inhibitor is an SCFA.

23. The method of claim 22, wherein the SCFA is selected from butyric acid, propanoic acid, or a pharmaceutically acceptable derivative or salts thereof.

24. The method of claim 21, wherein the HD AC inhibitor is a BCFA.

25. The method of claim 24, wherein the BCFA is isovaleric acid.

26. Tumor-infiltrating CD4+T-cells prepared by the method of any one of claims 21-25.636648851127. Reprogrammed CD4+T-cells which exhibit increased cytotoxicity in comparison to naive CD4+T-cells, wherein said CD4+T-cells additionally exhibit: i) upregulation of a migration marker and / or ii) downregulation of one or more pro-apoptotic proteins, as compared to native CD4+T-cells.

28. The reprogrammed CD4+T-cells of claim 27, wherein the migration protein is the CX3C motif chemokine receptor 1 (CX3CR1) or Lymphocyte Function-Associated Antigen (LFA-1).

29. The tumor-infiltrating CD4+T-cells of claim 26 and the reprogrammed CD4+T-cells of claims 27 or 28 formulated for administration with a pharmaceutically acceptable carrier.

30. A method of treating a patient with cancer comprising administering tumor-infiltrating CD4+T-cells as defined in claim 26 or reprogrammed CD4+T-cells as defined in claim 27-29 to the patient.

31. A method of treating a patient with cancer comprising administering to the patient HD AC inhibitor encapsulated within a delivery system modified to target CD4+T-cells.

31. The method of claim 30 or 31, wherein the cancer is an MHC-I deficient cancer.

32. The method of claim 30 or 31, wherein the cancer is selected from melanoma, head and neck squamous cell carcinoma (HNSCC), breast cancer, lung cancer, bladder cancer, prostate cancer, neuroblastoma, hematological malignancy and colorectal cancer.

33. The method of claim 31, wherein the delivery system is lipid nanoparticles (LNPs).

34. The method of claim 31 or 33, wherein the delivery system is modified to incorporate CD4- targeting antibodies on the surface thereof.

35. A therapeutic delivery system comprising a Class 1 HD AC inhibitor, wherein the delivery system is modified to incorporate CD4+T-cell targeting antibodies.

36. A delivery system as defined in claim 35, comprising lipid nanoparticles.6366488511

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