WO2024148402A1 - A method of preparing cells for adoptive cell therapy - Google Patents

Abstract

The present disclosure relates generally to methods for preparing T cell populations for adoptive cell therapy. In particular, disclosed herein are methods for preparing a population of T cells enriched for central memory T cells relative to effector T cells. The present disclosure further relates to populations of T cells enriched for central memory T cells prepared according to the methods described herein, and the use of populations of T cells enriched for central memory T cells for the treatment of cancer.

Description

A METHOD OF PREPARING CELLS FOR ADOPTIVE CELL THERAPY

Related Applications

[0001] The present application claims priority from Australian Provisional Patent Application No. 2023900063 filed on 12 January 2023, the entire content of which is incorporated by reference in its entirety.

Field

[0002] The present disclosure relates generally to methods for preparing T cell populations for adoptive cell therapy. In particular, disclosed herein are methods for preparing a population of T cells enriched for central memory T cells relative to effector T cells. The present disclosure further relates to populations of T cells enriched for central memory T cells prepared according to the methods described herein, and the use of populations of T cells enriched for central memory T cells for the treatment of cancer.

Background

[0003] Adoptive cell therapy (ACT) has emerged as one of the most promising approaches for the treatment of haematological cancers. However, ACT has performed poorly to date in clinical trials in patients with advanced solid tumors. There are many potential reasons for this, including heterogeneous target antigen expression, poor trafficking of adoptively transferred cells into the tumor and tumor microenvironment (TME)-induced immune suppression and poor persistence after adoptive transfer.

[0004] Persistence of endogenous or adoptively transferred T cells, e.g., CAR T-cells, is at least partially related to differentiation status. In particular, less differentiated T cells, e.g., T cells with stem-like memory or precursor phenotype, are correlated with improved therapeutic outcomes due to their enhanced capacity to expand and their long-term persistence (Chen et al., 2021, Cancer Discovery, 11(9): 2186-2199). Despite this, the majority of ACT clinical trials have engineered cell populations from a bulk population of T cells extracted from a patient, consisting of a mixture of CD4+ and CD8+ T cells across naive, central memory and effector linages. This is particularly problematic when treating cancer, given the high proportion of exhausted T cells that are comprised in the bulk population of T cells, which are characterized by the sustained expression of inhibitory receptors, altered metabolism fitness, low proliferative capacity and reduced secretion of effector cytokines (Hashimoto et al., 2018, Annual Review of Medicine, 69: 301-318).

[0005] Accordingly, there is a need to develop methods for improving T cell persistence by preparing cell populations with less differentiated phenotypes to improve therapeutic outcomes for patients, such as those receiving adoptive immunotherapies (e.g., CAR-T cell therapy).

Summary

[0006] In one aspect, the present disclosure provides a method of preparing a population of T cells enriched for central memory T cells relative to effector T cells, the method comprising: a. obtaining a population of T cells; b. activating the population of T cells; and c. contacting the population of T cells with an inhibitor of one or both of CREB-binding protein (CBP) and p300 for a time and under conditions suitable to promote memory differentiation.

[0007] In another aspect, there is provided a population of T cells enriched for central memory T cells prepared according to the methods described herein.

[0008] In another aspect, there is provided a pharmaceutical composition comprising the population of T cells enriched for central memory T cells described herein.

[0009] In another aspect, there is provided a pharmaceutical composition comprising population of T cells enriched for central memory T cells, wherein the composition comprises at least about 30% central memory T cells.

[0010] In another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the population of T cells enriched for central memory T cells, or the pharmaceutical composition described herein.

[0011] In another aspect, there is provided a use of the population of T cells enriched for central memory T cells, or the pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer. [0012] In another aspect, there is provided a method for manufacturing a population of transduced T cells for adoptive cell therapy, the method comprising: a. obtaining a population of T cells; b. activating the population of T cells; c. contacting the population of T cells with an inhibitor of one or both of CBP and p300 for a time and under conditions suitable to promote memory differentiation; d. following step (c), transducing the population of T cells with a construct comprising a nucleic acid sequence encoding a CAR or a TCR.

Brief Description of the Drawings

[0013] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the accompanying drawings.

[0014] Figure 1 shows the transcriptional profiling of epigenetic inhibition in T cells. (A) A schematic representation of the multiplexed analysis of cells (MAC) sequencing protocol. (B) A graphical representation of MAC-seq results showing central memory T cells (y-axis; % Tcm) across the compound screening library (x-axis).

[0015] Figure 2 shows transcriptome changes that match the phenotype identified by flow cytometry in naive murine T cells activated in the presence of various epigenetic targeting compounds. (A) A graphical representation of the transcriptome profile of central memory T cells (y-axis; % CD44+ CD62L+) harvested at 96 hours post treatment with various epigenetic targeting compounds (y-axis). (B) A graphical representation of the expression of phenotype signature genes (y-axis; phenotype % CD44+ CD62L+) and memory signature genes (x-axis) of murine central memory T cells (CD62L+ CD44+ T cells) harvested at 96 hours post-treatment with various epigenetic targeting compounds. (C) A graphical representation of the expression of effector signature genes (y-axis) and memory signature genes (x-axis) of murine central memory T cells (CD62L+ CD44+ T cells) harvested at 96 hours post treatment with various epigenetic targeting compounds.

[0016] Figure 3 shows that treating primary murine CD8+ T cells with A485 promotes memory differentiation in vitro. (A) A schematic representation of the in vitro T cell activation protocol. (B) A graphical representation of FACS-based analysis of untreated cells (left panel) and A485 treated cells (right panel) sorted based on the expression of CD44 (x- axis) and CD62L (y-axis). (C) A graphical representation of the FACS-based analysis in (B) showing the proportion of central memory T (Tcm) cells (y-axis; % CD62L+ CD44+) in untreated or A485 treated cells (x-axis). (D) A graphical representation of the FACS-based analysis in (B) showing the proportion of effector T (Teff) cells (y-axis; % CD62L- CD44+) in untreated or A485 treated cells (x-axis). (E) A graphical representation of the overall percentage of T cells (y-axis; % of CD8+) expressing high levels of CD44 and CD62L (Tcm) or high levels of CD44 (Teff) across a titrated range of A485 concentrations (x-axis; pM). (F) A graphical representation of intracellular staining (y-axis; MFI Alexa647) for H3K27ac and H3K18ac (x-axis) at 48 hours post activation and treatment with 1 pM A485. Pooled data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; unpaired Student's t-test for n = 3 (D and E) and representative of n = 1 (F).

[0017] Figure 4 shows that treating primary human CD8+ T cells with A485 promotes memory differentiation in vitro. (A) A graphical representation of FACS-based analysis of untreated cells (left panel) and A485 treated cells (right panel) gated on CD45RO+ for activation and sorted based on the expression of CCR7 (x-axis) and CD62L (y-axis) to determine central memory status. (B) A graphical representation of the FACS-based analysis in (A) showing the proportion of Tcm cells (y-axis; % CD45RO+ CD62L+ CCR7+) in untreated or A485 treated cells (x-axis). (C) A graphical representation of the FACS-based analysis in (A) showing the proportion of Teff cells (y-axis; % CD45RO+ CD62L- CCR7-) in untreated or A485 treated cells (x-axis).

[0018] Figure 5 shows that A485 effects on proliferation does not affect differentiation. (A) A graphical representation of cell proliferation by untreated CTV labelled murine primary T cells or CTV labelled murine primary T cells treated with A485 during activation at 48 hours (left panel) or 72 hours (right panel) post-activation. (B) A graphical representation of FACS-based analysis of untreated CTV labelled murine primary T cells (left panel) or CTV labelled murine primary T cells treated with A485 (right panel) sorted based on the expression of CTV (x-axis) and CD62L (y-axis). (C) A graphical representation of the proportion of central memory T cells per division (y-axis; % CD62L+ CD44+) across 6 divisions (x-axis) in untreated CTV labelled murine primary T cells or CTV labelled murine primary T cells treated with A485. [0019] Figure 6 shows memory differentiation in primary murine T cells following treatment with A485 during activation. (A) A graphical representation of FACS-based analysis of central memory T cells (y-axis; % Tcm) derived from murine primary T cells at up to 96 hours post-activation (x-axis; hours post-activation) following treatment with A485 during activation. (B-C) A graphical representation of the proportion of central memory T cells (y-axis; % CD62L+ CD44+) in a population of naive murine T cells activated and at indicated time points (x-axis; hours post activation), 0.5 pM A485 was either (B) added in or (C) washed out.

[0020] Figure 7 shows that P300/CBP bromodomain inhibition increases the proportion of central memory T cells in a population of primary murine T cells. A graphical representation of the memory status of primary T cells from three murine donors with reference to the proportion of central memory T cells (y-axis; % CD62L+ CD44+) following activation in the presence or absence of A485 or GNE781 (x-axis). Pooled data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; unpaired Student's t- test for n = 3.

[0021] Figure 8 shows that CBP regulates the memory differentiation process in primary T cells. (A) A schematic representation of the in vitro CRISPR/Cas9-mediated knockout of EP300 and CREBBP in T cells. (B) A graphical representation of the memory status of primary T cells from three murine donors with reference to the proportion of central memory T cells (y-axis; % CD62L+ CD44+) following single knockout of EP 300 or CREBBP and double knockout of EP300 and CREBBP, relative to the ROSA26 control (x- axis). (C) A graphical representation of the memory status of primary T cells from three human donors with reference to fold change of central memory T (Tcm) cells (y-axis; Fold change % CD62L+ CD44+) following single knockout of EP300 or CREBBP and double knockout of EP 300 and CREBBP, relative to the ROSA26 control (x-axis), including statistical analysis of combined data from triplicate samples from separate donors for central memory T cell populations (CD62L+ CCR7+). Pooled data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; unpaired Student's t-test for n = 3. (D) A photographic representation of a Western Blot analysis to confirm the knockout of EP300 and/or CREBBP. (E) A graphical representation of the memory status of primary T cells from three human donors with reference to fold change of central memory T cells (y-axis; Fold change CCR7+ CD62L+) following single knockout of EP300 or CREBBP and double knockout of EP300 and CREBBP, relative to the AAVS1 control (x-axis). (F) A graphical representation of the memory status of primary T cells from three human donors with reference to fold change of central memory T (Tcm) cells (y-axis; Fold change % CD45RO+ CD62L+ CCR7+) following single knockout of EP300 or CREBBP and double knockout of EP300 and CREBBP, relative to the AA VS1 control (x-axis), including statistical analysis of combined data from triplicate samples from separate donors for Tcm cell populations. Pooled data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; unpaired Student's t-test for n = 3. (G) A photographic representation of a capillary Western Blot analysis to confirm the knockout of EP300 and/or CREBBP at day 6 post electroporation. (H) A graphical representation of the quantified signal of the capillary Western Blot of (G) normalized to LAMIN-B1.

[0022] Figure 9 shows that pre-treatment with A485 increases T cell persistence in vivo. (A) A schematic representation of the experimental workflow showing murine CD45.1 OT- 1 cells were activated in the presence or absence of 0.5 pM A485. Cells were competed against each other in a 50/50 mix in vivo. (B-C) A graphical representation of the proportion of total CD8+ T cells in blood (y-axis) and time (x-axis; days) from mice administered (B) untreated, or (C) A485 pre-treated cells.

[0023] Figure 10 shows that treatment of T cells with A485 increases T cell persistence and recall capacity in vivo. (A) A schematic representation of the in vivo experimental protocol. (B) A graphical representation of the in vivo peripheral expansion of CD45.1 cells (y-axis; %) over time (x-axis; days) in CD8+ T cells treated with A485 or untreated CD8+ T cells. (C) A graphical representation of the number of CD45.1 cells per 200 pL of blood (y-axis) in CD8+ T cells treated with A485 or untreated CD8+ T cells following re-challenge with Listeria-Ova. Re -challenged cells were analyzed by FACS on day 5. (D-E) A graphical representation of the number of CD45.1 cells per 200 pL of (D) blood or (E) spleen (y-axis; %CD45.1 of total CD8) in CD8+ T cells treated with A485 or untreated CD8+ T cells following re-challenge with Listeria-Ova. Re-challenged cells were analyzed by FACS on day 5 or day 7. Pooled data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; unpaired Student's t-test for n = 4 (untreated) and n = 3 (A485 pre-treated). (F-G) A graphical representation of the level of effector cytokines (F) IFNy and (G) TNFa (y-axis; MFI) produced by harvested T cells treated with A485 or untreated CD8+ T cells (x-axis) following re -challenge with Listeria-Ova. Pooled data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; unpaired Student's t-test for n = 4 (untreated) and n = 3 (A485 pre-treated).

[0024] Figure 11 shows that treatment of T cells with A485 during activation improves anti-tumor activity in vivo. (A) A graphical representation of tumor size (y-axis; mm²) and time (x-axis; days post-inoculation) in B16F10-ova tumor bearing mice following inoculation with murine OT-1 T cells treated with A485 or untreated murine OT-1 T cells. (B) A graphical representation of tumor size (y-axis; mm²) and time (x-axis; days postinoculation) in E0771-Her2 tumor bearing mice following inoculation with murine aHer2 CAR-T cells treated with A485 or untreated murine aHer2 CAR-T cells. Data are represented as mean ± SEM. *p < 0.05, **p < 0.001, ****p < 0.0001; Two-way ANOVA for n = 5 (untreated) and n = 6 (A485 pre-treated). (C) A graphical representation of FACS- based analysis of the CAR construct tagged with mCherry (left panels) and CD62L and CD44 expression (right panels) for untreated murine primary T cells (top panels) or murine primary T cells treated with A485 (bottom panels). (D) A graphical representation of tumor size (y-axis; mm²) and time (x-axis; days post-inoculation) in OVCAR3-LeY tumor bearing mice following inoculation with human aLeY CAR-T cells pre-treated with A485 or untreated human aLeY CAR-T cells.

Detailed Description

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information. [0026] The articles "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells (e.g. , a population of cells); reference to "an agent" includes a single agent, as well as two or more agents; and so forth.

[0027] In the context of this specification, the term “about” is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.

[0028] Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0029] The term “optionally” is used herein to mean that the subsequent described feature may or may not be present or that the subsequently described event or circumstance may or may not occur. Hence the specification will be understood to include and encompass embodiments in which the feature is present and embodiments in which the feature is not present, and embodiment in which the event or circumstance occurs as well as embodiments in which it does not.

[0030] As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. [0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] The present disclosure is predicated, in part, on the surprising finding that epigenetic inhibitors of one or both of CREB-binding protein (CBP) and p300 are capable of modulating T cell differentiation by promoting T cell memory formation. This finding has been reduced to practice in methods of preparing T cell populations enriched for central memory T cells from populations of T cells. Such methods are particularly beneficial in the preparation of T cells for adoptive cell therapies, as it has been shown herein that the use of T cell populations enriched for central memory T cells improves T cell persistence and the anti-tumor effects of CAR-T cell therapies in vivo.

[0033] Accordingly, in an aspect disclosed herein, there is provided a method of preparing a population of T cells enriched for central memory T cells relative to effector T cells, the method comprising: a. obtaining a population of T cells; b. activating the population of T cells; and c. contacting the population of T cells with an inhibitor of one or both of CBP and p300 for a time and under conditions suitable to promote memory differentiation.

[0034] The term "T cell" as used herein refers to CD4+ and CD8+ T cells, and isolated subpopulations thereof. Suitable T cells would be known to persons skilled in the art, illustrative examples of which include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, activated T lymphocytes or tumor infiltrating lymphocytes (TILs). Illustrative populations of T cells suitable for use in particular embodiments include but are not limited to heterogeneous populations of CD4+ and CD8+ T cells across naive, central memory and effector linages, helper T cells (HTL; CD4⁺ T cell), cytotoxic T cells (CTL; CD8⁺ T cell), CD4⁺CD8⁺ T cell, CD4 CD8" T cell, or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include but are not limited to T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD 197, and HLA-DR and if desired, can be further isolated by positive or negative selection techniques. [0035] In an embodiment, the population of T cells comprises naive T cells (e.g. , CD25- , CD45RA+, CD45RO- and/or CD127+). In another embodiment, the population of T cells comprises naive T cells (e.g., CD25-, CD45RA+, CD45RO- and/or CD127+), effector T cells (e.g., CD25-, CD45RA-, CD45RO+, CD62L- and/or CD 127+) , central memory T cells (e.g., CD25+, CD45RA-, CCR7+ and/or CD45RO+), and combinations of the foregoing.

[0036] The terms "central memory T cell", "Tern", "CD62L+ CD44+ cells", "CD45RO+ CD62L+ CCR7+ cells", and "CD62L+ CCR7+ cells" are used interchangeably herein to refer to a central memory T cell, or populations thereof.

[0037] The terms "effector T cell", "Teff", "CD62L- CD44+ cells", "CD45RO+ CD62L- CCR7-" and "CD62L- CCR7-" are used interchangeably herein to refer to an effector T cell, or populations thereof.

[0038] The term "enriched" as used herein refers to the selective expansion and/or propagation of specific cell types, relative to other cell types in the population. In some embodiments, enrichment of a specific cell type may be evidenced as an increase in the proportion of specific cell types, as described elsewhere herein. In the context of the methods disclosed herein, "enrichment" of central memory T cells occurs relative to other T cells in the starting population of T cells, for example, relative to naive T cells, effector T cells, or combinations thereof.

[0039] The term "cell" as used herein refers to an individual cell, cell line, cell culture or population of cells that comprise the nucleic acid molecule or vectors described herein, or that is capable of expressing the fusion protein described herein. The term "population of cells" may refer to homogenous cell populations comprising central memory T cells, or heterogeneous cell populations that may comprise naive T cells, effector T cells and/or memory T cells. It is also contemplated herein the heterogeneous cell populations comprise the progeny of a single parental cell. Due to natural, accidental or deliberate mutation, the progeny cells may not necessarily be identical in morphology or in genome to the original parental cell.

[0040] The T cells contemplated herein may be derived from any species, particularly a vertebrate, and even more particularly a mammal. Suitable vertebrates that fall within the scope of the disclosure include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In a preferred embodiment, the T cells are obtained from a human.

[0041] In an embodiment, the T cell is an autologous T cell.

[0042] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later to be administered into the subject in accordance with the methods disclosed herein. Accordingly, in certain embodiments, T cells isolated from the subject may be contacted with the genome editing systems described herein and cultured ex vivo for a time and under conditions suitable for the integration of the heterologous nucleotide sequence, before being reinfused back into the subject in accordance with the method of treatment described herein.

[0043] In another embodiment, the T cell is an allogenic T cell.

[0044] The term "allogenic" as used herein refers to any material derived from a different animal of the same species as the subject to whom the material is administered.

[0045] In an embodiment, the population of T cells is obtained from peripheral blood mononuclear cells (PBMCs).

[0046] T cells may be obtained from PBMCs using any suitable method for isolating T cells known in the art. The term “isolated” as used herein refers to a T cell, which is substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment, e.g., whole blood. Methods for the isolation of T cells from whole blood would be known to persons skilled in the art, illustrative examples of which include the isolation of T cells from whole blood using the Ficoll-Paque method.

[0047] In an embodiment, the method further comprises transducing the population of T cells enriched for central memory T cells with a construct comprising a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0048] As used herein the terms “polynucleotide”, “nucleic acid” or “nucleic acid molecule” mean a single- or double-stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues or natural nucleotides, or mixtures thereof, and can include molecules comprising coding and non-coding sequences of a gene, sense and antisense sequences and complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

[0049] The term “nucleotide” as used herein refers to the nucleotides adenosine, guanosine, cytidine, thymidine and uridine, each of which comprise a nucleotide base attached to a ribose ring. A person skilled in the art will appreciate that the terms "adenine / adenosine", "uracil / uridine", "guanine / guanosine", "cytosine / cytidine" and "thymidine / thymine" (C) may be used interchangeably herein with the single letters A, U, G, T and T, respectively, which refer the nucleotide base comprised by the nucleotides.

[0050] The terms "non-naturally occurring", "engineered" or "recombinant" may be interchangeably used herein to refer to nucleotides or nucleic acid molecules that are distinguished from their naturally occurring counterparts. For example, the nucleic acid molecule of the present disclosure may be recombinant, synthetic, or comprise mixtures of naturally and non-naturally occurring nucleotides. Non-naturally occurring nucleotides or nucleotide analogs may be modified at the ribose, phosphate and/or base moiety.

[0051] As used herein, the terms “encode”, “encoding” and the like refer to the capacity of a nucleic acid molecule to provide for another nucleic acid or a polypeptide. For example, a nucleic acid molecule is said to "encode" a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid molecule may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms "encode," "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.

[0052] In an embodiment, the construct is a vector.

[0053] The vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell), or can be vectors that integrate into a host cell genome. Vectors may be replication competent or replication-deficient. Exemplary vectors include, but are not limited to, plasmids, cosmids, and viral vectors, such as adeno-associated virus (AAV) vectors, lentiviral, retroviral, adenoviral, herpesviral, parvoviral and hepatitis viral vectors. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. Preferably, however, the vector is suitable for use in biotechnology.

[0054] Vectors suitable for use in biotechnology would be known to persons skilled in the art, illustrative examples of which include viral vectors derived from adenovirus, adeno- associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, self-amplifying single-strand RNA (ssRNA) viruses such as alpha virus (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis, Ml), and flavivirus (e.g., Kunjin virus, West Nile virus, Dengue virus), rhabdovirus (e.g., rabies, vesicular stomatitis virus), measles virus, Newcastle Disease virus (NDV) and poxvirus as described by, for example, Lundstrom (2019, Diseases, 6: 42).

[0055] In an embodiment, the vector is a plasmid or a viral vector.

[0056] The population of T cells may be provided with the construct described herein using any suitable method known in the art. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. The skilled person would readily understand and adapt any such method taking consideration of whether the construct is provided as a polynucleotide or within a vector. The term "recombinant T cell" as used herein refers to a T cell which comprises the constructs or vectors described herein. The term "recombinant T cell" includes the specific T cell and the progeny of the T cell.

[0057] The terms “chimeric antigen receptor” or “CAR” as used herein mean a recombinant polypeptide comprising at least an antigen-binding domain that is linked, via hinge and transmembrane domains, to an intracellular signaling domain.

[0058] The antigen-binding domain is a functional portion of the CAR that is responsible for transmitting information within the cell to regulate cellular activity via defined signaling pathways. In an embodiment, the antigen-binding domain may comprise an antibody or antibody fragment thereof.

[0059] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific antibodies (e.g., bispecific antibodies), and single variable domain antibodies so long as they exhibit the desired biological activity. The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, Cm, CH2 and Cm- Each light chain comprises a light chain variable region (which may be abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CLI). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments disclosed herein, the FRs of an antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Included within the scope of the term “antibody” is an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called a, 5, 8, y, and p, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0060] An “antigen-binding fragment” may be provided by means of arrangement of one or more CDRs on non-antibody protein scaffolds. “Protein scaffold” as used herein includes but is not limited to an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions. The protein scaffold may be an Ig scaffold, for example an IgG, or IgA scaffold. The IgG scaffold may comprise some or all the domains of an antibody (i.e., CHI, CH2, CH3, VH, VL). The antigen binding protein may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be IgGl. The scaffold may consist of, or comprise, the Fc region of an antibody, or is a part thereof. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigenbinding fragment,” as used herein. An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain. In certain embodiments, an antigenbinding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody include: (i) VH-CHI; (ii) VH-CH2; (iii) VH-CHS; (iv) VH-CHI-CH2; (V) VH-CHI-CH2-CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2, (X) VL-CH3; (xi) VL-CHI-CH2; (xii) VL- CHI-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigenbinding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antigen-binding molecule format may be adapted for use in the context of an antigen-binding fragment of an antibody using routine techniques available in the art.

[0061] In an embodiment, the antigen-binding domain comprises an antibody fragment. For example, the antigen-binding domain may comprise a scFv consisting of a VL and VH sequence of a monoclonal antibody (mAb) specific for a tumor cell surface molecule (i.e., tumor antigen).

[0062] In an embodiment, the CAR comprises an antigen binding domain that binds specifically to a cancer-associated antigen. [0063] In an embodiment, the CAR comprises an antigen binding domain that binds specifically to an antigen selected from the group consisting of CD7, CD 19, CD20, CD22, CD30, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD133, CD138, CD171, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSCA, PSMA, Glycolipid F77, Her2, EGFR, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, Claudin 6, Claudin 18.2, Lewis Y (LeY), GRP-78, EphA2, CEA, CEACAM5, ROR1, FAP and combinations of the foregoing.

[0064] The terms "T-cell receptor" or "TCR" as used herein mean a recombinant or naturally-occurring heterodimeric polypeptide comprising an alpha polypeptide chain (i.e., alpha chain, a chain) and a beta polypeptide chain (i. e. , beta chain, (3 chain), which is capable of binding to a peptide antigen bound to MHC.

[0065] In an embodiment, the TCR binds specifically to an antigen selected from the group consisting of 707- AP, AFP, ART-4, BAGE, Bcr-abl, CAMEL, CAP-1, CASP-8, CDC27m, CDK4/m, CEA, CT, Cyp-B, DAM, EGFRvlll, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GplOO, HAGE, HER-2/neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT, hTRT, iCE, KIAA0205, LAGE (L antigen), LDLR/FUT, MAGE, MART-l/Melan-A, MCI R, Myosin/m, MUC1, MUM-1, MUM -2, MUM -3, NA88-A, NY-ESO-1, P15, pl90 minor, Pml/RARa, PRAME, PSA, PSMA, RAGE, RU1, RU2, SAGE, SART-1, SART-3, SSX1, SSX2, SSX3, SSX4, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2 and WT1.

[0066] The term "activation" as used herein refers to a process of stimulating T cells in preparation for transduction and expansion ex vivo. Suitable methods for the activation of T cells would be known to persons skilled in the art, illustrative examples of which include polyclonal stimulation by antibodies (e.g., anti-CD28, anti-CD3 antibodies), soluble activation proteins, lipid microbubbles, dissolvable microspheres and linked antibodies.

[0067] In an embodiment, the population of T cells is activated by an agent selected from the group consisting of an anti-CD28 antibody or a CD28 -binding fragment thereof, an anti-CD3 antibody or a CD3-binding fragment thereof, and combinations of the foregoing.

[0068] In an embodiment, the anti-CD28 antibody or a CD28-binding fragment thereof and/or the anti-CD3 antibody or a CD3 -binding fragment thereof are coupled to a bead. [0069] In an embodiment, the bead is a magnetic bead. Suitable magnetic beads would be known to persons skilled in the art, illustrative embodiments of which include superparamagnetic polymer beads (e.g., Dynabeads®).

[0070] Histone acetyltransferases" mediate histone acetylation both on the unstructured tails and the globular core domains of histones by transferring acetyl group from acetyl-CoA to specific lysine residues. The acetylation of histones by histone acetyltransferases results in a dispersed structure of chromatin, which becomes accessible to transcription factors.

[0071] cAMP-response element binding protein", "CREB-binding protein" or "CBP" is a HAT protein localized in the nucleus (i.e., a Type A HAT), which is capable of acetylating histones and non-histone substrates, including p53, GATA-1, and HMGI/Y.

[0072] Histone acetyltransferase p300", "ElA-associated protein p300" or "p300" is a histone acetyltransferases localized in the nucleus (z.e., a Type A histone acetyltransferases), which shares a high level of homology within structured domains, including the enzymatic HAT domains and their bromodomains. However, p300 and CBP are less homologous outside of the structured domains and have been shown to interact with different protein partners (Dancy et al., 2015, Chemical Reviews, 115: 2419-2452).

[0073] In an embodiment, the inhibitor of one or both of CBP and p300 is selected from the group consisting of a small molecule inhibitor, a bivalent protein degrader, a nanobody and a biological agent.

[0074] Small molecule inhibitors of CBP and/or p300 include bromodomain inhibitors (e.g., CCS1447, CBP30, FT-7051, NEO2734 and GNE-781) and HAT inhibitors (e.g., A485, A241, CBP/p300-IN-12).

[0075] In an embodiment, the small molecule inhibitor is selected from the group consisting of A485, GNE-781, and combinations of the foregoing.

[0076] In an embodiment, the small molecule inhibitor is A485.

[0077] A485 is a potent, selective CBP/p300 inhibitor with the structure of formula (I).

A485 has been shown to have an IC50 of 0.06 pM for the HAT domain of p300.

[0078] In an embodiment, the population of T cells is contacted with about 0.01 pM to about 0.5 pM A485. Accordingly, in an embodiment, the population of T cells is contacted with about 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM, 0.08 pM, 0.09 pM, 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM or 0.5 pM A485.

[0079] In an embodiment, the small molecule inhibitor is GNE-781.

[0080] GNE-781 is a potent and selective inhibitor of CBP with the structure of formula

(II). GNE-781 has been shown to have an IC50 of 0.94 nM for the bromodomain of CBP.

[0081] In an embodiment, the population of T cells is contacted with about 0.01 pM to about 0.5 pM GNE-781. Accordingly, in an embodiment, the population of T cells is contacted with about 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM, 0.08 pM, 0.09 pM, 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM or 0.5 pM GNE-781.

[0082] "Proteolysis targeting chimerics", "PROTACs", "bivalent protein degraders" or "degraders" are heterobifunctional molecules that degrade specific endogenous proteins

SUBSTITUTE SHEET (RULE 26) through the E3 ubiquitin ligase pathway (Potjewyd el al., 2020, Cell Chemical Biology, 27: 47-56). The bivalent protein degrader structurally connects the proteins of interest (POI)- binding ligand and the E3 ubiquitin ligase (E3) ligand through an appropriate linker. The structure / function of bivalent protein degraders is reviewed in depth by Qi et al., 2021 (Frontiers in Pharmacology, (!'. 692574).

10083] The term "nanobody" as used herein refers to heavy chain only antibodies (i.e., HcAbs), which comprise two heavy chains with a single variable domain as the antigenbinding region.

[0084] As used herein, the term "biological agent" refers to any agent that leads to the reduction or elimination of expression of a gene encoding CBP and/or p300. Suitable agents would be known to persons skilled in the art, illustrative examples of which include interfering RNA (e.g., siRNA, miRNA, shRNA), endonucleases, zinc finger nucleases (ZFN), TAL effector proteins (TALENS), transposases, site-specific recombinases and CRISPR endonucleases.

[0085] In an embodiment, the population of T cells is contacted with the activating agent simultaneous or concurrent with the inhibitor of one or both of CBP and p300. Accordingly, in some embodiments, the population of T cells enriched for central memory T cells is an activated population of T cells enriched for central memory T cells.

[0086] In an embodiment, the population of T cells is contacted with an inhibitor of p300.

[0087] In an embodiment, the population of T cells is contacted with an inhibitor of CBP.

[0088] In an embodiment, the population of T cells is contacted with an inhibitor of CBP and p300.

[0089] In an embodiment, the population of T cells is contacted with an inhibitor of CBP and an inhibitor of p300.

[0090] In an embodiment, the population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) comprises at least about 30% central memory T cells (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,

38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,

54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,

70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,

86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% central memory T cells).

[0091] In an embodiment, the population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) comprises a proportion of central memory T cells that is increased by at least about 50% (e.g. , 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% ) relative to a population of T cells (e.g., a population of activated T cells) that has not been contacted with an inhibitor of one or both of CBP and p300.

[0092] In an aspect disclosed herein, there is provided a population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) prepared according to the methods described herein.

Pharmaceutical compositions

[0093] In another aspect disclosed herein, there is provided a pharmaceutical composition comprising the population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) described herein.

[0094] In another aspect disclosed herein, there is provided a pharmaceutical composition comprising a population of T cells enriched for central memory T cells, wherein the composition comprises at least about 30% central memory T cells (e.g., 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,

47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,

63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,

79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99% or 100% central memory T cells). [0095] The term "pharmaceutical composition" as used herein refers to a composition that is in a form that allows the biological activity of the active ingredient (e.g., a T cell expressing a CAR or a TCR, as described herein) to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.

[0096] In an embodiment, the pharmaceutical composition comprises a population of the T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) in sufficient number to administer a dosage of 10⁴ to 10⁹ cells/kg body weight per dose. Accordingly, the pharmaceutical composition may comprise a population of the T cells enriched for central memory T cells in sufficient number to administer a dosage of 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹ cells/kg body weight per dose.

[0097] In an embodiment, the pharmaceutical composition comprises a population of the T cells enriched for central memory T cells e.g., an activated population of T cells enriched for central memory T cells) in sufficient number to administer a dosage of 10⁵ to 10⁶ cells/kg body weight per dose, including all integer values within those ranges.

[0098] In some embodiments, periodic re-administration of the pharmaceutical composition may be required to achieve a desirable therapeutic effect. The exact amounts and rates of administration of the pharmaceutical composition will depend on a number of factors, examples of which are described elsewhere herein, such as the subject’s age, body weight, general health, sex and dietary requirements, as well as any drugs or agents used in combination or coincidental with the administration of the composition. Where multiple divided doses are required, these may be administered hourly, daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. Alternatively, a continuous infusion strategy can be employed.

[0099] In an embodiment, the pharmaceutical composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.

[0100] The pharmaceutical compositions disclosed herein may be prepared according to conventional methods well known in the pharmaceutical industries, such as those described in Remington’s Pharmaceutical Handbook (Mack Publishing Co., NY, USA), comprising a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0101] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal and anti-bacterial agents, isotonic and absorption agents, and the like.

[0102] In an embodiment, the pharmaceutical composition further comprises one or more immune adjuvants.

[0103] The term “immune adjuvant” as used herein refers to a compound or substance that is capable of enhancing a subject’s immune response to the immunogen including, for example, the subject's antibody response to the immunogen. An immune adjuvant may therefore assist to enhance the immune response to an engineered T cell in a subject, compared to the administration of the engineered T cell or in the absence of the immune adjuvant.

[0104] Suitable immune adjuvants will be familiar to persons skilled in the art, illustrative examples of which include an inhibitor of the PDL-1 : PD-1 axis, a TLR3 agonist, a 4-1BB agonist, a TLR7 agonist, an inhibitor of TIM-3, and an inhibitor of CTLA-4.

[0105] It is further contemplated herein that the pharmaceutical composition may be coadministered with one or more other agents suitable for the treatment or amelioration of symptoms associated with cancer, such as a solid tumor, illustrative examples of which include surgery, chemotherapy (e.g., anastrozole, bicalutamide, bleomycin sulfate, busulfan, busulfan injection, capecitabine, N4-pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, fludarabine phosphate, 5- fluorouracil, flutamide, tezacitibine, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, L- asparaginase, leucovorin calcium, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, mylotarg, paclitaxel, phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride for injection, vinblastine, vincristine, and vinorelbine), radiation, immunosuppressive agents (e.g., cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506), antibodies, or other immunoablative agents (e.g., CAMPATH), targeted agents, steroids, and peptide vaccines.

[0106] Such combinations may be administered simultaneous with the pharmaceutical composition or concurrently with the pharmaceutical composition.

Methods of treatment and associated therapeutic uses

[0107] It is further contemplated that the population of T cells enriched for central memory T cells (e. g. , an activated population of T cells enriched for central memory T cells) and pharmaceutical compositions described herein may be adapted for the treatment of cancer. Accordingly, in an aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the population of T cells enriched for central memory T cells (e.g. , an activated population of T cells enriched for central memory T cells), or the pharmaceutical composition described herein.

[0108] In another aspect, the present disclosure provides the population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) or the pharmaceutical composition described herein for use in the treatment of cancer.

[0109] In yet another aspect, the present disclosure provides the use of the population of T cells enriched for central memory T cells e.g., an activated population of T cells enriched for central memory T cells) or the pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer.

[0110] The therapeutic regimen for the treatment of cancer can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type, size, stage and receptor status of the tumor in addition to the age, weight and general health of the subject. Another determinative factor may be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease. Similarly, for a subject identified as having a more advanced stage of cancer, for example, stage III or IV disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject that has a less advanced stage of cancer. loi n] The term “cancer” as used herein means any condition associated with aberrant cell proliferation. Such conditions will be known to persons skilled in the art. In an embodiment, the cancer is a primary cancer e.g., a tumor). In another embodiment, the cancer is a metastatic cancer.

[0112] Examples of various cancers are described elsewhere herein and include breast cancer, colorectal cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, sarcoma and the like. The terms "cancer" and "tumor" may be used interchangeably herein, e.g., encompassing both solid and diffuse or circulating tumors.

[0113] In an embodiment, the cancer is a solid tumor. Suitable solid tumors would be known to persons skilled in the art, illustrative examples of which include breast cancer, melanoma, carcinoid, cervical cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, endometrial cancer, renal cancer, glioma, skin cancer, head and neck cancer, stomach cancer, liver cancer, testis cancer, lung cancer, thyroid cancer, lymphoma and urothelial cancer.

[0114] In another embodiment, the cancer is selected from the group consisting of melanoma, breast cancer and ovarian cancer.

[0115] The term “subject” as used herein refers to any mammal, including livestock and other farm animals (such as cattle, goats, sheep, horses, pigs and chickens), performance animals (such as racehorses), companion animals (such as cats and dogs), laboratory test animals and humans. In an embodiment, the subject is a human. In an embodiment, the subject is an adult. In another embodiment, the subject is a child.

[0116] In an embodiment, the population of T cells enriched for central memory T cells {e.g., an activated population of T cells enriched for central memory T cells) is autologous to the subject. [0117] In another embodiment, the population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells) is allogenic to the subject.

[0118] As used herein, the term “effective amount” typically refers to an amount of the population of T cells enriched for central memory T cells (e.g., an activated population of T cells enriched for central memory T cells), or pharmaceutical composition described herein that is sufficient to affect one or more beneficial or desired therapeutic outcomes (e.g., reduction in tumor size). Said beneficial or desired therapeutic outcomes may be measured using clinical techniques known in the art, illustrative examples of which include the measurement of imaging biomarkers, tumor size (e.g., as measured by anatomical imaging modalities, such as CT or MRI), quantification of the presence of inflammatory mediators (e.g., Interleukin- 1, TNF, TGF-(3, etc.). An “effective amount” can be provided in one or more administrations. The exact amount required may vary depending on factors such as the nature and severity of the cancer to be treated, and the age and general health of the subject.

[0119] The terms “treat”, "treating", “treatment” and the like are used interchangeably herein to mean relieving, reducing, alleviating, ameliorating or otherwise inhibiting the severity and/or progression of cancer, or a symptom thereof, in a subject. It is to be understood that the terms “treat”, "treating", “treatment” and the like, as used herein, do not imply that a subject is treated until clinical symptoms of cancer have been eliminated or are no longer evident (e.g., elimination of solid tumor mass and associated metastatic lesions, if any). Said treatment may also reduce the severity of cancer by preventing progression or alleviating the symptoms associated with cancer.

[0120] The terms “prevent”, “preventing”, “prevention” and the like are used interchangeably herein to mean inhibit, hinder, retard, reduce or otherwise delay the development of cancer and/or progression of cancer, or a symptom thereof, in a subject. In the context of the present disclosure, the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and/or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and/or be temporal in nature. Methods of manufacturing a population of T cells for adoptive cell therapy

[0121] In an aspect disclosed herein, there is provided a method of manufacturing a population of transduced T cells for adoptive cell therapy, the method comprising: a. obtaining a population of T cells; b. activating the population of T cells; c. contacting the population of T cells with an inhibitor of one or both of CBP and p300 for a time and under conditions suitable to promote memory differentiation; and d. following step (c), transducing the population of T cells with a construct comprising a nucleic acid sequence encoding a CAR or a TCR.

|0122] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0123] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the present disclosure without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0124] The present disclosure will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the disclosure.

Examples

General methods

Animals

[0125] NSG, C57BL/6 human-Her2 (hHer2) transgenic mice and C57BL/6 OT-I CD45.1 transgenic mice were bred in the animal facility at the Peter MacCallum Cancer Centre (Melbourne, Victoria, Australia). C57BL/6 wild type mice were purchased from the Walter and Eliza Hall Institute (WEHI, Parkville, Victoria, Australia). All experiments were performed on mice from the age of 6 to 20 weeks, which were housed in PC2 specific pathogen-free conditions.

Isolation and culture of mouse CD8+ T cells

[0126] Naive CD 8+ T cells were isolated from murine spleens using the EasySep™ Mouse Naive CD8+ T Cell Isolation Kit (StemCell #19858), re-suspended in T cell media containing 600U of r-IL2 and distributed in 6 well plates (1.2 xlO⁵ cells per 3 mL, per well) pre-treated with CD3 (5 pg/mL) and CD28 (1 pg/mL) per well. OT-1 T cells were obtained by isolating splenocytes from C57BL/6 OT-I CD45.1 mice. Splenocytes were cultured T cell media containing 600U of r-IL2 and the ovalbumin K^b-binding peptide, SIINFEKL.

Isolation and culture of human CD8+ T cells

[0127] Fresh whole blood samples were collected and mixed 50:50 with PBS. 15 mL of Ficoll was added slowly to create a gradient then centrifuged. The peripheral blood mononuclear cell (PBMC) interface was collected and transferred into a new tube, before treatment with Ammonium-Chloride-Potassium (ACK) lysis buffer. Cells were then washed, counted and stored in -80°C. Bulk activation of PBMCs was performed by resuspending 50 xlO⁶ cells in 50 mL of T cell media containing 100U of r-IL2 and CD3 (30 ng/mL).

[0128] Naive CD8+ T cells were isolated with the EasySep™ Human Naive CD8+ T Cell Isolation Kit II (StemCell #17968). Isolated naive CD8+ T cells were re-suspended in T cell media containing 100U of r-IL2 and activated with Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Gibco #11161D) in accordance with the manufacturer's instructions. Multiplexed analysis of cells (MAC) sequencing

[0129] Naive T cells were isolated and seeded into a CD3/CD28 coated 96-well plate at 2500 cells per well. T cells were cultured with various epigenetic targeting compounds at a final concentration of 1 pM. At 6 hours, cells were spun down, washed with ice-cold PBS and cell pellets were frozen at -80°C. At 96 hours, the plate designated for FACS analysis was used to establish differentiation status and cell number. 2500 cells were harvested from the plates designated for RNA harvest and washed with ice-cold PBS, before cell pellets were frozen at -80°C. Once thawed, 15 pL of lysis buffer was added to the cell pellet and incubated for 15 minutes at room temperature at 900 rpm. 1 pL of 10 nM barcoded-MACseq primers was combined with 12.5 pL of the lysate and 7.5 pL reverse transcriptase mix and incubated for 2 hours at 42°C. All wells with barcoded cDNA were then combined into one tube. cDNA concentration and clean-up was then performed using the DNA Clean & ConcentratorTM-100 and RNAClean XP and eluted in 22 pL of nuclease free water. cDNA was amplified using KAPA HiFi HotStart ReadyMix and the quality was assessed on the D5000 Screentape (TapeStation, Agilent). Libraries were prepared for tagmentation using TDE1 enzyme and TD buffer and KAPA HiFi HotStart ReadyMix and custom primers (MAC-seq P5 PCR and MAC-seq Indexing Mix) for amplification. Final libraries were purified using DNA AMPure XP beads and assessed on the DNA 1000 tape. Libraries were sequenced on the Illumina NextSeq 500 using the custom sequencing primers and High Output Kit v2.5 75 Cycles (Illumina) with paired-end configuration.

CAR-T cell production

[0130] GP-E86 cells were engineered to express retroviral vectors comprising the anti- Her2 CAR as well as a truncated human nerve growth factor receptor (NGFR). Briefly, splenocytes from wild-type C57BL/6 mice were activated in T cell media containing CD3 (5 pg/mL), CD28 (5 pg/mL), IL-2 (100 U/mL) and IL-7 (200 pg/mL) at 5 x 10⁶ cells per mL. 24 hours later, Ficoll was added slowly to create a gradient then centrifuged. The T cell interface was collected and transferred to a new tube. Retroviral supernatant from the GP- E86 cells was added to 6 well plates pre-treated with retronectin (40 pg/4 mL per well). The viral supernatant was centrifuged onto the retronectin for 30 minutes 1200 x g. T cells were re-suspended in viral supernatant, containing IL-2 (100 U/mL) and IL-7 (200 pg/mL), and added to plates at 7-10 x 10⁶ cells in 5 mL per well and spun down for 90 minutes at 1200 x g. This step was repeated the next day. Transduced CAR-T cells were used on day 7 post activation.

[0131] PG13 cells were engineered to express a second-generation scFv-anti-Lewis-Y CAR (CD28 and CD3 as well as a truncated human CD34). Human PBMCs were activated in bulk as described elsewhere herein. 48 hours post-activation, retroviral supernatant from the PG13 cells was added to 6 well plates pre-treated with retronectin (40 pg/4 mL per well). The viral supernatant was centrifuged onto the retronectin for 30 minutes 1200 x g. T cells were re-suspended in viral supernatant, containing IL-2 (100 U/mL) and added to the plates at 7-10 x 10⁶ cells in 5 mL per well and spun down for 90 minutes at 1200 x g. This step was repeated the next day. Transduced CAR-T cells were used on day 7 post activation.

Cell proliferation assay

[0132] Naive CD8+ T cells were labelled with 5 rnM CellTrace™ Violet (CTV, Molecular Probes, Thermo Fischer Scientific) in PBS at a final concentration of 1.25 pM for 18 min at 37°C. Cells were then washed with ice cold T cell media, plated and activated. Cells for staining were collected in a 96-well V-bottom plate in FACS buffer (2% FCS in PBS) and stained with the relevant antibody for 30 minutes on ice, followed by two washes with FACS buffer.

[0133] For intercellular stains, cells were re-suspended in 200 uL of Fixation Buffer (Biolegend # 420801) for 20 minutes on ice followed by a spin. Cells were re-suspended in lx Intracellular Staining Permeabilization Wash Buffer (Biolegend #421002), which was repeated three times. Cells were then stained with relevant antibody diluted in the permeabilization wash buffer. Cells are washed in the permeabilization wash buffer twice. Prior to analysis Precision Count Beads™ (Biolegend #424902) were directly added to the sample. Cells were analyzed on a Fortessa™-X20 or FACSymphony™ A3/A5 (BD Biosciences Pharmigen, Franklin Lakes, NJ, USA). Flow cytometry data was analyzed using FlowJo software (version 10.4.0, Tree Star Incorporated, Ashland, OR, USA).

Persistence and listeria-ova infection assays

[0134] Spleens were collected from untreated or treated (0.5 pM A485) OT-1+ CD45.1 cg/cg or cg/wt mice. T cells were isolated and activated as described elsewhere herein. Isolated T cells were cultured ex vivo for 5 days, washed and mixed at a 1:1 ratio and phenotypically analyzed on FACSymphony™ A5. Thereafter, T cells were injected intravenously into C57BL/6 mice (5 x 10⁶ cells/200 uL per mice). Retro-orbital bloods were obtained every 7 days from the injected mice, processed and analyzed on the FACSymphony™ A5. After 28 days spleen, lymph node and liver were harvested, processed and analyzed on the FACSymphony™ A5.

[0135] 5 x 10⁴ cells of CD45.1 T cells (as % of live splenocytes) were injected intravenously into C57BL/6 mice. These mice were then injected with Listeria-Ova (2.5 x 10⁴ c.f.u./mouse) through retro-orbital i.v. Blood was collected 5 days post Listeria and on day 7 spleen, lymph nodes and bloods were harvested, processed and analyzed on the FACSymphony™ A5.

In vivo analysis

[0136] B 16-F10 ova cells were injected subcutaneously into the flank of C57BL/6 mice (2 x 10⁵ cells/200 uL). Untreated or treated (0.5 pM A485) T cells from OT+ CD45.1 cg/cg mice were isolated, activated and cultured ex vivo for 5 days then washed and phenotypically analyzed on FACSymphony™ A5. T cells were then injected intravenously into C57BL/6 mice (5 x 10⁶ cells/200 uL per mice) on day 10 and 13 post-tumor inoculation. Tumor growth was monitored every 2-3 days by measuring width and length. Mice were sacrificed when tumor area reached >100 mm², or if there were apparent signs of disease.

[0137] E0771-Her2 cells were injected under anesthesia in the mammary fat pad of female C57BL/6 hHer2 transgenic mice (2 x 10⁵/100 uL). 7 days post-tumor injection the mice received 4-Gy total body irradiation. Mice then received 1 x 10⁷ anti-Her2 CAR T cells on day 7 and 8 after tumor injection. Every day for 4 days the mice received IL-2 injections intraperitoneally (50.000U). Tumor growth was monitored every 2-3 days by measuring width and length. Mice were sacrificed when tumor area reached >100 mm², or if there were apparent signs of disease.

[0138] OVCAR3-LeY cells were injected subcutaneously into the flank of NSG mice (5 x 10⁵/200 pL). Once the tumor reached a size around 20 mm², the mice received 1-Gy total body irradiation. Mice then received 1 x 10⁷ anti LeY CAR T cells on day 7 and 8 after tumor injection. Every day for 4 days the mice received IL-2 injections intraperitoneally (50.000U). Tumor growth was monitored every 2-3 days by measuring width and length. Mice were sacrificed when tumor area reached >100 mm², or if there were apparent signs of disease.

CRISPR/Cas9 gene editing

[0139] P3 electroporation reaction buffer was prepared freshly by combining 3.6 uL of Supplement 1 to 16.4 uL of P3 Primary Cell Solution (Lonza 4D Nucleofector kit). The sgRNA/Cas9 ribonuclearprotein (RNP) complex was formed by combining 1 uL of Cas9 with 1 uL of sgRNA, or for double KO, 0.5 uL of each sgRNA and topped up with sterile PBS to 5 uL, and incubating for 10 minutes at room temperature to form the RNP complex. Isolated naive CD8 T cells were then spun down and re-suspended in 20 uL of the P3 reaction buffer per reaction and mixed with the RNP. The reaction was then transferred into the wells of the nucleofection strip and bubbles were removed. The nucleofector strip was placed into the Lonza nucleofector and the Pulse ND 100 or Mouse T cell, unstimulated pre-configured program was selected. After electroporation, 125 uL of pre-warmed T cell media was added to the wells and the strip was incubated for 15 minutes at 37°C. Cells were then plated and activated. FACS was performed to determine phenotype and lysates were taken for KO validation.

Statistical analysis

[0140] Statistical analyses were performed in Prism v9 software (GraphPad). A p-value < 0.05 was considered statically significant. Data analyses were conducted using unpaired Student's t-test to compare two data sets of data. Data presented as mean ± standard error of the mean (SEM). All statistical calculations for RNA-seq were performed in R software (version 4.1.0). Multiple comparison was used in RNA-seq analysis, and a Benjamini- Hochberg adjusted p-value < 0.05 was considered as statistically significant.

Example 1 Transcriptional profiling of epigenetic inhibition in T cells

[0141] Using MK2206, an AKT inhibitor previously one of the top regulators of T cell memory differentiation, as a positive control, a compound screening library was assessed by MAC-seq to identity other regulators of T cell memory differentiation. As shown in Figure 1, the top hit of this screen was A485, an inhibitor of the histone acetyl transferase domain of p300 and CBP. [0142] The transcriptome changes that were identified by MAC-seq matched the phenotype of cells identified by flow cytometry when comparing the expression of memory signature genes and effector signature genes (Figure 2).

Example 2

Pre-treatment with the histone acetyl transferase inhibitor, A485, increases the proportion of central memory T cells in vitro

[0143] To validate the compound screen results in Example 1, primary mouse and human T cells were activated in the presence of A485. As shown in Figures 3 and 4, A485 increased the proportion of memory T cells in both mouse and human cells in vitro.

[0144] To investigate the effects of A485 on T cell function in greater detail, a proliferation assay was conducted. It was shown that activating primary mouse T cells in the presence of A485 resulted in a modest delay in proliferation, together with a phenotype that is independent from the rate of cell division (Figure 5). To determine if the phenotype was hypo-acetylation dependent, the same proliferation assay was performed whereby A485 was either added in, or washed out of the culture medium. As shown in Figure 6A, it was demonstrated that memory differentiation as promoted by A485 was established within the first 24-28 hours of activation.

[0145] The effect of selectively targeting the bromodomain of CBP was also assessed by activating primary mouse T cells in the presence of different concentrations of GNE-781. As shown in Figure 7, naive murine T cells activated with GNE-781 at all concentrations tested increased the proportion of central memory T cells, similar to that of A485.

Example 3

Genetic deletion of CBP, or CBP and p300 increases the proportion of central memory T cells in vitro

[0146] A485 targets the histone acetyl transferase domain of both p300 and CBP, whereas GNE-781 targets the bromodomain of CBP. To investigate the independent roles of p300 and CBP in T cell memory formation, CRISPR Cas9 was utilized to knock out EP300, CREBBP or the combination of both EP300 and CREBBP in mouse and human primary T cells. While the single knockout of EP300 had modest effects on T cell memory formation, the effects shown using A485 were phenocopied in the single knockout of CREBBP and double knockout of EP300 and CREBBP in both mouse and human cells (Figure 8).

Example 4

A485 induces a central memory / persistence phenotype in vivo

[0147] True memory cells are able to persist longer overtime, as well as being able to reactivate, proliferate and differentiate into effector cells upon re-stimulation. To determine if the "memory-like" T cells generated following in vitro culture with A485 possess these characteristics, an in vivo persistence analysis was performed where untreated and A485 pretreated T cells were activated and injected into wild-type C57BL/6 in a competition (Figure 9) or re -challenge (Figure 10) setting. Using in vivo competition assay (Figure 8A), it was shown that pre-treating T cells resulted in increased persistence (Figure 9C) relative to control (Figure 9B).

[0148] As shown in Figure 10B, the A485 pre-treated T cells persist longer overtime, relative to the untreated T cells. To assess the re-call capacity of these cells, an equal amount of persisting T cells were injected in new wild-type C57BL/6 recipient mice and challenged with Listeria-Ova. Consistent with a true memory cell phenotype, the A485 pre-treated T cells had the capacity to respond to the antigen by reactivating and differentiate into effector cells (Figure 10C and 10D). Moreover, upon re-challenge A485 pre-treated cells had the ability to proliferate more than the conventional (i.e., untreated) T cells.

[0149] Harvested T cells were also stimulated with the ova peptide to assess the production of effector cytokines, IFNy (Figure 10F) and TFNa (Figure 10G).

Example 5

Pre-treatment of CAR-T cells with A485 improves anti-tumor efficacy relative to conventional CAR-T cells in vivo

[0150] To assess if there is an advantage to using T cell populations enriched for central memory T cells by A485 for the treatment of cancerous cells in vivo, a range of in vivo tumor models were used to assess the anti-tumor efficacy of OT-1, anti-Her2 CAR-T and anti-LeY CAR-T cells pre-treated with A485. In three different tumor models (i.e., B16-F10, E0771 and OVCAR3), it was shown that pre-treatment with A485 results in superior anti-tumor efficacy as compared to conventional (i.e., untreated) T cells (Figure 11). Conclusion

[0151] CAR-T cells have performed poorly to date in clinical trials treating patients with solid tumors. A key factor in this negative outcome is the poor long-term persistence of T cells following adoptive transfer. Previous approaches to improve T cell persistence include ex vivo manipulation (e.g., culturing with IL-7, L-15, antioxidants such as N- acetylcysteine), patient lymphodepletion, co-administration of PI3K inhibitors or checkpoint blockade inhibitors and optimization of CAR structure. However, optimization of the T cell populations for adoptive cell therapy by enriching for specific T cell subpopulations has been limited, at least in part due to the technical difficulties associated with the manufacture of T cell populations for adoptive cell therapy (e.g., CAR T-cell therapy). Therefore, despite studies indicating that the adoptive transfer of T cells enriched for sternness / central memory function improves persistence and patient outcomes, methods to enrich for such T cell subpopulations have not been generally adopted in clinical trials.

[0152] As described herein, this issue has been addressed, at least in part, by pretreating T cell populations with an inhibitor of one or both of CBP and p300. The data presented herein demonstrate that pre-treatment of T cell populations with an inhibitor of CBP and/or p300 promotes T cell central memory formation ex vivo, which resulted in increased persistence and cytotoxicity in vivo.

[0153] Taken together, these data enable methods of preparing a population of T cells enriched for central memory T cells. Importantly, the methods disclosed herein are useful in the manufacture of T cells for adoptive cell therapy, including engineered CAR-T cell therapy and TCR-T cell therapy.

[0154] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Claims (26)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of preparing a population of T cells enriched for central memory T cells relative to effector T cells, the method comprising: a. obtaining a population of T cells; b. activating the population of T cells; and c. contacting the population of T cells with an inhibitor of one or both of CREB- binding protein (CBP) and p300 for a time and under conditions suitable to promote central memory differentiation.

2. The method of claim 1, wherein the population of T cells is activated with an agent selected from the group consisting of an anti-CD28 antibody or a CD28-binding fragment thereof, an anti-CD3 antibody or a CD3-binding fragment thereof and combinations of the foregoing.

3. The method of claim 2, wherein the anti-CD28 antibody or a CD28-binding fragment thereof and/or the anti-CD3 antibody or a CD3-binding fragment thereof are conjugated to a bead.

4. The method of claim 3, wherein the bead is a magnetic bead.

5. The method of any one of claims 1 to 4, wherein the population of T cells is contacted with the inhibitor of one or both of CBP and p300 during activation in step (b).

6. The method of any one of claims 1 to 5, wherein the inhibitor of one or both of CBP and p300 is selected from the group consisting of a small molecule inhibitor, a bivalent protein degrader, a nanobody and a biological agent.

7. The method of any one of claims 1 to 6, wherein the inhibitor of one or both of CBP and p300 is a small molecule inhibitor.

8. The method of claim 7, wherein the small molecule inhibitor is selected from the group consisting of A485, GNE-781, and combinations of the foregoing.

9. The method of claim 8, wherein the population of T cells is contacted with from about 0.01 pM to about 0.5 pM A485.

10. The method of claim 8 or claim 9, wherein the population of T cells is contacted with from about 0.01 pM to about 0.5 pM GNE-781.

11. The method of any one of claims 1 to 10, wherein the population of T cells enriched for central memory T cells comprises at least about 30% central memory T cells.

12. The method of any one of claims 1 to 11, wherein the population of T cells is obtained from peripheral blood mononuclear cells (PBMCs).

13. The method of any one of claims 1 to 12, further comprising transducing the population of T cells enriched for central memory T cells with a construct comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

14. The method of claim 13, wherein the CAR comprises an antigen binding domain that binds specifically to a cancer-associated antigen.

15. The method of claim 14, wherein the CAR comprises an antigen binding domain that binds specifically to an antigen selected from the group consisting of CD7, CD19, CD20, CD22, CD30, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD133, CD138, CD171, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSCA, PSMA, Glycolipid F77, Her2, EGFR, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, Claudin 6, Claudin 18.2, Lewis Y (LeY), GRP-78, EphA2, CEA, CEACAM5, ROR1, FAP and combinations thereof.

16. The method of any one of claims 1 to 15, wherein the population of T cells is contacted with an inhibitor of CBP.

17. A population of T cells enriched for central memory T cells prepared according to the method of any one of claims 1 to 16.

18. A pharmaceutical composition comprising the population of T cells enriched for central memory T cells of claim 17.

19. A pharmaceutical composition comprising a population of T cells enriched for central memory T cells, wherein the composition comprises at least about 30% central memory T cells.

20. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the population of T cells enriched for central memory T cells of claim 17, or the pharmaceutical composition of claim 18 or claim

21. The method of claim 20, wherein the population of T cells enriched for central memory T cells are autologous to the subject.

22. The method of claim 20, wherein the population of T cells enriched for central memory T cells are allogenic to the subject.

23. The method of any one of claims 20 to 22, wherein the cancer is a solid tumor.

24. The population of T cells enriched for central memory T cells of claim 17, or the pharmaceutical composition of claim 18 or claim 19 for use in the treatment of cancer.

25. Use of the population of T cells enriched for central memory T cells of claim 17, or the pharmaceutical composition of claim 18 or claim 19 in the manufacture of a medicament for the treatment of cancer.

26. A method of manufacturing a population of transduced T cells for adoptive cell therapy, the method comprising: a. obtaining a population of T cells; b. activating the population of T cells; c. contacting the population of T cells with an inhibitor of one or both of CBP and p300 for a time and under conditions suitable to promote memory differentiation; d. following step (c), transducing the population of T cells with a construct comprising a nucleic acid sequence encoding a CAR or a TCR.