Methods to identify and selectively expand tumor antigen-specific t cells

Abstract

Disclosed are methods of selectively expanding a number of T cells expressing an exogenous T cell receptor (TCR) having antigenic specificity for a target antigen and methods of selectively expanding a number of T cells each having antigenic specificity for a target antigen. Also disclosed are methods of isolating a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity for the target antigen; methods of preparing a population of cells that express a TCR, or an antigen-binding portion thereof, having antigenic specificity for a target antigen; methods of preparing a pharmaceutical composition comprising a selectively expanded number of T cells each having antigenic specificity for a target antigen; and methods of treating or preventing a condition in a mammal.

Description

METHODS TO IDENTIFY AND SELECTIVELY EXPAND TUMOR ANTIGENSPECIFIC T CELLSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 572,693, filed April 1, 2024, which is incorporated by reference in its entirety herein.STATEMENT REGARDINGFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number ZIA- BC-010984 by the National Institutes of Health. National Cancer Institute. The Government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 24,307 Byte Extensible Markup Language (XML) file named “772583. xml,” dated February 27, 2025.BACKGROUND OF THE INVENTION

[0004] Adoptive cell therapies (ACT) targeting tumor antigens can successfully treat some cancer patients. To produce a T cell product to administer to a patient, T cells may be expanded in vitro using a conventional rapid expansion protocol (REP), in which T cells are nonspecifically activated and cultured using, e.g., the anti-CD3 antibody. However, REP can reduce the frequency of tumor antigen-reactive T cells and also lead to one or both of T-cell exhaustion and differentiation. This can make it difficult to isolate a tumor-reactive T-cell receptor or to produce a T-cell infusion product containing high numbers of antigen-reactive T cells, leading to ineffective ACT. Accordingly, there exists a need for improved methods of preparing T cell products to administer to patients.BRIEF SUMMARY OF THE INVENTION

[0005] An aspect of the invention provides a method of selectively expanding a number of T cells expressing an exogenous T cell receptor (TCR) having antigenic specificity for a target antigen, the method comprising: introducing a nucleic acid into peripheral blood mononuclear cells (PBMC), wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR having antigenic specificity for the target antigen, to produce T cells expressing the exogenous TCR; inducing autologous antigen presenting cells (APCs) to present the target antigen; stimulating, in the presence of interleukin (IL)-2 with or without IL-21 in vitro, the T cells expressing the exogenous TCR with the APCs that present the target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity for the target antigen; and non-specifically expanding the number of T cells.

[0006] Another aspect of the invention provides a method of selectively expanding a number of T cells expressing an exogenous TCR having antigenic specificity for a target antigen, the method comprising: introducing a nucleic acid into PBMC, wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR having antigenic specificity for the target antigen, to produce T cells expressing the exogenous TCR; and inducing autologous APCs to present the target antigen; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, the T cells expressing the exogenous TCR with the APCs that present the target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity for the target antigen; wherein the method does not comprise non-specifically expanding the number of T cells.

[0007] Another aspect of the invention provides a method of selectively expanding a number of T cells each having antigenic specificity for a target antigen, the method comprising: inducing APCs of the mammal to present one or more target antigens; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, thereby producing a selectivelyexpanded number of T cells each having antigenic specificity for the one or more target antigens; and non-specifically expanding the number of T cells.

[0008] Another aspect of the invention provides a method of selectively expanding a number of T cells each having antigenic specificity for a target antigen, the method comprising: inducing autologous APCs of the mammal to present one or more target antigens; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, wherein the T cells having antigenic speci ficity for the tumor antigen receive proliferation signals, the number of T cells having antigenic specificity for the target antigen expand and the number of T cells that do not have antigenic specificity’ for the target antigen do not expand, thereby producing a selectively expanded number of T cells each having antigenic specificity for the one or more target antigens; wherein the method does not comprise non-specifically expanding the number of T cells.

[0009] Still another aspect of the invention provides a method of isolating a TCR, or an antigen-binding portion thereof, having antigenic specificity for the target antigen, the method comprising: selectively expanding a number of T cells each having antigenic specificity’ for one or more target antigens according to any of the inventive methods described herein; and isolating a TCR. or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity for one of the target antigens.

[0010] Another aspect of the invention provides a method of preparing a population of cells that express a TCR. or an antigen-binding portion thereof, having antigenic specificity' for a target antigen, the method comprising: isolating a TCR. or an antigen-binding portion thereof, according to any of the inventive methods described herein, and introducing the nucleotide sequence encoding the isolated TCR, or the antigen-binding portion thereof, into PBMC to obtain cells that express the TCR, or the antigen-binding portion thereof.

[0011] Another aspect of the invention provides a T cell modified to express one or both of BACH2 and KLF2 or a population of cells comprising the T cell or a pharmaceutical composition comprising the T cell or population of cells.

[0012] Additional aspects of the invention provide related methods of preparing a pharmaceutical composition and methods of treating or preventing a condition.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0013] Figure 1A is a schematic showing the conventional way of TIL expansion, screening and the REP for the development of a TIL infusion product.

[0014] Figure IB is a graph showing the frequencies of neoantigen-reactive TIL before and after a REP. TIL fragment cultures with neoantigen reactivities before and after the REP were co-cultured with autologous dendritic cells pulsed with mutated peptides. Percent reactive cells were assessed by flow cytometric measurement of 4-lBB+or OX-40+cells. Shaded bars represent the TIL infusion product. Unshaded bars represent the TIL fragment culture.

[0015] Figure 1C is a schematic showing NeoExpand using neoantigen-loaded APCs for the expansion of neoantigen-reactive TILs (or T cells from other sources with tumor reactivity, which include lymph node and peripheral blood).

[0016] Figure 2A shows the results of a representative example of neoantigen-reactive TIL enrichment by neoantigenic stimulation: enrichment of p53R175H-reactive cells from 4141 TILs via NeoExpand for 14 days was compared with the culture with IL-2 for the same duration. The result of the expansion of p53R175H-reactive cells was determined by flow cytometric measurement of 4-1BB and OX-40 following an overnight co-culture against the same stimulus of p53R175H. Peptide-pulsed, HLA-engineered COS7 cells were used as APCs. ME minimal epitope.

[0017] Figure 2B is a graph showing the frequencies of the two p53R175H-reactive clonotypes among reactive cells in Figure 2A.

[0018] Figure 2C is a graph showing the results of a peptide titration assay testing specificity7of 4141 NeoExpand TCR isolated and shown in Table 2. 4-1BB was measured following an overnight co-culture of TCR-engineered healthy donor PBLs with A*02- engineered COS7 cell.

[0019] Figure 2D shows the results of HLA testing of 4141 NeoExpand TCR. Healthy donor PBLs expressing 4141 NeoExpand TCR were co-cultured with COS7 cells transfected with individual HLAs expressed by patient 4141. IFN-y secretion was measured by an ELISpot assay.

[0020] Figure 2E is a graph showing the frequencies of 4141 '‘known” clonotype and 4141 NeoExpand clonotype in the patient infusion product were determined by CDR3B sequencing.

[0021] Figure 2F is a schematic showing the steps of an in vivo functional test of 4141 NeoExpand TCR.

[0022] Figure 2G is a graph showing the results (tumor measurement) of an in vivo functional test of 4141 NeoExpand TCR (n=5). Replicates from ACTs of two healthy donor PBLs are shown. Statistical analysis by two-way ANOVA. ***p<0.001.

[0023] Figure 3A shows the results of flow cytometric measurement of p53R273C reactive cells following NeoExpand.

[0024] Figure 3B is a graph showing the results of a peptide titration assay testing specificity of 4386 NeoExpand TCR isolated and shown in Table 3C. IFN-y secretion was measured following an overnight co-culture of TCR-engineered healthy donor PBLs with 4386 DCs pulsed with WT or mutant p53 peptides.

[0025] Figure 3C is a Venn diagram showing the number of patients with a positive neoantigen screen (left) or the number of neoantigen-reactive CD4+or CD8+T-cell clonotypes identified following the conventional TIL expansion or NeoExpand (right).

[0026] Figure 4A is a schematic of 4196, 4385, and 4391 TIL expansion by the REP with OKT3 or NeoExpand for mouse xenograft ACT studies.

[0027] Figures 4B-4D are graphs comparing REP and NeoExpand. 4196 TILs (4B), 4385 TILs (4C) and 4391 TILs (4D) were expanded by IL-2 alone, REP or NeoExpand: Fold changes of total CD3+cells, frequencies of neoantigen-reactive TILs. and fold changes of neoantigen-reactive TILs were evaluated by tetramer staining (4B) or by 4- IBB and OX-40 measurement (4C, 4D). Fold changes were calculated based on the number of input cells.

[0028] Figure 4E is a graph showing the results of an experiment in which ten TIL samples were tested to compare the efficiency of neoantigen-reactive TIL expansion between the REP and NeoExpand. Statistical analysis by Wilcoxon matched-pairs signed rank test. **p<0.01.

[0029] Figures 5A-5B show the results of single-cell transcriptome (scRNA-seq) analysis of 4196 TIL following NeoExpand or REP. Fig. 5 A shows the results of a Uniform Manifold Approximation and Projection (UMAP) analysis of 4196 TILs (left) and p53R175H-reactive cells (right). Clusters 3, 4, and 10 containing high numbers of p53R175H-reactive cells are encircled. Fig. 5B shows the gene expression of markers of stem-like memory T cells and exhausted T cells.

[0030] Figure 5C is a graph showing the relative frequencies of p53R175H-reactive cells in clusters 3, 4, and 10 and their clonal composition.

[0031] Figures 5D-5F show the results of scRNA-seq analysis of 4391 TIL following NeoExpand or REP. Fig. 5D shows the results of UMAP analysis of 4391 TILs (left) and RASG12V -reactive cells (right). Fig. 5E shows gene expression of markers of stem-like memory' T cells and exhausted T cells.

[0032] Figure 5F is a graph showing the relative frequencies of RASG12V-reactive cells in cluster 9 and their clonal composition.

[0033] Figure 6A is a schematic showing the testing 4196 TILs against TYK-nu cancer cells or 4385 and 4391 TILs against 4391 PDX cells in an in vivo xenograft model.

[0034] Figures 6B-6D are graphs showing tumor growth in mice injected with 4196 TILs (6B). with 4385 TILs (6C), or with 4391 TILs (6C) (n=5). Mice in 6B and 6C were injected with 2 x 107TILs per mouse and mice in 6D were injected with 1X107TILs per mouse. The 4196 experiment was independently replicated once. Statistical analysis by two-way ANOVA *p<0.05, ***p<0.001.

[0035] Figure 7 are graphs showing the fold expansion of CD8+mTCR+cells by NeoExpand with HLA-engineered C0S7 cells pulsed with different peptide concentrations. The TCRs exogenously expressed contained murine constant region sequences (mTCR), which were detected by the mTCR-specific antibody. The IL-2 condition without the neoantigenic stimulation was included as a negative control. Statistical analysis by one-way ANOVA, excluding the IL-2 conditions. NS, not significant (n=3). The table on the right lists the two mutant KRAS or p53-reactive TCRs and their HLA restrictions.

[0036] Figure 8A is a graph showing HLA specific activation of 4432 TILs following RAS G12D-specific stimulation. Autologous DCs or HLA-A*02 or A* 11 -engineered C0S7 cells were used as APC. Reactive cell frequencies (4-lBB+OX-40+cells) are reported following an overnight co-culture.

[0037] Figure 8B is a graph showing peptide titration of the HL A-A* 11 -restricted RAS G12D-reactive TCR isolated from 4432 TILs by NeoExpand.

[0038] Figure 8C is a schematic showing the in vivo functional test of 4432 RAS G12D- reactive TCR.

[0039] Figure 8D is a graph showing the tumor measurement of NSG mice implanted with 4069 PDX following ACT of 4432 TCR-engineered healthy donor PBL (n=5).Statistical analysis by two-way ANOVA, ***p<0.001.

[0040] Figures 9A-9D show graphs showing peptide titration of 4 p53Rl 75H-reactive TCRs isolated following NeoExpand. HLA-A*02+T2 cells were used as APCs. The TCRsare 4196-IVS-A (9A), -C (9B), -D (9C). and -E (9D). The TCRs were found in 4196 TILs as a part of the study shown in Figures 5 A-5C.

[0041] Figure 9E is a graph showing the results of an in vitro tumor cell killing assay. Fifty thousand healthy donor PBLs transduced with one of the four p53R175H-reactive or irrelevant TCRs were co-cultured with 10.000 TYK-nu cells over 72h (n=4). Statistical analysis by two-way ANOVA, ***p<0.001. Figures 9A-9E show that the TCRs newly and exclusively identified following the inventive methods are specific and can exert anti-tumor function.

[0042] Figures 10A-10D show graphs showing the peptide titration of four RAS G12V- reactive TCRs isolated following NeoExpand from 4391 TILs. C0S7 cells stably expressing HLA-C*01 :02 were pulsed with either wild-type (WT) or RAS G12V minimal epitope (ME), and were used in the overnight co-culture. The TCRs are 4391-B2 (10A), 4391-C1 (10B), 4391-K (IOC), and 4391-L2 (10D).

[0043] Figures 11 A-l 1C are graphs showing the GSEA of clusters 3 (11 A), 4 (1 IB) and 10 (1 1C) against publicly available scRNA-seq data. Figures 11 A-l IE show results of single cell RNA seq analysis of 4196 TILs.

[0044] Figure 12 is a graph showing the results of an experiment in which 4196 TILs were stained with phenotypic markers and were pre-gated for CD8 and A*02-tetramer containing the p53R175H 9-mer epitope. Two biological replicates are shown. EM effector memory (CD62L-CD45RO+); CM central memory (CD62L+CD45RO+).

[0045] Figure 13 shows graphs showing the results of scRNA-seq analysis of 4391 TILs. Fig. 13 shows the GSEA of cluster 9 of 4391 TILs against publicly available scRNA-seq data.

[0046] Figure 14 is a schematic showing sample-by sample experimental procedures.

[0047] Figure 15 shows the expression of 41BB and / or 0X40 measured by flow cy tometry' following co-culture of effector cells with target cells. Effector cells were PBMCs expressing the 4304 TCR. Target cells were DCs pulsed with tumor lysate. T cells cocultured with DC pulsed with KRAS G12V 25-mer peptide in the high concentration of 1 pg / ml or medium concentration of 10 ng / ml served as a positive control. T-cells co-cultured with DC or cultured alone served as a negative control.

[0048] Figure 16A is a representative graph showing reactivity measured by 4- IBB and / or 0X40 upregulation measured by flow cytometry following 14 days of NeoExpand(post-NeoExpand) in comparison to the reactivity existing in the same TIL population before the IVS (Pre NeoExpand).

[0049] Figure 16B is a graph showing the percentage of reactive cells detected before and after NeoExpand, as described for Figure 16A, for 10 additional patients' TIL samples.

[0050] Figure 17 is a graph showing expansion of the numbers of (percentage of mTCR+CD8+T cells) detected following NeoExpand using EBV-B cells or HLA-engineered COS cells or expansion of the numbers of T cells using REP.

[0051] Figures 18A-18D are graphs showing the fold expansion of CD8+mTCR+T cells following co-culture of APCs with T cells engineered with the 4148 (18A). 4373 (18B), 4391 (18C), or 4424 (18D) TCR. The APCs were pulsed with the indicated concentration of minimal epitope peptide. T cells cultured with IL-2 alone served as a control. T cells cultured with COS7 cells engineered with HLA and TMG served as a control. T cells expanded by REP also served as a control.

[0052] Figures 19A-19C are graphs showing (i) the frequency of CD8+mTCR+cells (%) (19A); (ii) total CD3+cell fold expansion (bulk) (19B); and (iii) fold expansion of CD8+mTCR+cells (19C) following co-culture of 4373 TCR-engineered T cells with the indicated APCs. The NeoExpand co-cultures were carried out in the presence of IL-2 and IL- 21. T cells cultured with IL-2 (no stimulation) and T cells expanded by REP served as controls.

[0053] Figures 19D and 19E are graphs presenting the percentage (19D) and the fold expansion (19E) of CD4+mTCR+cells for an aggregate of 5 different TCRs isolated from CD4+cells.

[0054] Figure 20 shows dot-plot graphs of the results of a flow cytometry assay following the co-cultures of 4373 TCR-engineered T cells with the APCs under the conditions described for Figures 19A-19C. The mTCR+CD8+gated cells were analyzed and evaluated for the effector / memory phenotype by testing the expression of CD45RO and CD62L as in the sample quadrant. EM (effector memory cells); CM (central memory cells); TN / TSCM (naive / stem cell memory); EMRA (terminally differentiated effector memory re-expressing CD45RA).

[0055] Figure 21 shows dot-plot graphs of the results of a flow cytometry assay following the co-cultures of 4373 TCR-engineered T cells with the APCs under the conditions described for Figures 19A-19C. The mTCR+CD8+gated cells were analyzed and evaluated for the expression of CD69 and CD39 as in the sample quadrant.

[0056] Figure 22A presents a schematic showing a combination of NeoExpand and REP according to an aspect of the invention.

[0057] Figures 22B-22C are graphs showing (i) frequency of CD8+mTCR+cells (%) (22B); and (ii) fold expansion of CDS mTCR cells (22C) measured following the culture conditions indicated in the Figures. Figures 22B and 22C present the percentage and the fold expansion of CD8+mTCR+cells, respectively, out of live CD3+cells on the day the co-culture was initiated (PRE) or 2 weeks following expansion of each one of the samples.

[0058] Figures 22D-22E are graphs presenting the percentage (22D) and the fold expansion (22E) of TCR-engineered cells expressing the relevant CD4 or CD8 co-receptor (mTCR+Co-R+) measured following the culture conditions indicated in the Figures.

[0059] Figures 23A-23C are graphs showing (i) frequency of tumor-reactive cells (%) (23 A); (ii) fold expansion of tumor-reactive cells (23B); and (iii) frequency of CD39' tumor- reactive cells (%) (23C) measured following the culture conditions indicated in the Figures.

[0060] Figures 24A-24C are graphs showing (i) frequency of tumor-reactive cells (%) (24A); (ii) fold expansion of tumor-reactive cells (%) (24B); and (iii) frequency of CD39' tumor-reactive cells (%) (24C) measured following the culture conditions indicated in the Figures.

[0061] Figure 25 A is a graph showing the percentage of 4-lBB+or OX-40+of CD4+cells(%) measured by flow cytometry of TIL samples before and after NeoExpand or REP. after overnight incubation with autologous DCs pulsed with the peptide pool. DMSO and T cell only conditions were included as negative controls.

[0062] Figure 25B is a graph showing the percentage of 4-lBB+or OX-40+of CD4+cells (%) measured by flow cytometry of 11 TIL samples before and after NeoExpand.

[0063] Figure 26A is a graph showing the percentage of 4-1 BB+or OX-40+of CD8+cells (%) measured by flow cytometry of TIL samples before and after NeoExpand or REP, after overnight incubation with autologous DCs pulsed with the peptide pool. DMSO and T cell only conditions were included as negative controls.

[0064] Figure 26B is a graph showing the percentage of 4-1 BB+or OX-40+of CD8+cells (%) measured by flow cytometry of 11 TIL samples before and after NeoExpand.

[0065] Figure 27 shows dot-plot graphs showing the percentage of reactive CD8+TILs measured by upregulation of 4- IBB and 0X40 in a flow cytometry assay following overnight co-culture with APC pulsed with pool of 25-mer mutated peptides or. as a negative control, with APC loaded with DMSO (peptide solvent) or culture without additional cells (T cellonly). Reactivity was tested immediately before start of NeoExpand (TIL pre-expansion) and 28 days after start of NeoExpand (Tumor Lysate NeoExpand 28 days).

[0066] Figure 28A is a graph showing the frequency of reactive cells (%) measured before NeoExpand (pre-expansion), after NeoExpand alone (transduction with empty vector), or after NeoExpand with BACH2 transduction.

[0067] Figure 28B is a graph showing the fold expansion of reactive cells measured after NeoExpand alone (transduction with empty vector) or after NeoExpand with BACH2 transduction.

[0068] Figure 28C is a graph showing the total number of cells measured after REP ofTIL which had been transduced with BACH2 or an empty’ vector (mock).

[0069] Figure 29 is a schematic illustrating a method of expanding the numbers of T cells with extremely rare CD8 reactivities using NeoExpand followed by bead selection and REP according to an aspect of the invention.

[0070] Figure 30 includes dot plots showing the results of flow cytometry assays measuring the numbers of cells expressing the mTCR beta chain following two 14 day cycles of (i) REP only, (ii) NeoExpand and REP (OKT3) only, or (iii) NeoExpand followed by bead selection then REP.

[0071] Figures 31 A-3 IB are graphs showing the percentage of neoantigen-reactive TIL fragment cultures detected for TIL from fragment 7 (31 A) and fragment 15 (3 IB) following (i) REP only, (ii) NeoExpand and REP (OKT3) only, or (iii) NeoExpand followed by bead selection then REP.

[0072] Figures 32A-32B are graphs showing the percentage of CD39'CD69' (32A) or CD39+CD69+(32B) cells measured following transduction of TIL transduced with KLF2 or an empty vector (mock).

[0073] Figure 32C is a graph showing the tumor size measured at the indicated number of days following administration of the indicated number of T cells transduced with KLF2 and an anti-mutated p53 TCR, the anti-mutated p53 TCR alone, or an empty vector (mock) to tumor-bearing mice.DETAILED DESCRIPTION OF THE INVENTION

[0074] It has been discovered that stimulating, in the presence of IL-2 with or without IL- 21 in vitro, T cells having antigenic specificity for a target antigen with APCs that present the target antigen selectively expand the number of T cells having antigenic specificity for thetarget antigen. The selective expansion achieved with the inventive methods may be superior to that which is achieved with conventional REP.

[0075] The inventive methods described herein can provide selective grow th of T cells having antigenic specificity' for a target antigen, including, e.g., TCR-engineered peripheral lymphocytes (PBL) and TILs. Conventional methods for in vitro T-cell culture, such as REP, can lead to outgrowth of bystander cells and, therefore, can reduce the frequency of target antigen-reactive cells. Alternative methods, such as flow activated cell sorting, can be difficult to employ in a clinical setting due to the complexity7of the methods and the low number of cells that can be processed at a time.

[0076] In the experiments described in the Examples herein, it has been demonstrated that, by7providing specific antigenic stimulus to cells of interest (i.e., TCR-engineered T cells or T cells, e.g., TILs having antigenic specificity' for a tumor antigen (e.g., neoantigen- reactive TILs, or “neoTILs”)), relevant T cells can be effectively expanded. The selective growth of T cells, in particular neoTILs. may allow sensitive identification of novel TCRs having antigenic specificity for a target antigen (e.g., neoantigen-reactive TCRs, e.g., neoTCRs). For instance, 25 patients whose tumor harbored either p53 or KRAS mutations were screened for the presence of neoantigens. The conventional method identified 8 patients with either p53 or KRAS neoantigens. The inventive method identified 16 patients with p53 or KRAS neoantigens. Conventional screening identified 14 TCRs (3 CD4; 11 CD8) and the inventive methods identified 42 TCRs (14 CD4; 28 CD8). Notably, neoantigens and neoTCRs identified by the inventive methods included all the neoantigens and TCRs identified by the conventional screening. These data suggest that the inventive methods capture and enrich novel and rare neoTIL clones, increasing their clonal diversity.

[0077] The inventive methods can bring about a stem-like memory T cell phenotype. This phenoty pe has been linked to better in vivo anti-tumor function and increased persistence. By flow cytometry and single cell transcriptome analysis, the experiments described in the Examples herein demonstrate that there may be an increase in the portion of T cells that maintain or acquire a stem-like memory T cell phenotype following the inventive methods when compared to the number of T cells expanded using REP.

[0078] Functionally, the experiments described in the Examples herein show that TILs selectively expanded by the inventive methods better treat xenograft tumors established in NOD SCID I12rg (NSG) mice as compared to those expanded by REP.

[0079] Unlike conventional REP that can nonspecifically expand T cells with the “wrong” co-receptor (i.e., CD4 or CD8), the inventive methods may increase neoTCR+T cells with the “correct” co-receptor, i.e., expansion of CD4+neoTCR+T cells for a CD4+cell- derived TCR and expansion of CD8+neoTCR+T cells for a CD8+cell-derived TCR. The inventive methods may, advantageously, provide sensitive identification of both CD4 and CD8 TCRs.

[0080] The inventive methods may provide any one or more of a variety of advantages. For example, the inventive methods may provide for the selective expansion of the numbers of T cells expressing the exogenous TCR over the number of cells which do not express the exogenous TCR. The inventive methods may provide populations of cells with a larger proportion of cells which express the exogenous TCR as compared to populations of cells prepared by methods which do not selectively expand the number of T cells as described herein. Without being bound to a particular theory' or mechanism, it is believed that populations of cells with a larger proportion of cells which express the exogenous TCR may provide one or both of improved destruction of target cells (e.g., cancer cells) and treatment of a condition (e.g., cancer) as compared to populations of cells with a smaller proportion of cells which express the exogenous TCR. Stimulating TCR-engineered T cells with APCs that express target antigens achieves selective growth of target antigen-reactive T cells, which allows generation of TCR-engineered T-cell products enriched for target antigen reactivity.

[0081] An aspect of the invention provides a method of selectively expanding a number of T cells expressing an exogenous TCR having antigenic specificity for a target antigen, the method comprising: introducing a nucleic acid into PBMC, wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR having antigenic specificity for the target antigen, to produce T cells expressing the exogenous TCR; inducing autologous APCs to present the target antigen; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, the T cells expressing the exogenous TCR with the APCs that present the target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity' for the target antigen; and non-specifically expanding the number of T cells.

[0082] An aspect of the invention provides a method of selectively expanding a number of T cells expressing an exogenous TCR having antigenic specificity for a target antigen, themethod comprising: introducing a nucleic acid into PBMC. wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR having antigenic specificity for the target antigen, to produce T cells expressing the exogenous TCR; and inducing autologous APCs to present the target antigen; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, the T cells expressing the exogenous TCR with the APCs that present the target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity for the target antigen; wherein the method does not comprise non-specifically expanding the number of T cells.

[0083] An aspect of the invention provides a method of selectively expanding a number of T cells expressing an exogenous TCR having antigenic specificity for a target antigen. The method may comprise introducing a nucleic acid into PBMC, wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR, to produce T cells expressing the exogenous TCR. By “exogenous” is meant that the TCR is not native to (naturally- occurring on) the cell. The exogenous TCR may be a recombinant TCR. A recombinant TCR is a TCR which has been generated through recombinant expression of one or more exogenous TCR a-, 0-. y-, and / or 5-chain encoding genes. A recombinant TCR can comprise polypeptide chains derived entirely from a single mammalian species, or the recombinant TCR can be a chimeric or hybrid TCR comprised of amino acid sequences derived from TCRs from two different mammalian species. For example, the TCR can comprise a variable region derived from a human TCR, and a constant region of a murine TCR. Any exogenous TCR having antigenic specificity for a target antigen may be useful in the inventive methods. The TCR generally comprises two polypeptides (i.e., polypeptide chains), such as an a-chain of a TCR, a 0-chain of a TCR, a y-chain of a TCR, a 5-chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The target antigen-specific TCR can comprise any amino acid sequence, provided that the TCR can specifically bind to and immunologically recognize a target antigen or epitope thereof.

[0084] Introducing a nucleic acid comprising a nucleotide sequence (e.g., a recombinant expression vector) encoding the exogenous TCR into PBMC may be carried out in any of a variety of different ways known in the art as described in. e.g., Green et al. (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 4th Ed. (2012). Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into PBMC include transformation, transduction, transfection, and electroporation.

[0085] The TCR may have antigenic specificity for a target antigen. The target antigen may be a tumor antigen, an antigen associated with a condition, a viral antigen, or a bacterial antigen. The antigen associated with a condition may be an antigen associated with an autoimmune disease. Examples of viral antigens include, but are not limited to, human immunodeficiency virus antigens, respiratory syncytial virus antigens, hepatitis C virus antigens and Epstein-Barr virus antigens. The target antigen may be a tumor antigen. Tumor antigens include tumor associated antigens and cancer germline antigens whose expression is relatively limited to tumor cells and neoantigens that arise from somatic mutations exclusively in tumor cells. The tumor antigen may be, for example, a tumor-associated antigen, a cancer germline antigen, or a neoantigen. Tumor-associated antigens (TAA), have elevated levels on tumor cells, but are also expressed at lower levels on healthy cells. TAAs include, for example, differentiation antigens (such as melanocyte differentiation antigens, e.g., gplOO, tyrosinase, and Melan-A), and overexpressed cellular antigens (such as HER2). Cancer germline antigen genes are a large family of genes whose expression is mainly limited to germ cells and cancer (e.g., MAGE-A1, MAGE-A2, MAGE-A3, NY-ESO-1, and SSX). Neoantigens are a class of cancer antigens which arise from cancer-specific mutations in expressed protein. The term “neoantigen” relates to a peptide or protein expressed by a cancer cell (e.g., tumor cell) that includes one or more amino acid modifications compared to the corresponding wild-type (non-mutated) peptide or protein that is expressed by a normal (non-cancerous) cell. A neoantigen may be patient-specific or it can be from shared mutations (e.g., RAS or p53).

[0086] The method may comprise inducing autologous APCs to present the target antigen. The APCs may be dendritic cells (DCs); B cells; peripheral blood mononuclear cells (PBMC); autologous tumor cells; a human leukocyte antigen (HLA)-engineered cell line; tumor organoid cells, or tumor xenograft cells. In an aspect of the invention, the APCs may be an engineered cell line. The number of cells of a cell line may be expanded indefinitely and may be inexpensive to grow . A library of cell lines expressing common HLA can be generated to expand the number of T-cells. Cell lines can be further engineered to boost T cell expansion (e.g., to express 4-1BBL). For example, COS7 cells (a monkey kidney cell line lacking HLA expression) may be transduced with the applicable HLA molecule and target antigen(s) and may serve as an APC in the inventive methods. As another example,primary B cells transformed by EBV and transduced with target antigen(s) may’ serve as an APC in the inventive methods. In an aspect of the invention, the APCs may be nonengineered cells. In this regard, for example, the APCs may be autologous or HLA-matched DC. The DC may be prepared by in vitro differentiation of monocytes from PBMC. As another example, the APCs may be autologous or HLA-matched B cells. The B cells may be either directly isolated from PBMCs or expanded in vitro through stimulation with the CD40 ligand following isolation. As another example, the APCs may be autologous or HLA- matched PBMC. The PBMCs may be prepared by purification of whole blood or leukapheresis.

[0087] In an aspect of the invention, inducing the APCs to present the target antigen comprises pulsing the APCs with a peptide, wherein the peptide comprises a target antigen amino acid sequence. In this regard, the APCs may be cultured with a peptide comprising the target antigen amino acid sequence to display the mutated amino acid sequence, bound to an MHC molecule, on the cell membrane. Methods of pulsing APCs are known in the art and are described in, e.g., Solheim (Ed.), Antigen Processing and Presentation Protocols (Methods in Molecular Biology), Human Press, (2010).

[0088] In an aspect of the invention, inducing the APCs to present the target antigen comprises introducing a nucleotide sequence encoding the target antigen into the APCs. The nucleotide sequence is introduced into the APCs so that the APCs express and display the target antigen amino acid sequence, bound to an MHC molecule, on the cell membrane. The nucleotide sequence encoding the target antigen amino acid sequence may be RNA or DNA. Introducing a nucleotide sequence into APCs may be carried out in any of a variety of different ways known in the art as described in, e.g., Solheim et al. supra. Non-limiting examples of techniques that are useful for introducing a nucleotide sequence into APCs include transformation, transduction, transfection, and electroporation.

[0089] In an aspect of the invention, tumor antigens isolated from tumor cells (e.g., resected tumor or cultured tumor cells), such as by lysing tumor cells, can be used to stimulate T cells having antigenic specificity for the tumor antigen, for example, as described in Examples 8 and 9. In an aspect of the invention, inducing the APCs to present the target antigen comprises pulsing the APCs with whole or lysed autologous tumor, whole or lysed tumor cells, or whole or lysed organoid derived from autologous tumor.

[0090] The method may further comprise stimulating, in the presence of IL-2 with or without IL-21 in vitro, the T cells expressing the exogenous TCR with the APCs that presentthe target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity for the target antigen. The method may comprise co-culturing the T cells expressing the exogenous TCR and APCs so that the exogenous TCR encounters the target antigen presented by the APCs in such a manner that the exogenous TCR specifically binds to and immunologically recognizes the target antigen presented by the APCs, thereby initiating proliferation of the T cells expressing the exogenous TCR and selectively expanding the number of T cells expressing the exogenous TCR over the number of T cells not expressing the exogenous TCR. In an aspect of the invention, the T cells are co-cultured in direct contact with the APCs.

[0091] It has been discovered that, if the IL-2 employed in the inventive methods is high dose IL-2, the expansion of the numbers of T cells having antigenic specificity for a target antigen can be enhanced. A dose of IL-2 that is considered a '‘high dose’’ of IL-2 may be, for example, 1000 to 6000 lU / ml and may vary depending on the type of cell. For example, for PBMCs, a high dose of IL-2 may be from 1000 lU / ml to 6000 lU / ml. In an aspect of the invention, the dose of IL-2 employed in the inventive methods for PBMC may start with a regular (non-high) dose of from 0 to 300 lU / ml IL-2 on the day of co-culture and may gradually increase up to a high dose of 1000 to 6000 lU / ml. For TIL, a high dose of IL-2 may be from 1500 lU / ml to 6000 lU / ml. In an aspect of the invention, the dose of IL-2 employed in the inventive methods for TIL may start with a regular (non-high) dose of from 0 to 500 lU / ml IL-2 on the day of co-culture, then gradually increase to 1000 to 3000 lU / mL IL-2 and then may gradually increase up to a high dose of 1500 to 6000 lU / ml IL-2.

[0092] In an aspect of the invention, the stimulating is carried out for a period of 10 to 28 days. For example, the stimulating may be carried out for a period of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days, or range of days defined by any two of the foregoing values.

[0093] In an aspect of the invention, the method does not comprise screening the PBMC or the T cells expressing the exogenous TCR for antigenic specificity for the target antigen before or during the stimulating.

[0094] The inventive methods of selectively expanding a number of T cells expressing an exogenous TCR having antigenic specificity for a target antigen can be combined with beadselection to enrich for extremely rare T cells with antigenic specificity for a target antigen. Extremely rare T cells may be present at a frequency of less than 0. 1 % in a bodily sample (e.g., blood, or tumor). The bead selection advantageously reduces the number of unreactive bystander cells that could otherwise proliferate in response to non-specific expansion.

[0095] In this regard, in an aspect of the invention, the inventive methods further comprise introducing a nucleotide sequence encoding a cell surface marker into the APCs to obtain APCs that express the cell surface marker. Methods of introducing a nucleotide sequence into APCs are described herein with respect to other aspects of the invention. The cell surface marker may be any cell surface marker that allows for bead selection. For example, the cell surface marker may be NGFR.

[0096] The method may further comprise adding magnetic beads to the APCs and the T cells during or after the stimulating, w herein the magnetic beads are coupled to a binding partner that specifically binds to the cell surface marker. The binding partner may be any moiety that specifically binds to the cell surface marker. Examples of binding partners include specific antibodies, lectins, and enzymes.

[0097] The method may further comprise co-culturing the magnetic beads with the APCs and the T cells during or after the stimulating, thereby producing a complex comprising the APCs, the T cells, and the magnetic beads, wherein the binding partner coupled to the magnetic beads is bound to the cell surface marker expressed by the APCs. and wherein the T cells expressing the exogenous TCR are bound to the target antigen presented by the APCs. The T cells and the APCs may be mixed with the magnetic beads so that the binding partner coupled to the magnetic beads specifically bind to the cell surface marker on the APCs. In addition, T cells expressing the exogenous TCR bind to the target antigen presented by the APCs.

[0098] The method may further comprise applying a magnetic field to the complex to physically separate the complex from other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs. A mixed population of T cells may result following the introducing of the nucleic acid encoding the exogenous TCR into the PBMCs, in which some of the resulting T cells express the exogenous TCR, and some do not. The magnetic field physically separates the T cells that express the exogenous TCR and are bound to the target antigen presented by the APCs from other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs.

[0099] Optionally, the method may further comprise non-specifically expanding the number of T cells, as described herein with respect to other aspects of the invention. Tn this aspect of the invention, the method may further comprise non-specifically expanding the number of T cells of the complex that express the exogenous TCR and are physically separated from the other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs.

[0100] Another aspect of the invention provides a method of selectively expanding a number of T cells each having antigenic specificity7for a target antigen. The T cells having antigenic specificity for the target antigen may express an endogenous TCR that has antigenic specificity for the target antigen. An endogenous TCR is a natively expressed TCR that an unmodified T cell has naturally developed while successfully undergoing negative selection in the thymus. Cells that express such an endogenous TCR may include, for example, TIL, or T cells isolated from any organ, including, but not limited to. lymph node and peripheral blood. Stimulating T cells (e.g., TIL) with APCs that express target antigen(s) achieves selective growth of target antigen-reactive T cells, which allows generation of T-cell products enriched for target antigen reactivity7and facilitates target antigen-reactive TCR isolation.

[0101] An aspect of the invention provides a method of selectively expanding a number of T cells each having antigenic specificity7for a target antigen, the method comprising: inducing autologous APCs of the mammal to present one or more target antigens; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, wherein the T cells having antigenic specificity for the tumor antigen receive proliferation signals, the number of T cells having antigenic specificity for the target antigen expand and the number of T cells that do not have antigenic specificity for the target antigen do not expand, thereby producing a selectively expanded number of T cells each having antigenic specificity7for the one or more target antigens; and non-specifically expanding the number of T cells.

[0102] An aspect of the invention provides a method of selectively expanding a number of T cells each having antigenic specificity for a target antigen, the method comprising: inducing autologous APCs of the mammal to present one or more target antigens; and stimulating, in the presence of IL-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, wherein the T cells having antigenic specificity for the tumor antigen receive proliferation signals, the number of T cells having antigenic specificity for the target antigen expand and the number of T cells that donot have antigenic specificity for the target antigen do not expand, thereby producing a selectively expanded number of T cells each having antigenic specificity for the one or more target antigens; wherein the method does not comprise non-specifically expanding the number of T cells.

[0103] The method may comprise inducing autologous APCs of the mammal to present one or more target antigens. The one or more target antigens may be as described herein with respect to other aspects of the invention. Inducing the APCs to present the one or more target antigens may be carried out as described herein with respect to other aspects of the invention. In an aspect of the invention, inducing the APCs to present the one or more target antigens comprises (i) pulsing the APCs with a peptide comprising a target antigen amino acid sequence or a pool of peptides, each peptide in the pool comprising a different target antigen amino acid sequence; (ii) pulsing the APCs with lysis of the tumor expressing different tumor antigens; (iii) introducing one or more nucleotide sequences encoding one or more different target antigens into the APCs; or (iv) pulsing the APCs with whole or lysed autologous tumor, whole or lysed tumor cells, or whole or lysed organoid derived from autologous tumor.

[0104] In an aspect of the invention, the one or more target antigens is a plurality of different target antigens. In an aspect of the invention, the number of different target antigens in the plurality of different target antigens is more than 2 different target antigens and can range from 2 to 1000 different target antigens. For example, the number of different target antigens may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 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, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126. 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137. 138, 139, 140, 141, 142. 143, 144, 145, 146, 147, 148, 149. 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, or a range defined by any two of the foregoing values. For example, the number of different target antigens in the plurality of different target antigens may range from 10 to 190, 20 to 180 30 to 170, 40 to 160. 50 to 150, 60 to 140, 70 to 130, 80 to 120, or 90 to 110 different target antigens.

[0105] The method may further comprise stimulating, in the presence of IL-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, thereby producing a selectively expanded number of T cells each having antigenic specificity for the one or more target antigens. The stimulating of the T cells from the mammal with the APCs may be carried out as described herein with respect to other aspects of the invention. The T cells may be, for example, tumor infiltrating lymphocytes (TIL). Other sources of T cells with target antigen reactivity may include lymph node and peripheral blood. In an aspect of the invention, the T cells are differentiated or exhausted TIL. In an aspect of the invention, the method does not comprise screening the T cells for antigenic specificity for the one or more target antigens before or during the stimulating.

[0106] Another aspect of the invention provides a method of isolating a TCR, or an antigen-binding portion thereof, having antigenic specificity for the target antigen. The method may comprise selectively expanding a number of T cells each having antigenic specificity for one or more tumor antigens according to any of the methods described herein with respect to other aspects of the invention.

[0107] The method may further comprise isolating a TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the TCR, or antigenbinding portion thereof, has antigenic specificity for one of the target antigens. The "‘the antigen-binding portion” of the TCR, as used herein, refers to any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the antigen-binding portion specifically binds to the target antigen as described herein with respect to other aspects of the invention. The term “antigen-binding portion” refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Antigen-binding portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to the target antigen, to a similar extent, the same extent, or to a higher extent, as compared to the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, 70%, 80%, 90%, 95%, or more, of the parent TCR.

[0108] The antigen-binding portion can comprise an antigen-binding portion of either or both of the a and (3 chains of the TCR, such as a portion comprising one or more of the complementarity determining region (CDR)1, CDR2, and CDR3 of the variable region(s) of the a chain and / or P chain of the TCR. In an aspect of the invention, the antigen-binding portion can comprise the amino acid sequence of the CDR1 of the a chain (CDRla), theCDR2 of the a chain (CDR2a), the CDR3 of the a chain (CDR3a). the CDR1 of the P chain (CDR1 ), the CDR2 of the P chain (CDR2P), the CDR3 of the P chain (CDR3P), or any combination thereof. Preferably, the antigen-binding portion comprises the amino acid sequences of CDRla, CDR2a. and CDR3a; the amino acid sequences of CDRip, CDR2P, and CDR3P; or the amino acid sequences of all of CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P of the TCR.

[0109] In an aspect of the invention, the antigen-binding portion can comprise, for instance, the variable region of the TCR comprising a combination of the CDR regions set forth above. In this regard, the antigen-binding portion can comprise the amino acid sequence of the variable region of the a chain (Va). the amino acid sequence of the variable region of the P chain (VP), or the amino acid sequences of both of the Va and VP of the TCR.

[0110] In an aspect of the invention, the antigen-binding portion may comprise a combination of a variable region and a constant region. In this regard, the antigen-binding portion can comprise the entire length of the a or p chain, or both of the a and P chains, of the TCR.

[0111] Isolating a nucleic acid comprising a nucleotide sequence that encodes the TCR, or the antigen-binding portion thereof, from the selected autologous T cells may be carried out in any suitable manner known in the art. For example, the method may comprise isolating RNA from the autologous T cells and sequencing the TCR, or the antigen-binding portion thereof, using established molecular cloning techniques and reagents such as, for example, 5’ Rapid Amplification of cDNA Ends (RACE) polymerase chain reaction (PCR) using TCR-a and -P chain constant primers.

[0112] The method may comprise isolating a CD4 TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the CD4 TCR, or antigenbinding portion thereof, has antigenic specificity for one of the target antigens.

[0113] The method may further comprise isolating a CD8 TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the CD8 TCR, or antigen-binding portion thereof, has antigenic specificity for one of the target antigens.

[0114] The TCR, or the antigen-binding portion thereof, isolated by the inventive methods may be useful for preparing cells for adoptive cell therapies. In this regard, another aspect of the invention provides a method of preparing a population of cells that express a TCR. or an antigen-binding portion thereof, having antigenic specificity for a target antigen.The method may comprise isolating a TCR. or an antigen-binding portion thereof, according to any of the methods described herein.

[0115] The method may further comprise introducing a nucleic acid comprising a nucleotide sequence encoding the isolated TCR. or the antigen-binding portion thereof, into PBMC to obtain cells that express the TCR, or the antigen-binding portion thereof. Introducing a nucleic acid comprising the nucleotide sequence (e.g., a recombinant expression vector) encoding the isolated TCR, or the antigen-binding portion thereof, into PBMC may be carried out in any of a variety of different ways known in the art as described in, e.g.. Green et al. supra. Non-limiting examples of techniques that are useful for introducing a nucleic acid comprising a nucleotide sequence into PBMC include transformation, transduction, transfection, and electroporation.

[0116] In an aspect of the invention, the inventive methods further comprise selectively introducing exogenous nucleic acid(s) into T cells by selectively stimulating T cells that have antigenic specificity for the target antigen, and selectively delivering the exogenous nucleic acid(s) to T cells with antigenic specificity for the target antigen.

[0117] In an aspect of the invention, the nucleic acid(s) comprise nucleotide sequence(s) encoding one or more of BTB Domain And CNC Homolog 2 (BACH2), KLF Transcription Factor 2 (KLF2). and IL-12. BACH2 and KLF2 are transcription factors involved in T-cell function, sternness, maturation and proliferation. With this, specific and robust proliferation of T cells having antigenic specificity for a target antigen may be achieved, thereby overcoming exhausted or differentiated T cells’ limited proliferation capacity. When inserted into selectively expanded T cells, BACH2 may increase the proliferation of selectively expanded T cells, e.g., neoTILs. This may allow effective and sensitive identification of rare neoTCRs. Additionally, robust expansion of T cells with the desired memory / stemness phenotype may be observed. When inserted into selectively expanded T cells, KLF2 does not affect the growth of the T cells, but it may enhance the effector function of T cells. The experiments described in the Examples herein show that KLF2+T cells kill tumor cells more efficiently in various in vitro and in vivo tumor models than the control T cells.

[0118] An aspect of the invention provides a T cell modified to express one or both of BACH2 and KLF2. In an aspect of the invention, a T cell has been modified to express one or both of BACH2 and KLF2 when an exogenous nucleotide sequence encoding one or both of BACH2 and KLF2 has been introduced into the T cell, e.g., using transfection, transformation, transduction, electroporation, a transposon, or a genome editing technique. Inan aspect of the invention, the genome editing technique to introduce the nucleotide sequence uses a zinc finger nuclease, transcription activator-like effector nuclease (TALENs), a CRISPR / Cas system, or engineered meganuclease.

[0119] For purposes herein, the T cell modified to express one or both of BACH2 and KLF2 can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell is a human T cell. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to. CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e g., Thl and Th2 cells, CD4+ T cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory' T cells (e.g., central memory' T cells and effector memory' T cells), naive T cells, regulatory T cells and the like.

[0120] Also provided by the invention is a population of cells comprising the T cell modified to express one or both of BACH2 and KLF2. The population of cells can be a heterogeneous population comprising the T cell modified to express one or both of BACH2 and KLF2, in addition to at least one other cell, e.g., a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of T cells modified to express one or both of BACH2 and KLF2. The population also can be a clonal population of cells, in which all cells of the population are clones of a single T cell modified to express one or both of BACH2 and KLF2, such that all cells of the population comprise the T cell modified to express one or both of BACH2 and KLF2. In one embodiment of the invention, the population of cells is a clonal population comprising T cells modified to express one or both of BACH2 and KLF2.

[0121] Another aspect of the invention provides a pharmaceutical composition comprising the T cell modified to express one or both of BACH2 and KLF2 or a population of cells comprising such T cell. The pharmaceutical composition may be as described herein with respect to other aspects of the invention.

[0122] Another aspect of the invention provides a method of treating or preventing a condition in a mammal, the method comprising administering to the mammal the T cellmodified to express one or both of BACH2 and KLF2 or population of cells or the pharmaceutical composition in an amount effective to treat or prevent the condition in the mammal.

[0123] IL-12 is a pro-inflammatory cytokine that can impact anti-tumor immunity. IL-12 has demonstrated strong anti-tumor immunity in various experimental models by modulating the tumor microenvironment. Despite its potential to address multiple barriers in immunotherapies for solid tumors, initial clinical applications of IL- 12 revealed severe systemic toxicities. Non-specific engineering of T cells with IL-12 can potentially lead to increased adverse effects of IL-12. Without being bound to a particular theory or mechanism, it is believed that, if T cells having antigenic specificity for a target antigen are specifically- engineered with IL- 12 using selective expansion according to the inventive methods, the side effects of IL-12 can potentially be mitigated.

[0124] Viral transduction of T cells may require prior stimulation (activation) to open chromatin for effective viral integration into the genome. Conventionally, anti-CD3 antibody, OKT3, and feeders or CD3-CD28 antibody-conjugated beads may be used to non- specifically activate T cells. In contrast, by specifically stimulating target antigen-reactive T cells using selective expansion according to the inventive methods, genes of interest, particularly those that can improve T-cell function or phenotype, can be specifically inserted into target antigen-reactive T cells.

[0125] The nucleic acid(s) may be introduced into the selectively expanded number of T cells as described herein with respect to other aspects of the invention. The nucleic acid(s) may be introduced into the selectively expanded number of T cells up to 5 days after initiation of the stimulating. For example, the nucleic acid may be introduced into the selectively expanded number of T cells on the same day as the stimulating, 1 , 2, 3, 4, or 5 days after initiation of the stimulating, or a range defined by any two of the foregoing values.

[0126] The numbers of T cells having antigenic specificity for a target antigen can be expanded by combining selective expansion according to the inventive methods with REP. This combination may overcome the non-specific nature of REP while taking advantage of the robust T-cell expansion of REP. In an aspect of the invention, the inventive methods further comprise non-specifically expanding the number of T cells. Non-specifically expanding the number of T cells may be carried out using a conventional rapid expansion protocol (REP) (also referred to as “rapid expansion”). Conventional REP is described in. for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent 11,401,503; Dudley etal., J. Immunother 26:332-42 (2003); and Riddell et al., J. Immunol. Methods. 128: 189-201 (1990). Non-specifically expanding the number of T cells comprises culturing the selectively expanded number of T cells in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) an antibody, or an antigen binding portion thereof, which specifically binds to the human CD3 complex (e.g., OK.T3). The culturing of the selectively expanded number of T cells in the presence of (i), (ii), and (iii) may be carried out from 12 hours to three days after initiation of the stimulating. For example, the culturing of the selectively expanded number of T cells in the presence of (i). (ii), and (iii) may be carried out 0 hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a range defined by any two of the foregoing values, after initiation of the stimulating. In an aspect of the invention, non-specifically expanding the number of T cells may be carried out at the same time as the stimulating or no earlier than 12 hours, no earlier than 24 hours, no earlier than 36 hours, no earlier than 48 hours, no earlier than 72 hours, or no earlier than a range defined by any two of the foregoing values, after initiation of the stimulating.

[0127] While selective expansion according to the inventive methods can be combined with non-specific expansion, the non-specific expansion can be optional. In an aspect of the invention, the method does not comprise non-specifically expanding the number of T cells.

[0128] The inventive methods of selectively expanding the number of T cells can be combined with bead selection to enrich for extremely rare T cells with antigenic specificity for a target antigen, as described herein with respect to other aspects of the invention. In this regard, an aspect of the invention provides a method of selectively expanding a number of T cells each having antigenic specificity for a target antigen, further comprising introducing a nucleotide sequence encoding a cell surface marker into the APCs to obtain APCs that express the cell surface marker; adding magnetic beads to the APCs and the T cells during or after the stimulating, wherein the magnetic beads are coupled to a binding partner that specifically binds to the cell surface marker; co-culturing the magnetic beads with the APCs and the T cells during or after the stimulating, thereby producing a complex comprising the APCs, the T cells, and the magnetic beads, wherein the binding partner coupled to the magnetic beads is bound to the cell surface marker expressed by the APCs, and wherein the T cells expressing the exogenous TCR are bound to the target antigen presented by the APCs; and applying a magnetic field to the complex to physically separate the complex from other T cells that do not express the exogenous TCR and are not bound to the target antigen presentedby the APCs. Optionally, the method may further comprise non-specifically expanding the number of T cells of the complex that express the exogenous TCR and are physically separated from the other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs. Introducing the nucleotide sequence, adding magnetic beads, co-culturing the magnetic beads with the APCs and the T cells, applying the magnetic field, and non-specifically expanding the number of T cells may be carried out as described herein with respect to other aspects of the invention.

[0129] Stimulating the T cells expressing the exogenous TCR with the APCs that present the target antigen advantageously selectively expands the number of T cells having antigenic specificity for a target antigen over the number of T cells not having antigenic speci ficity for a target antigen. In this regard, the inventive methods may, advantageously, provide populations of cells with a larger proportion of cells which have antigenic specificity for a target antigen as compared to methods of expanding the number of cells which do not stimulate the T cells with the APCs that present the target antigen. In an aspect of the invention, the method increases the number of T cells having antigenic specificity for a target antigen by 10-fold to 1,000-fold or more. For example, the inventive methods may increase the number of T cells having antigenic specificity for a target antigen by 10-fold to 1,000- fold, 50-fold to 850-fold, 100-fold to 900-fold, 150-fold to 850-fold, 200-fold to 800-fold, 250-fold to 750-fold. 300-fold to 700-fold, 350-fold to 650-fold, or 400-fold to 600-fold. For example, the inventive methods may increase the number of T cells having antigenic specificity7for a target antigen by 10-fold, 50-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold. 400-fold, 450-fold, 500-fold, 550-fold, 600-fold, 650-fold, 700-fold, 750- fold, 800-fold, 850-fold, 900-fold. 950-fold. 1,000-fold, or a range defined by any two of the foregoing values. The foregoing fold expansion may be achieved over a period of 10 to 19 days, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 days, or a range defined by any two of the foregoing values. The fold expansion achieved by the inventive methods may be highly variable and may be donor-dependent.

[0130] In an aspect of the invention, the method produces a selectively expanded population of cells, wherein 10% to 95% of the cells in the selectively expanded population have antigenic specificity' for a target antigen. In this regard, the method may produce a selectively expanded population of cells, wherein 10% to 95%, 15% to 90%, 20% to 85%, 25% to 80%. 30% to 75%. 35% to 70%. or 40% to 65% of the cells in the selectively expanded population have antigenic specificity for a target antigen. The method mayproduce a selectively expanded population of cells, wherein 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or a range defined by any two of the foregoing values, of the cells in the selectively expanded population have antigenic specificity for a target antigen.

[0131] The inventive methods may, advantageously produce any number of T cells having antigenic specificity for a target antigen which may be suitable for any of a variety of applications. In an aspect of the invention, the method may produce I x 106to 1 x 1011or more T cells which have antigenic specificity for a target antigen. For example, in small vessels (e.g., a T25 flask), the inventive methods may produce I x 106to 1 x 107T cells which have antigenic specificity for a target antigen. In a larger vessel (e.g., a T175 flask), the inventive methods may produce 5 x IO6to 3 x 107T cells which have antigenic specificity for a target antigen. In other vessels (e.g., a GREX flask, available from Wilson Wolf Manufacturing, New Brighton, MN), the inventive methods may produce 1 x 109to 1 x 1010T cells which have antigenic specificity for a target antigen. A population of 1 x 106to I x 1010T cells which have antigenic specificity’ for a target antigen may be useful for small- scale screening experiments. Larger numbers of cells which have antigenic specificity for a target antigen, e.g.. 1.5 x 1010or more may also be obtained using the inventive methods, e.g., for clinical applications. The number of T cells which have antigenic specificity for a target antigen produced by the inventive methods may be highly vanable and may be donordependent.

[0132] The inventive methods may provide any of a variety of advantages. For example, the inventive methods may produce an increase in any one or more of the following as compared to T cells the number of which was expanded by REP: (i) a proportion of T cells having a stem-like memory T cell phenoty pe; (ii) a proportion of T cells having antigenic specificity for the target antigen; (iii) diversity’ of T cells having antigenic specificity for the target antigen; (iv) in vivo anti-tumor function of T cells having antigenic specificity for a tumor antigen; (v) a ratio of the T cells having antigenic specificity for the target antigen with the relevant CD4 or CD8 co-receptor; and (vi) a total number of the T cells having antigenic specificity for the target antigen with the relevant CD4 or CD8 co-receptor.

[0133] The inventive methods may, advantageously, be used to isolate target antigen specific T cells or TCRs for several further applications including, for example, any one or more of: to grow and enrich target antigen-specific T cells, e.g., neoTIL for adoptive cell transfer; to isolate target antigen-specific TCRs that can be transduced into autologous T cellsfor adoptive cell transfer; to isolate T cells or TCRs against known mutated driver genes (p53 or RAS) or patient-specific neoantigens; to build a 1 i brary of TCRs to treat broad populations with cancer across different histologies; and to grow TCR-engineered PBLs to a large number with enhanced phenotypes and frequencies of target antigen-reactive T cells for adoptive transfer.

[0134] Another aspect of the invention provides a method of preparing a pharmaceutical composition comprising a selectively expanded number of T cells each having antigenic specificity for a target antigen. The method may comprise selectively expanding a number of T cells according to the inventive methods described herein. The method may further comprise combining the selectively expanded number of T cells with a pharmaceutically acceptable carrier.

[0135] Another aspect of the invention provides a method of preparing a pharmaceutical composition comprising a selectively expanded number of T cells each having antigenic specificity for a target antigen. The method may comprise preparing a population of cells that express a TCR, or an antigen binding portion thereof, according to the inventive methods described herein. The method may further comprise combining the population of cells that express a TCR. or an antigen binding portion thereof, with a pharmaceutically acceptable carrier.

[0136] With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for the particular population of cells under consideration. Such pharmaceutically acceptable carriers are w ell-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity under the conditions of use.

[0137] The choice of carrier will be determined in part by the particular population of cells, as w ell as by the particular method used to administer the population of cells. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for oral, parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intratumoral, intrathecal, or interperitoneal administration. More than one route can be used to administer the population of cells, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

[0138] Preferably, the population of cells is administered by injection, e.g.. intravenously. When the population of cells is to be administered, the pharmaceutically acceptable carrierfor the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w / v of NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA- LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an aspect, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0139] It is contemplated that the populations of cells and pharmaceutical compositions can be used in methods of treating or preventing a condition in a mammal. Without being bound to a particular theory or mechanism, the selectively expanded number of T cells are believed to bind specifically to the target antigen, such that a TCR expressed by the cell, is able to mediate an immune response against a target cell expressing the target antigen. In this regard, another aspect of the invention provides a method of treating or preventing a condition in a mammal. The method may comprise selectively expanding a number of T cells according to the inventive methods or preparing a pharmaceutical composition according to the inventive methods. The method may further comprise administering the selectively expanded a number of T cells or pharmaceutical composition to the mammal in an amount effective to treat or prevent the condition in the mammal. In an aspect of the invention, the condition is cancer, an autoimmune disease, a viral infection, or a bacterial infection. Examples of viral infections include, but are not limited to, those caused by human immunodeficiency viruses, respiratory syncytial virus, hepatitis C virus and Epstein-Barr virus.

[0140] Another aspect of the invention provides a method of treating or preventing a condition in a mammal. The method may comprise preparing a population of cells that express a TCR, or an antigen binding portion thereof, according to any of the inventive methods described herein or preparing a pharmaceutical composition according to any of the inventive methods described herein. The method may further comprise administering the population of cells that express a TCR, or antigen binding portion thereof, or pharmaceutical composition to the mammal in an amount effective to treat or prevent the condition in the mammal.

[0141] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary’ skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of the condition in a mammal.Furthermore, the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the condition being treated or prevented. For example, treatment or prevention can include promoting the regression of a tumor. Also, for purposes herein, "prevention" can encompass delaying the onset of the condition, or a symptom or condition thereof.

[0142] For purposes of the invention, the amount or dose of the population of cells or pharmaceutical composition administered (e.g., numbers of cells when the population of cells is administered) should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the mammal over a reasonable time frame. For example, the dose of the population of cells or pharmaceutical composition should be sufficient to bind to a target antigen, or detect, treat or prevent a condition in a period of from about 2 hours or longer, e.g., 12 to 24 or more hours, from the time of administration. In certain aspects, the time period could be even longer. The dose will be determined by the efficacy of the particular population of cells or pharmaceutical composition administered and the condition of the mammal (e.g., human), as well as the body weight of the mammal (e.g., human) to be treated.

[0143] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which target cells are lysed or IFN-yis secreted by T cells upon administration of a given dose of such T cells to a mammal among a set of mammals of which is each given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-yis secreted upon administration of a certain dose can be assayed by methods known in the art.

[0144] The dose of the population of cells or pharmaceutical composition also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular population of cells or pharmaceutical composition. Typically, the attending physician will decide the dosage of the population of cells or pharmaceutical composition with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, population of cells or pharmaceutical composition to be administered, route of administration, and the severity of the condition being treated.

[0145] For purposes of the inventive methods, wherein populations of cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0146] The cancer may, advantageously, be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear. cancer of the oral cavity, cancer of the vagina, cancer of the vulva, cholangiocarcinoma, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer. non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, urinary bladder cancer, solid tumors, and liquid tumors. Preferably, the cancer is an epithelial cancer. In an aspect, the cancer is cholangiocarcinoma, melanoma, colon cancer, or rectal cancer. In an aspect of the invention, the cancer expresses the one or more tumor antigens.

[0147] The mammal referred to in the inventive methods can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). Preferably, the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). Preferably, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). A more preferred mammal is the human. In an especially preferred aspect, the mammal is the patient expressing the one or more tumor antigens.

[0148] The following examples further illustrate the invention but, of course, should not be constmed as in any way limiting its scope.EXAMPLES

[0149] The following materials and methods were employed to carry out the experiments described in Examples 1-7.Human subjects and clinical protocols

[0150] Written, informed consent was obtained from all study participants, and all studies were conducted in accordance with The Declaration of Helsinki, The Belmont Report, and the U.S. Common Rule. This study was approved by the Investigational Review Board at the National Cancer Institute (NCI) in accordance with an assurance filed with and approved by the U.S. Department of Health and Human Services and was registered at clinicaltrials.gov under NCT00068003, NCT01174121, and NCT03412877. TILs or fresh tumor digests were generated from 25 patients with chemorefractory metastatic epithelial cancers enrolled in tissue procurement protocol NCT00068003. Metastases and leukaphereses were collected from each patient at the time of recruitment. TILs were expanded ex vivo for 2 to 4 weeks, frozen, and kept in liquid nitrogen until use. Leukaphereses were instantly cryopreserved and kept in liquid nitrogen until use. Healthy donors were recruited under the tissue procurement protocol NCT00068003 and underwent leukaphereses.

[0151] Adults ages 18 to 70 with upper or lower gastrointestinal, pancreatic or breast cancer refractory' to standard chemotherapy were recruited to either NCT01174121 or NCT03412877. Infusion products from 8 patients who were treated with ACT of autologous TILs (NCT01174121) were examined to determine the clonal architecture of neoantigen- reactive TILs, and their neoantigen reactivity has been previously reported (Parkhurst et al.. Cancer Discov.. 9: 1022-35 (2019); Lowery et al., Science 2022; 375:877-84; Zacharakis et al., J. Clin. Oncol., 2022; 40:1741-54).Generation of antigen-presenting cells (APCs)Primary immature dendritic cells (DC)

[0152] Generation of autologous immature DCs has been previously described (Tran et al., N. Engl. J. Med., 2016; 375:2255-62). Briefly, peripheral blood monocytes from patient apheresis were isolated using the plastic adherence method. Frozen apheresis was thawed, washed and resuspended in AIM-V media (Thermo Fisher, Cat. 12055083) with 1 pg / mL DNase (Stemcell Technology. Cat. 07900) at 10X6 cells / cm2After 90 minutes of incubation at 37°C, 5% CO2, non-adherent cells were removed and adherent cells were vigorously washed 3 times with PBS. After another incubation with AIM-V media for 60 minutes, adherent cells were washed again and were cultured for 4-5 days with DC media consisting of RPMH640 (Thermo Fisher. Cat. 21870092), 5% human serum (Gemini Bio, Cat.H122013 or Valley Biomedical, Cat. HP1022HI). 1% Penicillin-Streptomycin (Thermo Fisher, Cat. 15070063), 1% Glutamax (Thermo Fisher, Cat. 35050061), 800 lU / ml GM-CSF (Leukine; Partner Therapeutics) and 200 U / ml IL-4 (Peprotech, Cat. 200-04). Immature DCs were collected for fresh uses or cryopreserved for further uses.Primary autologous B cells

[0153] Primary autologous B cells were generated as previously described (Gros et al.. Nat. Med., 2016; 22:433-8). Briefly. B cells were isolated from autologous apheresis by positive selection using CD19+microbeads (Miltenyi Biotec, Cat. 130-050-301) and were coincubated with irradiated NIH3T3 cells constitutively expressing human CD40 ligand in the presence of 200 U / ml IL-4. B cells were harvested between day 4 or 6 after the initial stimulation and were restimulated up to 3 times, cryopreserved or freshly used. When used after cry opreservation. B cells were thawed into B cell medium 16-24 h before use. B cell medium comprised of Iscove's modified Dulbecco's medium (IMDM) (Thermo Fisher, Cat. 12440053) supplemented with 10% human serum, 1% Penicillin-Streptomycin, 1% Glutamax and 200 U / ml IL-4.Transformation of patient-derived B cells using Epstein-Barr virus (EBV)

[0154] Transformation of patient-derived B cells (EBV-B) was performed using supernatant from B95-8 cells containing EBV (ATCC, Cat. VR-1492) according to the manufacturer’s instruction without using feeder cells. Either thawed apheresis or CD 19+B cells following bead selection were used for transformation.HLA engineering of COS7 cells

[0155] A library of HLAs was individually introduced into an MSGV1 backbone for retrovirus generation. HLA sequences were collected from IPD-IMGT / HLA (versions 3.35 to 3.51), codon optimized, and cloned into an MS GV 1 vector using Nhel and EcoRI (custom cloning by Genscript). Class II HLAs were cloned in as a pair and were spaced with a P2A site. Retroviral supernatant was generated in HEK293 cells constitutively expressing Gag and Pol as described previously (Kim et al., Cancer Immunol. Res., 2022; 10:932-46). COS7 cells were transduced using RETRONECTIN reagent (Takara Bio, Cat. T100B), expanded and sorted by fluorescence-activated cell sorting (FACS) (using individual HLA-specificantibodies [Pure Protein] or pan-antibodies against HLA-DP [BD Biosciences. Cat. 566825], HLA- DQ [BD Biosciences, Cat. 347453] or HLA-DR [BD Biosciences, Cat. 347367]) or selected by antibiotics.TMG engineering ofCOS7 or EBV-B cells

[0156] For constitutive expression of tandem minigenes (TMG), WT or mutant p53 or RAS TMG sequences from previous studies (Kim et al., Cancer Immunol. Res., 2022; 10:932-46; Levin et al., Clin. Cancer Res., 2021; 27: 5084-95) were individually or together (with a P2A site in the middle) cloned into an MSGV 1 vector (Genscript) with a blasticidin resistance gene. HLA-engineered COS7 cells or EBV-B cells were retrovirally transduced as described above. Blasticidin-resistant cells were selected under 5-10 pg / mL blasticidin treatment for 1 week and were maintained with 5 pg / mL blasticidin. Selected APCs were functionally validated using T cells expressing known TCRs targeting p53 or RAS neoantigens by co-culturing T cells and TMG-expressing APCs overnight and measuring 4- 1BB expression by flow cytometry and / or interferon gamma (IFN-y) secretion by an IFN-y ELISPOT assay (Mabtech. Cat. 3420-2H).Transient transfection of TMG RNA into APCs

[0157] Autologous DCs or B cells, or HLA-engineered COS7 cells were transiently transfected with TMG RNA. The sequence of mutant TP53 and RAS TMGs were previously reported in Malekzadeh et al., J. Clin. Invest., 2019; 129:1 109-14 and Levin et al., Clin. Cancer Res., 2021; 27:5084-95, respectively. Synthesis and transfection of TP53 or RAS TMG RNAs were performed as previously described (Kim et al., Cancer Immunol. Res., 2022; 10:932-46; Levin et al., Clin. Cancer Res., 2021; 27:5084-95). Up to 3.5 million DCs or B cells were centrifuged and resuspended in 100 pL of OPTI-MEM reduced-serum medium (Thermo Fisher, Cat. 11058021) and electroporated with 5-10 pg TMG RNA using 2-mm cuvette and a BTX ECM 830 Square Wave Electroporation System (BTX Cat. 45- 2052) at 150 V for 10 milliseconds (DC) or for 20 milliseconds (B cells). Alternatively, COS7 cells or DCs were transfected with TMG RNA using LIPOFECTAM1NE MESSENGERMAX transfection reagent (Thermo Fisher, Cat. LMRNA015) according to the manufacturer’s instruction. Electroporated or transfected APCs were used the next day for co-culture.Peptide pulsing ofAPCs

[0158] 25-mer peptides containing single amino acid mutations in the middle or minimal epitopes were synthesized and purified to >90% purity by the high-performance liquid chromatography (Genscript, custom synthesis). The concentration of peptides used in NeoExpand ranged from 10 ng / rnL to 500 ng / mL (see also Fig. 7). The sequences of the peptides used throughout the study are available in Table 1.TABLE 1Flow cytometry and antibodies

[0159] T cells following a co-culture with APCs were stained with antibodies specific for the following human markers and mTCR: CD4 FITC (clone RPA-T4; 1 :20, catalog no. 555346), 0X40 PE (clone ACT35; 1 :20, catalog no. 555838), CD8 PE-cy7 (clone RPA-T8; 1:25, catalog no. 560917,), 4-1BB APC (clone 4B4-1; 1 :20, catalog no. 550890), and CD3 APC-Cy7 (SK7; 1:25, catalog no. 341090,) with or without mTCR|3-BV421 staining (catalog no. 562839; all from BD Biosciences). 4196 TILs were stained with the following two panels of antibodies in conjunction with tetramer staining: panel 1 : CD3 APC-Cy7, CD8 PE- Cy7, CD4 FITC (same as above), CD39 PE (clone Al; 1:40, catalog no. 328208, BioLegend), CD69 BV650 (clone FN50; 1 :25, catalog no. 563835, BD Biosciences), PD-1 BV421 (clone EH12.1, BD Biosciences. Cat. 562516) and tetramer-APC (custom generated). Panel 2: CD62L BV421 (clone DREG-56; 1 :50, catalog no. 304828, BioLegend), CD8 BV650 (clone RPA-T8, 1:20, catalog no. 301042, Biolegend), TIM3 BB515 (clone FN50; 1 :20, catalog no. 565568, BD Biosciences), TIGIT PE-Cy7 (clone A15153G. 1:20, catalog no. 372714, Biolegend), CD45RO APC (Clone UCHL1; 1:20, catalog no. 559865, BD Biosciences), CD4 APC-H7 (Clone SK3; 1 :20, catalog no. 641398, BD Biosciences) and tetramer-PE (custom generated). Analytic flow cytometry was performed on LSRFORTESSA cell analyzer, or FACSYMPHONY cell analy zer (BD Biosciences) with analysis by FLOWJO software (version 10.6.2, TreeStar). All cells were gated via lymphocytes [forward scatter (FSC) and side scatter (SSC)J) and live cells by exclusion of cells stained with propidium iodide (catalog no. P1304MP, Thermo Fisher), or DAPI (Biolegend, cat. 422801). For TCR isolation 4-lBB+and / or OX40+cells were sorted separately through CD3 CD4 CD8 (for CD4) and CD3 CD4 CD8 (for CD8) gates using SH800S or MA900 (Sony Biotechnology). For single-cell transcriptome analysis of patient 4196 and 4391 TILs, CD8+cells were sorted using MA900 (Sony Biotechnology).TCR transduction of healthy donor PBLs

[0160] Transduction of healthy donor autologous PBLs was performed as previously described (Kim et al., Cancer Immunol. Res., 2022; 10:932-46). Healthy donor-aphereses were thawed, counted and were stimulated in 50 / 50 media [RPMI1640 media containing 10% human serum, 1% GLUTAMAX supplement, 12.5 mmol / L HEPES (Thermo Fisher, Cat. 15630080), 1% Penicillin-Streptomycin, and 5 pg / mL gentamicin (Quality Biological, Cat. 120-099-661) mixed with AIM-V at 1 : 1 ratio] supplemented with 50 ng / rnL anti-CD3 antibody (Miltenyi Biotec. Cat. 130-050-301) and 300 IU IL-2 (Aldesleukin, Clinigen) for 48 hours. Retroviral supernatants were loaded into RETRONECTIN reagent (Takara Bio, Cat. T100B)-coated 24 or 6 well plates and were spun for 2 h at 32°C at 2,000 g. Next, stimulated PBLs were added into the virus-loaded plates, spun for 10 to 20 minutes at 32°C at 1,500 RPM with minimal acceleration and brake. Transduced T cells were cultured for up to 1 month in 50 / 50 media supplemented with 300 IU IL-2. At day 4-6 post-transduction, T cells were collected and examined for exogenous TCR expression by flow cytometry.Generation of TILs

[0161] TILs used in this study were generated using the previously described method (Dudley et al., J. Immunother., 2003; 26:332-42). Resected tumors were removed of normal tissues immediately after surgical excision. Areas of firm, solid tumor were selected for processing and sized to about 1 to 3 mm per section. Individual fragments were placed in a 24-well plate in 2 mL of the T-cell culture media [RPMI1640 containing 10% human serum, 1% GLUTAMAX supplement, 12.5 mmol / L HEPES, 1% Penicillin-Streptomycin, and 5 pg / mL gentamicin without AIM-V] containing high-dose IL2 (6000 lU / mL, Chiron). Fragments were cultured at 37°C at 5% CO2 for 5 days. On day 5, culture media were replenished with fresh media and IL-2 (6,000 lU / mL) and reassessed every 2 to 3 days. When cultures exceeded 10X6 cells / mL or were nearly confluent, the wells were split 1: 1. Each fragment was maintained as a separate culture.NeoExpandAntigen loading onto APCs

[0162] When peptides were used for antigen loading, 10 ng / mL to 100 ng / mL minimal predicted epitope peptides or 100 ng / mL to 500 ng / mL 24-25 mer-long peptides with a mutation in the middle were pulsed onto APCs for 2 to 4 hours. Peptide-loaded APCs were then washed with PBS and were used for co-culture. TMGs were either transfected or constitutively expressed in various APCs as described above.NeoExpand coculture of antigen-loaded APCs and T cells

[0163] TCR-engineered T cells or TILs were counted and were incubated with antigen- loaded APCs at 4: 1 to 1 : 10 effector to target ratio. For the first three days of NeoExpand coculture, cells were cultured in 50 / 50 media supplemented with 30 ng / mL IL-21 (Peprotech, Cat. 200-21) and 0 to 50 IU IL-2 for TCR-engineered PBLs or 300 IU IL-2 for TILs. After initial feeding the cells were fed every 3 days with 50 / 50 media containing 30 ng / mL IL-21 and 300 IU IL-2 for TCR-engineered PBLs or 1,000 IU IL-2 for TILs. At the end of 14-19 days of NeoExpand coculture, T cells were collected for testing the frequency of TCR- engineered T cells or neoantigen-reactive TILs. The stimulation of T cells with APCs that present the tumor antigen in the presence of IL-2 with or without IL-21 is referred to herein as “NeoExpand. ”Determination of the frequency of TCR-engineered T cells (A) or neoantigen-reactive TILs (B)A. TCR-engineered T ceils

[0164] During and after NeoExpand, the frequency of TCR-engineered T cells was determined by flow cytometry. Because all the neoantigen-reactive TCRs used in this study contained murine TCR constant region sequences, exogenous TCR expression was tracked by staining with an mTCR antibody.B. Neoantigen-reactive TILs

[0165] The frequency of neoantigen-reactive TILs was determined by one or more of the following methods:

[0166] 1. TILs following NeoExpand were subjected to additional co-culture with APCs expressing a candidate antigen(s) for 18 hours. T cells recognizing neoantigens were determined by flow cytometry measuring the upregulation of 4- IBB or OX-40 and by IFNy ELISPOT assays. Twenty thousand to hundred thousand APCs were co-cultured with twentythousand to hundred thousand TILs in IFNy ELISPOT plates [96-well plates with a poly vinylidene difluoride (PVDF) membrane; EMD Millipore, Cat. MAIPSWUIO], Phorbol 12-myristate 13-acetate (PMA; 81 nmol / L) and ionomycin (1.34 pmol / L) (Thermo Fisher, Cat. 00-4970-93,) were included as a positive control. Co-cultured cells were stained and analyzed by flow cytometry, and IFNy ELISpot plates were processed using the Human IFNy ELISPOT BASIC® kit [horseradish peroxidase (HRP); Mabtech. Cat. 3420-2H) according to the manufacturer's instructions.

[0167] 2. Once the sequences of TCRB of neoantigen-reactive TILs clonotypes were identified, their frequencies were determined by TCRB survey sequencing using bulk genomic DNA by Adaptive Biotechnologies.

[0168] 3. For CD8+neoantigen-reactive TILs clones, when the HLA restriction element and the minimal epitope sequences were available, tetramers were synthesized. Tetramer generation was previously reported (Kim et al., Cancer Immunol. Res., 2022; 10:932-46). TILs following NeoExpand were stained with tetramers and were analyzed by flow cytometry.

[0169] 4. 4196, and 4391 TILs described in Figs. 6A-6D were analyzed using the single cell transcriptome analysis which included Single Cell Immune Profiling for TCRAB sequence identification.Rapid expansion protocol (REP)

[0170] Non-specific T cell stimulation for T cell expansion through REP has been described before (Jin et al., J. Immunother., 2012; 35:283-92). Briefly. T cells were incubated with 30 ng / mL OKT3, 3,000 IU IL2 and irradiated allogeneic feeders (50-100 times the number of T cells) in T- 175 flaks or G-Rex®24 well, G-Rex®6 well plates or G- Rex®100 flasks (WILSON WOLF, Cat. 80192M. 80240M and 80500, respectively). After 5 days, half the media was removed and replaced with fresh 50 / 50 media containing 300 IU IL- 2 for TCR-engineered T cells or 3,000 IU IL-2 for TILs.Cell lines

[0171] Commercially available C0S7, TYK-nu, and PDX line 4391, have been described before (Kim et al., Cancer Immunol. Res., 2022; 10:932-46; Levin et al., Clin. Cancer Res., 2021; 27:5084-95). Pancreatic cancer patient-derived xenograft (PDX) line 4069 was established as follows. A freshly resected tumor metastasis from patient 4069 with metastatic pancreatic cancer was dissected into small fragments of 2 mm in diameter. One fragment was implanted subcutaneously into the flank of an NSG mouse using a 20-gauge needle. Tumor growth was measured weekly and when the tumor reached 1 cm in diameter, it was harvested and subsequently passaged into another NSG mouse. When the tumor grew in the mouse the second time, it was harvested and mechanically dissociated using gentleMACS® Dissociator (Miltenyi Biotech, Cat. 130-093-235) using the “mouse implanted tumor 1.01” program. The resulting cell suspension was filtered through a 100-pm cell strainer and washed once before being placed in a tissue culture flask. Tumor cell culture media consisted of RPMI1640 supplemented with 10% FBS (Cytiva, Cat. SH30071.03HI or Gemini Bio, Cat. 100-106), IX nonessential amino acid (Thermo Fisher, Cat. 11140050), 1 mmol / L sodium pyruvate (Thermo Fisher, Cat. 11360070). 1% Penicillin-Streptomycin, 1% Glutamax, 10 pg / mL gentamicin, and 55 pmol / L 2-mercaptoethanol (Thermo Fisher, Cat. 31350010). Media were replaced even' 3 to 7 days and cells were passaged when confluence reached 70%. The presence of KRASG12Dmutation and HLA-A*11 in 4069 PDX cells was initially determined by whole exome sequencing of the freshly resected tumor and later validated by RT-PCR and sanger sequencing of the RNA from the established PDX line.Xenograft tumor treatment by ACT

[0172] Animal experiments were approved by the Institutional Animal Care and Use Committees of the NCI and performed in accordance with the National Institutes of Health (NIH) guidelines. Immunodeficient NSG or NCG (NOD-Prkdcem26Cd52I12rgem26Cd22 / NjuCrl) mice were obtained from NCI or Charles River, respectively. Six to eight weeks-old female mice were used for all the xenograft experiments. One to three million tumor cells were subcutaneously implanted into the flank of NSG or NCG mice. In 2 to 3 weeks, when the tumor size reached ~30mm2, mice were randomized, intravenously injected with TCR- engineered PBLs or TILs and monitored for tumor grow th. PBS was used as a vehicle for T- cell injection. At the time of T-cell injection and two times additionally, the mice wereintraperitoneally injected with 180,000 IU of recombinant human IL-2 in 500 pL of PBS. Tumor growth was measured once or twice a week, and tumor size was calculated as the product of two perpendicular measurements. All experiments were conducted in a blinded manner. Retroviral transduction of healthy donor PBLs was performed as described above. The TCR-engineered PBLs in Fig. 2G were injected at day 14 post-transduction. The TCR- engineered PBLs in Fig. 8D were sorted for CD8+mTCR+cells, expanded for 14 days with REP and injected into mice. Neoantigen-reactive TIL injection in Figs. 6A-6D was performed at day 15 after REP or NeoExpand without a sort.Sample preparation and sequencing for single-cell transcriptome and TCR sequencing analysis

[0173] Single cell transcriptome analyses were performed as described before (Kim et al., Cancer Immunol. Res.. 2022; 10:932-46). Live CD8+cells were FACS sorted (see Antibodies, flow cytometry, and FACS) from 4196 and 4391 TILs following the IL-2 culture, NeoExpand or REP. The sorted T cells were resuspended in PBS at the concentration of 5 * 105cells / mL. and loaded onto a Chromium Controller (10X Genomics, Cat. 1000204) for single-cell sample preparation. One to two channels per reaction were used to prepare each sample for sequencing following the manufacturer's protocol. Ten thousand T cells per channel were loaded onto the Chromium Controller with the target-cell recovery of 6,000 single cells. The single-cell cDNA samples were first universally amplified by running 16 cycles of PCR using a thermocycler (Bio-Rad, Cat. T100) and the Chromium Next GEM® Single Cell 5' Reagent Kits V2 (10X Genomics, Cat. PN-1000265) according to the manufacturer's instructions. cDNAs for TCR (VDJ) sequencing were further amplified by two additional PCR reactions using TCR-specific primers according to the manufacturer's protocols (10X Genomics, Cat. PN-1000252,). The whole transcriptomes from the same cDNA samples were amplified after cDNA fragmentation per the manufacture's protocol. The processed single-cell cDNA samples were sequenced using an Illumina NextSeq® 550 sequencer (High Output Kit v2.5; Readl : 26 b.p; Read2: 98 b.p.; Illumina, Cat. 20024912). The whole transcriptome libraries were sequenced using the Illumina NextSeq® 2000-P3 kit (Read 1 : 26 b.p.; Read 2: 90 b.p.; Illumina, Cat. 20040561).EXAMPLE 1

[0174] This example demonstrates that reductions in neoantigen-reactive TIL frequencies occur during the conventional REP with OKT3.

[0175] Methods to identify TIL recognizing neoantigens expressed by solid epithelial cancers and the expansion of those TIL for use in ACT of patients with cancer have previously been reported. Briefly, single or multiple tumor metastases are dissected to establish and expand multiple TIL fragment cultures with high dose IL-2 (6,000 lU / mL) for neoantigen screening (Parkhurst et al., Cancer Discov., 2019; 9: 1022-35; Zacharakis et al., J. Clin. Oncol., 2022; 40:1741-54). TIL fragment cultures that recognize neoantigens are then further expanded to >lxlO10cells by the REP where TILs are stimulated with an anti-CD3 antibody (OKT3), IL-2 and irradiated allogeneic peripheral blood mononuclear cells as feeders (Fig. 1A), a culture method widely used in the field (Rosenberg et al.. Science 2015; 348:62-8; Tran et al., N. Engl. J. Med., 2016;375:2255-62; Zacharakis et al., Nat. Med., 2018;24:724-30; Stevanovic et al., Science, 2017;356:200-5; Zacharakis et al., J. Clin.Oncol.. 2022;40: 1741-54; Pillai et al., Am. J. Cancer Res., 2022;12:3967-84; Rohaan et al., N. Engl. J. Med.. 2022;387:2113-25; Samaik et al., J. Clin. Oncol.. 2021;39:2656-66;Stevanovic et al., Clin. Cancer Res., 2019;25: 1486-93). It was tested whether prolonged exposure to high-dose IL-2 and / or nonspecific stimulation during the REP by OKT3 led to expansion of bystander cells to reduce the frequency of neoantigen-reactive TILs. Ten different neoantigen reactivities identified from three breast cancer TIL infusion products used in clinical trial NCT01174121 (Zacharakis et al., J. Clin. Oncol., 2022; 40: 1741-54) were analyzed before and after the REP. All of the 10 reactivities showed a decrease in their frequencies in the infusion products relative to the individual fragment cultures before the REP (Fig. IB). Some reactivities, including those against mutated BTF3, RCLL TTI2 and p53, showed greater than 10-fold decrease making them nearly undetectable at the end of expansion.EXAMPLE 2

[0176] This example demonstrates the development of NeoExpand for neoantigenic stimulation of TILs.

[0177] To selectively expand neoantigen-reactive TILs, an in vitro TIL culture method was developed, termed “NeoExpand,” that involved the specific stimulation of TILs againstpreviously identified or candidate neoantigens. As a starting material, either TIL fragment cultures established individually as described above, a pool of TIL fragment cultures or TILs from fresh tumor digests were used (Figure 1C). Fresh tumor digests were either directly used or were briefly cultured for less than a week with IL-2. To provide neoantigen-specific stimulation, a variety of APC, including autologous DCs, B cells and HLA-engineered cell lines, such as COS7 cells, were tested (Figure 1C). Antigens were introduced into APCs either transiently by transfection of mutated tandem minigene (TMG) RNA or constitutively by virally expressing TMGs. Additionally, APCs were loaded with antigens by pulsing long (24-25 mer) peptides or predicted minimal epitope peptides. From all candidate mutated epitopes identified from whole exome sequencing of tumor vs. normal tissues, a small number (<10) of minimal epitopes were prioritized based on NetMHCpan4.0 (Jurtz et al., J. Immunol., 2017;199:3360-8), MHCflunyl.6 (O’Donnell et al., Cell Syst., 2018;7: 129-32 e4), and a machine learning-based prediction model (Gartner et al., Nat. Cancer, 2021;2:563-74). Although minimal epitopes were used, the nature of the neoantigenic stimulation was sequence-agnostic as the 24 or 25 amino acids containing mutations in the form of peptides or TMG RNAs were intracellularly processed to be presented by APCs. To determine optimal concentrations of peptides, the efficiency of NeoExpand was examined on TCR- engineered T cells as a model. Healthy donor PBLs transduced with two different p53 or RAS neoantigen-reactive TCRs identified previously (Kim et al.. Cancer Immunol. Res.. 2022;10:932-46; Levin et al., Clin. Cancer Res., 2021;27:5084-95) were co-cultured with HLA-engineered COS7 cells pulsed with a range of peptide concentrations (Fig. 7). The use of peptide between 10 and 1,000 ng / ml appeared to have a negligible effect on the growth of TCR-transduced PBL, indicating flexibility in terms of the amount of antigens required for effective neoantigenic stimulation (Fig. 7). Finally, TILs were co-cultured with antigen- loaded APCs in the presence of IL-2 and IL-21. IL-21 was added during NeoExpand, because it has been shown to preserve the proliferation capacity of antigen-experienced T cells while counteracting differentiation induced by IL-2 (Wolfl et al., Nat. Protoc., 2014;9:950-66; Cafri et al., Nat. Commun., 2019;10:449; Hinrichs et al., Blood, 2008;l 11 :5326-33; Li et al., J. Immunol., 2005;175:2261-9).

[0178] For the expansion of TILs targeting shared neoantigens, such as p53 or KRAS, peptides and / or TMGs can be prepared in advance and the entire process of NeoExpand can take approximately two weeks. Although not discussed in this study, for the expansion ofTILs targeting private neoantigens, newly synthesizing peptides and TMGs can add additional 4 to 6 weeks to the timeline.EXAMPLE 3

[0179] This example demonstrates the expansion of CDS and CD4+neoantigen-reactive TIL clonal repertoire and sensitive identification of neoantigen-reactive TCRs following neoantigenic stimulation.

[0180] The effect of neoantigenic stimulation was tested on TILs to facilitate the identification of neoantigen-reactive TCRs and to develop TIL ACT products with improved neoantigen-reactivity, phenotype and functions to potentially replace the conventional REP with OKT3. As an example, Figures 2A-2G show neoantigenic stimulation of TILs from a colorectal cancer patient (4141), whose tumor harbored a p53R175Hmutation. A pool of 4141 TIL fragment cultures were ex vivo expanded with or without neoantigenic stimulation using HLA-engineered COS7 cells as APCs. Following NeoExpand, a dramatic expansion of p53R175H-reactive TILs was observed whereas conventional culture with high dose IL-2 did not lead to expansion of p53R175H-reactive T cells (Figure 2A). From the reactive TILs, one previously identified (Malekzadeh et al., J. Clin. Invest., 2019;129: 1109-14) and one novel TCR were isolated (Fig. 2B, Table 2). The novel TCR showed specificity' for mutant p53 but not wild-type p53 (Fig. 2C) and was restricted by HLA-A*02:01 (Fig. 2D). This p53R175H- reactive “NeoExpand” clonotype was found at a very low level (<0.01%) in the patient’s infusion product (Fig. 2E). again indicating that the conventional TIL culture, including the REP with OKT3, failed to expand this neoantigen-reactive TIL clonotype. The therapeutic function of this new TCR was tested using a human ovarian cancer xenograft model (Kim et al., Cancer Immunol. Res., 2022;10:932-46). Ten million PBLs from two different healthy donors were transduced with the new 4141 NeoExpand TCR and injected into immunocompromised NOD-s z IL2Rgnu11(NSG) mice bearing human ovarian cancer TYK- nu cells naturally expressing both p53R175Hand HLA-A*02 (Fig 2F). The two groups of mice that received PBLs engineered with 4141 NeoExpand TCR showed a significant delay in tumor grow th (Fig. 2G).TABLE 2

[0181] Additional examples of neoantigen-reactive TIL identification by NeoExpand are shown in Figure 8A-8D. From a colorectal cancer TIL generated from tumor cells that expressed both HLA-A*02 and A*11 and a KRASG12Dmutation (4432), NeoExpand was conducted using COS7 cells engineered with HLA-A*02 or A*11 as APCs. KRASG12D- reactive CD8+T cells were identified only in the TIL stimulated with COS7 cells expressing HLA-A* 11 but not with A*02-engineered COS7 cells, indicating that the neoantigenic stimulation led to T-cell expansion in an HLA-specific manner (Fig. 8A). The conventional culture without the neoantigenic stimulation failed to expand this clonotype and no reactivity was identified (data not shown). The TCR isolated from the KRASG12D-reactive clonotype showed specificity for KRASG12Dbut not the wild-type peptide (Fig. 8B). To test the in vivo anti-tumor function of this novel TCR, a new xenograft model was developed using allogeneic pancreatic cancer patient-derived xenograft (PDX) cells (4069) that naturally expressed KRASG12Dand HLA-A* 11 :01. NSG mice were injected with one million 4069 PDX cells and two weeks later received ACT of 6 million healthy donor PBLs expressing 4432 NeoExpand TCR (Fig. 8C). This RASG12D-reactive TCR showed anti-tumor efficacy, causing complete tumor regression in this model (Fig. 8D). Collectively, in these two examples, novel neoantigen-reactive TCRs were isolated following neoantigenic stimulation and the TCRs exhibited specificity for neoantigens and in vivo functionality.

[0182] Next, it was tested whether CD4+neoantigen-reactive TILs could also be selectively expanded by neoantigen-specific stimulation. Figures 3A-3C and Tables 3A-3E show an example where a CD4+neoantigen-reactive clonoty pe that w as progressively declining in numbers was expanded by neoantigenic stimulation. Initially by the conventional expansion with IL-2 followed by the neoantigen screening, p53R273C-reactive cells were identified in fragment culture 7 of 4386 breast cancer TIL with robust IFN-y secretion against mutant p53 TMG or p53R273C(Table 3A). A TIL infusion product wasgenerated by further expanding reactive cultures, including fragment culture 7. by the REP with 0KT3. When the final infusion product was tested for neoantigen reactivity, however, a loss of the p53 reactivity based on reduced IFN-y secretion was noted (Table 3B). It was examined whether the decreasing p53R273-reactive cells could be expanded by neoantigenic stimulation. From the pool of all the 4386 cultures, NeoExpand was carried out using autologous DCs as APCs. Following the NeoExpand procedure, expansion of pSS82730- reactive cells was noted (Fig. 3A). From the reactive cells, a single TCR was isolated (Table 3C). When reconstructed and expressed in healthy donor PBLs, the TCR showed specificity for mutant p53 (Fig. 3B) and HLA restriction of DPAl*01:03-DPBl*04:02 (Table 3D), which is found in over 60% of Hispanic populations in the US and South American countries (Gonzalez-Galarza et al.. Nucleic Acids Res.^ 2020;48:D783-D8). The mutant p53-reactive clonotype was not detected in fragment culture 7 following the REP but was detected in other fragment cultures at a low level, which might have been the source of T cells stimulated by NeoExpand (Table 3E).TABLE 3A4386 TIL fragment culture 7

[0183] Table 3A shows IFN-y secretion of 4386 TIL fragment culture 7 against the p53R273C neoantigen. An ELISpot assay was performed following co-culture of 4386 TIL fragment culture 7 with autologous DCs electroporated with p53 TMG or pulsed with p53R273C 25 mer. The mock transfected (TMG mock) condition and DMSO as the vehicle control for peptide treatment were included as negative controls. “Positive result” indicates that IFN-y secretion was detected. “Negative result” indicates that IFN-y secretion was not detected.TABLE 3B4386 TIL infusion product

[0184] Table 3B shows IFN-y secretion of 4386 TIL infusion product against p53 TMG or the p53R273C peptide as described with respect to Table 3A. ‘'Positive result” indicates that IFN-y secretion was detected. “Negative result” indicates that IFN-y secretion was not detected.TABLE 3CTABLE 3D

[0185] Table 3D shows the results of HLA testing of the 4386 NeoExpand TCR. Healthy donor PBLs expressing 4386 NeoExpand TCR were co-cultured with COS7 cells transfected with both A and B molecules of class II HLAs expressed by patient 4386. IFN-y secretion was measured by an ELISpot assay. ‘"Positive result” indicates that IFN-y secretion was detected. “Negative result” indicates that IFN-y secretion was not detected.TABLE 3E

[0186] Table 3E shows the frequencies of the 4386 NeoExpand clonotype in the peripheral blood before ACT, individual TIL fragments and the infusion product (RX).EXAMPLE 4

[0187] This example demonstrates that NeoExpand broadens CD4+and CD8+neoantigen-reactive TIL clonal repertoire.

[0188] Iteratively, NeoExpand was performed on 25 TIL samples whose tumor expressed p53 or RAS mutations, and the result was compared to the screening result following the conventional TIL expansion without neoantigenic stimulation. Out of 25 TIL samples from different patients, the conventional expansion and screening identified 9 reactivities against mutant p53 or RAS, while NeoExpand enabled the identification of 16 reactivities, which included all the 9 reactivities found through the conventional screening (Fig. 3C and Table 4). All of the TCR sequences isolated from the neoantigen-reactive TIL clonotypes were reconstructed into retrovirus for functional testing. When all the different, functionally validated neoantigen-reactive TIL clonotypes were enumerated, the conventional screening identified 14 clonotypes (3 CD4; 11 CD8) and NeoExpand identified 42 clonotypes (14 CD4; 28 CD8) (Fig. 3C). TILs from tumors expressing both p53 and RAS mutations (4424, 4426, and 4430) were stimulated against both neoantigens but only single reactivities against either p53 or RAS neoantigens were identified (Table 4). This indicated that unlike naive T cells (Wolfl et al., Nat. Protoc., 2014;9:950-66), neoantigen-reactive TILs could not be induced to generate a novel reactivity. These data in conjunction with the examples in Figures 2A-2G, Table 2, Figures 3A-3C, and Tables 3A-3E demonstrate that neoantigenic stimulation can facilitate effective neoantigen-reactive TCR isolation, including both CD4+and CD8+TCRs, by expanding the neoantigen-reactive TIL clonal repertoire.TABLE 4N=no; Y=yes; na=not applicableEXAMPLE 5

[0189] This example demonstrates effective neoantigen-reactive TIL expansion by neoantigenic stimulation for use in ACT.

[0190] Next, the translational potential for NeoExpand as a method to grow TILs for patient treatment was investigated by comparing it to the conventional REP that has been commonly used to generate a large number of T cells for ACT. As exemplified in Figure IB, non-specific stimulation of T cells by OKT3 could reduce the frequencies of neoantigen- reactive TILs. Therefore, it was tested whether neoantigenic stimulation could address decreases in frequencies of neoantigen-reactive TIL during ex vivo expansion while achieving exponential growth of TILs. As proof-of-principle, TILs from patients 4196. 4385, and 4391 with metastatic colorectal cancers were used to compare NeoExpand and the conventional REP. These TIL samples were selected based on their availability as well as compatibility with the existing mouse models for functional testing. As in Fig. 4A, TILs were grown either by NeoExpand or the REP with OKT3. The p53R175H-reactive cells from 4196 TILs were counted by staining them with an HLA-A*02 tetramer containing the p53R175Hepitope. Due to unavailability of the HLA-C*01 :02 tetramers, to enumerate neoantigen-reactive T cells within 4385 and 4391 TILs, the expanded TILs underwent another co-culture with HLA- engineered COS 7 cells pulsed with the RASG12Dminimal epitope peptide. The REP achieved total CD3+T-cell fold-expansion greater than that of NeoExpand in 4196 TILs (Fig. 4B, top right); however, the NeoExpand TILs showed higher frequencies and fold-expansion of neoantigen-reactive T cells than the REP culture (Fig. 4B, bottom left and right).Furthermore, following neoantigenic stimulation of 4196 TILs, 4 novel p53R175H-reactive clonotypes were identified in addition to the three known clonotypes. 6-11. 12-6 and 38-10, that were previously identified following a conventional TIL culture (Lo et al., Cancer Immunol. Res., 2019;7:534-43) (Figs. 9A-9D). When reconstructed, these 4 new TCRs demonstrated in vitro tumor lysis of TYK-nu cells (p53R175H+; HLA-A*02+) (Fig. 9E). Similar to 4196 TILs, the REP led to greater fold-expansion of the bulk CD3+T cells of 4385 and 4391 TILs than NeoExpand (Fig. 4C. D, leftmost). However, NeoExpand achieved greater fold-expansion of neoantigen-reactive TILs than the REP (Fig. 4C, D, middle and rightmost). In the case of 4391 TIL, following NeoExpand, 1 previously identified TCR (Levin et al., Clin. Cancer Res., 2021;27:5084-95) and 4 novel RASG12V-reactive clonotypes w ere identified. The 4 novel TCRs showed mutant RAS specificity with no wild-typereactivity (Figs. 10A-10D). In aggregate of 11 TIL samples tested for NeoExpand and the conventional REP, including the three samples discussed above, the fold-expansion of neoantigen-reactive TILs by NeoExpand was significantly greater than that of the REP (Fig. 4E).TABLE 5EXAMPLE 6

[0191] This example demonstrates the phenotypic characterization of TILs before and after neoantigenic stimulation or REP by single-cell transcriptome analysis (scRNA-seq).

[0192] The T cell cultures from patients 4196 and 4391 were further characterized by scRNA-seq analysis. 4385 TILs were not analyzed due to their highly monoclonal (80%) composition of neoantigen-reactive TILs following NeoExpand, which lacked the clonal complexity of fresh TILs and would not be representative. The UMAP (uniform manifold approximation and projection) analysis revealed 14 clusters with distinct transcriptome signatures among the 4196 TIL (Fig. 5 A, left). The p53R175H-reactive neoantigen-reactive TIL clonotypes were mainly found in clusters 3. 4 and 10 (Fig. 5 A, right). Gene setenrichment analysis (GSEA) revealed that the gene expression profile of cluster 4 bore high similarity to the signatures of CD39 CD69" stem-like cells described by Krishna et al., (Science, 2020;370: 1328-34) or stem-like memory cells described by Caushi et al., (Nature, 2021;596: 126-32) (Fig. 11A-11C, Table 5), which also expressed genes associated with stemlike memory T cells, such as IL7R, KLF2, SELL (CD62L), and TCF7 (TCF1) with little expression of exhaustion markers, such as ENTPD1 (CD39) or HAVCR2 (TIM3) (Fig. 5B). When p53R175H-reactive clonotypes in cluster 4 were enumerated, most of the reactive cells were from either the pre-expansion TIL before NeoExpand / REP (PRE) or the NeoExpand culture (Fig. 5C), indicating depletion of the p53 neoantigen-reactive cells with the stem-like memory phenotype when expanded conventionally by the REP with OKT3. In contrast, clusters 3 and 10 resembled the gene expression profiles of differentiated effector cells (Caushi et al., Nature, 2021;596: 126-32; Yost et al., Nat. Med., 2019;25: 1251-9) (Fig. 5B and 11 A-l 1C) and contained similar numbers between the different culture conditions (Fig. 5C). This finding was further substantiated by flow cytometric analysis of tetramer 4196 TILs. which showed expansion of a central memory (CD62L+CD45RO+) population — T cells thought to harbor a long-term repopulating ability with stem like features (Graef et al., Immunity, 2014;41: 116-26) — following NeoExpand (Fig. 12). The same population following the REP was 4.2-fold lower than that of NeoExpand. An scRNA-seq analysis of mutant RAS-targeting 4391 TILs also identified clusters with high numbers of RASG12V- reactive neoantigen-reactive TILs (Fig. 5D). Cluster 9 that contained high numbers of the neoantigen-reactive TILs resembled the phenoty pe of the stem-like memory cells (Caushi et al., Nature, 2021;596: 126-32) or the CD39 CD69’ stem-like cells (Krishna et al., Science, 2020;370: 1328-34) (Fig. 13. Table 5) and expressed high levels of TCF1 and CD62L and low amounts of CD39 (Fig. 5E). TOX, a transcription factor associated with T-cell exhaustion (Khan et al., Nature, 2019;571:211-8), was generally high in 4391 TILs, including cluster 9 and indicated that these cells despite their stem-like gene expression profdes might be different from naive T cells that do not express high levels of TOX (Fig. 5E). Neoantigen- reactive TILs within cluster 9 were made up almost exclusively of the cells generated through NeoExpand (Fig. 5F). Other exhaustion-associated genes that had been considered markers for neoantigen-reactive TILs, such as CD39, TIM3 or PD1, showed heterogenous patterns of expression and the clusters expanded in response to neoantigenic stimulation tended to express lower levels of these genes, implying their less exhausted phenotypes than the TILs expanded by the REP (Figs. 5B, 5E).TABLE 6EXAMPLE 7

[0193] This example demonstrates the functional characterization of TILs expanded by neoantigenic stimulation or the REP using in vivo xenograft ACT models.

[0194] The three TILs expanded viaNeoExpand or REP (Fig. 4A) were functionally compared using in vivo xenograft models. NSG mice were subcutaneously implanted with TYK-nu cells (p53R175H+; HLA-A*02:01+) or 4391 colorectal cancer patient-derived xenograft (PDX) cells (KRASG12V t; HLA-C*01:02 ). These tumor cells naturally expressed the neoantigens and HLA molecules corresponding to 4196 (p53) or 4385 and 4391 TILs (KRAS). When tumors were established, the NSG mice were injected with 4196, 4385 or 4391 TILs expanded through NeoExpand or the REP with OKT3 (Fig. 6A). The mice treated with 20 million 4196 (Fig. 6B) or 4385 TILs (Fig. 6C) expanded viaNeoExpand showed significant tumor regression while TILs expanded by the REP failed to do so when compared to the vehicle controls. The mice treated with 10 million 4391 TILs expanded via NeoExpand did not show tumor regression but a significant delay in tumor grow th relative to that of REP (Fig. 6D).EXAMPLE 8

[0195] This example demonstrates that T cells recognize tumor antigen isolated directly from a tumor and presented by APCs.

[0196] Tumor expressing KRASG12Vresected from Patient 4571 was kept at a temperature of -80°C. A piece of the tumor w as then resuspended and homogenized in deionized water. The homogenized tumor was then lysed by multiple cycles of fast freezingthawing (liquid nitrogen and 37°C water bath) and rough mixing by vortex and / or by sonication. The tumor lysate was pulsed on allogeneic DC from Patient 4203, which expressed HLA-DRBl*01. The DC were washed 36 hours later. PBMCs expressing the 4304 TCR, which recognizes KRASG12Vrestricted by HLA-DRBl*01, were co-cultured with the DC overnight. Flow cytometry measured reactivity by upregulation of 4- IBB and / or 0X40. T cells co-cultured with DC pulsed with KRASG12V25-mer peptide in the high concentration of 1 pg / ml or medium concentration of 10 ng / ml served as a positive control. T-cells co-cultured w ith DC or cultured alone served as a negative control. The results are shown in Figure 15.EXAMPLE 9

[0197] This example demonstrates NeoExpand using organoid from the patient’s tumor.

[0198] Tumor organoid cells were lysed as described for the tumor in Example 8. DC from Patient 4540 were pulsed with a lysate of autologous tumor organoid. 24 hours later, the DC were washed, and TILs from Patient 4540 were co-cultured with the DC. The reactivity was measured by 4-1BB and / or 0X40 upregulation by flow cytometry of the cells following 14 days of NeoExpand (post-NeoExpand) in comparison to the reactivity existing in the same TIL population before the IVS (Pre Neoexpand). The results are shown in Figure 16A. 25- mer peptides providing a pool of the mutations expressed by the patient’s tumor were pulsed on DC and served as the specific stimulus for this test. T cells co-cultured with DC pulsed with DMSO (DMSO) or the T cells’ 4-1BB and 0X40 baseline expression (T only) served as negative control.

[0199] The same process was repeated for 10 additional TIL samples (total of 11 samples). The data demonstrate significant enrichment of tumor-reactive cells following the NeoExpand culture using organoid lysates, compared to the pre-existing reactivities in the pre-expansion TIL samples (Fig. 16B). For purposes of the experiment described for Fig. 16B, tumor-reactive cells are defined as 4-1 BB+ or OX-40+ cells of CD8+ cells for CD8+ cell reactivity or 4-1BB+ or OX-40+ cells of CD4+ cells for CD4+ cell reactivity.EXAMPLE 10

[0200] This example demonstrates expansion of the number of TCR-engineered cells using EBV-B cells or HLA-engineered COS cells in NeoExpand.

[0201] 40,000 HLA and TMG-engineered COS7 or 100,000 TMG-engineered EBV-B were co-cultured with 10,000 T cells for 14 days with IL-2 and IL-21. The TMG encoded the respective targets in Table 7. As a control, the number of T cells was expanded using REP. At the end of the 14-day co-culture, the frequency of TCR-engineered (mTCR+) and co- receptor (CD8+) cells was evaluated by flow cytometry. Exogenously expressed TCRs contain the murine TCR constant region sequences for improved pairing between alpha and beta TCR subunits and can be labeled with a murine TCR-specific antibody (referred to as mTCR+). The TCRs used in this experiment are shown in Table 7.TABLE 7

[0202] The results showed that, in 3 out of 4 cases, NeoExpand was more effective in expanding the number of TCR-engineered and CD8+T cells as compared to REP (Fig. 17). Both EBV-B and COS7 cells were effective as APCs.EXAMPLE 11

[0203] This example demonstrates that various concentrations of peptides can expand TCR-engineered T cells.

[0204] 20,000 HLA-engineered COS7 cells (APCs) were co-cultured with 20,000 TCR- engineered T cells using NeoExpand, REP alone, or IL-2 alone. The TCRs used in this experiment are the 4148, 4373, and 4391 TCRs shown in Table 7, in addition to the 4424 TCR, which recognizes p53 R175H presented by HLA-A*02:01. The COS7 cells were pulsed with minimal epitopes or engineered with TMG (n=3). Fold expansion following NeoExpand was measured using flow cytometry. The results demonstrate that various peptide concentrations can be used to achieve effective expansion of tumor-reactive T cells (Figs. 18A-18D).EXAMPLE 12

[0205] This example demonstrates that NeoExpand enriches TCR-engineered T cells using various autologous or allogeneic primary cells as APCs.

[0206] In this experiment, NeoExpand with various autologous and allogeneic APCs was tested and compared with the conventional REP. Patient-donor T cells were engineered with the 4373 TCR and then grow n in T cell media supplemented with IL-2 (with no stimulation), or the numbers of engineered cells were expanded by REP or NeoExpand.

[0207] For the NeoExpand cultures, APCs were pulsed with KRAS G12D 10-mer peptide. APCs were autologous DCs, irradiated autologous PBMC, autologous B cells, or allogeneic B cells. Engineered T cells were co-cultured with the APCs in the presence of IL- 2 and IL-21. Samples were analyzed at day 7, 10 and 14 of co-culture.

[0208] Because the 4373 TCR was isolated from CD8+cells, the frequency of CD8+mTCR+cells was measured. Most of the NeoExpand conditions showed an increase in CD8+mTCR+cells, while the REP or IL-2 (no stimulation) samples showed progressive reductions in CD8+mTCR+cells (Fig. 19A). Historically, T-cell stimulation has been frequently conducted with DCs or B cells. To the best of the inventors’ knowledge, this is the first demonstration that autologous PBMCs can effectively stimulate T cells to induce selective growth of TCR-engineered T cells. PBMCs may be an advantageous APC type because they do not require lengthy and complicated in vitro expansion compared with DC or B cells.

[0209] Irrespective of transduction status, bulk CD3+T cells were enumerated during and after expansion. In this analysis, the conventional REP generated the highest number of T cells (Fig. 19B).

[0210] When the fold expansion of CD8+mTCR+cells was measured following the 14 days of expansion, all of the NeoExpand conditions showed a significantly greater fold expansion of CD8+mTCR+cells relative to the IL-2 condition and equivalent expansion compared to the REP (Fig. 19C).

[0211] In a set of five TCRs derived from CD4+cells, a similar pattern emerged.NeoExpand irrespective of the type of APCs enriched the frequency of CD4+mTCR+cells more significantly than the REP. However, the fold expansion of CD4+mTCR+cells was no different between all the expansion conditions (Fig. 19D, 19E).EXAMPLE 13

[0212] This example demonstrates that NeoExpand improves T-cell phenotype.

[0213] Following the co-cultures of the 4373 TCR-engineered cells described in Example12, CD8+mTCR+cells were phenotypically analyzed by flow cytometry’. T cells can be subdivided by CD45RO and CD62L expression. In particular, CD62L+CD45R0‘ cells harbor stem-like properties. When compared to REP, the NeoExpand condition with autologous PBMCs contained more TN / TSCM (Fig. 20). T cells with the TN / TSCM phenotype exhibit enhanced T-cell function with highly proliferative capacity and improved in vivo persistence. These data indicate that NeoExpand can improve a T-cell phenotype to increase T-cell sternness.

[0214] CD39+CD69+T cells are highly differentiated, whereas CD39'CD69‘ cells maintain sternness (Krishna et al., Science, 370(6522): 1328-1334 (2020)). When the 4373TCR-engineered cells were evaluated, the NeoExpand condition with autologous PBMCs showed a greater frequency of stemlike CD39 CD69" cells as compared to REP, substantiating that NeoExpand improves T-cell phenotype to maintain sternness. When CD39, an established T-cell exhaustion marker, was analyzed individually, the NeoExpand condition with autologous PBMCs also showed a higher frequency of CD39’ cells as compared to REP, indicating reduced T-cell exhaustion by NeoExpand (Fig. 21).EXAMPLE 14

[0215] This example demonstrates that NeoExpand can be combined with REP to maximize the expansion of T cells engineered with an exogenous TCR without compromising the improved frequency and phenotype of mTCR-engineered and relevant CD4 or CD8 co-receptor-expressing (mTCR+Co-R+) cells.

[0216] Patient-donor T cells were engineered with the 4391 TCR of Table 7. The numbers of engineered T cells were then expanded separately in gas-permeable flasks by REP, NeoExpand using PBMCs as the APC pre-loaded with KRAS-G12V 9-mer antigen (referred to as "PBMC") or NeoExpand in combination with REP (referred to as “PBMC+REP”) (Fig. 22A). NeoExpand was carried out with or without the addition of the REP for 16-24 hours following initiation of the co-culture of the engineered T cells with the APC. In this experiment, only autologous PBMCs were used as APCs for NeoExpand. Samples were analyzed at day 14 for bulk CD3+T cell fold expansion. Cells were analyzed and evaluated for expression of CD3 / CD8 / CD4 and mTCR by flow cytometry.

[0217] NeoExpand with PBMCs irrespective of REP more effectively enriched the frequency of CD8+mTCR+cells compared to the conventional REP or the pre-expansion sample (PRE) (Fig. 22B).

[0218] NeoExpand in combination with the REP achieved the greatest fold expansion of CD8+mTCR+cells than NeoExpand alone (Fig. 22C).

[0219] In an aggregate experiment that included 2 different TCRs derived from CD4+cells and 2 different TCRs derived from CD8+cells, NeoExpand with PBMCs irrespective of REP more effectively enriched the frequency of mTCR Co-R cells compared to the conventional REP or PRE (Fig. 22D).

[0220] Consistent with the Fig. 22C data, regarding the fold expansion of mTCR+Co-R+cells, NeoExpand in combination with REP achieved greater fold expansion than NeoExpand alone (PBMC) or the conventional REP (Fig. 22E).EXAMPLE 15

[0221] This example demonstrates that autologous PBMCs used as APCs in NeoExpand can serve as both APCs and feeder cells and that irradiated allogeneic feeders are dispensable, which does not compromise the improved frequency and phenotype of mTCR Co-R+cells.

[0222] T cell samples from 11 different patients were respectively transduced with one of 3 or 4 different TCRs. These TCRs were derived from either CD4+or CD8+cells. The numbers of transduced T cells were expanded through the conventional REP, NeoExpand without OKT3 or NeoExpand with OKT3, as described in Example 14. Cells were analyzed and evaluated for expression of CD3 / CD8 / CD4 and mTCR by flow cytometry.

[0223] NeoExpand with or without REP or OKT3 enriched the frequency of mTCR+Co- R+cells more effectively than the conventional REP or PRE (Fig. 23 A). There was no statistical difference between the three NeoExpand conditions.

[0224] NeoExpand with OKT3 or NeoExpand in combination with REP demonstrated a greater fold expansion of mTCR+Co-R+cells compared to the conventional REP and NeoExpand without OKT3 (Fig. 23B). There was no statistical difference between the two NeoExpand conditions (PBMC+OKT3 vs. PBMC+REP). These findings suggest that NeoExpand with OKT3 leverages the selective expansion capacity of NeoExpand combined with the robust growth-promoting effects of OKT3 characteristic of the conventional REP and allogeneic feeders are not required in this process.

[0225] For the purposes of the experiments described in this Example, mTCR+Co-R+cells are defined as CD8+mTCR+cells for TCRs derived from CD8+cells and as CD4+mTCR+cells for TCRs derived from CD4 cells.EXAMPLE 16

[0226] This example demonstrates that IL-21 does not affect the enrichment or fold expansion of mTCR+Co-R+cells during NeoExpand, but IL-21 does improve T cell phenotype during NeoExpand.

[0227] IL-21 was included in the NeoExpand stimulations described in the experiments of Examples 1-15. In this Example, the effect of IL-21 on the expansion capacity and the improved phenotype of T cells during NeoExpand was assessed.

[0228] T cell samples from 10 different patients were respectively transduced with one of 3 or 4 different TCRs. These TCRs were either CD4 or CD8. The numbers of engineered T cells were then expanded separately in gas-permeable flasks by REP or by NeoExpand using PBMCs as the APC pre-loaded with target tumor antigen. NeoExpand was carried out with and without IL-21. Cells were analyzed and evaluated for expression of CD3 / CD8 / CD4 and mTCR by flow cytometry.

[0229] These data demonstrate that both NeoExpand conditions, irrespective of the presence of IL-21, effectively enrich the frequency of mTCR+Co-R+cells (Fig. 24 A). These data indicate that IL-21 is not required for enrichment of tumor-reactive cells.

[0230] When the fold expansion of tumor-reactive cells was examined, there was no difference between the two NeoExpand conditions irrespective of IL-21 (Fig. 24B), indicating that IL-21 is not required for expansion of mTCR+Co-R+cells.

[0231] To examine the effect of IL-21 on a T-cell phenot pe during NeoExpand, the expression of CD39 was examined. Compared to the conventional REP and NeoExpand without IL-21, NeoExpand with IL-21 achieved a greater number of CD39negatlvetumor reactive cells (Fig. 24C), indicating that IL-21 is responsible for the improvement of T-cell phenoty pe induced by NeoExpand.

[0232] For the purposes of the experiments described in this Example, mTCR+Co-R+cells are defined as CD8+mTCR+cells for TCRs derived from CD8+cells and as CD4+mTCR+cells for TCRs derived from CD4 cells.

[0233] IL-21 was included in the NeoExpand stimulations in the experiments described in the Examples that follow.EXAMPLE 17

[0234] This example demonstrates that TILs from a cancer patient can be stimulated with a single pool of all peptides that a tumor sample expresses to induce selective expansion of tumor-reactive CD4+T cells by NeoExpand.

[0235] TILs from colon cancer patient 4546 were tested to compare NeoExpand and conventional REP. Autologous DCs were pulsed overnight with one pool of 171 different 25-mer peptides. For the NeoExpand culture condition, the pulsed DCs were co-cultured with the TILs for 14 days. In parallel, the same number of TILs were subjected to the REP using the standard protocol, involving an anti-CD3 antibody, high dose IL-2 and irradiated feeders.

[0236] TIL samples before and after the NeoExpand or the REP were tested for reactivity by measuring T-cell activation markers, 4-1 BB and OX-40, after overnight incubation with autologous DCs pulsed with the peptide pool. DMSO and T cell only conditions were included as negative controls. Following expansion, TILs cultured by NeoExpand were the only condition that showed increased reactivity (Fig. 25A).

[0237] A total of 11 patients’ TIL samples, including 4546 TILs, were subjected to NeoExpand and then were tested for reactivity, as described for Fig. 25 A. Following expansion, significant increases in neoantigen-reactive cells were observed (Fig. 25B).EXAMPLE 18

[0238] This example demonstrates that TILs from a cancer patient can be stimulated with a single pool of all predicted minimal peptides or tandem minigene (TMG) RNA that a tumor sample expresses to induce selective expansion of tumor-reactive CD8+T cells.

[0239] TILs from colon cancer patient 4554 were tested to compare NeoExpand and conventional REP. Autologous DCs were pulsed overnight with one pool of predicted 186 peptides (8- to 11-mer). Epitope prediction was conducted using MHCflurry (O'Donnell et al.. Cell Cyst., 1 l(l):42-48 (2020)), and the cutoff for significant binding was “rank <0.5%”. For the NeoExpand culture condition, pulsed DCs were co-cultured with the TILs for 14 days. In parallel, the same number of TILs were subjected to REP using the standard protocol, involving an anti-CD3 antibody, high dose IL-2 and irradiated feeders.

[0240] TIL samples before and after the NeoExpand or the REP were tested for reactivity by measuring T-cell activation markers, 4-1 BB and OX-40, after overnight incubation with autologous DCs pulsed with the peptide pool of 186 epitope peptides. DMSO and T cell only conditions were included as negative controls. Significant expansion of neoantigen-reactive cells was seen with the TILs expanded by NeoExpand with the peptide pool (Fig. 26A).

[0241] The same process that was earned out for Fig. 26A was repeated using autologous DCs engineered with TMG RNA instead of autologous DCs pulsed with the peptide pool. Following expansion, significant increases in neoantigen-reactive cells were observed only with the NeoExpand condition (Fig. 26B).

[0242] The approach described in Examples 17 and 18 can significantly streamline the NeoExpand culture process, reducing time and resources required for NeoExpand. The experiments described in Examples 17 and 18 show that the number of antigens targeted with NeoExpand can be expanded to include all somatic non-synonymous mutations identified indifferent patient tumors. The finding described in Examples 17 and 18 reveal that a single pool of peptides, often exceeding 100, can be successfully utilized to stimulate TIL, resulting in a greater number of tumor-reactive cells compared to conventional REP. Additionally, these data show that using NeoExpand to target the entire tumor mutation pool identifies additional tumor antigens that conventional screening methods fail to detect. This capability highlights the broad applicability of NeoExpand, making it a versatile tool for targeting a wide range of tumors, regardless of their specific mutation profiles. To the best knowledge of the inventors, this is the first demonstration that antigens as many as 171 peptides can be combined into a single peptide pool to effectively enrich antigen-specific T cells.EXAMPLE 19

[0243] This example demonstrates that lysates of freshly resected tumor cells can stimulate tumor-reactive TIL for NeoExpand.

[0244] Tumor organoid cells or freshly resected tumor tissues were cut, homogenized and resuspended in water before being transferred to a microtube. The tube was subjected to 10 cycles of freezing in liquid nitrogen, thawing in a 37 °C water bath, and vortexing for 30 seconds between cycles. APCs were pulsed with the lysate for 16-24 hours (h) prior to the NeoExpand co-culture.

[0245] Colon cancer TILs were subjected to NeoExpand using autologous PBMCs as APCs pulsed with fresh autologous tumor lysate. Following the NeoExpand culture, the frequency of CD8+TIL reactive against the pool of 25-mer mutant peptides was evaluated. The percentage of reactive CD8+TILs was measured following overnight co-culture with APC pulsed with a pool of 25-mer mutated peptides expressed by the patient’s cancer. The percentage of reactive CD8+TILs was measured by upregulation of 4-1BB and 0X40, as detected by flow cytometry’. As a negative control, TIL were co-cultured overnight with APC loaded with DMSO (peptide solvent) or cultured without additional cells (T cell only).Reactivity was measured immediately before the start of NeoExpand (TIL pre-expansion), 14 days after the start of NeoExpand and again 14 days after that (Tumor Lysate NeoExpand day 28). The NeoExpand condition showed a significant increase in 4-lBB+OX-40+cells (i.e., tumor-reactive cells) (Fig. 27).

[0246] Using tumor lysates of patient-derived tumor organoids or freshly resected tumors to conduct NeoExpand may, advantageously, reduce the time and labor required to conduct NeoExpand compared to using peptides or TMG RNA because they do not require thesequencing of tumors or synthesis of peptides or TMG RNAs. The experiments described in Examples 9 and 19 demonstrate that both patient-derived tumor organoids and freshly resected tumors can be processed to generate tumor lysates that can be pulsed onto APCs for effective and selective expansion of tumor-reactive T cells using NeoExpand.EXAMPLE 20

[0247] This example demonstrates that NeoExpand with BACH2 significantly increases the fold expansion of the number of tumor-reactive TILs when compared to NeoExpand without BACH2.

[0248] A vector was created which encoded BACH2 and green fluorescent protein (GFP) as a reporter (“the BACH2 vector”). TILs were transduced with the BACH2 vector or an empty vector as a negative control. GFP+cells (i.e. BACH2+) were sorted and expanded by the conventional REP. At the end of the REP, the total number of cells was counted.Expression of BACH2 significantly increased the number of TILs during the REP (Fig. 28C).

[0249] At the end of T-cell transduction with the BACH2 vector and REP, phenotypes were analyzed by single-transcriptome analysis. By gene expression. T cells transduced with BACH2 showed an increase in the CD62L level that is associated with stem-like T cells, which are thought to be more functional and give rise to daughter cells that can mount stronger anti-tumor responses than differentiated CD62L" cells.

[0250] Conventionally, prior to genetic engineering, T cells are non-specifically stimulated with OKT3 or CD3-CD28 beads. Non-specific expression of BACH2 in TILs may result in growth of bystander cells. To prevent this, neoantigen-reactive T cells within bulk TILs were specifically stimulated using NeoExpand. TIL samples from 11 patients with cancer were stimulated with a single pool of peptides using NeoExpand and subsequently were transduced with the BACH2 vector or an empty vector and cultured for 14 days.

[0251] The frequency of reactive cells was measured before and after NeoExpand with or without BACH2 expression. The reactivity of the 1 1 TIL samples significantly increased following NeoExpand and NeoExpand and BACH2 transduction (Fig. 28A). Significant differences between the NeoExpand alone vs. NeoExpand followed by BACH2 transduction were not detected. These data demonstrate that NeoExpand significantly increases the frequency of tumor-reactive TILs and adding BACH2 did not induce any significant changes in the frequency.

[0252] When the total number of tumor-reactive cells were counted and compared to the starting cell number, expression of BACH2 significantly increased the fold expansion of neoantigen-reactive TILs when compared to the NeoExpand alone conditions (Fig. 28B). These data demonstrate that NeoExpand can be used to specifically engineer neoantigen- reactive T cells with BACH2 to enhance their proliferation.EXAMPLE 21

[0253] This example demonstrates a method of expanding the numbers of T cells with extremely rare T-cell reactivities using NeoExpand followed by bead selection and REP.

[0254] While REP can increase total cell yield, REP can come at the cost of expanding the number of bystander (non-reactive) cells due to the non-specific nature of OKT3 stimulus. Additionally, the limit of detection for tumor-reactive cells is determined by the sensitivity of flow cytometry used in neoantigen screening, which is approximately 0. 1 to 1%.

[0255] Therefore, it was sought to develop a method that enriches extremely rare (frequency <0.1%) tumor-reactive cells by yielding even purer tumor-reactive cell populations as compared to NeoExpand alone. This method employs NeoExpand followed by a positive bead selection to selectively pull out rare T-cell reactivities (Figure 29). This bead selection reduces the number of unreactive bystander cells that could otherwise proliferate in response to OKT3.

[0256] Autologous PBL were transduced with truncated human nerve growth factor receptor (NGFR) and then pulsed with peptides for known or suspected T cell reactivities. NGFR is a marker that allows for bead selection. Next, the peptide pulsed target cells (expressing NGFR) were irradiated. T cells (TIL, PBL or lymph node suspensions) were subjected to NeoExpand by co-culturing with the irradiated, peptide-pulsed, NGFR- expressing target cells for 2-16 hours. After NeoExpand. NGFR+cells were removed with positive magnetic bead selection and transferred to flasks with mixed allogeneic feeders and 30 ng / mL OKT3 in 3,000 lU / mL IL-2 (i.e. REP). NGFR can be replaced with any other cell surface marker as long as the marker of interest does not negatively impact the function or the viability of APCs.EXAMPLE 22

[0257] This example demonstrates that NeoExpand followed by bead selection then REP yields significant enrichment of extremely rare cells.

[0258] In this experiment (Fig. 30), TCR-transduced, CD8+T cells were artificially diluted with known reactivity7(the transgenic TCR has a mouse TCR0 chain) to 3 cells per 10,000. This starting frequency of 0.03% mimics rare, putative tumor-reactive T cells in peripheral blood, lymph node or tumor. The diluted cells were subjected to a first 14 day cycle of (i) REP only, (ii) NeoExpand and REP (OKT3) only, or (iii) NeoExpand followed by bead selection then REP, as described in Example 21. A portion (1 / 35) of the cells that were subjected to NeoExpand followed by bead selection were subjected a second 14 day cycle of (i) REP only, (ii) NeoExpand and REP (OK.T3). or (iii) NeoExpand followed by bead selection then REP.

[0259] After two serial 14-day cycles of NeoExpand followed by bead selection and REP, this rare reactivity was expanded approximately 3 million-fold and enriched 1000-fold. This experiment was repeated with different TCRs yielding comparable results. One observation was that this bead enrichment method appears to be most efficient at low frequencies. For example, in the first 14-day cycle shown in Figure 30, the T cells are enriched from a frequency of 0.03% to approximately 3.5%, which was an approximately 115-fold enrichment. However, the second 14 day cycle increased the 3.5% TCR+cell product to 32%, which was an approximately a 9-fold enrichment.EXAMPLE 23

[0260] This example demonstrates that NeoExpand followed by bead selection then REP effectively expands rare tumor-reactive TIL populations.

[0261] A patient TIL product (from colorectal cancer Patient 4617) had neoantigen reactivity against HAUS4 in a TIL fragment (culture 7) and against CAPZA2 in another TIL fragment (culture 15), but at a low frequency. TILs were subjected to (i) REP only, (ii) NeoExpand and REP (OKT3) only, or (iii) NeoExpand followed by bead selection then REP, as described in Example 21, using HAUS4 and CAPZA2 mutant minimal peptides.

[0262] The proportion of reactive CD8+T cells in TIL fragment 7 increased from 9% (REP only) to 48% (NeoExpand followed by bead selection then REP) (Fig. 31 A). For fragment 15. the percentage of reactive cells increased from 2% (REP only) to 19%(NeoExpand followed by bead selection then REP) (Fig. 3 IB). These data demonstrate that NeoExpand followed by bead selection then REP provides maximal enrichment and expansion in TIL.EXAMPLE 24

[0263] This example demonstrates that expression of KLF2 functionally enhances antitumor functions of T cells in a murine model.

[0264] KLF2 was expressed in tumor-reactive TIL from multiple epithelial cancer patients to evaluate its impact on phenotypes.

[0265] Single-cell trans criptomic analysis of 6 neoantigen-reactive KLF2-engineered selected TIL samples demonstrated increases in “stem-Like” signatures, including upregulation of sternness-associated genes (e.g. IL7-R, LTB, ZNF683) and downregulation of T-cell exhaustion markers (e.g. LAG3, ENTPD1, TOX) among TILs transduced with KLF2.

[0266] Flow cytometric analyses of tumor-reactive TILs expressing KLF2 showed a significant increase in the stem-like population (CD39 CD69", range: 3-80-fold, P=0.001, paired T test) and a decrease in the terminally differentiated population (CD39 CD69 . range: 1-16-fold, P=0.017, paired T test) (Figs. 32A-32B).

[0267] To determine anti -tumor function of T cells engineered with KLF2, peripheral blood T cells from a healthy donor were transduced with KLF2 and a TCR targeting mutant p53 R175H. The KLF-expressing T cells exhibited significant tumor regression of TYK-nu human ovarian cancer in vivo in a mouse model, even at a very low cell dose of 3e5 cells / mouse, when compared to the mock control (P=0.001, 2-way ANOVA, n=5 mice per group) (Fig. 32C). At the 6e5 cells / mouse dose, KLF2+cells significantly outperformed the TCR only condition.

[0268] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0269] The use of the terms “a” and “an7’ and "the" and “at least one’7and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherw ise indicated herein or clearly contradicted by context. The terms “comprising,’' “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to.”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0270] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of selectively expanding a number of T cells expressing an exogenous T cell receptor (TCR) having antigenic specificity for a target antigen, the method comprising: introducing a nucleic acid into peripheral blood mononuclear cells (PBMC), wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR having antigenic specificity for the target antigen, to produce T cells expressing the exogenous TCR; inducing autologous antigen presenting cells (APCs) to present the target antigen; stimulating, in the presence of interleukin (IL)-2 with or without IL-21 In vitro, the T cells expressing the exogenous TCR with the APCs that present the target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity for the target antigen; and non-specifically expanding the number of T cells.

2. The method of claim 1, further comprising: introducing a nucleotide sequence encoding a cell surface marker into the APCs to obtain APCs that express the cell surface marker; adding magnetic beads to the APCs and the T cells during or after the stimulating, wherein the magnetic beads are coupled to a binding partner that specifically binds to the cell surface marker; co-culturing the magnetic beads with the APCs and the T cells dunng or after the stimulating, thereby producing a complex comprising the APCs, the T cells, and the magnetic beads, wherein the binding partner coupled to the magnetic beads is bound to the cell surface marker expressed by the APCs, and wherein the T cells expressing the exogenous TCR are bound to the target antigen presented by the APCs; and applying a magnetic field to the complex to physically separate the complex from other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs, wherein non-specifically expanding the number of T cells comprises non-specifically expanding the number of T cells of the complex that express the exogenous TCR and arephysically separated from the other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs.

3. The method of claim 1 or 2. wherein non-specifically expanding the number of T cells comprises culturing the selectively expanded number of T cells in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) an antibody, or an antigen binding portion thereof, which specifically binds to the human CD3 complex.

4. The method of claim 3. wherein culturing the selectively expanded number of T cells in the presence of (i), (ii), and (iii) is carried out from 0 hour to three days after the stimulating.

5. A method of selectively expanding a number of T cells expressing an exogenous T cell receptor (TCR) having antigenic specificity for a target antigen, the method comprising: introducing a nucleic acid into peripheral blood mononuclear cells (PBMC), wherein the nucleic acid comprises a nucleotide sequence encoding the exogenous TCR having antigenic specificity for the target antigen, to produce T cells expressing the exogenous TCR; inducing autologous antigen presenting cells (APCs) to present the target antigen; and stimulating, in the presence of interleukin (IL)-2 with or without IL-21 in vitro, the T cells expressing the exogenous TCR with the APCs that present the target antigen, wherein the T cells expressing the exogenous TCR receive proliferation signals, the number of T cells expressing the exogenous TCR expand and the number of T cells that do not express the exogenous TCR do not expand, thereby producing a selectively expanded number of T cells expressing the exogenous TCR having antigenic specificity for the target antigen; wherein the method does not comprise non-specifically expanding the number of T cells.

6. The method of claim 5, further comprising: introducing a nucleotide sequence encoding a cell surface marker into the APCs to obtain APCs that express the cell surface marker;adding magnetic beads to the APCs and the T cells during or after the stimulating, wherein the magnetic beads are coupled to a binding partner that specifically binds to the cell surface marker; co-culturing the magnetic beads with the APCs and the T cells during or after the stimulating, thereby producing a complex comprising the APCs, the T cells, and the magnetic beads, wherein the binding partner coupled to the magnetic beads is bound to the cell surface marker expressed by the APCs, and wherein the T cells expressing the exogenous TCR are bound to the target antigen presented by the APCs; and applying a magnetic field to the complex to physically separate the complex from other T cells that do not express the exogenous TCR and are not bound to the target antigen presented by the APCs.

7. The method of any one of claims 1-6, wherein the method does not comprise screening the PBMC or the T cells expressing the exogenous TCR for antigenic specificity for the target antigen before or during the stimulating.

8. The method of any one of claims 1-7, wherein inducing the APCs to present the target antigen comprises:(i) pulsing the APCs with a peptide, wherein the peptide comprises a target antigen amino acid sequence;(ii) introducing a nucleotide sequence encoding the target antigen into the APCs; or(iii) pulsing the APCs with whole or lysed autologous tumor, whole or lysed tumor cells, or whole or lysed organoid derived from autologous tumor.

9. A method of selectively expanding a number of T cells each having antigenic specificity for a target antigen, the method comprising: inducing autologous antigen presenting cells (APCs) of the mammal to present one or more target antigens; stimulating, in the presence of interleukin (IL)-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, wherein the T cells having antigenic specificity for the tumor antigen receive proliferation signals, the number of T cells having antigenic specificity for the target antigen expand and the number of T cells that do not have antigenic specificity for the target antigen do not expand, therebyproducing a selectively expanded number of T cells each having antigenic specificity for the one or more target antigens; and non-specifically expanding the number of T cells.

10. The method of claim 9, further comprising: introducing a nucleotide sequence encoding a cell surface marker into the APCs to obtain APCs that express the cell surface marker; adding magnetic beads to the APCs and the T cells during or after the stimulating, wherein the magnetic beads are coupled to a binding partner that specifically binds to the cell surface marker; co-culturing the magnetic beads with the APCs and the T cells during or after the stimulating, thereby producing a complex comprising the APCs, the T cells, and the magnetic beads, wherein the binding partner coupled to the magnetic beads is bound to the cell surface marker expressed by the APCs, and wherein the T cells having antigenic specificity for the one or more target antigens are bound to the target antigen presented by the APCs; and applying a magnetic field to the complex to physically separate the complex from other T cells that do not have antigenic specificity for the one or more target antigens and are not bound to the target antigen presented by the APCs, wherein non-specifically expanding the number of T cells comprises non-specifically expanding the number of T cells of the complex that have antigenic specificity for the one or more target antigens and are physically separated from the other T cells that do not have antigenic specificity for the one or more target antigens and are not bound to the target antigen presented by the APCs.

11. The method of claim 9 or 10, wherein non-specifically expanding the number of T cells comprises culturing the selectively expanded number of T cells in the presence of (i) one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells, (ii) one or more cytokines, and (iii) an antibody, or an antigen binding portion thereof, which specifically binds to the human CD3 complex.

12. The method of claim 11. wherein culturing the selectively expanded number of T cells in the presence of (i). (ii). and (iii) is carried out from 0 hour to three days after the stimulating.

13. A method of selectively expanding a number of T cells each having antigenic specificity for a target antigen, the method comprising: inducing autologous antigen presenting cells (APCs) of the mammal to present one or more target antigens: and stimulating, in the presence of interleukin (IL)-2 with or without IL-21 in vitro, T cells from the mammal with the APCs that present the one or more target antigens, wherein the T cells having antigenic specificity for the tumor antigen receive proliferation signals, the number of T cells having antigenic specificity for the target antigen expand and the number of T cells that do not have antigenic specificity for the target antigen do not expand, thereby producing a selectively expanded number of T cells each having antigenic specificity’ for the one or more target antigens; wherein the method does not comprise non-specifically expanding the number of T cells.

14. The method of claim 13, further comprising: introducing a nucleotide sequence encoding a cell surface marker into the APCs to obtain APCs that express the cell surface marker; adding magnetic beads to the APCs and the T cells dunng or after the stimulating, wherein the magnetic beads are coupled to a binding partner that specifically binds to the cell surface marker; co-culturing the magnetic beads with the APCs and the T cells during or after the stimulating, thereby producing a complex comprising the APCs, the T cells, and the magnetic beads, wherein the binding partner coupled to the magnetic beads is bound to the cell surface marker expressed by the APCs, and wherein the T cells having antigenic specificity7for the one or more target antigens are bound to the target antigen presented by the APCs; and applying a magnetic field to the complex to physically separate the complex from other T cells that do not have antigenic specificity for the one or more target antigens and are not bound to the target antigen presented by the APCs.

15. The method of any one of claims 9-14, wherein the T cells are isolated from tumor, lymph node, or peripheral blood.

16. The method of any one of claims 9-15, wherein the method does not comprise screening the T cells for antigenic specificity for the one or more target antigens before or during the stimulating.

17. The method of any one of claims 9-16, wherein inducing the APCs to present the one or more target antigens comprises:(i) pulsing the APCs with a peptide comprising a target antigen amino acid sequence or a pool of peptides, each peptide in the pool comprising a different target antigen amino acid sequence;(ii) pulsing the APCs with lysis of a tumor expressing different tumor antigens;(iii) introducing one or more nucleotide sequences encoding one or more different target antigens into the APCs; or(iv) pulsing the APCs with whole or lysed autologous tumor, whole or lysed tumor cells, or whole or lysed organoid derived from autologous tumor.

18. A method of isolating a T cell receptor (TCR), or an antigen-binding portion thereof, having antigenic specificity for the target antigen, the method comprising: selectively expanding a number of T cells each having antigenic specificity for one or more target antigens according to the method any one of claims 9-17; and isolating a TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the TCR, or antigen-binding portion thereof, has antigenic specificity for one of the target antigens.

19. The method of claim 18, wherein the method comprises: isolating a CD4 TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the CD4 TCR, or antigen-binding portion thereof, has antigenic specificity for one of the target antigens; and isolating a CD8 TCR, or an antigen-binding portion thereof, from the selectively expanded number of T cells, wherein the CD8 TCR, or antigen-binding portion thereof, has antigenic specificity7for one of the target antigens.

20. A method of preparing a population of cells that express a TCR, or an antigenbinding portion thereof, having antigenic specificity for a target antigen, the method comprising: isolating a TCR, or an antigen-binding portion thereof, according to the method of claim 18 or 19. and introducing the nucleotide sequence encoding the isolated TCR, or the antigenbinding portion thereof, into peripheral blood mononuclear cells (PBMC) to obtain cells that express the TCR, or the antigen-binding portion thereof.

21. The method of any one of claims 1-20, wherein the one or more target antigens is 2 or more different target antigens.

22. The method of any one of claims 1-21, wherein the stimulating is carried out for a period of 10 to 28 days.

23. The method of any one of claims 1-22, further comprising selectively introducing exogenous nucleic acid(s) into T cells by selectively stimulating T cells that have antigenic specificity for the target antigen, and selectively delivering the exogenous nucleic acid(s) to T cells with antigenic specificity for the target antigen.

24. The method of claim 23, wherein the nucleic acid(s) comprise nucleotide sequence(s) encoding one or more of IL- 12, BACH2 and KLF2.

25. The method of claim 23 or 24, wherein introducing the nucleic acid into the selectively expanded number of T cells is carried out up to 5 days after initiation of the stimulating.

26. The method of any one of claims 1-25, wherein the APCs are dendritic cells (DCs); B cells; peripheral blood mononuclear cells (PBMC); autologous tumor cells; a human leukocyte antigen (HLA)-engineered cell line; tumor organoid cells, or tumor xenograft cells.

27. The method of any one of claims 1-26, wherein the method selectively expands the number of T cells by 10-fold to 1000-fold.

28. The method of any one of claims 1-27, wherein the method produces an increase in any one or more of (i)-(vi) as compared to T cells the number of which was expanded by rapid expansion protocol (REP):(i) a proportion of T cells having a stem-like memory T cell phenotype;(ii) a proportion of T cells having antigenic specificity for the target antigen;(iii) diversity of T cells having antigenic specificity for the target antigen;(iv) in vivo anti-tumor function of T cells having antigenic specificity for a tumor antigen;(v) a ratio of the T cells having antigenic specificity for the target antigen with the relevant CD4 or CD8 co-receptor; and(vi) a total number of the T cells having antigenic specificity for the target antigen with the relevant CD4 or CD8 co-receptor.

29. A method of preparing a pharmaceutical composition comprising a selectively expanded number of T cells each having antigenic specificity for a target antigen, the method comprising: selectively expanding a number of T cells according to the method of any one of claims 1-17 and 21-28; and combining the selectively expanded number of T cells with a pharmaceutically acceptable carrier.

30. A method of treating or preventing a condition in a mammal, the method comprising: selectively expanding a number of T cells according to the method of any one of claims 1-17 and 21-28 or preparing a pharmaceutical composition according to the method of claim 29; and administering the selectively expanded a number of T cells or pharmaceutical composition to the mammal in an amount effective to treat or prevent the condition in the mammal.

31. A method of preparing a pharmaceutical composition comprising a selectively expanded number of T cells each having antigenic specificity for a target antigen, the method comprising: preparing a population of cells that express a TCR, or an antigen binding portion thereof, according to the method of claim 20; and combining the population of cells that express a TCR, or an antigen binding portion thereof, with a pharmaceutically acceptable carrier.

32. A method of treating or preventing a condition in a mammal, the method comprising: preparing a population of cells that express a TCR, or an antigen binding portion thereof, according to the method of claim 20 or preparing a pharmaceutical composition according to the method of claim 31; and administering the population of cells that express a TCR, or antigen binding portion thereof, or pharmaceutical composition to the mammal in an amount effective to treat or prevent the condition in the mammal.

33. The method of any one of claims 1-32. wherein the target antigen is a tumor antigen, an antigen associated with a condition, a viral antigen, or a bacterial antigen.

34. A T cell modified to express one or both of BACH2 and KLF2 or a population of cells comprising the T cell.

35. A pharmaceutical composition comprising the T cell or population of cells of claim 34.

36. A method of treating or preventing a condition in a mammal, the method comprising administering to the mammal the T cell or population of cells of claim 34 or the pharmaceutical composition of claim 35 in an amount effective to treat or prevent the condition in the mammal.

37. The method of any one of claims 30, 32, and 36, wherein the condition is cancer, an autoimmune disease, a viral infection, or a bacterial infection.

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