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WO2025212473A1 - Procédés d'identification et d'expansion sélective de lymphocytes t spécifiques d'un antigène tumoral - Google Patents

Procédés d'identification et d'expansion sélective de lymphocytes t spécifiques d'un antigène tumoral

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Publication number
WO2025212473A1
WO2025212473A1 PCT/US2025/022247 US2025022247W WO2025212473A1 WO 2025212473 A1 WO2025212473 A1 WO 2025212473A1 US 2025022247 W US2025022247 W US 2025022247W WO 2025212473 A1 WO2025212473 A1 WO 2025212473A1
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Prior art keywords
cells
apcs
tcr
antigen
target antigen
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English (en)
Inventor
Noam LEVIN
Sanghyun Kim
Steven A. Rosenberg
Nicholas D KLEMEN
Sri Krishna
Lior M. LEVY
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US Department of Health and Human Services
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US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4201—Neoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/50—Colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)

Definitions

  • Adoptive cell therapies targeting tumor antigens can successfully treat some cancer patients.
  • 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.
  • REP rapid expansion protocol
  • 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
  • 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
  • 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
  • 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 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.
  • 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.
  • 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.
  • Additional aspects of the invention provide related methods of preparing a pharmaceutical composition and methods of treating or preventing a condition.
  • 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.
  • 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).
  • Figure 2B is a graph showing the frequencies of the two p53R175H-reactive clonotypes among reactive cells in Figure 2A.
  • Figure 2C is a graph showing the results of a peptide titration assay testing specificity 7 of 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.
  • 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.
  • 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.
  • Figure 4A is a schematic of 4196, 4385, and 4391 TIL expansion by the REP with OKT3 or NeoExpand for mouse xenograft ACT studies.
  • 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.
  • 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.
  • Figure 5F is a graph showing the relative frequencies of RASG12V-reactive cells in cluster 9 and their clonal composition.
  • 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.
  • FIG. 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.
  • the table on the right lists the two mutant KRAS or p53-reactive TCRs and their HLA restrictions.
  • 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.
  • 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.
  • Figure 8C is a schematic showing the in vivo functional test of 4432 RAS G12D- reactive TCR.
  • FIGS 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 TCRs are 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.
  • FIGs 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 nonaive/ stem cell memory
  • EMRA terminal differentiated effector memory re-expressing CD45RA.
  • 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.
  • Figure 22A presents a schematic showing a combination of NeoExpand and REP according to an aspect of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 28C is a graph showing the total number of cells measured after REP of
  • 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.
  • 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.
  • 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 the target antigen.
  • the selective expansion achieved with the inventive methods may be superior to that which is achieved with conventional REP.
  • 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.
  • a target antigen including, e.g., TCR-engineered peripheral lymphocytes (PBL) and TILs.
  • PBL peripheral lymphocytes
  • TILs 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 complexity 7 of the methods and the low number of cells that can be processed at a time.
  • 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.
  • 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).
  • neoantigens and neoTCRs identified by the inventive methods included all the neoantigens and TCRs identified by the conventional screening.
  • 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.
  • flow cytometry and single cell transcriptome analysis 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.
  • 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.
  • 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.
  • inventive methods may provide any one or more of a variety of advantages.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the tumor antigen may be, for example, a tumor-associated antigen, a cancer germline antigen, or a neoantigen.
  • Tumor-associated antigens 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).
  • differentiation antigens such as melanocyte differentiation antigens, e.g., gplOO, tyrosinase, and Melan-A
  • 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).
  • 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.
  • the T cells are co-cultured in direct contact with the APCs.
  • 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.
  • a high dose of IL-2 may be from 1000 lU/ml to 6000 lU/ml.
  • 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.
  • a high dose of IL-2 may be from 1500 lU/ml to 6000 lU/ml.
  • 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.
  • 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 bead selection 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.
  • 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.
  • the cell surface marker may be NGFR.
  • 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.
  • T cells expressing the exogenous TCR bind to the target antigen presented by the APCs.
  • 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.
  • Stimulating T cells e.g., TIL
  • 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 reactivity 7 and facilitates target antigen-reactive TCR isolation.
  • 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.
  • 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 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.
  • KLF2 + T cells kill tumor cells more efficiently in various in vitro and in vivo tumor models than the control T cells.
  • An aspect of the invention provides a T cell modified to express one or both of BACH2 and KLF2.
  • 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.
  • 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.
  • TALENs transcription activator-like effector nuclease
  • 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.
  • 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.
  • 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.
  • the population of cells is a clonal population comprising T cells modified to express one or both of BACH2 and KLF2.
  • 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.
  • 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.
  • 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.
  • 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.
  • the non-specific expansion can be optional.
  • the method does not comprise non-specifically expanding the number of T cells.
  • 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.
  • 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 presented by the APCs
  • 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.
  • 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.
  • the method increases the number of T cells having antigenic specificity for a target antigen by 10-fold to 1,000-fold or more.
  • the method may produce 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.
  • 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.
  • the method may produce I x 10 6 to 1 x 10 11 or more T cells which have antigenic specificity for a target antigen.
  • the inventive methods may produce I x 10 6 to 1 x 10 7 T cells which have antigenic specificity for a target antigen.
  • the inventive methods may produce 5 x IO 6 to 3 x 10 7 T cells which have antigenic specificity for a target antigen.
  • 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.
  • 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.
  • 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.
  • the population of cells is administered by injection, e.g.. intravenously.
  • the pharmaceutically acceptable carrier for 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.
  • the pharmaceutically acceptable carrier is supplemented with human serum albumin.
  • the populations of cells and pharmaceutical compositions can be used in methods of treating or preventing a condition in a mammal.
  • 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.
  • 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.
  • the condition is cancer, an autoimmune disease, a viral infection, or a bacterial infection.
  • viral infections include, but are not limited to, those caused by human immunodeficiency viruses, respiratory syncytial virus, hepatitis C virus and Epstein-Barr virus.
  • 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.
  • inventive methods can provide any amount of any level of treatment or prevention of the condition in a mammal.
  • 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.
  • treatment or prevention can include promoting the regression of a tumor.
  • prevention can encompass delaying the onset of the condition, or a symptom or condition thereof.
  • the cells can be cells that are allogeneic or autologous to the mammal.
  • the cells are autologous to the mammal.
  • 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.
  • 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.
  • the cancer is an epithelial cancer.
  • the cancer is cholangiocarcinoma, melanoma, colon cancer, or rectal cancer.
  • the cancer expresses the one or more tumor antigens.
  • 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.
  • B cells 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.
  • IMDM Iscove's modified Dulbecco's medium
  • Transformation of patient-derived B cells 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 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-specific antibodies [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.
  • FACS fluorescence-activated cell sorting
  • 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
  • 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
  • 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 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • NeoExpand. 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. ”
  • TILs 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.
  • PVDF poly vinylidene difluoride
  • 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 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
  • 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.
  • mice were obtained from NCI or Charles River, respectively.
  • NCI or Charles River 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 ⁇ 30mm 2 , 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.
  • the mice were intraperitoneally 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.
  • 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).
  • 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 >lxlO 10 cells 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.
  • NeoExpand an in vitro TIL culture method was developed, termed “NeoExpand,” that involved the specific stimulation of TILs against previously identified or candidate neoantigens.
  • NeoExpand an in vitro TIL culture method
  • 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.
  • 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.
  • Healthy donor PBLs transduced with two different p53 or RAS neoantigen-reactive TCRs identified previously 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).
  • 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).
  • peptides and/or TMGs can be prepared in advance and the entire process of NeoExpand can take approximately two weeks.
  • newly synthesizing peptides and TMGs can add additional 4 to 6 weeks to the timeline.
  • This example demonstrates the expansion of CDS and CD4 + neoantigen-reactive TIL clonal repertoire and sensitive identification of neoantigen-reactive TCRs following neoantigenic stimulation.
  • FIGS. 2A-2G show neoantigenic stimulation of TILs from a colorectal cancer patient (4141), whose tumor harbored a p53 R175H mutation.
  • a pool of 4141 TIL fragment cultures were ex vivo expanded with or without neoantigenic stimulation using HLA-engineered COS7 cells as APCs.
  • NeoExpand was carried out using autologous DCs as APCs. Following the NeoExpand procedure, expansion of pSS 82730 - 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.
  • 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 3B
  • 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 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 shows the frequencies of the 4386 NeoExpand clonotype in the peripheral blood before ACT, individual TIL fragments and the infusion product (RX).
  • 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.
  • 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.
  • neoantigen-reactive TILs could not be induced to generate a novel reactivity.
  • 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.
  • This example demonstrates effective neoantigen-reactive TIL expansion by neoantigenic stimulation for use in ACT.
  • 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.
  • 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.
  • TILs from patients 4196. 4385, and 4391 with metastatic colorectal cancers were used to compare NeoExpand and the conventional REP.
  • TIL samples were selected based on their availability as well as compatibility with the existing mouse models for functional testing.
  • TILs were grown either by NeoExpand or the REP with OKT3.
  • the p53 R175H -reactive cells from 4196 TILs were counted by staining them with an HLA-A*02 tetramer containing the p53 R175H epitope.
  • the expanded TILs underwent another co-culture with HLA- engineered COS 7 cells pulsed with the RAS G12D minimal 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).
  • This example demonstrates the phenotypic characterization of TILs before and after neoantigenic stimulation or REP by single-cell transcriptome analysis (scRNA-seq).
  • scRNA-seq single-cell transcriptome analysis
  • 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.
  • TILs expanded viaNeoExpand or REP were functionally compared using in vivo xenograft models.
  • NSG mice were subcutaneously implanted with TYK-nu cells (p53 R175H+ ; HLA-A*02:01 + ) or 4391 colorectal cancer patient-derived xenograft (PDX) cells (KRAS G12V t ; HLA-C*01:02 ).
  • PDX colorectal cancer patient-derived xenograft
  • This example demonstrates that T cells recognize tumor antigen isolated directly from a tumor and presented by APCs.
  • Tumor expressing KRAS G12V resected 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 KRAS G12V restricted by HLA-DRBl*01, were co-cultured with the DC overnight.
  • This example demonstrates NeoExpand using organoid from the patient’s tumor.
  • 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.
  • 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 number of T cells was expanded using REP.
  • 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
  • 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.
  • 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).
  • NeoExpand in combination with the REP achieved the greatest fold expansion of CD8 + mTCR + cells than NeoExpand alone (Fig. 22C).
  • 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).
  • NeoExpand in combination with REP achieved greater fold expansion than NeoExpand alone (PBMC) or the conventional REP (Fig. 22E).
  • 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.
  • TMG tandem minigene
  • TCR-transduced, CD8 + T cells were artificially diluted with known reactivity 7 (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 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.
  • KLF2 was expressed in tumor-reactive TIL from multiple epithelial cancer patients to evaluate its impact on phenotypes.
  • peripheral blood T cells from a healthy donor were transduced with KLF2 and a TCR targeting mutant p53 R175H.
  • KLF2 + cells significantly outperformed the TCR only condition.

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Abstract

L'invention concerne des procédés d'expansion sélective d'un certain nombre de lymphocytes T exprimant un récepteur de lymphocytes T (TCR) exogène ayant une spécificité antigénique pour un antigène cible et des procédés d'expansion sélective d'un certain nombre de lymphocytes T ayant chacun une spécificité antigénique pour un antigène cible. L'invention concerne également des procédés d'isolement d'un récepteur de lymphocytes T (TCR), ou d'une portion se liant à l'antigène de celui-ci, ayant une spécificité antigénique pour l'antigène cible ; des procédés de préparation d'une population de cellules qui expriment un TCR, ou une portion se liant à l'antigène de celui-ci, ayant une spécificité antigénique pour un antigène cible ; des procédés de préparation d'une composition pharmaceutique comprenant un nombre sélectivement étendu de lymphocytes T ayant chacun une spécificité antigénique pour un antigène cible ; et des procédés de traitement ou de prévention d'une affection chez un mammifère.
PCT/US2025/022247 2024-04-01 2025-03-31 Procédés d'identification et d'expansion sélective de lymphocytes t spécifiques d'un antigène tumoral Pending WO2025212473A1 (fr)

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