Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes

Abstract

Background: Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment.

Methods: We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand.

Results: We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4⁺ and CD8⁺ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models.

Conclusions: Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation.

Trial registration number: NCT00068003, NCT01174121, and NCT03412877.

Keywords: Adoptive cell therapy - ACT; T cell Receptor - TCR; Tumor infiltrating lymphocyte - TIL.

Figure 1

Conventional TIL expansion and the development of NeoExpand for selective neoantigenic stimulation of TILs. (A) Schematic showing the conventional way of TIL expansion, screening and the rapid expansion for the development of a TIL infusion product. (B) Frequencies of neoantigen-reactive TIL before (ie, TIL fragment culture) and after a rapid expansion with OKT3 (ie, TIL infusion product). TIL fragment cultures with neoantigen reactivities before and after the rapid expansion were co-cultured with autologous dendritic cells pulsed with mutated peptides indicated above. Per cent reactive cells were assessed by flow cytometric measurement of 4-1BB⁺ or OX-40⁺ cells. (C) Schematic of NeoExpand using neoantigen-loaded APCs for the expansion of neoantigen-reactive TILs. APC, antigen presenting cell; DC, dendritic cell; IL, interleukin; TCR, T-cell receptor; TIL, tumor infiltrating lymphocytes.

Figure 2

Expansion of CD8⁺ neoantigen-reactive TILs targeting a p53 neoantigen following neoantigenic stimulation. (A) Representative example of neoantigen-reactive TIL enrichment by neoantigenic stimulation: enrichment of p53^R175H-reactive cells from 4,141 TILs via NeoExpand was determined by flow cytometric measurement of 4-1BB and OX-40 following an overnight co-culture. Peptide-pulsed, HLA-engineered COS7 cells were used as antigen-presenting cells. ME minimal epitope. (B) CDR3A and CDR3B TCR sequences of reactive cells in (A). (C) Frequencies of the two p53^R175H-reactive clonotypes among reactive cells in (A). (D) Peptide titration assay testing specificity of 4,141 NeoExpand TCR isolated in (B). 4-1BB was measured following an overnight co-culture of TCR-engineered healthy donor PBLs with A*02-engineered COS7 cell. (E) HLA testing of 4,141 NeoExpand TCR. Healthy donor PBLs expressing 4,141 NeoExpand TCR were co-cultured with COS7 cells transfected with individual HLAs expressed by patient 4,141. IFN-γ secretion was measured by an ELISpot assay. (F) Frequencies of 4,141 “known” clonotype and 4141 NeoExpand clonotype in the patient infusion product were determined by CDR3B sequencing. (G, H) In vivo functional test of 4,141 NeoExpand TCR. Summary diagram (G) and tumor measurement (H) (n=5). Replicates from ACTs of two healthy donor PBLs are shown. Statistical analysis by two-way analysis of variance. ***p<0.001. ACT, adoptive cell therapies; DMSO, dimethyl sulfoxide; HLA, human leukocyte antigen; IFN, interferon; mTCR, murine TCR; PBL, peripheral blood lymphocytes; TCR, T-cell receptor; TIL, tumor infiltrating lymphocytes; WT, wild-type.

Figure 3

Neoantigenic stimulation selectively expands neoantigen-reactive TILs, allowing sensitive TCR isolation. (A) IFN-γ secretion of 4,386 TIL fragment culture seven against the p53^R273C neoantigen. An ELISpot assay was performed following co-culture of 4,386 TIL fragment culture seven with autologous DCs electroporated with p53 TMG or pulsed with p53^R273C 25 mer. The mock transfected (TMG mock) condition and DMSO as the vehicle control for peptide treatment were included as negative controls. (B) IFN-γ secretion of 4,386 TIL infusion product against p53 TMG or the p53^R273C peptide was measured as described in (A). (C) Flow cytometric measurement of p53^R273C-reactive cells following NeoExpand. (D) Isolation of p53^R273C-reactive TCR. (E) Peptide titration assay testing specificity of 4,386 NeoExpand TCR isolated in (D). IFN-γ secretion was measured following an overnight co-culture of TCR-engineered healthy donor PBLs with 4,386 DCs pulsed with WT or mutant p53 peptides. (F) HLA testing of 4,386 NeoExpand TCR. Healthy donor PBLs expressing 4,386 NeoExpand TCR were co-cultured with COS7 cells transfected with both A and B molecules of class II HLAs expressed by patient 4386. IFN-γ secretion was measured by an ELISpot assay. (G) Frequencies of the 4,386 NeoExpand clonotype before and after the TIL adoptive cell therapy. (H) 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). See also online supplemental table S1. DMSO, dimethyl sulfoxide; HLA, human leukocyte antigen; IFN, interferon; PBL, peripheral blood lymphocytes; PMA, phorbol 12-myristate 13-acetate; RX, infusion product; TCR, T-cell receptor; TIL, tumor infiltrating lymphocytes; TMG, tandem minigene; WT, wild-type.

Figure 4

Superior expansion of neoantigen-reactive TILs by neoantigenic stimulation relative to the conventional rapid expansion. (A) Schematic of 4,196, 4,385, and 4,391 TIL expansion by the rapid expansion with OKT3 or NeoExpand for mouse xenograft adoptive cell therapies studies. (B–D) Comparison of the rapid expansion and NeoExpand. 4,196 TILs (B), 4,385 TILs (C), and 4,391 TILs (D) were expanded by IL-2 alone, rapid expansion 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 (B) or by 4-1BB and OX-40 measurement (C,D). Fold changes were calculated based on the number of input cells. (E) 10 TIL samples were tested to compare the efficiency of neoantigen-reactive TIL expansion between the rapid expansion and NeoExpand. Statistical analysis by Wilcoxon matched-pairs signed rank test. **p<0.01. HLA, human leukocyte antigen; IL, interleukin; scRNA-seq, single-cell RNA sequencing; TCR, T-cell receptor; TIL, tumor infiltrating lymphocytes

Figure 5

ScRNA-seq analysis reveals expansion of neoantigen-reactive TILs with stem-like memory phenotypes following neoantigenic stimulation. (A–C) ScRNA-seq analysis of 4,196 TIL following NeoExpand or rapid expansion. (A) UMAP analysis of 4,196 TILs (left) and p53^R175H-reactive cells (right). Clusters 3, 4, and 10 containing high numbers of p53^R175H-reactive cells are marked in red. (B) Gene expression of markers of stem-like memory T cells and exhausted T cells. (C) Relative frequencies of p53^R175H-reactive cells in clusters 3, 4, and 10. (D–F) ScRNA-seq analysis of 4391 TIL following NeoExpand or rapid expansion. (D) UMAP analysis of 4391 TILs (left) and RAS^G12V-reactive cells (right). (E) Gene expression of markers of stem-like memory T cells and exhausted T cells. (F) Relative frequencies of RAS^G12V-reactive cells in cluster 9. See also online supplemental table S4 for phenotypic annotation of the clusters. scRNA-seq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection; TCR, T-cell receptor; TILs, tumor infiltrating lymphocytes.

Figure 6

In vivo functional test comparing the conventional rapid expansion and NeoExpand. (A) Diagram showing in vivo xenograft models testing 4,196 TILs against TYK-nu cancer cells or 4,385 and 4,391 TILs against 4391 PDX cells. (B–D) Tumor growth measurement of mice injected with 4,196 TILs (B) with 4,385 TILs (C) or with 4,391 TILs (C) (n=5). Mice in (B) and (C) were injected with 2×10⁷ TILs per mouse and mice in (D) were injected with 1×10⁷ TILs per mouse. The 4196 experiment was independently replicated once. Statistical analysis by two-way analysis of variance *p<0.05, ***p<0.001. ACT, adoptive cell therapies; PDX, patient-derived xenograft; TILs, tumor infiltrating lymphocytes.