Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy

Abstract

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.

Keywords: Cancer immunotherapy; Immunology; T-cell receptor.

Conflict of interest statement

Conflict of interest: The authors have filed patent applications related to KRASG12V (US application no. 62/560,930) and TP53G245S (US application no. 62/565,383).

Figures

Figure 1. Illustration of the new high-throughput approach for enrichment, culturing, and screening strategy of TILs.

(1) Tumor cell digests were thawed and rested overnight in complete media in the absence of exogenous cytokines. (2a) A piece of the tumor underwent whole-exome sequencing (WES) and RNA sequencing to identify nonsynonymous mutations. Based on mutation calls, 25mer peptides encompassing the mutations at position 13 were synthesized. (2b) Cells were washed, labeled, and sorted based on PD-1 and/or activation markers (CD134 or CD137) expression (pink area represents that gate used in the sort). (3) Sorted cells were cultured in 96-well plates at 3 cells/well in the presence of irradiated allogeneic feeder cells, 3,000 IU/ml IL-2, and anti-CD3ε (OKT3) for expansion. (4) Peptide pools were pulsed on autologous APCs that served as a target in a coculture with sorted cells that grow in the microwell cultures. To minimize the assays, cells from 2 or 3 cultures were combined in the assay wells. (5a) Cultures that showed recognition against peptide pools were further expanded for future testing. (5b) Cells from Pt.4097 coculture assay were labeled and reactive T cells were single-cell sorted into 96-well plates containing lysis buffer and PCR primers for TCR sequencing.

Figure 2. Sorting strategy, functional screen assays, and identification of neoantigen-reactive TCRs in Pt.4078.

(A) Gating strategy used in flow cytometry–based sort for CD3+PD-1+ and/or CD134+ TILs. (B–D) ELISPOT assays measuring IFN-γ secretion of microwell cultures upon coculture with target cells. (B) Following expansion, pools of 2 cultures were tested against autologous DCs pulsed with 2 peptide pools (PP), indicated by symbols. (C) Cultures from the reactive pools were tested separately against autologous DCs pulsed with all suspected peptide pools. (D) IFN-γ ELISPOT and CD137 flow cytometry analysis showing reactivity of the TIL cultures following coculture with autologous DCs pulsed with single 25mer peptides from each peptide pool. (E) Allogeneic T cells retrovirally transduced with neoantigen-reactive TCRs cocultured with autologous DCs pulsed with serially diluted mutated and WT 25mer peptides. ‘>’ denotes greater than 500 spots. All data are representative of at least 3 independent experiments except in A.

Figure 3. Rapid sequencing of neoantigen-reactive TCRs from Pt.4097.

(A and B) Pools of TIL cultures incubated with DCs pulsed with pooled peptide pools (PP), indicated by symbols. (A) Summary of TIL culture pools showing secretion of IFN-γ in ELISPOT. (B) Cells from ELISPOT coculture wells were collected, labeled, and single cells expressing T cell activation markers were sorted (as shown in the bottom right panel) into a 96-well PCR plate containing lysis buffer and PCR primers for TCRα and -β. (C) CDR3β of the sequenced culture well. Bolded are the origin of the reactive TCR, based on functional assays done with the individual cultures (data not shown). (D) Expanded cultures from the indicated TIL microwells coincubated with autologous DCs pulsed with single peptides from the pools that the cultures showed recognition against in previous experiments, representative of at least 2 independent experiments. ‘>’ denotes greater than 500 spots.

Figure 4. Isolation and characterization of 3 TP53G245S neoantigen-reactive TCRs from Pt.4127 sorted TIL microwell cultures.

TILs from tumor digest were sorted and expanded based on the expression of PD-1 and/or CD134. (A) Cells from 2 cultures were combined and cocultured with DCs that were pulsed with pools of mutated peptides. Showing cultures displayed enhanced IFN-γ secretion against peptide pools 9 and 10 (pool 9 encompassing TP53 mutated peptide) in (B) IFN-γ ELISPOT assay at 16 hours. Following expansion, 2 × 104 cells from individual TIL cultures that showed specific reactivity against peptide pool 9 (not shown) were cocultured with 1 × 105 DCs pulsed with single peptides present in the pool. (C and D) TCRs targeting TP53G245S were sequenced, synthesized, and virally delivered into allogeneic PBMCs to assess reactivity and specificity. (C) TCR-transduced cells were coincubated with COS7 cells that were transfected with plasmids encoding the patient’s HLA class II and pulsed with mutated 25mers. (D) Cells were coincubated with autologous DCs pulsed with serially diluted TP53G245S and WT peptides. ‘>’ denotes greater than 500 spots. All data are representative of at least 2 independent experiments except in A.

Figure 5. Characterization of a highly potent HLA-DRB1*07:01–restricted TCR isolated from a metastatic lesion of endometrial cancer.

CD3+PD-1+ and/or CD134+ TILs were sorted, expanded at 3 cells/well, and cultures that grew were tested. (A) TIL microwell culture that showed recognition against DCs pulsed with pooled peptide pools (PP) were expanded and IFN-γ secretion was assessed following coculture for 16–20 hours with DCs pulsed with single peptide pools, and (B) single peptides from PP1. (C and D) The functionality of autologous PBMCs virally transduced with the TCR isolated from neoantigen-reactive culture was measured following incubation with (C) DCs liposomally transfected with full-length RNA encoding for KRASWT, KRASG12V, and KRASG12D, and (D) DCs loaded with supernatant from lysed cell lines that underwent 5 cycles of freezing and thawing at 1:5:10 ratio (T cells/DCs/cell lines). (E) Autologous DCs pulsed with the mutated peptide were incubated with HLA-blocking antibodies for 2 hours prior to the addition of the PBMCs expressing the TCR. (F) Effector cells expressing the TCRs were incubated with DCs (pulsed with the mutated peptide) from donors matched at one of the DRB1 alleles or with DCs from a complete DRB1 mismatch. ‘>’ denotes greater than 500 spots. All data are representative of at least 3 independent experiments.