Neoantigen identification strategies enable personalized immunotherapy in refractory solid tumors

Abstract

Background: Recent genomic and bioinformatic technological advances have made it possible to dissect the immune response to personalized neoantigens encoded by tumor-specific mutations. However, timely and efficient identification of neoantigens is still one of the major obstacles to using personalized neoantigen-based cancer immunotherapy.

Methods: Two different pipelines of neoantigens identification were established in this study: (1) Clinical grade targeted sequencing was performed in patients with refractory solid tumor, and mutant peptides with high variant allele frequency and predicted high HLA-binding affinity were de novo synthesized. (2) An inventory-shared neoantigen peptide library of common solid tumors was constructed, and patients' hotspot mutations were matched to the neoantigen peptide library. The candidate neoepitopes were identified by recalling memory T-cell responses in vitro. Subsequently, neoantigen-loaded dendritic cell vaccines and neoantigen-reactive T cells were generated for personalized immunotherapy in six patients.

Results: Immunogenic neo-epitopes were recognized by autologous T cells in 3 of 4 patients who utilized the de novo synthesis mode and in 6 of 13 patients who performed shared neoantigen peptide library, respectively. A metastatic thymoma patient achieved a complete and durable response beyond 29 months after treatment. Immune-related partial response was observed in another patient with metastatic pancreatic cancer. The remaining four patients achieved the prolonged stabilization of disease with a median PFS of 8.6 months.

Conclusions: The current study provided feasible pipelines for neoantigen identification. Implementing these strategies to individually tailor neoantigens could facilitate the neoantigen-based translational immunotherapy research.TRIAL REGSITRATION. ChiCTR.org ChiCTR-OIC-16010092, ChiCTR-OIC-17011275, ChiCTR-OIC-17011913; ClinicalTrials.gov NCT03171220.

Funding: This work was funded by grants from the National Key Research and Development Program of China (Grant No. 2017YFC1308900), the National Major Projects for "Major New Drugs Innovation and Development" (Grant No.2018ZX09301048-003), the National Natural Science Foundation of China (Grant No. 81672367, 81572329, 81572601), and the Key Research and Development Program of Jiangsu Province (No. BE2017607).

Keywords: Antigen; Cancer immunotherapy; Immunology; Oncology; T cells.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Identification of personalized neoantigen in patient A008 with metastatic pancreatic cancer.

Autologous PBMCs were stimulated with candidate mutant peptides every 3 days in the presence of IL-2, and on day 10 T cell responses to each antigen were measured by flow cytometric analysis for 4-1BB upregulation on CD8+ T and CD4+ T cells (gated on CD3) (A) and IFN-γ ELISPOT assay (B). The no-peptide (media) stimulation was tested as control. Data are representative of 3 independent experiments.

Figure 2. Characterization and immunogenicity testing of neoantigen in patient A017 with metastatic thymoma.

(A–C) Three HLA-A*0201–restricted candidate mutant peptides and 9 irrelevant mutant peptides from the shared neoepitope peptide library were selected to assess the T cell–specific antigen response. After 10-day recall memory T cell assay, IFN-γ ELISPOT (A) and flow cytometry (B and C) were performed to measure the IFN-γ and 4-1BB expression (gated on CD3). (D) T2 cells were cocultured with the mutant CDC73 (CDC73-MT) and the corresponding wild-type (CDC73-WT) peptides to assess the binding affinity to HLA-A*0201. The HLA-A*0201–restricted CMV-pp65-495, EBV-LMP2a-356, and EBV-LMP2a-426 peptides were used as positive control; the HLA-A*1101–restricted KRAS-G12C peptide was used as negative control. The fluorescence index is shown for each peptide. (E) IFN-γ release measured by cytometric bead array after overnight coculture of T cells with T2 cells that were pulsed with the indicated concentrations of mutant peptides and corresponding wild-type peptides. (F) NRTs (bulk T cells) were cocultured with CFSE-labeled T2 cells that were pulsed with mutant CDC73 peptide or T2 cells not pulsed with peptide at an effector/target (E/T) ratio of 2.5:1, 5:1, 10:1, 20:1, and 40:1, respectively. After 6 hours, propidium iodide (PI) was added and the PI+CFSE+ T cells were analyzed by FACS. A–C are representative of 3 independent experiments. In D–F, data are presented as mean ± SEM (n = 3); *P < 0.05, **P < 0.01 by 2-tailed Student’s t test.

Figure 3. Identification of personalized neoantigen in patient A004 with advanced gastric cancer.

(A) Autologous PBMCs were stimulated with 8 candidate mutant peptides for 10 days, after which IFN-γ ELISPOT assays were performed to assess the T cell–specific antigen response. (B) FACS was used to detect 4-1BB upregulation on CD8+ T cells (gated on CD3). Phytohemagglutinin (PHA) was used as positive control, and no-peptide stimulation was tested as negative control. Data are representative of 3 independent experiments.

Figure 4. Frequency of somatic mutations and predicted epitopes in 17 patients with advanced solid tumor.

(A) A large clinical-grade targeted sequencing panel of 416 cancer-related genes was performed in 17 patients with advanced solid cancer. Tumor-specific somatic mutations were identified. The frequency of somatic missense mutations of each patient is shown. (B) The frequency of neoantigen epitopes was predicted for each patient’s nonsynonymous single-nucleotide variations of the restricting HLA class I alleles (HLA-A, HLA-B, and HLA-C). “+” indicates screened tumor samples in which neoantigen-specific T cell responses were detected; “–” indicates the 1 screened tumor sample in which no neoantigen-specific T cell response was detected.

Figure 5. The proportion of patients covered by the selected 29 hotspot mutations and the shared neoantigen peptide library (TCGA).

(A)The proportion of cancer patients harboring the selected 29 hotspots in the TCGA database (9.49%–89.56%). (B)The proportion of patients in the TCGA database covered by the shared neoantigen peptide library (5.11%–83.8%).

Figure 6. Immune and clinical responses to personalized immunotherapy in patient A017 with metastatic thymoma.

(A) Treatment scheme: PBMCs were collected to generate neoantigen-loaded DC vaccines and NRTs in the laboratory. Before cell infusion, the patient was preconditioned with an immunomodulatory chemotherapy comprising 1000 mg/m2 gemcitabine on day 1 and day 6 and 250 mg/m2 cyclophosphamide on day 6. Approximately 1 × 107 DC vaccines were inoculated (i.n.) subcutaneously on day 7, followed by subcutaneous injection of 150 μg GM-CSF for 5 days. Approximately 1 × 1010 bulk T cells composed of 1 × 109 NRTs were intravenously infused on day 17, followed by continuous intravenous (c.i.v.) injection of 4.0 million IU (MIU) IL-2 for 5 days. (B) CT scans were performed before and approximately 2.5 months, 6 months, and 9 months after personalized immunotherapy; representative radiological data are shown. (C) IFN-γ ELISPOT showed changes in peptide-specific IFN-γ secretion by patient PBMCs before and 6 months after treatment following 10-day culture with mutant CDC73 (CDC73-MT) or control. (D and E) Cytometric bead array assays demonstrated IFN-γ secretion by PBMCs before and 6 months after treatment following 10-day culture with tumor-associated antigens and control. (D) ***P < 0.001, 2-tailed Student’s t test, n = 3. (E) Epitope spreading was demonstrated. Data from representative experiments are depicted (n = 3).

Figure 7. Tumor regression after treatment with KRAS-G12D–based personalized immunotherapy in patient C003.

(A) Treatment scheme: PBMCs were collected to generate neoantigen-loaded DC vaccines and NRTs in the laboratory. Before vaccination, the patient was preconditioned with an immunomodulatory chemotherapy comprising 1000 mg/m2 gemcitabine on day 1 and day 6 and 250 mg/m2 cyclophosphamide on day 6. DC vaccines were inoculated subcutaneously on day 7, followed by subcutaneous injection of 150 μg GM-CSF for 5 days. Before NRT infusion, partial lesions received low-dose radiation (0.5 Gy twice daily for 2 days [#]); NRTs were administered on day 17, followed by c.i.v. infusion of 4.0 MIU IL-2 for 5 days. (B) PET-CT scans were performed before and approximately 2.5 months after treatment; representative images are shown. (C) Representative data of immunogenic neoepitope identification using shared neoantigen peptide library.