mRNA vaccine-induced neoantigen-specific T cell immunity in patients with gastrointestinal cancer

Abstract

BACKGROUNDTherapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination.METHODSWe recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients' tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer.RESULTSThe vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRASG12D mutation. We observed no objective clinical responses in the 4 patients treated in this trial.CONCLUSIONThis vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers.TRIAL REGISTRATIONPhase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA.FUNDINGCenter for Clinical Research, NCI, NIH.

Keywords: Cancer immunotherapy; Oncology; Vaccines.

Figure 1. CONSORT diagram of the clinical study.

Description of the clinical trial.

Figure 2. Vaccine design, prior treatments, and safety.

(A) Illustration of the pipeline used to select the vaccine antigens. (B) The basic concatemer vaccine structure. (C) Overview of the vaccination schedule and immune monitoring. W_n, week number. (D) Patient-specific timeline of clinical trial progression. (E) Summary of preexisting, immunogenic, and nonimmunogenic vaccine antigens. (F) Percentage of neoantigen-specific CD8⁺ and CD4⁺ T cells from all patients. (G) Number of neoantigen-specific reactivities found in each patient.

Figure 3. Immune monitoring for patient 4251.

(A) T cells were negatively selected from PBMCs and subjected to IVS using either TMG-transfected or peptide-loaded DCs. DCs alone served as a negative control. IVS cultures were then restimulated with DCs loaded with single peptides and tested either by flow cytometry for 4-1BB expression or IFN-γ secretion using an ELISPOT assay. Data from before vaccine administration, after 4 vaccines, and after 7 vaccines following peptide or TMG restimulation are presented. Positive responses were defined as a 3-fold increase in IFN-γ and 4-1BB or OX40 expression above the DMSO control levels. All positive responses are indicated with black arrows. (B) Positive T cell cultures showing at least a 3-fold increase compared with the DMSO sample from A were cocultured for 18 hours with autologous DCs that were loaded with WT or mutated long peptide (Mut). Cells were tested for antigen recognition by flow cytometry evidence of 4-1BB upregulation (results are representative of 1 of 2 experiments).

Figure 4. Immune monitoring for patient 4251.

(A) COL6A3 cells were restimulated and sorted according to 4-1BB upregulation into 96-well plates for single-cell TCR sequencing. (B) TCR-transduced PBLs were cocultured with DCs pulsed with a serial dilution of COL6A3-mutated (Mut) or WT peptides (results are representative of 1 of 2 experiments). (C) COS-7 cells were transfected with each of the patient’s class I HLAs, loaded with peptides, and cocultured with COL6A3- and OR10H1-specific cells. Reactivity was determined by upregulation of the 4-1BB surface marker.

Figure 5. Immune monitoring for patient 4271.

(A) T cells were negatively selected from PBMCs, and IVS was performed using either TMG-transfected or peptide-loaded DCs. DCs alone served as a negative control. IVS cultures were then restimulated with DCs loaded with single peptides and tested either by flow cytometry for 4-1BB upregulation or by ELISPOT assays for IFN-γ secretion. Data are presented for before vaccine administration, after 4 vaccines, and after 8 vaccines following peptide restimulation. Positive responses were defined as a 3-fold increase in IFN-γ and 4-1BB or OX40 expression above the DMSO control level. All positive responses are indicated with black arrows. (B) Positive T cell cultures showing at least a 3-fold increase compared with the DMSO sample level from A were cocultured for 18 hours with autologous DCs that were loaded with WT or mutant long peptides. Cells were tested for antigen recognition by flow cytometry for 4-1BB expression (results are representative of 1 of 2 experiments). (C) Neoantigen-specific cells were restimulated and sorted according to 4-1BB upregulation into 96-well plates for single-cell TCR sequencing. PBMCs were collected before and after vaccination and sent for TCR VB sequencing. The data show the frequency of neoantigen-specific cells before and after vaccination.

Figure 6. KRAS^G12D-specific TCR detection, generation, and analysis.

(A) T cells were negatively selected from PBMCs, and IVS was performed using full-length KRAS RNA, KRAS TMGs, or peptide-loaded DCs. IVS cultures were restimulated with autologous DCs loaded with 10 μg/mL WT or G12D long peptide (LP), DMSO, or PMA (positive control). Reactivity was tested by ELISPOT assay for IFN-γ secretion (A) and flow cytometry for 4-1BB/OX40 upregulation (B). (C) Positive cells were restimulated and sorted according to 4-1BB upregulation into 96-well plates for single-cell TCR sequencing. (D) TCR Vα and TCR Vβ sequences found by single-cell TCR sequencing. TCR Vβ and TCR Vα pairs were cloned into a retroviral vector, transduced into autologous PBLs, and tested for reactivity with different concentrations of KRAS^G12D or WT long peptide–loaded autologous DCs.

Figure 7. Immune monitoring for patient 4289.

(A) T cells were negatively selected from PBMCs, and IVS using either TMG-transfected or peptide-loaded DCs was performed. DCs alone served as a negative control. IVS cultures were then restimulated with DCs loaded with single peptides and tested either by flow cytometry for 4-1BB expression or ELISPOT assay for IFN-γ secretion. Data from before vaccination and after 4 vaccines are presented following peptide restimulation. Positive responses were defined as a 3-fold increase in IFN-γ and 4-1BB or OX40 expression above the DMSO control level. All positive responses are indicated with black arrows. (B) Positive T cell cultures showing at least a 3-fold increase from the DMSO sample level from A were cocultured for 18 hours with autologous DCs that were loaded with WT or mutant long peptide (results are representative of 1 of 2 experiments). Cells were tested for antigen recognition by flow cytometric analysis of 4-1BB expression.

Figure 8. Immune monitoring for patient 4289.

(A) Positive T cell cultures from Figure 7A were cocultured for 18 hours with autologous DCs that were loaded with mutation-predicted minimal epitopes. Cells were tested for antigen recognition by flow cytometric analysis of 4-1BB expression. (B) Neoantigen-specific cells were restimulated and sorted according to 4-1BB upregulation into 96-well plates for single-cell TCR sequencing. PBMCs were collected before and after vaccination for TCR Vβ sequencing.