Neoantigen vaccination induces clinical and immunologic responses in non-small cell lung cancer patients harboring EGFR mutations

Abstract

Background: Neoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are predicted to be shared between patients, and none to date have demonstrated therapeutic value in the context of vaccination.

Methods: We report here a phase I trial of personalized NeoAg peptide vaccination (PPV) of 24 stage III/IV non-small cell lung cancer (NSCLC) patients who had previously progressed following multiple conventional therapies, including surgery, radiation, chemotherapy, and tyrosine kinase inhibitors (TKIs). Primary endpoints of the trial evaluated feasibility, tolerability, and safety of the personalized vaccination approach, and secondary trial endpoints assessed tumor-specific immune reactivity and clinical responses. Of the 16 patients with epidermal growth factor receptor (EGFR) mutations, nine continued TKI therapy concurrent with PPV and seven patients received PPV alone.

Results: Out of 29 patients enrolled in the trial, 24 were immunized with personalized NeoAg peptides. Aside from transient rash, fatigue and/or fever observed in three patients, no other treatment-related adverse events were observed. Median progression-free survival and overall survival of the 24 vaccinated patients were 6.0 and 8.9 months, respectively. Within 3-4 months following initiation of PPV, seven RECIST-based objective clinical responses including one complete response were observed. Notably, all seven clinical responders had EGFR-mutated tumors, including four patients that had continued TKI therapy concurrently with PPV. Immune monitoring showed that five of the seven responding patients demonstrated vaccine-induced T cell responses against EGFR NeoAg peptides. Furthermore, two highly shared EGFR mutations (L858R and T790M) were shown to be immunogenic in four of the responding patients, all of whom demonstrated increases in peripheral blood neoantigen-specific CD8+ T cell frequencies during the course of PPV.

Conclusions: These results show that personalized NeoAg vaccination is feasible and safe for advanced-stage NSCLC patients. The clinical and immune responses observed following PPV suggest that EGFR mutations constitute shared, immunogenic neoantigens with promising immunotherapeutic potential for large subsets of NSCLC patients. Furthermore, PPV with concurrent EGFR inhibitor therapy was well tolerated and may have contributed to the induction of PPV-induced T cell responses.

Keywords: EGFR; EGFR inhibitor; neoantigen vaccine; non-small cell lung cancer; tumor regression.

Figure 1

PPV trial design and patient outcomes. (A) Neoantigen peptide vaccine manufacturing pipeline leading to immunization of patients with advanced non-small cell lung cancer (NSCLC). DNA from lung tumor biopsies was sequenced using a panel of 508 tumor-associated genes, while high-resolution HLA typing was performed on patient peripheral blood. Neoantigen vaccine peptides were selected largely based on HLA class I and class II peptide binding predictions (methods). Each patient was immunized weekly with a unique saline-based mixture of short and long neoantigen peptides divided into two cocktails and administered into opposite extremities for 12 weeks. Green arrows represent weeks when vaccination was received. Blood was drawn at weeks 0, 4, 8 and 12 as indicated with red syringe symbols. (B) Clinical event timeline for the 24 NSCLC patients who received PPV. Green bars: duration of PPV immunization. Orange bars: duration of EGFR inhibitor therapy. (C) Tumor histology of the 24 NSCLC study patients as divided into three subgroups based on EGFR mutation status and use of EGFR inhibitor during vaccination. (D) Progression-free survival curve of all 24 PPV patients. (E) Measurements of the overall tumor burden (sum of all target lesions) of PPV patients over the course of treatment. The clinical response of each patient is indicated by color: red: PD; black: SD; green: CR or PR. Tumor burden at the time point ‘pre’ indicates the tumor burden measured prior to patient disease progression. Tumor burden at the time point ‘0’ indicates the tumor burden immediately prior to the start of PPV treatment.*Patient 1 developed pleural effusion at 12 weeks. **Follow-up CT scan of patient 11 was taken at 24 weeks. No tumor measurements are shown for patient 2 due to their having no measurable tumors. Follow-up CT scans were not available for patients 13 and 15, but other clinical follow-up information regarding response and survival was obtained. (F) Response summary of immunized patients by group showing progression-free survival, overall survival, and clinical response as assessed using RECIST V.1.1 criteria 12–18 weeks following initiation of PPV. Response of patients 9, 19 and 20 were assessed at 6–8 weeks after PPV initiation due to on-treatment death from disease progression. Survival curve was generated using Kaplan-Meier estimate. CR, complete response; EGFR, epidermal growth factor receptor; HLA, human leukocyte antigen; MUT, mutated; PD, progressive disease; PPV, personalized NeoAg peptide vaccine; PR, partial response; RECIST, Response Evaluation Criteria In Solid Tumors; SD, stable disease.

Figure 2

Patient clinical responses following personalized neoantigen peptide vaccination. (A) CT scans showing regression of two lung lesions from complete responder patient 17. (B) Tissue biopsy confirmed that the remaining lung CT signal was composed of only fibrotic tissue containing no viable tumor cells. (C) Patient 17 bone metastases evaluated by T2-weighted MRI was absent 18 weeks after the start of neoantigen vaccination (yellow arrow). This bone metastasis was considered as a non-targeted lesion according to RECIST (V.1, bone lesion measurability). (D) Two additional patients in group 2 had objective clinical responses to PPV. Patient 11 experienced lung tumor regression in addition to resolution of obstructive atelectasis 24 weeks after PPV initiation (yellow arrow and red circle), while a pneumothorax showed no change (green arrow). Subcutaneous neck metastases of patient 14 showed significant regression 12 weeks after the start of PPV treatment (yellow arrow). (E) CT scans showing lung tumor regressions in patients 5, 8, 12, and 22, all of whom had partial clinical responses following PPV treatment. (F) Change in overall tumor burden of PPV study patients 3 to 4 months post-PPV compared with pretreatment baseline. Response of patients 9, 19 and 20 were assessed at 6–8 weeks after PPV initiation due to on-treatment death from disease progression. *Patient 1 developed pleural effusion at 12 weeks. CR, complete response; PD, progression disease; PPV, personalized NeoAg peptide vaccination; PR, partial response; RECIST, Response Evaluation Criteria In Solid Tumors; SD, stable disease.

Figure 3

EGFR neoantigen peptides are immunogenic, shared and show distinctive HLA class I binding preferences. (A) Summary figure showing the total vaccine peptide-specific immune reactivity for each patient (IFN-gamma ComboScore, see Methods) along with their associated group, histology, and clinical outcome. (B) Deconvolution of individual vaccine peptide reactivities by IFN-γ ELISA for patients 5, 8, 14 and 17 revealed dominant immune responses in patients 5, 8, and 14 against the HLA-A*1101 restricted EGFR-L858R peptide KITDFGRAK. Complete responder patient 17 similarly showed a dominant response against the HLA*C1502 restricted EGFR-T790M peptide LTSTVQLIM. Individual peptide reactivities for other PPV study patients are shown in supplemental data (online supplemental figure S6). (C and D) Summaries of IFN-γ ELISPOT assay (three replicates) and HLA tetramer-based staining determined that PBMC frequencies of HLA-A*1101/KITDFGRAK-specific CD8⁺ T cells in patients 5, 8 and 14, and HLA-C*1502/LTSTVQLIM-specific CD8⁺ T cells in patient 17 increased significantly over the course of PPV. *P<0.05; **p<0.01; ***p<0.001. (E) Summary of results of immune monitoring of study patients. Patient ID in green font indicates responding patients. (F) ELISPOT assay showed post-PPV PBMC from patients 5, 8, 14, and 17 specifically recognized mutated EGFR neoantigen peptides but not the corresponding wild-type (WT) EGFR peptides. (G) EGFR protein sequences and predicted HLA peptide binding affinities of the mutant EGFR-L858R (green) and T790M (blue) peptides and corresponding WT epitopes. (H and I) Neoantigens derived from the most prevalent EGFR mutations, L858R (green) and exon 19 deletions (Ex19del, orange) show distinctive binding preferences for HLA class I allotypes within the A3 superfamily, whereas other less prevalent EGFR point mutations (S768I, T790M, and L861Q, blue) show binding preferences for HLA-A2 and C3 superfamily members. (J) Expanded view showing individual HLA class I allotypes with the highest number of predicted binding EGFR neoantigens (<500 nM affinity) for the most prevalent shared EGFR mutations in lung cancer. Black arrows indicate the A*1101-restricted KITDFGRAK peptide and C*1502-restricted LTSTVQLIM peptide. Statistical comparisons were measured compared with pretreatment. Two-tailed unpaired t-tests or two-way analysis of variance test with multiple group comparison adjustment (Dunnett’s test) were used to analyze the statistical significance between groups. P<0.05 was considered significantly different. EGFR, epidermal growth factor receptor; ELISPOT, enzyme-linked immunospot; HLA, human leukocyte antigen; I, individual peptide; IFN-γ, interferon-gamma; ND, not determined; P, pooled peptides; PBMC, peripheral blood mononuclear cell; PPV, personalized NeoAg peptide vaccination.

Figure 4

Immunomodulation by EGFR inhibitors (EGFRi) promotes immune cell infiltration, tumor antigen presentation, and T cell activation. H1975 (EGFR-L858R/T790M) and H1299 (EGFR-WT) cell lines were treated with the EGFRi osimertinib, and RNAseq analysis was performed at 0, 12, or 24 hours post-treatment. (A) Relative transcript expression levels of genes associated with cell division, cell cycle, apoptosis and cell survival decreased in H1975 cells following EGFRi treatment. (B) Gene expression pathway changes in EGFRi-treated H1975 and H1299 cell lines. Red: upregulation; blue: downregulation. (C) EGFRi upregulated expression of immune-related genes associated with antigen presentation and immune cell trafficking in H1975 cells. (D) EGFRi treatment of H1975 cells downregulated genes associated with EGFR signaling and proliferation rate while upregulating genes associated with TRAIL signaling. (E and F) Luminex analysis of H1975 cell supernatants confirmed changes of 10 chemokines and cytokines at the protein level. Statistical comparisons were measured compared with control. (G) Migration assay showed that EGFRi treatment of H1975 cells increased the migration of PBMC monocytes and CD4⁺ T cells and activated CD8⁺ tumor-infiltrating lymphocytes (TILs) towards H1975 cell supernatants. (H) HLA class I surface expression increased in H1975 but not H1299 cells following EGFRi treatment. (I) Tumor antigen-specific CD8⁺ T cells showed significantly increased IFN-γ secretion in response to recognition of cognate antigen on EGFRi-treated H1975 cells compared with untreated cells. (J) Proposed mechanistic model to explain how EGFRi treatment might synergize with PPV to enhance immune cell trafficking and T cell activation at the tumor site. Two-tailed unpaired t-test or Mann-Whitney U test was used to analyze the statistical significance between groups. P<0.05 was considered significantly different. *P<0.05; **p<0.01. EGFR, epidermal growth factor receptor; IFN-γ, interferon-gamma; PBMC, peripheral blood mononuclear cell; PPV, personalized NeoAg peptide vaccination.