Personalized therapy with peptide-based neoantigen vaccine (EVX-01) including a novel adjuvant, CAF®09b, in patients with metastatic melanoma

Abstract

The majority of neoantigens arise from unique mutations that are not shared between individual patients, making neoantigen-directed immunotherapy a fully personalized treatment approach. Novel technical advances in next-generation sequencing of tumor samples and artificial intelligence (AI) allow fast and systematic prediction of tumor neoantigens. This study investigates feasibility, safety, immunity, and anti-tumor potential of the personalized peptide-based neoantigen vaccine, EVX-01, including the novel CD8⁺ T-cell inducing adjuvant, CAF®09b, in patients with metastatic melanoma (NTC03715985). The AI platform PIONEER^TM was used for identification of tumor-derived neoantigens to be included in a peptide-based personalized therapeutic cancer vaccine. EVX-01 immunotherapy consisted of 6 administrations with 5-10 PIONEER^TM-predicted neoantigens as synthetic peptides combined with the novel liposome-based Cationic Adjuvant Formulation 09b (CAF®09b) to strengthen T-cell responses. EVX-01 was combined with immune checkpoint inhibitors to augment the activity of EVX-01-induced immune responses. The primary endpoint was safety, exploratory endpoints included feasibility, immunologic and objective responses. This interim analysis reports the results from the first dose-level cohort of five patients. We documented a short vaccine manufacturing time of 48-55 days which enabled the initiation of EVX-01 treatment within 60 days from baseline biopsy. No severe adverse events were observed. EVX-01 elicited long-lasting EVX-01-specific T-cell responses in all patients. Competitive manufacturing time was demonstrated. EVX-01 was shown to be safe and able to elicit immune responses targeting tumor neoantigens with encouraging early indications of a clinical and meaningful antitumor efficacy, warranting further study.

Keywords: Personalized therapy; cancer vaccine; immune response; immunotherapy; neoantigen.

Figure 1.

Study timelines. (A) Biopsy, PET/CT scan, and blood samples was collected at baseline. Treatment with CPI was either initiated shortly after biopsy or had already been initiated for at least 4 months before biopsy. EVX-01 vaccination was administered around week 6–8 (as quickly as possible) and every second week for a total of 6 vaccinations. Tumor biopsies were performed again if possible, at TP2 and TP4. Radiographic imaging was performed every 12 weeks, and blood samples were collected TP 1 to TP4 and every time a scan was performed. (B) An overview of the mechanisms in tumor cells and surrounding immune cells that are believed to be required/desirable for a neoepitope to have a clinical effect. 1) Tumor-specific mutations are detected using WES sequencing data from the tumor sample and normal sample. 2) The expression levels of each mutation are determined by analyzing tumor RNA sequencing data. 3) The tumor-specific mutations are translated into protein space, generating neopeptide sequences. 4–5) Neopeptide sequences predicted to be presented by the patient’s specific HLA class I and class II molecules are identified. Neoepitopes must be presented by MHC class I and class II in order to be recognized by T-cells. 6) The subset of neoepitopes that are clonal, meaning present in all tumor cells, are prioritized as this allows the elicited immune response to eradicate the whole tumor, as well as potential metastases in the patient. (Arts in 1B obtained from https://smart.servier.com/). (C) Overview of patient inclusion, CPI initiation, baseline biopsy, time before vaccine treatment and follow-up information of the first five patients. The blue and red arrows indicate time points for either i.p vaccinations or i.m vaccinations, respectively. The depiction of disease condition and patient status are indicated with various colors.

Figure 2.

EVX-01-specific T-cell responses shown by Elispot on PBMCs.(A) PBMCs were prestimulated with the EVX-01 peptides in addition to IL-2 for ten to thirteen days before screening for peptide recognition of T-cells using IFNγ ELISPOT assay. After 10 days EVX-01 single peptides and/or EVX-01 peptide pool were added to prestimulated PBMCs. A positive ELISPOT response was defined when the number of spots for tested peptides exceeded the background spot number plus 3 times the standard deviation of the background (irrelevant peptide) and at least 10 spots over background (*).(B) Peptide specific T-cell response was determined for patient 1, 2, 3 and 5. Patient 4 was left out due to technical consideration and insufficient PBMC availability. The red columns represent irrelevant peptides. Highlighted columns represent positive ELISPOT response. For each patient we observed specific vaccine induced T-cell response after both 3 and 6 vaccinations. Patient 1, 2 and 3 continued to show T-cell activation at follow-up (14, 10 and 1,5 months after the last vaccination respectively).

Figure 3.

EVX-01 induced mainly CD4⁺ T-cell responses in PBMCs after vaccination. EVX-01-specific CD4⁺ T-cells were identified in all five patients at multiple timepoints after vaccination. EVX-01-specific CD8⁺ T-cells were identified in patient 5 at time point 3 (TP3). Prestimulated PBMCs were restimulated with EVX-01 peptide pool or irrelevant peptide (negative control) for 8 hours and subsequently analyzed by flow cytometry. T-cell reactivity was defined as the percentage of live CD8⁺ or CD4⁺ T-cells staining positive for at least two of three markers (TNF, IFNγ, CD107a). TP1 = Baseline; TP2 = CPI; TP3 = 3x vaccination; TP4 = 6x vaccination; FU = follow-up. Vertical hatched line separates timepoints before and after vaccinations.

Figure 4.

EVX-01-specific T-cell responses were detected in SKILs (skin-test infiltrating lymphocytes) after vaccination of patient 2. (A) IFNγ-ELISPOT responses were detected in SKILs isolated from patient 2 (TP4, after 6 vaccinations) after overnight co-culture with EVX-01 peptide pool and individual peptides. 100,000 SKILs with 10,000 autologous monocytes were seeded per well. (B) Representative example of ELISPOT-wells. (C) EVX-01-specific CD4⁺ T-cells were identified in the SKILs isolated from patient 2 (TP4, after 6 vaccinations). SKILs were restimulated with EVX-01 peptide pool or irrelevant peptide (negative control) for 8 hours. T-cell reactivity was defined as the percentage of live CD8⁺ or CD4⁺ T-cells staining positive for at least two of four markers (CD137, TNF, IFNγ, CD107a).