Intradermal vaccination of HPV-16 E6 synthetic peptides conjugated to an optimized Toll-like receptor 2 ligand shows safety and potent T cell immunogenicity in patients with HPV-16 positive (pre-)malignant lesions

Abstract

Background: Amplivant is a molecularly optimized Toll-like receptor 2 ligand that can be covalently conjugated to tumor peptide antigens. In preclinical models, amplivant-adjuvanted synthetic long peptides (SLPs) strongly enhanced antigen presentation by dendritic cells, T cell priming and induction of effective antitumor responses. The current study is a first-in-human trial to investigate safety and immunogenicity of amplivant conjugated to human papillomavirus (HPV) 16-SLP.

Methods: A dose escalation phase I vaccination trial was performed in 25 patients treated for HPV16 positive (pre-)malignant lesions. Amplivant was conjugated to two SLPs derived from the two most immunodominant regions of the HPV16 E6 oncoprotein. The vaccine, containing a mix of these two conjugates in watery solution without any other formulation, was injected intradermally three times with a 3-week interval in four dose groups (1, 5, 20 or 50 µg per conjugated peptide). Safety data were collected during the study. Peptide-specific T cell immune responses were determined in blood samples taken before, during and after vaccination using complementary immunological assays.

Results: Toxicity after three amplivant-conjugated HPV16-SLP vaccinations was limited to grade 1 or 2, observed as predominantly mild skin inflammation at the vaccination site and sometimes mild flu-like symptoms. Adverse events varied from none in the lowest dose group to mild/moderate vaccine-related inflammation in all patients and flu-like symptoms in three out of seven patients in the highest dose group, after at least one injection. In the lowest dose group, vaccine-induced T cell responses were observed in the blood of three out of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T cell response after vaccination. These HPV16-specific T cell responses lasted until the end of the trial.

Conclusions: Amplivant-conjugated SLPs can safely be used as an intradermal therapeutic vaccine to induce robust HPV16-specific T cell immunity in patients previously treated for HPV16 positive (pre-) malignancies. Increased vaccine dose was associated with a higher number of mild adverse events and with stronger systemic T cell immunity.

Trial registration numbers: NCT02821494 and 2014-000658-12.

Keywords: Adjuvants, Immunologic; Immunogenicity, Vaccine; Immunotherapy; Vaccination.

Conflict of interest statement

Competing interests: FO, CJMM, DVF and GAvdM are inventors of a patent application related to the work in this article entitled 'Adjuvant compound', with publication number WO 2013/051936 and filing date October 4, 2012. CJMM and WJK receive a salary from ISA Pharmaceuticals BV and are in possession of ISA stock appreciation rights and are inventors on patents that are licensed to or owned by ISA Pharmaceuticals BV, dealing with synthetic long peptide vaccines. SHB is named as an inventor on the patent for the use of synthetic long peptides as vaccine. SHB serves as a paid member of the strategy board of ISA Pharmaceuticals and received honoraria as a consultant for PCI Biotech, IO Biotech and DC prime.

© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

A schematic overview of the vaccination scheme. Individuals received the vaccine injections within a dose escalation of 1, 5, 20 or 50 µg/peptide – conjugate in patients with HPV16+ tumors or premalignant lesions at weeks 0, 3 and 6. Before, after two injections, three and 20 weeks after last injection, blood was drawn for immunomonitoring. Safety was assessed throughout the whole trial using the CTCAE. CTCAE, Common Terminology Criteria for Adverse Events; HPV, human papillomavirus.

Figure 2

Proliferative and cytokine response rate increases with dose of conjugated peptide vaccine. (A) An example (patient #25) of proliferative response indicated by stimulation index (SI) in the left graph as determined by LST of fresh PBMC. In the middle and right graph the IFNγ and IL-5 production, respectively, as measured by CBA in the LST of the same patient. Peptide pool E6.2 and E6.4 (and to a lesser extent E6.3) contain the conjugated peptides as present in the vaccine. The time points of tested PBMC samples were baseline, week 6 (postvaccination 2), week 9 (postvaccination 3) and week 26 (follow-up). The cut-off in the left graph indicates the counts per minutes in negative control (cells in medium only) plus 3 × SD SI above this cut-off are considered a positive response. In the middle and right graph, the cut-off indicates the threshold of the CBA. (B) A summary of the responses for all patients is presented in a heat map. (C) The percentage of positive proliferative (SI) responses (calculated as number of positive responses divided by the total tested peptide pools times 100) per time point and per vaccine dose level (cohort) is depicted in the upper graph. The numbers above the bars indicate the number of patients. A two-way analysis of variance test was performed to determine the differences between the cohorts. (D) The MoM of SI (LST) was determined per patients per vaccine dose level (cohort) and each dot represents a patient. The Mann-Whithney test was used to determine differences between the dose levels. (E) The cytokines produced on recognition of HPV16 peptide pools and measured by CBA are displayed in a heat map. No cytokine production was measurable on E6.1 or the E7 peptides stimulation and therefore not shown here. CBA, cytometric bead array; LST, lymphocyte stimulation test; MoM, median of maximal response; PBMC, peripheral blood mononuclear cell; SI, stimulation index.

Figure 3

Frequency of IFNγ producing T cells increased with dose of vaccine. (A) An example of the 4-day IFNγ ELISpot results showing patient #19 against the two peptides in the vaccine (pept A: HPV16 E671-95 and pept B: HPV16 E6127-158). The specific spot counts are given per 105 cells. The cut-off defines a spot count of 10 above which a response is positive. (B) An overview of the ELISpot responses to the two vaccine peptides for all patients. (C) The percentage of positive ELISpot responses (calculated as number of positive responses divided by the total tested peptide pools times 100) per time point and per vaccine dose level (cohort) is shown. The numbers above the bars indicate the number of patients. A two-way analysis of variance test was performed to determine the differences between the cohorts. (D) Patients are grouped according to their dose of vaccine and the median of max (MoM, see Methods), is depicted for each patient by a dot. Statistical significant differences are indicated (Mann-Whitney test).

Figure 4

Both CD4+ and CD8+ T cell responses are induced by the conjugated peptide vaccine. A selected (based on responses in the 4-day IFNγ ELISpot assay as well as availability of PBMCs) group of six patients were analyzed in the intracellular cytokine staining (ICS) after a 10-day prestimulation. The PBMCs were only tested for the two vaccine peptides (pept A: HPV16 E671-95 and pept B: HPV16 E6127-158) and SEB was taken along as a positive control. (A) The individual frequencies of the peptide responses (indicated cytokine) per patient as percentage of CD4+ (upper graphs) or CD8+ (lower graphs) T cells are given. (B) A heat map of the ICS responses for both CD4+ (upper) and CD8+ (lower) T cells is displayed. The dose of the vaccine and patient number (ID) is given. PBMCs, peripheral blood mononuclear cells; SEB, staphylococcal enterotoxin B.