Photochemical Internalization Enhanced Vaccination Is Safe, and Gives Promising Cellular Immune Responses to an HPV Peptide-Based Vaccine in a Phase I Clinical Study in Healthy Volunteers

Abstract

Background and aims: Photochemical internalization (PCI) is a technology for inducing release of endocytosed antigens into the cell cytosol via a light-induced process. Preclinical experiments have shown that PCI improves MHC class I antigen presentation, resulting in strongly enhanced CD8+ T-cell responses to polypeptide antigens. In PCI vaccination a mixture of the photosensitizing compound fimaporfin, vaccine antigens, and an adjuvant is administered intradermally followed by illumination of the vaccination site. This work describes an open label, phase I study in healthy volunteers, to assess the safety, tolerability, and immune response to PCI vaccination in combination with the adjuvant poly-ICLC (Hiltonol) (ClinicalTrials.gov Identifier: NCT02947854).

Methods: The primary objective of the study was to assess the safety and local tolerance of PCI mediated vaccination, and to identify a safe fimaporfin dose for later clinical studies. A secondary objective was to analyze the immunological responses to the vaccination. Each subject received 3 doses of HPV16 E7 peptide antigens and two doses of Keyhole Limpet Hemocyanin (KLH) protein. A control group received Hiltonol and vaccine antigens only, whereas the PCI groups in addition received fimaporfin + light. Local and systemic adverse effects were assessed by standard criteria, and cellular and humoral immune responses were analyzed by ELISpot, flow cytometry, and ELISA assays.

Results: 96 healthy volunteers were vaccinated with fimaporfin doses of 0.75-50 µg. Doses below 17.5 µg were safe and tolerable, higher doses exhibited local tolerability issues in some study subjects, mainly erythema, and pain during illumination. There were few, and only mild and expected systemic adverse events. The employment of PCI increased the number of subjects exhibiting a T-cell response to the HPV peptide vaccine about 10-fold over what was achieved with the antigen/Hiltonol combination without PCI. Moreover, the use of PCI seemed to result in a more consistent and multifunctional CD8+ T-cell response. An enhancement of the humoral immune response to KLH vaccination was also observed.

Conclusions: Using PCI in combination with Hiltonol for intradermal vaccination is safe at fimaporfin doses below 17.5 µg, and gives encouraging immune responses to peptide and protein based vaccination.

Keywords: immunologic adjuvant; multifunctional T-cells, phase I study photochemical enhancement of T-cell responses; peptide vaccines; photochemical internalization; vaccine delivery.

Conflict of interest statement

TO and AH are employees of PCI Biotech AS and own shares and share options in the Company. AH and PS are inventors on several patents and patent Applications on the PCI Technology. HO and SJ work as consultants for PCI Biotech, and SJ is working for ZellNet Consulting, Inc. VE is an employee of PCI Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Otterhaug, Janetzki, Welters, Håkerud, Nedberg, Edwards, Boekestijn, Loof, Selbo, Olivecrona, van der Burg and Høgset.

Figures

Figure 1

Patient disposition. The flow of study subjects through the different phases of the study is outlined. Each study subject participated in one group only. The arrows depict the temporal relationships for the consecutive treatment of the different groups. As described in detail in the main text the study started with a safety run-in part (gray boxes) where a dose of 50 µg fimaporfin was found to be tolerated. The main study (white boxes) was then started with this dose, but because of the appearance of vaccination site ulcers in some subjects the dose was reduced to 25 µg. Also this dose was not tolerated, due to the occurrence of ulcers and pain during illumination. The fimaporfin dose was therefore reduced to 2.5 µg. This dose was well tolerated, and the dose was escalated (green boxes) up to 17.5 µg, which was not tolerated due to the development of erythema of a size that exceeded the cohort stopping criteria. Due to initial promising signs of immune responses in the 2.5 µg dose cohort, also the lower doses of 0.75 and 1.5 µg fimaporfin were included in the study (light red boxes). In parallel with the first dose cohorts a control group, receiving the vaccine without fimaporfin, was performed (yellow boxes). Based on the results obtained during the study the control group and several of the fimaporfin dose groups were extended with six or four additional study subjects (as indicated on the figure), meaning the at the end of the study a total of 12 subjects had been treated in the control group and 10, 12 and 12 in the 2.5, 5 and 7.5 µg dose groups, respectively.

Figure 2

Treatment and blood sampling schedule. Subjects could receive three doses of HPV vaccine, and two doses of KLH vaccine given at 2 weeks intervals. Blood samples were collected pre- and post-vaccination at the time points indicated by red arrows.

Figure 3

Development of vaccination site erythema induced discontinuation of treatment in the 17.5 µg dose cohort. The figure shows an example of erythema > 10 cm developed after PCI-mediated vaccination in the 17.5 µg fimaporfin dose group. Vaccination was performed on the belly, and the picture was taken after the second vaccination, 24 h after illumination. The black circle denotes the illuminated area.

Figure 4

Antibody response to KLH vaccination. The presence of anti-KLH IgG was analysed by ELISA as described under Materials and Methods. Vaccinations were performed at days 1 and 15. (A) Geometrical mean (+/- SEM) of IgG titres in different fimaporfin dose groups. (B) IgG titres at the different time points in single study subjects in the control (black lines) and the 12.5 µg fimaporfin (red lines) groups.

Figure 5

ELISpot responses against HPV16 E7 by cohorts and timepoint. PBMCs from the study subjects were isolated before and at different time points after vaccination, and subjected to ELISpot analysis after re-stimulation with the HPV E7 peptides used for the vaccination, as described under Materials and Methods. The spot counts for each sample were background corrected for simplified interpretation by subtracting the mean of the negative control wells from each donor from each antigen measurement from that donor. If the difference was <0, it was set to a value of 0. Each panel shows the result for an individual dose group. The lines indicate median values with the upper and lower quartiles indicated by the boxes. Mean values are shown as black diamonds. Group 25 µg A received the normal light dose of 1 J/cm2 at an irradiance of 5 mW/cm2, group 25 µg B received a light dose of 0.5 J/cm2 at an irradiance of 2.5 mW/cm2.

Figure 6

Percentage of responders to HPV16 E7 vaccination. (A) Based on the IFN-γ ELISpot analysis (re-stimulation with the HPV16 E7 peptides used for vaccination), the percentage of responders to the vaccination within each dosage group was calculated using as response definition: HPV response at any time after day 1; no pre-existing response. 2-fold empirical rule with median sport counts > 20 spots per 200,000 cells (see also Materials and Methods). (B) The kinetics of the response for each dose group, using the DFR(2x) response criterion (see Materials and Methods).

Figure 7

CD8+ T-cell responses to HPV16 E7 peptides. The expression of IFN-γ, IL-2, TNF-α, and CD154 was assessed in CD8+ T-cells following stimulation with HPV16 E7 22-mer peptides (described under Materials and Methods) for each analyzed sample. Results for the three time-points [d(ay)29, d43, d57] are indicated by the colored bars: d29 dark blue, d43 light blue, and d57 green. All measurements have been background-subtracted.

Figure 8

Analysis of CD8+ T-cell multifunctionality. PBMCs were incubated with pools of HPV peptides for 10 days, re-stimulated with HPV E7 22-mer peptides, stained with antibodies recognizing various surface and functional markers (CD3, CD4, CD8, IFN-γ, TNF-α, IL-2, CD154), and analysed by flow cytometry as described under Materials and Methods. (A) Assessment of the co-expression of CD154, IFN-γ, IL-2, and TNF-α. The pie charts illustrate the relative representation of cells co-expressing all four, different combinations of three, different combinations of two, or only a single of these functional markers within total functional cells (i.e. all cells that are positive for CD154 and/or IFN-γ and/or IL-2 and/or TNF), for those samples that had measurable CD8+ T-cell responses to HPV16 E7 peptide re-stimulation as defined in Materials and Methods. The data are not background-subtracted. (B) Percentage of CD8-cells expressing multiple (≥3) functional markers. Results are shown for each subject defined as a responder under the criteria described in Materials and Methods.