Therapeutic Cancer Vaccination With a Peptide Derived From the Calreticulin Exon 9 Mutations Induces Strong Cellular Immune Responses in Patients With CALR-Mutant Chronic Myeloproliferative Neoplasms

Abstract

Background: The calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR-mutant MPN.

Methods: The safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALRmut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446).

Results: Patients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe and tolerable as only two grade three AE were detected, and none of these were considered to be related to the vaccine. A decline in platelet counts relative to the platelets counts at baseline was detected during the first 100 days, however this did not translate into neither a clinical nor a molecular response in any of the patients. Immunomonitoring revealed that four of 10 patients had an in vitro interferon (IFN)-γ ELISPOT response to the CALRLong36 peptide at baseline, and four additional patients displayed a response in ELISPOT upon receiving three or more vaccines. The amplitude of the immune response increased during the entire vaccination schedule for patients with essential thrombocythemia. In contrast, the immune response in patients with primary myelofibrosis did not increase after three vaccines.

Conclusion: Therapeutic cancer vaccination with peptide vaccines derived from mutant CALR with montanide as an adjuvant, is safe and tolerable. The vaccines did not induce any clinical responses. However, the majority of patients displayed a marked T-cell response to the vaccine upon completion of the trial. This suggests that vaccines directed against mutant CALR may be used with other cancer therapeutic modalities to enhance the anti-tumor immune response.

Keywords: calreticulin; cancer immune therapy; cancer vaccines; myeloproliferative neoplasms; neo-antigen.

Figure 1

Variation in platelet counts and CALRmut variant allele frequency (VAF) during the trial. (A) Platelet counts (10⁹/L) for each patient during the trial. (B) To get a better impression of the cumulative change in platelet counts, the relative change in platelet counts from baseline was calculated for each patient. Each dot represents the mean change in platelet count in percent relative to the platelet count at baseline. Error bars depict the standard error of the mean. Statistical testing was performed with the Wilcoxon signed-rank test. (C) Changes in the CALRmut VAF over time.

Figure 2

Immune responses to CALRLong36 in IFN-γ ELISPOT. (A) Heat map depicting the responses to CALRLong36 in patient peripheral blood mononuclear cells at each time point for all patients. The number of CALRLong36-specific cells was calculated by subtracting the mean spots in the control wells from the mean spots in the peptide-stimulated wells. The analysis was only performed in duplicates for patient 1 and 5 at the 6^th vaccination and for patient 9 at the 15^th vaccination, which prevented us from performing statistical analysis of the results. All other experiments were performed in triplicates. * Indicates a statistically significant response according to the DFR-rule. ** Indicates a statistically significant response according to the DFR(2x)-rule (38). (B) Representative image from (A, C) ELISPOT responses over time in patients with essential thrombocythemia (ET) and patients with myelofibrosis (MF) with each dot representing the mean of normalized spots. Error bars depict the standard error of the mean. Statistical testing was performed using the Mann-Whitney test.

Figure 3

Responses against CALRLong36 identified by intracellular cytokine staining in patients with a response in ELISPOT. (A) Responses in CD4⁺-gated T cells with IFN-γ secreting cells (top), IFN-γ/TNF-α secreting cells (middle). and TNF-α secreting cells (bottom). (B) Responses in CD8⁺-gated T cells with IFN-γ secreting cells (top), IFN-γ/TNF-α secreting cells (middle), and TNF-α secreting cells (bottom). (C) An example of a CD4⁺ T-cell response. (D) An example of a CD8⁺ T-cell response.

Figure 4

Responses in skin-infiltrating lymphocytes (SKILs) against CALRLong36. SKILs were harvested as described in the Materials and Methods section and were then expanded in either high-dose IL-2 (6,000 U/mL) culture medium or low-dose IL-2 (100 U/mL) culture medium. Cells were harvested and analyzed by IFN-γ ELISPOT for a response against CALRLong36. (A) Normalized numbers of cells specific to CALRLong36 in SKILs from patient 1, 3, 4, 6 and 8 cultured in low dose IL-2. (B) Response in SKILs from patients 6 expanded in low dose IL-2. (C) Normalized numbers of cells specific to CALRLong36 in SKILs from patient 1, 3, 4, 5, 6, 8 and 9 cultured in high dose IL-2. (D) Response in SKILs from patients 6 expanded in high dose IL-2.

Figure 5

Phenotyping of peripheral blood mononuclear cells (PBMC) and T cells by fluorescence-activated cell sorting. (A) Quantification of the mean PBMC subsets in peripheral blood of vaccinated patients. (B) Quantification of CD4⁺ central memory T cells (T_CM), CD4⁺ naïve T cells (T_naïve), CD4⁺ effector memory T cells (T_EM), and CD4⁺ effector memory T cells re-expressing CD45RA (T_EMRA). (C) Quantification of CD8⁺ central memory T cells (T_CM), CD8⁺ naïve T cells (T_naïve), CD8⁺ effector memory T cells (T_EM), and CD8⁺ effector memory T cells re-expressing CD45RA (T_EMRA). (D) Expression levels of PD-1 on T cells and CD4⁺ and CD8⁺ T cells, and number of regulatory T cells (Treg). Statistical testing was performed using the Wilcoxon signed-rank test. Bars represent standard error of the mean. * denotes p ≤ 0.05, ** denotes p ≤ 0.01.

Figure 6

Changes in levels of PD-1 expression in CD4⁺ and CD8⁺ T cells in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) during the vaccination schedule. (A) PD-1 expression on CD4⁺ T cells. (B) PD-1 expression on CD8⁺ T cells. Statistical testing was performed using the Mann-Whitney test. Bars represent standard error of the mean.

Figure 7

Phenotyping of NK cells and other cells fractions in PBMC by fluorescence-activated cell sorting. (A) Levels of different NK cell subsets. (B) Alterations in the levels of monocytic MDSC (mMDSC) and plasmacytoid dendritic cells (pDC). (C) Quantification of the CD4⁺/CD8⁺ ratio during the trial. Statistical testing was performed using the Wilcoxon signed-rank test. Bars represent standard error of the mean. ** denotes p ≤ 0.01.