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a CTL epitope and possessing immunotherapeutic poten- tial. Disis et al. reported that vaccination with a CTL-epi- tope LP derived from human epidermal growth factor receptor 2 (EGFR2, better known as HER2) generated robust and persistent tumor-specific T-cell immunity in patients with metastatic breast cancer.8 Recent clinical stud- ies using a telomerase-derived LP encompassing CTL-epi- topes (GV1001) showed an increase in survival of cancer patients when given in combination with radio- and chemotherapy.9 The success of LP therapeutic vaccines can be attributed to the fact that it could induce close collabora- tion between cells of the innate immune system, in partic- ular antigen-presenting dendritic cells (DC) and cells of the adaptive immune system, especially CD4+ T-helper (Th) cells and CD8+ CTL.10 LP must be taken up and processed by antigen-presenting cells (APC) before they are presented. Professional APC, such as DC, can manage pools of LP and are capable of properly excising multiple HLA class I and II peptide epitopes for presentation at the cell surface.11-13 Therefore, injection of LP will ensure the induction of both CD4+ and CD8+ T cells to available epitopes, each of which can contribute to the anti-tumor response. CD8+ CTL exert key cytotoxicity to tumor cells. CD4+ T cells are necessary elements of cellular immunity for priming tumor-specific CTL and influencing the differentiation and expansion of tumor antigen-specific CTL. Thus, an ideal peptide vaccine for cancer immunotherapy may be optimally composed of a single LP spanning epitopes for both Th cells and CTL.
In this study, we identified a human DKK1-derived LP
(DKK13-76-LP) and explored its potential as a vaccine to
induce human DKK1-specific CD4+ Th and CD8+ CTL
responses. We found that DKK13-76-LP successfully induced
Th1-cell responses in individuals expressing several com-
mon HLA allelic variants, including HLA-A, HLA-B, HLA-C,
HLA-DR alleles, and that an efficient cross-presentation of
the DKK1 -LP also induced DKK1-specific CTL response. 3-76
Methods
Patients and samples
Peripheral blood samples from patients with MM and healthy donors were used. This study was approved by the Institutional Review Board of the Cleveland Clinic, and informed consent was obtained in accordance with the Declaration of Helsinki.
Selection of HLA class I- and class II-binding peptides
To predict possible promiscuous HLA class I and II-binding pep- tides on human DKK1, the amino acid sequence of the human DKK1 protein was analyzed by Immune Epitope Database (IEBD) recommended methods.14 The program identified a 74 amino acid LP, DKK13-76, that contains multiple peptide motifs (Figure 1) with high affinity for common and major MHC class I and class II mol- ecules, representing 95% of humans.14
All peptides, including long and short MHC class I and class II binding peptides, were synthesized by Biosynthesis (Lewisville, TX, USA). The purity of synthetic peptides, confirmed by reversed-phase high-performance liquid chromatography and mass spectrometry, was over 98%. Synthetic peptides were dis- solved in dimethyl sulfoxide (DMSO; Sigma, St Louis, MO, USA), and stored at -20°C until use.
Generation of dendritic cells
Monocyte-derived mature DC were generated from human peripheral blood mononuclear cells (PBMC).11,15 The quality of
DC was judged based on their expression of CD11c, CD40, CD80, CD86, and MHC class II molecules.16 Detailed informa- tion is provided in the Online Supplementary Appendix.
Determination of in vivo immunogenicity of DKK1 peptides
HLA-A*0201-transgenic (Tg[HLA-A2.1])17 and HLA-DR*4- transgenic mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA).18 Mice were maintained at the animal facility and studies were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic.
For immunization, peptides were diluted in phosphate buffered saline at room temperature, mixed, and emulsified with an equal volume of incomplete Freund's adjuvant (Sigma). Groups of three mice were immunized subcutaneously at the tail base with 100 L of emulsion containing 100 g of peptides. All of the mice were immunized at least three times. Two weeks after the immunization, mice were killed and splenocytes were isolated for in vitro studies. The same experiments were repeated three times.
Generation of DKK1-specific CD4+ and CD8+ T-cell responses
DKK1-specific T cells were generated from PBMC of HLA- A*0201+ and HLA-DR*4+ blood donors and patients with MM by repeated stimulations of autologous T cells with DKK1 pep- tide-loaded mature DC. Further details are available in the Online Supplementary Appendix.
Cytotoxicity assay
The standard 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit was used to measure the cytolytic activity of T cells on target cells. Further details are available in the Online Supplementary Appendix.
Statistical analyses
Statistical analysis was performed with Student t-test. P<0.05 was considered statistically significant. Results are presented as mean ± standard deviation unless otherwise indicated.
Results
Identification of long peptide containing multiple T-cell epitopes on Dickkopf-1 protein
To identify LP comprising the most potential MHC class I and II binding epitopes on human DKK1 protein, we examined the amino acid sequence of DKK1 using the fol- lowing websites: http://www.bimas.cit.nih.gov/molbio/hla bind/,http://www.imtech.res.in/raghava/propred/, www.immuneepitope.org to predict the epitopes. We focused on regions with multiple MHC class I and class II epitope binding prospects. As a result, we identified an LP, DKK13-76 , that contains 74 amino acids and multiple epi- topes that can potentially bind with all major MHC class I (e.g., HLA-A, B, or C) and class II molecules (e.g., HLA-
haematologica | 2021; 106(3)
DKK1 long peptide for myeloma immunotherapy
Assessment of DKK1-specific T-cell responses
The frequency of peptide-specific, IFN-g-secreting CD4+ T cells was analyzed using 3x104 bulk CD4+ T cells stimulated with equal numbers of peptide-pulsed autologous PBMC or, alternatively, 5x104 bulk CD4+ T cells stimulated with 1x104 peptide-pulsed DC expressing HLA-DR or -DP molecules. Further details are available in the Online Supplementary Appendix.
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