Preclinical therapeutic vaccination strategies in malignancies with focus on B-cell chronic lymphocytic leukemia

A therapeutic strategy that may potentially delay progression or prevent relapse of B-cell chronic lymphocytic leukemia (B-CLL), is anti-leukemic vaccine therapy. This thesis focuses on comparing different strategies for loading antigen on dendritic cells (DC) with the aim of delineating the most effective antigen presentation platform for stimulating anti-leukemic T cells. We have studied different methods of loading DC with tumor preparations i.e. DC-tumor cell fusion, apoptotic tumor cells (Apo-DC), tumor cell lysate (DC-lysate) and total tumor RNA (DC-RNA). A defined, synthetic peptide derived from telomerase has also been investigated as an alternative to the whole tumor cell antigen approach.

We examined autologous T cell activation by Apo-DC and compared it to the T-cell stimulatory capacity of DC that were fused with tumor cells. Following overnight incubation 22.6 ± 6.2% (mean ± SEM) of DC had endocytosed apoptotic leukemic cells while the frequency of DC-leukemic cell hybrids was 10.5 ± 2.6%. A T-cell proliferative response was observed in four out of five CIL patients when using Apo-DC whereas fusion hybrids lacked the ability to elicit a proliferative response. Both preparations were able to induce an IFN-gamma response.

In the second study the T cell stimulation capability of Apo-DC was compared with that of DC-RNA and DClysate. In all six CIL patients tested, Apo-DC induced greater HLA-restricted T-cell responses than DC pulsed with tumor lysate or RNA. ELISpot assay revealed high IFN-gamma secretion by T-cells when Apo-DC was used to stimulate autologous T cells. Real-time PCR confirmed higher expression of IFN-gamma and IL-2 mRNA in T-cells stimulated with Apo-DC. Our data suggest that cellular vaccines based on DC loaded with apoptotic bodies may be suitable for vaccination trial in patients with B-CLL.

In the third study we pulsed monocyte-derived DC with lysate from the melanoma cell line, A-375 and used it for proliferation assay and to repeatedly stimulate T-cells from a HLA-A2 positive normal donor. The resultant T-cells were examined for cytotoxic activity against A-375 targets as well as the HLA A2positive melanoma cell line DFW. We also examined the ability of lysate-pulsed DC to present melanoma-associated antigens to T-cells. The results demonstrate that lysate from allogeneic tumor cells may be used as source of antigens to stimulate tumor-specific T-cells in melanoma.

In the fourth study we investigated the feasibility of large-scale generation of monocyte-derived DC from B-CLL patients as well as the effects of freezing and thawing on the function of DC loaded with autologous tumor cell preparations. Based on the results obtained from eight B-CLL patients, it appeared that the total yield of monocytic precursors, as well as the purity, was higher with immunomagnetic separation compared to counterflow elutriation. DC pulsed with autologous apoptotic tumor cells before cryopreservation retained their morphology, surface phenotype, as well as ability to stimulate T cell proliferation and cytokine production (IFN-gamma). Our results demonstrate that monocyte precursors can be successfully isolated and that an adequate number of DC required for clinical therapy can be generated from blood.

In the fifth study, human telomerase reverse transcriptase (hTERT) was identified using RT-PCR in 19 out of 25 patients with B-CLL. DC were generated from PBMC of 7 telomerase-positive and three telomerase-negative B-CLL patients as well as three healthy donors and used for hTERT-specific CTL expansion by pulsing DC with a 16 amino acid long peptide from hTERT and with a Ras peptide of comparable length as control. In six out of seven patients, DC pulsed with the hTERT peptide could generate cytotoxic T lymphocytes (CTLs) against the autologous leukemic cells upon two rounds of restimulation which was not noted with telomerase negative patients or with the Ras control peptide as the antigen.

In conclusion, the data obtained in this study showed that B-CLL patients with telomerase-positive leukemic cells have spontaneously occurring telomerase-specific T cells which can be expanded in vitro to lyse the leukemic cells. Telomerase might therefore be a valid target structure for vaccine development in B-CLL.

List of scientific papers

I. Kokhaei P, Rezvany MR, Virving L, Choudhury A, Rabbani H, Osterborg A, Mellstedt H (2003). Dendritic cells loaded with apoptotic tumour cells induce a stronger T-cell response than dendritic cell-tumour hybrids in B-CLL. Leukemia. 17(5): 894-9.
https://doi.org/10.1038/sj.leu.2402913

II. Kokhaei P, Choudhury A, Mahdian R, Lundin J, Moshfegh A, Osterborg A, Mellstedt H (2004). Apoptotic tumor cells are superior to tumor cell lysate, and tumor cell RNA in induction of autologous T cell response in B-CLL. Leukemia. 18(11): 1810-5.
https://doi.org/10.1038/sj.leu.2403517

III. Mahdian R, Kokhaei P, Moteeian NH, Choudhury A, Mellstedt H (2006). Dendritic cells, pulsed with lysate of allogeneic tumor cells, are capable of stimulating MHC-restricted antigen-specific antitumor T cells. Medical Oncology.
https://doi.org/10.1385/MO:23:2:273

IV. Kokhaei P, Adamson L, Palma M, Osterborg A, Choudhury A, Mellstedt H (2006). Generation of dendritic cell-based vaccine for therapy of B-CLL paients. Comparison of two methods for enriching monocytic precursors. [Submitted]

V. Kokhaei P, Palma M, Hansson L, Osterborg A, Choudhury A, Mellstedt H (2006). Telomerase (hTERT 611-626) - a tumor antigen in B cell chronic lymphocytic leukemia (B-CLL) inducing spontaneously leukemia cell specific cytotoxic T lymphocytes. [Manuscript]