Conjugation of new DNA vaccine with polyethylenimine induces cellular immune response and tumor regression in neuroblastoma mouse model.

AIM: To estimate immunogenicity and antitumor effect of new DNA vaccine against neuroblastoma using tyrosine hydroxylase as an antigen and linear polyethylenimine (PEI) 20 kDa as a synthetic DNA carrier in syngeneic mouse tumor model. MATERIALS AND METHODS: DNA vaccine was made by cloning the tyrosine hydroxylase minigene fused to the potato virus X coat protein gene into the expression vector. The A/J mice were vaccinated by three intramuscular injections. For immunogenicity study, immune response was estimated by target cells cytotoxicity assay, interferon-gamma production in enzyme-linked immunospot assay and antigen-specific antibodies in 14 days after the final vaccination. Antitumor effect was assessed by measurement of tumor volume and event-free survival rate in mice with engrafted NB41A3 murine neuroblastoma cells following three intramuscular injections of the vaccine: 7 days before, 5 and 10 days after tumor engraftment. The immune response was also assessed on the 30th day after tumor engraftment. RESULTS: The immunogenicity and antitumor effect of the vaccine in the form of aqueous solution of DNA and DNA-PEI conjugate were compared. Splenocytes cytotoxicity was the highest in the group of DNA-PEI vaccines (37.3 ± 6.9% lysis of target cells) compared with the unconjugated DNA vaccine (26.2 ± 4.0%) and placebo control (21.9 ± 3.7%). The production of interferon-gamma in the enzyme-linked immunospot assay was about ten times higher in the DNA-PEI group than in the other groups. The vaccine slowed or prevented the growth of the tumor. Mice vaccinated with the DNA-PEI vaccine had significantly better survival compared to control group (p < 0.0003). CONCLUSIONS: DNA vaccine against tyrosine hydroxylase, administered as a DNA-PEI 20 kDa conjugate, slows down the growth of neuroblastoma cells engrafted to mice.

Phase I clinical trial of idiotypic DNA vaccine administered as a complex with polyethylenimine to patients with B-cell lymphoma.

We report on the design of a phase I, non-randomized, open-label study of idiotypic DNA vaccination in patients with B-cell non-Hodgkin's lymphoma (ISRCTN31090206). The study uses DNA fusion gene vaccination encoding patient-specific single chain variable fragment, or idiotype, linked to an immunostimulatory sequence. Two types of immunostimulatory sequence are being explored: potato virus X coat protein and human chemokine MIP3α. Linear polyethylenimine with low molecular weight (8 kDa) is used as a synthetic vehicle for vaccine delivery. Humoral and T-cellular immune responses to vaccination will be measured by ELISA and ELISPOT, respectively. The primary study endpoints are safety, tolerability and immunogenicity of DNA-PEI vaccination.

Response monitoring in follicular lymphoma by synchronous evaluation of minimal residual disease and diffusion-weighted MRI.

AIM: To demonstrate quantitative assessment of tumor response to treatment in patients with follicular lymphoma using parallel monitoring of minimal residual disease (MRD) and diffusion-weighted MRI (MRI-DWI) derived apparent diffusion coefficient (ADC). MATERIALS AND METHODS: Two patients with follicular lymphoma were undergone synchronous evaluation of MRD and MRI-DWI at definite time points before, during and after chemotherapy. MRD level was calculated in diagnostic and follow up samples relative to the highest level of amplification of the target. Allele-specific primer for clonal IgH gene rearrangement was used as a target for real-time quantitative polymerase chain reaction (PCR). 1.5 Tesla scanner was used for MRI-DWI. The largest non necrotic lymph node was chosen for serial ADC measurement. RESULTS: In first patient MRD reduced drastically in blood after chemotherapy but persisted at low level in bone marrow. Whole body MRI-DWI demonstrated regression of most of tumor lesions except one -marginally enlarged iliac lymph node and allowed to predict tumor progression in this particular anatomical site based on low ADC value after treatment. In the second patient all three methods (MRD, ADC and radiologic evaluation) gave concordant result of complete tumor response and patient remained in a clinical remission during follow-up time. CONCLUSION: Quantitative PCR measurement may detect very low level of MRD in patients with follicular lymphoma which may have prognostic value. Combination of both PCR-based MRD and quantitative evaluation of MRI-DWI derived ADC provides safe added-value disease monitoring in lymphoma.

Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination.

AIM: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. METHODS: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. RESULTS: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. CONCLUSION: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6-7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.

Rearrangements of IgH, TCRD and TCRG genes as clonality marker of childhood acute lymphoblastic leukemia.

AIM: Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific targets for clonality studies and monitoring of acute lymphoblastic leukemia (ALL). THE AIMS of the study were to select the optimum panel of primers, evaluate incidence of particular types of monoclonal and oligoclonal gene rearrangements and observe alteration of rearrangement profile between diagnosis of ALL and subsequent relapse(s). METHODS: We used polymerase chain reaction (PCR) for amplification of junctional region of rearranged IgH, TCRD and TCRG genes in combination with heteroduplex analysis in polyacrylamide gel. RESULTS: TCRD gene rearrangements were detected in 64%, TCRG - in 45%, and IgH - in 79% of B-precursor ALL patients. For patients with T-ALL, TCRD gene rearrangements were found in 47%, TCRG gene - in 66%, and IgH - in 19% of cases. Evaluation of biallelic and oligoclonal rearrangements was performed in the study. The highest incidence of oligoclonal rearrangements - 25% was shown for IgH gene in patients with B-precursor ALL. Seven pair cases of patients with de novo leukemia and relapses were analyzed and revealed subclonal deviation in rearrangements of IgH or TCR genes during disease evolution. CONCLUSION: We propose a panel of 13 types of rearrangements (primer pairs) sufficient for tumor cell clonality detection in 96% of patients with ALL. Applications of PCR-based analysis of rearranged IgH, TCRD and TCRG genes for discrimination of mono- and oligoclonality and identification of the origin of relapse were demonstrated.

[Detection of rearranged immunoglobulin and T-cell receptor genes as a method for characterization of tumor cell clonality in acute lymphoblast leukemia in children]. / Vyiavlenie rearrazhirovki genov immunoglobulinov i T-kletochnogo retseptora kak metod kharakteristiki klonal'nosti opukholevykh kletok pri ostrykh limfoleikozakh u detei.

Selection of primers for polymerase chain reaction (PCR) as used in detection of the reconstructed genes of immunoglobulins and T-cells receptor in tumor cells in children with acute lymphatic leukemia is described in the paper. PCR potentialities and limitations in characterizing the monoclonality observed in primary acute lymphatic leukemia are demonstrated. Cross linkages of the heavy chain of immunoglobulins and of T-cell receptor were found between T- and B-linear acute lymphatic leukemia forms, but not between acute lymphatic leukemia forms and myeloleukemia in children.