[Anticancer therapeutic effect of SEA-linked and membrane-bound HSP70-expressed intestine-carcinoma vaccine].

OBJECTIVE: To develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect. METHODS: The pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined. RESULTS: The laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged. CONCLUSION: A new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.

[Preparation of mB7.1-GPI and SEA-TM dual-anchored tumor cell vaccine and its antitumor effect].

OBJECTIVE: To prepare the SEA-TM and mB7.1-GPI dual-anchored EL-4 cell vaccine and to investigate its antitumor effects. METHODS: mB7.1-GPI-anchored EL-4 cell vaccine, SEA-TM-anchored EL-4 cell vaccine, SEA-TM and mB7.1-GPI dual-anchored EL-4 cell vaccine were prepared. In vitro the biological activities of these vaccines were measured using a lymphocyte proliferation assay and cytokine release assay on splenocytes derived from C57BL/6 mice. The splenocytes were co-cultured with EL-4 or EL-4/mB7.1-GPI or EL-4/SEA-TM or EL-4/SEA-TM + mB7.1-GPI (treated with Mitomycin C). Lymphocyte proliferation was determined with MTT assay, the concentrations of cytokines (IL-2 and IFN-gamma) were measured using a ELISA technique. Forty C57BL/6 mice were inoculated with EL-4 cells, after 3 days the mice were randomly divided into 5 groups with 8 in each and were treated with PBS, EL-4 cell vaccine, EL-4/mB7.1-GPI cell vaccine, EL-4/SEA-TM cell vaccine and EL-4/SEA-TM + mB7.1-GPI cell vaccine respectively, vaccines were injected three time with two-day interval. Animals were observed daily, tumor sizes were measured every third day. Twenty-five days after tumor challenge, 3 mice in each group were sacrificed and splenic lymphocytes were isolated to examine the activity of natural killer cells (NK) and cytolytic T lymphocytes (CTL). The survival of the remaining 5 mice in each group was observed till the 90th day. RESULTS: mB7.1-GPI or/and TM-SEA fusion protein was stably anchored onto the surface of EL-4 tumor cells. EL-4/mB7.1-GPI or EL-4/SEA-TM had a stronger ability to stimulate lymphocyte proliferation and IL-2 and IFN-gamma production than EL-4 (P < 0.05); while EL-4/SEA-TM + mB7.1-GPI showed a further increased ability than EL-4/mB7.1-GPI and EL-4/SEA-TM in stimulating lymphocyte proliferation and cytokine production in vitro (P < 0.05). Volume of tumor was smaller and survival time of mice was longer in EL-4/mB7.1-GPI vaccine group, EL-4/SEA-TM vaccine group and EL-4/SEA-TM + mB7.1-GPI vaccine group, comparing with PBS group and EL-4 cell vaccine group (P < 0.05). Tumor volume was much smaller and survival time of mice was much longer in EL-4/mB7.1-GPI + mB7.1-GPI vaccine group, comparing with EL-4/SEA-TM vaccine group and EL-4/mB7.1-GPI vaccine group (P < 0.05). Lymphocytes derived from the mice treated with EL-4/SEA-TM + mB7.1-GPI showed much higher NK activity and CTL activity than those derived from EL-4/mB7.1-GPI vaccine group and EL-4/SEA-TM vaccine group (P < 0.05), meanwhile the NK activity and CTL activity of EL-4/mB7.1-GPI vaccine group and EL-4/SEA-TM vaccine group was higher than EL-4 vaccine group (P < 0.05). CONCLUSION: mB7.1-GPI or/and SEA-TM fusion protein was stably anchored onto the surface of EL-4 tumor cells. The tumor cell vaccines prepared from these cells exhibited antitumor effect. The mB7.1-GPI and SEA-TM dual-anchored tumor cell vaccine had much stronger antitumor effect than the single-anchored tumor cell vaccine.

[Construction of a chimeric SEA-hPLAP-1 cDNA with gene splicing by overlap extension].

OBJECTIVE: To construct a chimeric SEA-hPLAP-1 cDNA with gene splicing by overlap extension. METHODS: The SEA gene and a DNA fragment encoding the signal for GPI-anchor attachment of hPLAP -1 were amplified by PCR. The two amplified gene sequence was annealed to form a chimeric GPI- anchored SEA molecule with gene splicing by overlap extension. The resulting chimera was cloned in pGEM-T vector and verified by sequencing analysis. RESULT: A chimeric SEA-hPLAP-1 cDNA was successfully constructed with gene splicing by overlap extension. CONCLUSION: Gene splicing by overlap extension is a successful specific PCR technique for gene recombination.

[Cloning of transmembrane domain sequence of EGFR gene]

OBJECTIVE: To clone the transmembrane (TM) domain sequence of EGFR gene and lay a good foundation for constructing the transmembrane expression vector of recombinant superantigens and cytokines. METHODS: A pair of primers special to the sequence encoding TM domain of EGFR gene were synthesized, TM domain fragment was cloned by RT-PCR, and the PCR product of TM domain sequence was ligated with the pGEM-T vector and confirmed by DNA sequencing. RESULTS: TM domain sequence was successfully cloned and verified by DNA sequencing. CONCLUSION: The successful cloning of TM domain sequence provides a basis for the construction of transmembrane fusion protein of Superantigen-TM or Cytokines-TM in cancer biotherapy.