The thesis presents the results of experimental investigation into the toxic, immuno-biologic and anticancer effects of the xenogeneic embryo proteins and explores the possibilities for utilizing chicken embryo proteins as a component of xenogeneic cancer vaccines.
Chicken embryo proteins (CEP) were extracted from 7-days embryos with the 0.1% EDTA solution. Blood serum of unimmunized mice bearing Lewis Lung Carcinoma (LLC), Ehrlich Carcinoma (EC), melanoma B-16 or sarcoma 37 (S37) cross-reacted with the CEP in ELISA. It indicates that CEP solution contains similar antigens. The zymography of the CEP showed that it contained MMP-2 (zymogene and active forms). The ELISA proved that it included VEGF too. The application of the CEP did not cause toxic effect, what was studied by blood analysis, body weight and the percentage of viable immune cells. The CEP immunization of intact C57Bl or Balb/c mice induced CEP-specific IgG, which were able to cross-react with LLC, EC or melanoma B-16 but not with S37 antigens.
It was shown that immunization with the CEP had an anticancer effect. Applied before the tumor cells injection, the CEP elicited tumor growth delay (p<0.05 and p=0.07 in the case of LLC and EC, respectively), but it was inefficient in the case of S37. Different schedules of immunizations after tumor challenge were tested in order to choose the most efficient one. It was shown that the most significant anticancer effects appeared when immunization started shortly after the tumor challenge, but the most efficient schedules differed depending on the experimental tumor. In the case of EC, 50% tumor growth inhibition (p<0.05, days 13-16 of tumor growth) was reached when the immunizations was performed on day 2, 5 and 8 after the tumor transplantation. For LLC model, tumor growth inhibition by 53% (р<0.05, day 14) and metastases inhibition index of 77% was reached when the immunizations was done on day 1, 7 and 14 after the tumor challenge. A potent and long-lasting antimetastatic effect was reached, when the CEP was applied after the LLC tumor surgical removal. The indices of metastases inhibition, compared to the control mice, which underwent only surgery, reached 96.9% and 97.8% on days 18 and 34 after the tumor removal respectively. When applied to mice bearing S37 (independently on the immunization scheme), the CEP was not able to influence tumor growth. Immune effects of CEP application to LLC- or EC-bearing mice were different. For the LLC model, CEP application elicited both NK and CTL activation: compared to the untreated tumor-bearing mice, NK cytotoxic activity (CTA) of immunized mice was by 61.5% higher on day 7 after the tumor challenge (p<0.05); lymphocyte proliferation induced with the antigens of LLC cells was by 94.4, 49.7, and 163.4% higher on day 14, 21 and 28 respectively; antibody-dependent lymphocytes CTA was 388.9, 114.0 and 246.5% higher on day 14, 21 and 28 respectively. In the immunized group, the increase in NK CTA coincided with the sharp IFN-ɣ increase in blood serum.
Immunization with the CEP after surgical LLC removal protected NK from surgery-induced down-regulation in an early postoperative period (day 7) and increased spontaneous lymphocytes blast-transformation (day 7-21). Applied to EC-bearing mice, the CEP elicited antibodies production, macrophages’ (Mph’s) direct and antibodies-dependent CTA. The Mph’s CTA and antibodies-dependent CTA were significantly increased in immunized mice on day 7. The number of antibodies-producing immunized mice as well as the level of CEP-specific antibodies was higher (p<0.05) than that in the control nonimmunized group on day 21-28. Moreover, immunization with CEP had protecting effects on the immune system of mice. On day 28 of the tumor growth in the immunized mice only Mph’s antibodies-dependent CTA was significantly suppressed. On the other hand, in the control nonimmunized EC-bearing group significant suppression of immune cells functions started on day 14 of the tumor growth and was constantly ameliorating towards the last time point observation. In this group, the suppression of almost all immune cells functions (Mph’s CTA, Mph’s antibodies-dependent CTA, NK CTA, lymphocyte’s antibodies-dependent CTA) was accompanied by continuous increase of circulating immune complexes and IL-10 in blood serum.
