Research Of Adoptive Transferring Allogenic RetroNectin Activated Killer In Renal Cancer In Vitro And In Vivo

Obejeetive Cytokines and target therapy are the first line treatments for metastatic renal cell carcinoma(mRCC).Adoptive immunotherapy is effective for mRCC patients with first line treatment failure and could used as salvage regimen.Infusion of a large amount of killer cells is critical factor for achieving curative effect of adoptive immunotherapy.The quantity of killer cells could be significantly increased cultured form peripheral blood monocytes(PBMC) with combining RetroNectin,CD3mAb and Interleukin-2(IL-2)(RetroNectin activated killer,RAK) ex vivo.In this experiment, RAKs cultured form perpheral blood of healthy donors would be investigated for its expansion,activation status,anticancer effect on human renal tumor in nude mice in vivo and cytotoxicity effect on RCC cells in vitro.The data would be accumulated for applying licens of treatment for mRCC with allogenic RAK(Allo-RAK).Methodsâ‘ RAK cells and CD3AK(anti-CD3-activated killer) were cultured form perpheral blood monocytes of(PBMC) stimulated by RetroNectin,CD3mAb and IL-2 and by CD3mAb and IL-2 respectively.RAK and CD3AK cell numbers and expansion folds were countered by Typan Blue.Surface markers of RAKs of CD3,CD4, CDS,CD25,CD16 and CD56 before and after culture were examined with fluorescent antibody by flow cytometry.â‘¡RAKs were cultured form perpheral blood monocytes of healthy donor and RCC patients stimulated by RetroNectin,CD3mAb and IL-2.The expansion folds was observed and surface markers of RAKs were examined with fluorescent antibody of CD3,CD4,CD8 and CD25 by flow cytometry.Interferon gamma (IFN-γ)and tumor necrosis factor(TNF-α) in RAK culture supernatant were measured by enzyme-linked immunosorbent assay kits.â‘¢HLA-A2 positive human renal cell line RCC-LSL was selected by PCR.Nude mice inoculated with RCC-LSL subcutaneously were divided into four groups and treated with high dose AIIo-RAK(1×10⁸ cells per day), low dose Allo-RAK(5×10⁷ cells per day),IL-2 and saline for six consecutive days.The tumor sizes were measured continuously and tumors were weighted on 35th day after inoculation.â‘£Two healthy donors and two renal cell carcinoma patients of HLA-A2 positive were selected by PCR.Two strains of primary renal cancer cells were cultured form the selected patients.Cytotoxicity effect of RAKs cultured form selected candidates on HLA-A2 positive RCC cell lines RCC-LSL and RCC-FTL and on two strains of primary renal cancer cells was quantitatively analyzed by LDH assay in vitro.Resultsâ‘ RAK cells formed colonies in which sixty-seven percent were CD₃⁺CD₈⁺T cells cultured after 14 days.CD₃⁺CD₂₅⁺ cells and expression of CD₂₅⁺ increased significantly in RAKs.The expansion folds of RAKs(423 times) cultured at 14 days was higher than that of CD3AK cells(50 times)(p＜0.05).â‘¡The expansion folds were 471 and 318 and the proportion of CD₃⁺CD₈⁺ were 66.68%and 55.45% respectively of RAKs cultured form healthy donors and RCC patients cultured for 14 days.The difference were statistically significant(p＜0.05).No difference between two groups of IFN-γand TNF-αin culture supematant(p value was 0.32 and 0.40 respectively).â‘¢The increase of average sizes of tmors of both high and low dose AIIo-RAK group was lower than that of IL-2 group and saline group.The average tumor weights of high dose Allo-RAK group(0.333±0.263 g) was significantly lower than that of low dose AIIo-RAK group(1.354±0.193 g),IL-2 group(1.113±0.254 g) and saline group(1.100±0.560 g)(p value was 0.040,0.026 and 0.048 respectively).The tumor inhibition rate of high dose Allo-RAK group was 69.7%.The average tumor weight of low dose Allo-RAK group was also lower than that of IL-2 group and saline group,but there was no significant difference(p value was 0.413 and 0.253 respectively).â‘£Allo-RAKs had significant cytotoxicity effects on both RCC cell lines RCC-LSL and RCC-FTL and on two strains of primary renal cancer cells.The lysis rates of RCC cells were 6.80%～71.69%,17.25%～79.98%and 27.92%～83.48%as the effector target ratio were 37.5:1,75:1 and 150:1.Conclusions The quantity of effector cells could be significantly increased cultured form PBMC with combining RetroNectin with CD3mAb and IL-2 ex vivo.The expansion ability and proportion of effector cells of RAKs form healthy donors were higher than that from RCC patients.The AIIo-RAKs were showed cytotoxicity effect on RCC cells in vitro;Allo-RAK form healthy donors had the function of killing human RCC cells and inhibiting tumor growth in tumor beating nude mice in vivo.This study suggests allo-RAK might be an effective measure for mRCC patients.