A Double Hit To Kill B Lymphoma Cells Targeted By MBAFF

Backgroud: Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217~224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy.Objective: We previously reported Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. In this study, we want to evaluate the effect of this targeted liposomes in SCID mice model.Methods: SCID mice received i.v. injection from the tail vein with 2×107 Raji cells in 0.2 ml of PBS. After 24 h, mice were injected with free VCR, targeted liposomal VCR, nontargeted liposomal VCR or free mBAFF while control group received PBS. The injected dose for both VCR and mBAFF was 1.5 mg/kg. Mice were monitored routinely for weight loss and survival times. Mice were euthanized when they developed hind leg paralysis and subsequently sacrificed and subjected to gross pathological examination. Their peripheral blood was smeared and stained with hematoxylin and eosin. Deceased animals were subjected to a gross immunohistochemical analyses to detect tumor dissemination.Results: (1) The SCID mice model of human Raji cells was established successfully. (2) Survive time assay indicated that the targeted liposomes loaded with vincristine exhibited significant targeting and specificity to tumor growth inhibition and were superior to conventional liposomes and free drugs.Conclusion: In summary, we have clearly shown that the targeted liposomes can exhibit an improved therapeutic efficacy on B-cell malignancies. This strategy may have potential applications in the treatment of many blood dyscrasias of B-cell origin.Part II A Double Hit to kill B lymphoma cells targeted by mBAFF fused with PinX1Backgroud: We have previously confirmed mBAFF may hold promise as a novel ligand for targeted anticancer therapy of B lymphocyte-related malignant hematologic diseases. Additionally, PinX1 or its TID can effectively inhibit telomerase, induce cells to enter crisis and inhibit tumor formation in nude mice, So PinX1 or its TID might be useful for cancer therapy. We investigated whether PnX1/c/n-9L-mBAFF is more efficient than PnX1/c/n or mBAFF in mBAFF receptor-expressing human B lymphoma cells.Objective: Here, a novel class of antitumor fusion proteins was explored to mediate tumor-specific cell growth inhibition both in vitro and in vivo.Methods: In this study, we firstly constructed several soluble mBAFF, PnX1/c/n cDNA and a synthetic polyarginine tract(9L)/G4S-9L-G4S.Then Fragments encoding PnX1/c/n, 9L/G4S-9L-G4S, mBAFF were cloned in the expression vector PET28a. After being sequenced, the construct PnX1/c/n-9l/G4S-9L-G4S/mBAFF were expressed in BL21DE3 respectively. The recombinant proteins were purified with Ni2+-NTA chromatography .Then investigate the in vitro targeting and cytotoxicity of the targeted construct. Finally, to evaluate the effect of this targeted fusion proteins in SCID mice model. Results:(1) The recombinant plasmids PnX1/c/n-9l/G4S-9L-G4S/mBAFF were successfully constructed and transformed into E.coli BL21DE3 respectively. The recombinant proteins were expressed at high level in the present of IPTG and identified by Western blot analysis. SDS-PAGE analysis showed that BL21DE3 were mainly located in cytoplasm of E.coli.(2) The proteins were purified by Ni2+-NTA chromatography under the native condition and dialysed against PBS. SDS-PAGE and densitometric scanning analysis showed that the target proteins were highly purified and their purity was more than 90%. Immunofluorescence staining demonstrated PnX1/c/n-9l/G4S-9L-G4S and PnX1/c/n-9l/ G4S-9L-G4S/mBAFF colocalized in the nucleus, especially in nucleoli and at telomere speckles.(3) In vitro cytotoxicity assay indicated that PnX1c-G4S-9L-G4S-mBAFF showed significantly higher levels of cytotoxicity towards Raji cells than the other recombinant proteins. PnX1/c-9l/G4S-9L-G4S/mBAFF can inhibit telomerase activity, shorten telomeres.(4) Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with PnX1c-G4S-9L-G4S-mBAFF compared to either PnX1c or PnX1c-mBAFF.Conclusion: PnX1c-G4S-9L-G4S-mBAFF showed significantly higher levels of cytotoxicity towards Raji cells than the other recombinant proteins in vitro and in vivo. These studies suggest the potential of the PnX1c-G4S-9L-G4S-mBAFF in targeted therapy of B-cell malignancies.