Screening And Identification Of Anti-tumor Molecule Base On APRIL Mutates

Malignant tumors are a great threat to human health, and to seek effective molecules and appropriate methods to counteract them has long been an effort of researchers. APRIL, also called TALL-2 (TNF and apoptosis ligand-related leukocyte-expressed ligand 2), was discovered and cloned in 1998 as a new member of the TNF superfamily. The expressed APRIL is cleaved by furin convertase, and part of the extracellular domain falls off the membrane, resulting in the formation of natural sAPRIL (soluble APRIL, i.e., amino acids 105-250). Full-length (i.e., membrane-bound) APRIL and sAPRIL are basically of the same biological activity. It was shown that amino acids 105-250 constitute the main active domain of human APRIL. Recent studies have also demonstrated that overexpression of APRIL plays a peculiar role in the proliferation and survival of malignant tumor cells as well as in the promotion of tumorigenesis. To effectively inhibit the functional activity of APRIL may provide a new method to treat relevant tumors.The present study was based on the status of research of APRIL-inhibiting molecules, the hypothesis formulated by Dalum et al who held that mutant cytokines modified by heterologous T helper cell (Th) epitopes may act as new antigens inducing endogenous, cross-reactive antibodies against natural cytokines, and precise evidence obtained from studies related to anti-TNF-a. In the present study, cDNA encoding natural human sAPRIL was cloned and used as template to clone sAPRIL cDNA mutants (msAPRIL cDNA) with deletion of different domains by PCR. Then HEL Th epitope-encoding DNA sequences were recombined with 5'- or 3 '-terminal of msAPRIL cDNA, and recombinant DNA was cloned into prokaryotic expression vectors pQE-80L to express the fusion protein composed of msAPRIL and HEL Th epitope. Fusion proteins were purified, and were confirmed to lose the functional activity of normal sAPRIL. Fusion proteins were used to immunize normal mice, and the induction of cross-reactive anti-human sAPRIL antibody by fusion proteins and the inhibition of the antibodies on the enhancement of tumor cell proliferation by human sAPRIL were observed. Fusion proteins that may induce high titres of anti-human sAPRIL antibodies were used to immunize nude mice, the immunity of which was reestablished by human peripheral blood mononuclear cells. The induction of cross-reactive anti-human sAPRIL antibodies by fusion proteins, the inhibition of the antibodies on the enhancement of tumor cell proliferation by human sAPRIL and the therapeutic effect of the antibodies on leukemia mice were observed.Based on the analyses above, we carried out the study in attempt to seek novel candidate molecules with multiple advantages for immunotherapy (even prevention of recurrence) of APRIL-related tumors. The main results are as follows:1. Human sAPRIL-encoding cDNA (aa105-aa250) was cloned by RT-PCR from human peripheral blood nucleated cells and constructed into pQE-80L. Sequence analysis confirmed that the sequence of the cloned sAPRIL cDNA was identical to that registered in GenBank.2. We cloned sAPRIL cDNA mutants (msAPRIL cDNA) with deletion of various domains by PCR using pQE-80L/sAPRIL as template. HEL Th epitope modified sAPRIL cDNA mutants were obtained by linking HEL Th epitopes to the deleted ends using nucleic acid ligation technique.3. The resulting HEL Th epitope modified sAPRIL cDNA mutants were cloned into the prokaryotic express vector pQE-80L, and sequence analysis confirmed the sequences of the obtained prokaryotic express vectors carrying cDNA mutants were correct.4. To obtain fusion proteins, we induced the expression of sAPRIL and fusion proteins in E. coli. Western blot confirmed the expression of target fusion proteins.5. To obtain purified proteins, the expressed proteins were subjected to SDS-PAGE which confirmed that the proteins mainly located in inclusion bodies. Lysates of inclusion bodies were purified by using Ni-NTA column, and the target protein eluent was subjected to renaturation by step-wise dialysis and lyophilization to obtain the purified proteins.6. To screen mutant proteins with no biological activity of sAPRIL, the MTT assay was used. The results showed that recombinant sAPRIL and mutant fusion proteins with HEL Th epitopes at both N- and C-terminals can stimulate in vitro proliferation of Raji cells, while mutant sAPRIL modified by HEL Th epitopes with deletions at the N- and C-terminals of 45bp (C, D) or 90bp deletion (E, F) cannot. The results demonstrated that mutants C, D, E and F had no sAPRIL activity and HEL Th epitopes did not affect sAPRIL activity.7. To test whether mutant proteins with no biological activity induce polyclonal antibodies in mice, mice were immunized by purified proteins C, D, E and F and immunoadjuvant. ELISA and Western blot analyses confirmed that the fusion proteins may induce polyclonal antibodies in mice. Meanwhile, antisera against proteins C, D, E and F were obtained.8. To clarify the effect of polyclonal antibodies on the biological activity of sAPRIL, the inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results indicated that the antisera may inhibit Raji and Jurkat cell proliferation stimulated by sAPRIL, however, antiserum against protein F did not inhibit Raji cell proliferation stimulated by sAPRIL. Among the four antisera, the antiserum against mutant protein D was the strongest in inhibiting the proliferation of the two kinds of cells.9. To verify whether mutant protein D may induce cross-reactive human sAPRIL polyclonal antibodies in SCID mice leaky phenotype and the influence of these polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL, we immunized SCID mice leaky phenotype with mutant D, identified the resulting antibodies by ELISA and Western blot, and assessed by the MTT assay the effect of these polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL. The results indicated that cross-reactive human sAPRIL polyclonal antibodies were obtained successfully, which may inhibit Raji and Jurkat cell proliferation stimulated by sAPRIL. The results preliminarily demonstrated that we have screened novel candidate molecules for proliferation inducing ligand-based tumor immunotherapy.10. To clarify the efficacy of the purified mutant protein D in leukemia animal models, we first assayed the influence of sAPRIL and mutant protein D on the proliferation of P388 leukemic cells. The results indicated that sAPRIL may promote the proliferation of P388 leukemic cells in a dose-dependent manner. Mutant protein D stimulated the proliferative activity of P388 leukemic cells, and prolonged the survival of mice with P388 induced leukemia; however, the therapy was ineffective in light of T/C value, a common parameter to evaluate the efficacy of a drug for tumor.