Antitumor Activity And Mechanisms Of DC-targeting DNA Vaccines

Although DNA vaccine holds a great potential for cancer immunotherapy,effective long-lasting antitumoral immunity sufficient for inducing objective responses in cancer patients remains to be achieved. Clinical trials for DNA vaccines have shown that immune responses can be generated in humans, but they also highlight the need for increased potency if this vaccine technology is to be effective. A critical initial step in DNA vaccination is that encoded proteins need to be taken up,processed,and presented by dendritic cells(DC),which are most potent antigen-presenting cells that govern many aspects of the immune response. Dendritic cells are effective in activating host immune response through initiation of na(?)ve T cell activation,and they play an important role in induction of host anti-tumor specific immunity. Following intradermal or intramuscular injection of a plasmid DNA vaccine in mice,the encoded gene is expressed in transfected keratinocytes and myocytes at the site of injection as well as a small number of DC. Keratinocytes and myocytes are poorly effective at presenting antigen and priming naive immune cells due to lack of expression of MHC class II and costimulatory molecules,and they do not have ready access to T cells in lymphoid tissues, as is the case for DCs. There are two ways for DCs to obtain the immune antigen expressed by DNA vaccine, one of which is DCs absorb the immune antigen directly, but only a small percentage of the DCs injected in current trials actually migrate from the injection site into the draining lymph node to present the antigen to T cells. Also, the local DCs are able to absorb the immune antigen released by skin and muscle cells, or to devour the dead skin and muscle cells which express the immune antigen. The latter is thought as the main way.To increase the efficacies of DNA vaccine,we prepared dendritic cel(lDC)-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragmen(tscFv)from NLDC-145(scFv^NLDC-145), a monoclonal antibody binding the mouse DC-restricted surface molecule DEC-205,and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models. Compared with untargeted HER2/neu DNA vaccines,scFv^NLDC-145-HER2/neu vaccination markedly enhanced the HER2/neu-specific cellular and hormonal immune responses,resulting in much more efficient protection against tumor challenge and induction of long-lasting memory immune response in HER2/neu-expressing transplantable breast cancer models. More importantly, in combination with temporary depletion of regulatory T cells by low-dose cyclophosphamide, vaccination with scFv^NLDC-145-HER2/neu induced the regression of established tumor in HER2-positive transplantable breast cancer model and significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice.Part One: Construction and expression of DNA vaccines1. Construction of DNA vaccines. The genes encoding the variable regions of the heavy (VH) and light (VL) chains of scFv^NLDC-145 were synthesized according to the published sequences. The sequence encoding for the extracellular domain of human HER2 or its rat homologue neu was amplified from cDNA of SK-BR-3 and TUBO cell lines.After that, overlapping PCR was performed to generate scFv^NLDC-145-HER2 and scFv^NLDC-145-neu. The PCR products of scFv^NLDC-145-HER2 and scFv^NLDC-145-neu were cloned into pGEM-Teasy vector to generate pGEM-scFv^NLDC-145-HER2 and pGEM-scFv^NLDC-145-neu, and sequence verified. scFv^NLDC-145-HER2 and scFv^NLDC-145-neu gene removed from pGEM-scFv^NLDC-145-HER2 and pGEM-scFv^NLDC-145-neu were doublely digested by HindIII and XbaI endonucleases and cloned into pcDNA3.1(+) expression vector previously digested with the same enzyme to creat pcDNA3.1- scFv^NLDC-145-HER2 and pcDNA3.1- scFv^NLDC-145-neu, and sequence verified.2. Expression of DNA vaccines. The pcDNA3.1-scFv^NLDC-145-HER2 and pcDNA3.1- scFv^NLDC-145-neu plasmids were transfected into 293T cells by the use of Lipofactamine 2000 according to the manufacturer's protocol. After 72 hours, the supernatants were collected and assayed for fusion expression by Western blotting, and their molecular weights were a little bit larger than predicted, indicating certain extent of glycosylation.Part two: In vivo targeting assay1. Construction of pcDNA3.1-scFv^NLDC-145-EGFP. We generated pcDNA3.1-scFvNL DC-145-EGFP by replacing the HER2 fragment with EGFP sequence cloned from pEGFP-N1 plasmid.2. scFv^NLDC-145 targets antigen to DC in vivo. The 50μg pcDNA3.1-scFv^NLDC-145-E GFP and pcDNA3.1-EGFP plasmids in 50μL PBS were injected into the upper leg muscle of the left hind limb of the mice followed by in vivo electroporation as described previously. The GFP fluorescence in the CD11c-positive DC was analyzed by flow cytometry . Part three:Antitumor Activity of DNA vaccines in BALB/c mice1. Protection of mice from challenge with HER2-expressing tumor cells. For prophylactic vaccination, female BALB/c mice were vaccinated on days -21 and -7 by intramuscular injections of 50μg pcDNA3.1-scFv^NLDC-145-HER2/neu, pcDNA3.1-HER2 /neu plasmid DNA in 50μL PBS as described above. As control, 50μL pcDNA3.1 were injected. On day 0, animals were inoculated subcutaneously (s.c.) with 2×10⁵ D2F2/E2 tumor cells in the opposite flank. Tumor growth was monitored with a caliper by measuring two perpendicular tumor diameters every week. The date show that scFv^NLDC-145-HER2 vaccination protected mice from D2F2/E2 tumor challenge, resulting in 100% survival in all mice during the observation period (120 days after vaccination).2. Therapeutic efficacy of scFv^NLDC-145-HER2 vaccine. We next evaluated the therapeutic effect of scFv^NLDC-145-HER2 vaccination on established tumors in D2F2/E2 breast tumor model. BALB/c mice were subcutaneously inoculated with D2F2/E2 tumor cells. On day 7, animals with tumors sizing 40 mm³ were randomized into groups treated with scFv^NLDC-145-HER2 or respective controls. Treatment was repeated once 2 week later. The date show that scFv^NLDC-145-HER2 vaccination substantially slowed tumor development and protected up to 20% of the mice from tumor growth at the end of experiment (120 days after tumor inoculation). We therefore tested DC-targeted vaccines in combination with Treg depletion by low-dose CTX. As we expected, this combination significantly improved the therapeutic effects of scFv^NLDC-145-HER2 vaccine; at the end of experiment, 80% mice rejected the established tumor. Untargeted DC vaccines failed to exert therapeutic effects although this vaccine in combination with CTX mildly delayed tumor growth.Part four: Evaluation of T-cell and antibody responses1. Induction of HER2-specific T cells. To analyze the nature of the immune responses induced by scFv^NLDC-145-HER2, splenocytes were isolated from the vaccinated mice and cultured in the presence of recombinant HER2 or TRP2 protein for 3 days in vitro. Splenocytes obtained from scFv^NLDC-145-HER2-treated mice showed vigorous proliferation upon restimulation with recombinant HER2 protein, but not TRP2 protein. A slightly increased proliferation was also detected in the splenocytes from scFv^NLDC-145-neu-vaccinated mice. In contrast, no evident T-cell proliferation could be observed when mice were vaccinated with untargeted HER2 or neu. The supernatants of stimulated T cells were tested for the presence of cytokines by ELISA. Splenocytes obtained from scFv^NLDC-145-HER2-vaccinated mice produced substantial amounts of TNF-αand IFN-γ,similarly, a mildly higher level of IFN-γand TNF-αcytokine was also detected in the supernatant from scFv^NLDC-145-neu-vaccinated mice. Next, we checked for the induction of HER2-specific CD4+ and CD8⁺ T cells. The results show that a much higher percentage of CD4⁺ and CD8+ T cells producing IFN-γand TNF-αwas detected upon in vitro restimulation with HER2-derived synthetic p63-71 peptide TYLPTNASL in splenocytes from scFv^NLDC-145-HER2-vaccinated mice. 2. Induction of HER2-specific antibody. Vaccination with scFv^NLDC-145-HER2induced a high titer of HER2-specific antibody specifically binding to recombinant HER2 protein in ELISA experiments. Detailed analysis of antibody isotype demonstrated that antibody induced by scFv^NLDC-145-HER2 vaccine was mainly IgG2a, which is consistent with the cytokine profile of splenocytes.Part five: Antitumoral activity of the scFv^NLDC-145-neu DNA vaccine in immunotolerant BALB-neuT mice1. Preventive efficacy of scFv^NLDC-145-neu vaccine in BALB-neuT mice. We first evaluated the preventive efficacy of scFv^NLDC-145-neu vaccine using transplantable neu-expressing TUBO tumor model in BALB-neuT mice. BALB-neuT mice received twice scFv^NLDC-145-neu or control vaccination at 14 days interval. One week after last vaccination, the animals were challenged with TUBO tumor cells. The date show that the animals receiving scFv^NLDC-145-neu vaccination were significantly protected against a subsequent challenge with TUBO cells. Sixty days after tumor challenge, 80% mice in this group remained tumor free and the other groups all died.2. The effect of scFv^NLDC-145-neu vaccination in the prevention of spontaneous mammary tumors in BALB-neuT mice. The scFv^NLDC-145-neu was given to the mice at week 8 from birth when diffuse atypical hyperplasia is already evident in the mammary glands but before in situ carcinoma is evident and repeated at week 10. Mice in one group also received CTX injection 4 days before the first vaccination. As we expected, scFv^NLDC-145-neu/CTX vaccination resulted in a significant prolongation of tumor-free survival. This corresponded with a marked delay (⁴ weeks) in the appearance of macroscopically detectable tumors in the mammary glands of these mice. By week 38, all of the mice that were vaccinated with scFv^NLDC-145-neu/CTX remained alive. In contrast, by week 26, all of the mice in the control groups had large tumors and required euthanasia. We again observed a mildly protective effect in scFv^NLDC-145-neu vaccination group, in which the survival of most mice delayed to week 30.ConclusionThe results demonstrate that DC-targeted DNA vaccines for in vivo direct delivery of tumor antigens to DC can induce potent antigen-specific cellular and humoral immune responses, when in combination with systemic depletion of regulatory T cells, mount an impressively therapeutic antitumoral activity,providing a rationale for further development of this approach for cancer treatment.