DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice

Six to 8-week-old female BALB/c (H-2^d) mice were purchased from the Animal Experimental Center of the Second Military Medical University. BALB-neuT mice (H-2^d) expressing a transforming neu under the control of mouse mammary tumor virus promoter were obtained from Charles River Laboratories (Shanghai, China). Heterozygous 6- to 15-week-old virgin females expressing rat neu as verified by PCR were used throughout this work. All animal studies were approved by the Institutional Review Board of the Second Military Medical University, Shanghai, China.

Mouse thymoma cell line EL4, breast cancer cell line 4 T1, and 293 T cell line were purchased from ATCC (American Type Culture Collection, VA, USA). The cells were maintained in DMEM supplemented with 10% FCS, 4 mmol/L glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. D2F2/E2 and EL4/E2 stably expressing human wild-type HER2 were maintained in complete DMEM medium containing 0.4 mg/mL G418 (Sigma-Aldrich). TUBO cells are neu-expressing breast carcinoma cells established from a lobular carcinoma of a female BALB-neuT mouse [28], and maintained in DMEM containing 20% FCS. Murine lymphocytes were cultured in RPMI-1640 containing 10% FCS, 2 mmol/L glutamine and 50 μmol/L 2-mercaptoethanol. All tissue culture reagents were purchased from Life Technologies unless described otherwise.

Peptides used in this study were obtained from Sigma-Aldrich. All peptides were > 95% pure as indicated by analytical HPLC. Lyophilized peptides were diluted in DMSO and stored at - 20°C until use. Recombinant HER2 and TRP2 protein were purchased from R&D Systems. Cyclophosphamide (CTX) were obtained from Sigma-Aldrich and reconstituted in sterile PBS (20 mg/mL) for in vivo injections. Monoclonal antibodies (mAbs) to the following antigens were purchased from eBiosciences (San Diego, CA): CD4 (GK 1.5) and CD8 (53–6.7) conjugated to fluorescein isothiocyanate (FITC); CD11c (N418) and Foxp3 (FJK-16 s) conjugated to phycoerythrin (PE); mAbs to PE-TNF-α (MP6-XT22) and PE-IFN-γ (XMG1.2) were purchased from BD PharMingen (San Jose, CA). Immunoglobulins with isotypes corresponding to the above mAbs and conjugated to the appropriate fluorochromes, were used as control for nonspecific binding.

The backbone for the construction of DNA vaccines was the mammalian expression vector pcDNA3.1 (Invitrogen). In this vector encoding vaccine proteins are expressed under the control of the CMV promoter as an in-frame fusion with an antibody kappa chain signal peptide (SP) sequence (amino acid MDFQVQIFSFLLISASVIISRG) for secretion and are followed by C-terminal His tag for detection. 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 [29]. Each VH fragment was bound to its VL partner by use of a spacer encoding a 15 amino-acid flexible linker (Gly₄Sert)₃, yielding scFv constructs scFv^NLDC-145. 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 using the following primers HER2-HindIII-s 5′-TTAAGCTTAG CACCCAAGTGTGCACCGGCAC-3′, HER2-XbaI-as 5′-TTTCTAGACAAACAGTGCCTGGCATT CACATAC-3′ and neu-HindIII-s 5′-TTAAGCTTGGAGCCGCGGGTACCCAAGTGTG-3′, neu-XbaI -as 5′-TTTCTAGATCCAAAGCAGGTCTCTGAGCTGTTTTGAG-3′.The resultant encoding sequences were then cloned in-frame downstream of the scFv^NLDC-145. For in vivo targeting assay, we generated pcDNA3.1-scFv^NLDC-145-EGFP by replacing the HER2 fragment with EGFP sequence cloned from pEGFP-N1 plasmid. The pcDNA3.1 vector encoding EGFP without DC-targeting scFv fragments as controls (pcDNA3.1 and pcDNA3.1-EGFP).

The different pcDNA3.1 constructs were transiently transfected in 293T cells using Lipofectamine 2000 according to the manual instruction (Invitrogen). The resultant supernatants were harvested at 72 hours post-transfection and concentrated and dialyzed using centrifugal filter devices (Amicon Ultra, 10K, millipore). Protein expression was analyzed by Western blotting using recombinant anti-His mAb (ab1187, Abcam).

The 50 μg pcDNA3.1-scFv^NLDC-145-EGFP and pcDNA3.1-EGFP plasmids in 50 μL PBS were injected into the upper leg muscle of the left hind limb of the mice followed by in vivo electroporation as described previously [30]. One day later, the lysate of the muscle tissues of injection site were prepared using RIPA buffer and subjected to the western blotting for detection of EGFP and scFv^NLDC-145-EGFP fusion protein by anti-EGFP antibody (ab111258, Abcam) as described above. The spleens were harvested from the injected mice at different time points (48, 60, 72 hours) and single-cell suspensions were prepared and stained with PE-conjugated anti-CD11c antibody or isotype control for 30 min. The GFP fluorescence in the CD11c-positive DC was analyzed by flow cytometry as described previously [14].

For prophylactic vaccination, female BALB/c mice or BALB-neuT mice were vaccinated on days -21 and -7 by intramuscular injections of 50 μg pcDNA3.1-scFv^NLDC-145-HER2/neu, pcDNA3.1-HER2/neu plasmid DNA in 50 μL PBS as described above. As control, 50 μL pcDNA3.1 or 50 μL PBS were injected. On day 0, animals were inoculated subcutaneously (s.c.) with 2 × 10⁵ D2F2/E2, D2F2 or TUBO tumor cells in the opposite flank. Tumor growth was monitored with a caliper by measuring two perpendicular tumor diameters every week, and tumor volumes were calculated according to the formula: length × (width)² × 0.5. For therapeutic vaccination, when the tumors were 3–4 mm in diameter (day 7), mice were injected intraperitoneally (i.p.) with cyclophosphamide (100 mg/kg) in 100 μL PBS. Four days later (day 11), animals were vaccinated as described above. Vaccination was repeated once 14 days later, and tumor growth was followed. If animals appeared moribund or the diameter of the tumors reached 15 mm, the mice were sacrificed and this was recorded as the date of death for survival studies. For rechallenging experiments, the long-term surviving mice were injected s.c. either with 2 ×10⁵ D2F2/E2, D2F2, or 4T1 tumor cells.

Preventive effects of the DNA vaccines were investigated in virgin female BALB-neuT mice that endogenously express neu in their mammary glands and develop neu-driven mammary carcinomas [28]. Animals were immunized twice at 8 and 10 week ages as described above. Mammary glands were inspected every week to monitor the appearance of tumors. Measurable/palpable masses >2 mm in diameter were regarded as tumors. Data are reported as tumor multiplicity (cumulative number of tumors per number of mice in each group) and shown as mean ±SE.

For detection of regulatory T cells (Treg), splenocytes from immunized mice were surface stained with FITC anti-mouse CD4 (GK1.5; eBioscience). After that, cells were washed with wash buffer (PBS with 1% fetal bovine serum and 0.09% sodium azide), fixed and permeabilized with the Cytofix/Cytoperm reagent (BD Bioscience) for 20 minutes at 4°C, after which they were washed in Perm/Wash buffer (BD Bioscience), and stained with PE anti-mouse Foxp3 (FJK-16 s; eBioscience) at 4°C for 30 minutes. Immunoglobulin G-PE and immunoglobulin G-FITC (mouse) were used as negative controls. All analysis was performed on the FACSCalibur (Becton Dickinson) flow cytometer.

For detection of HER2-specific cellular immune response, splenocytes from vaccinated mice were cultured in 96-well flat-bottomed plates in 100 μL of growth medium in the presence of 10 μg/mL recombinant HER2 or TRP2 protein in vitro. After 80 h, [³H] thymidine (1 μCi/well; Amersham) was added for the remaining 16 h of the assay. [³H] thymidine incorporation was analyzed by liquid scintillation counting as described previously [14]. The supernatants were also collected and assayed for production of IFN-γ, TNF-α, IL-4, IL-10 by ELISA kits (R&D Systems).

For detection of IFN-γ and TNF-α-producing CD4 or CD8 T cells, intracellular cytokine staining assays were performed. Briefly, splenocytes harvested from vaccinated mice were cultured in the presence of 10 μg/mL recombinant HER2 or TRP2 protein for 24 h. During the final 4 h of incubation, 10 μg/mL brefeldin A were added. After surface staining with FITC-CD4 and FITC-CD8, cells were permeabilized and stained with PE-IFN-γ and PE-TNF-α prior to analysis by flow cytometry as described above. For CTL measurements, ⁵¹Cr-release assays were performed as described previously [14].

Peripheral blood was collected from the tail vein of the mice, and 1:100 dilutions of sera were analyzed by ELISA with recombinant HER2 protein or by flow cytometry using D2F2/E2 and TUBO tumor cells as described previously [14]. Normal mouse serum served as negative control.

Differences among tumor growth kinetics, tumor multiplicity, and specific cytotoxicity were evaluated by ANOVA or the Student’s test. Values of P < 0.05 were considered significant. For survival studies, Kaplan-Meier survival curves were plotted and analyzed using Prism 5.00 software (GraphPad Software).

We obtained the genes encoding scFv^NLDC-145 by whole gene synthesis according to the published sequences [29]. The carboxyl terminus of the scFv^NLDC-145 was directly fused in-frame to the sequences encoding the extracellular domain of HER2 (amino acids 22–561) or neu (amino acids 22–582) amplified from SK-BR-3 or TUBO breast cell lines, followed by His tag (Figure 1A). To confirm the expression of these constructs, 293T cells were transiently transfected with these plasmids, and then supernatants were harvested 72 hours later and tested for protein secretion by Western blotting. As shown in Figure 1B, we detected the production of scFv^NLDC-145-HER2 and scFv^NLDC-145-neu proteins (lane 1, 2) or HER2 and neu fragments (lane 3, 4) in the supernatants by anti-His tag antibody respectively, and their molecular weights were slightly larger than predicted, indicating certain extent of glycosylation.

To test whether scFv^NLDC-145 moiety delivery encoded antigens into DC in vivo, we constructed pcDNA3.1-scFv^NLDC-145-EGFP and pcDNA3.1-EGFP plasmids (Additional file 1: Figure S1), which facilitated us to trace the final fate of antigens carried by scFv^NLDC-145. The animals were injected i.m. with scFv^NLDC-145-EGFP plasmids, sacrificed at different time points post-injection, and splenocytes were harvested and probed for GFP fluorescence by flow cytometry. We first confirmed the comparative expression of EGFP and scFv^NLDC-145-EGFP fusion protein with correct molecular weight in the muscle tissue of injection site by western blotting (Figure 1C, right). As shown in Figure 1D, approximate 50% CD11c-positive DC in the spleen from pcDNA3.1-scFv^NLDC-145-EGFP-treated animals showed GFP fluorescence at 48 h post-injection, and this percentage further went up, approaching about 65% at 60 h post-injection. Antigen loading was DC-specific since CD11c-negative non-DC cells showed few, if any, GFP fluorescence. A representative dot plot obtained at 60 h post-injection was shown in Figure 1C, left. In contrast, after injection of pcDNA3.1-EGFP plasmid, some fluorescence slightly above background levels were detectable, resulting in only 10% of DC being loaded (background level was 5%). The much lower percentage of GFP-positive DC in pcDNA3.1-EGFP-treated mice was not due to lower GFP expression efficiency of pcDNA3.1-EGFP plasmid since total GFP-positive cells in splenocytes were almost same from mice receiving injection of these two constructs.

To investigate whether immunization with DC-targeted vaccines induce antitumoral immunity and protect animals from subsequent tumor challenge, BALB/c mice were i.m. vaccinated twice at two week interval with various vaccines. Seven days after last immunization, the mice were subcutaneously challenged with HER2-positive D2F2/E2 tumor cells, and tumor development was monitored. As shown in Figure 2A, 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). Furthermore, animals treated with scFv^NLDC-145-neu exhibited slower tumor growth compared with those treated with untargeted HER2 or neu DNA vaccine or control pcDNA3.1, resulting in prolonged survival of these mice, which indicated the induction of cross-reactivity toward HER2 antigen by scFv^NLDC-145-neu vaccination. To examine whether HER2-specific responses induced by scFv^NLDC-145-HER2 vaccines were responsible for protection, a similar experiment was done using parental HER2-negative D2F2 tumor cells. Rapid tumor growth was observed in all animals regardless of either treatment (Figure 2B), suggesting that antitumor activity against HER2-expressing D2F2/E2 tumor cells was due to HER2-specific immune responses induced by scFv^NLDC-145-HER2. We also obtained a similar protective effect of scFv^NLDC-145-HER2 in C57BL/6 mice using paired EL/4 and EL4/E2 tumor models (data not shown).

To test whether immunologic memory was developed, long-term surviving mice initially vaccinated with scFv^NLDC-145-HER2 were rechallenged with D2F2/E2 tumor cells. The parental D2F2 cells or unrelated syngeneic 4 T1 cells were used as controls. As shown in Figure 2C, the mice rejected subsequent rechallenges with the D2F2/E2 tumor cells and remained tumor-free, however, the mice could not reject unrelated syngeneic 4 T1 tumor. Interestingly, mice were also resistant to the rechallenge with the parental D2F2 tumor cells with 70% of mice tumor-free and remaining mice displaying drastically slow tumor growth. In summary, these results indicate that scFv^NLDC-145-HER2 vaccination induced long-lasting HER2-specific antitumor immunity, which could protect mice from HER2-expressing tumor challenge.

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. As shown in Figure 3A, left, splenocytes obtained from scFv^NLDC-145-HER2-vaccinated mice showed vigorous proliferation upon stimulation 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-α and IFN-γ (Figure 3A, right); similarly, a mildly higher level of IFN-γ and TNF-α cytokine was also detected in the supernatant from scFv^NLDC-145-neu-vaccinated mice. We did not detect the secretion of IL-4 and IL-10 cytokines with immunosuppressive activity in all groups.

Next, we evaluated for the induction of HER2-specific CD4⁺ and CD8⁺ T cells. As shown in Figure 3C, a much higher percentage of CD4⁺ and CD8⁺ T cells producing IFN-γ and TNF-α was detected upon in vitro restimulation with recombinant HER2 protein in splenocytes from scFv^NLDC-145-HER2-vaccinated mice. These T cells were HER2-specific since no cells produced these two cytokines upon restimulation with recombinant TRP2 pretein. A representative dot plot was shown in Figure 3B. In addition, splenocytes from scFv^NLDC-145-HER2-vaccinated mice exhibited significantly higher target cell killing than did those from other group mice (Figure 3D, left). The specificity of the killing was validated by the inability of the splenocytes to kill parental D2F2 target cells and EL4/E2 target cells with a different H-2^b background (Figure 3D, right). The cytotoxic effect was mediated by CD8⁺ T cells, because the killing was inhibited by addition of anti-CD8, but not anti-CD4, antibody (data not shown).

We also evaluated the induction of HER2-specific antibody. As shown in Figure 4A, vaccination with scFv^NLDC-145-HER2 induced a significantly highly level of HER2-specific antibodies specifically binding to recombinant HER2 protein in ELISA assays. Detailed analysis of antibody isotype demonstrated that antibodies induced by scFv^NLDC-145-HER2 vaccine was mainly IgG2a, which was consistent with cytokine profile of splenocytes from scFv^NLDC-145-HER2-vaccinated mice (Figure 4A).

We also tested whether the sera of vaccinated mice contained antibodies binding to “natural” D2F2/E2 cells. As shown in Figure 4B, we detected D2F2/E2-specific antibodies in sera derived from mice vaccinated with scFv^NLDC-145-HER2, whereas no antibodies were found in other groups. In summary, the results show that scFv^NLDC-145-HER2-vaccinated mice developed antibodies that recognize epitopes expressed by D2F2/E2 cells and thus may also confer protection against tumor growth in vivo.

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. As shown in Figure 5A, scFv^NLDC-145-HER2 vaccination substantially slowed tumor development and protected up to 20% (2/10) of the mice from tumor growth at the end of experiment (120 days after tumor inoculation).

Since regulatory T cells (Treg) have been shown to mediate immune-tolerance towards tumor-antigens in various tumor models, we further tested whether systemic depletion of regulatory T cells would increase the therapeutic efficacy of DC-targeting vaccine. An approach with intraperitoneal injection of low-dose (100 mg/kg) cyclophosphamide (CTX) that was successfully applied in various models was utilized this study. As shown in Figure 5B, low-dose CTX injection in the D2F2/E2-bearing mice efficiently depleted Treg almost to the level in tumor-free mice 4 days post-injection, however, no direct killing effect on tumor cells were observed (data not shown). We therefore tested DC-targeted vaccines in combination with Treg depletion by low-dose CTX. As shown in Figure 5C, this combination significantly improved the therapeutic effects of scFv^NLDC-145-HER2 vaccine; at the end of experiment, 80% (16/20) mice rejected the established tumor and remaining 4 (20%) mice had stably small tumors (~30 mm³). These tumor-free mice also rejected the rechallenges with the same tumor cells (date not shown). Untargeted DC vaccines failed to exert therapeutic effects although this vaccine in combination with CTX mildly delayed tumor growth. The experiment was repeated with similar results (date not shown). The data indicate that DC-targeting vaccines are able to mount an impressively therapeutic antitumor effect when in combination with systemic Treg depletion.

Tumor models based on human HER2-expressing D2F2/E2 cells are useful to assess the basic functionality of cancer vaccines, however, such models do not fully reflect the situation of human cancer usually characterized by immunologic tolerance toward HER2. Therefore, we further tested DC-targeted DNA vaccines in female BALB-neuT mice that represent an immunotolerant model of spontaneous cancer [28]. In these experiments, we used scFv^NLDC-145-neu instead of scFv^NLDC-145-HER2 since the spontaneous tumors developed in BALB-neuT mice are driven by constitutively activated rat neu protein.

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. As shown in Figure 6A, the animals receiving scFv^NLDC-145-neu vaccination were significantly protected against a subsequent challenge with TUBO cells. Sixty days after tumor challenge, 90% (9/10) mice in this group remained tumor free and 1 mouse had small tumors (~35 mm³). The experiment was repeated with similar results (data not shown). Mechanistic investigation showed that scFv^NLDC-145-neu induced high levels of neu-specific CTLs and antibodies (Figure 6B,C).

The effect of scFv^NLDC-145-neu vaccination in the prevention of spontaneous mammary tumors that naturally arise in BALB-neuT mice was also evaluated. 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 [31] and repeated at week 10. Mice in one group also received CTX injection 4 days before the first vaccination. As shown in Figure 6D, 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.