Melanoma Cell Lysate Plus C-type CpG ODN Induced Anti-tumor Immunity

Theoretically, tumor cell lysate (TCL) prepared from whole tumor cells either autologous or allogenic could be developed into tumor vaccines capable of activating anti-tumor immune response against multiple tumor antigens. However, when used alone, TCL failed to induce immunity efficient to eradicate tumor, showing poor immunogenicity. To circumvent the disadvantage, a variety of approaches were tested. Initially, whole bacterial cell or bacterial components such as BCG or monophosphoryl lipid A were shown enhancing the immunogenicity of TCL in mice as well as clinical trials. With the development of molecular cloning, recombinant cytokines such as IL-2 and GM-CSF were evaluated as adjuvants of TCL. Systemic administration of recombinant IL-12 augmented the efficacy of whole TCL, inducing protective immunity against tumor challenge in mice. Autologous TCL plus recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) induced tumor regression in patients with metastatic melanoma. Upon the recognition of heat shock protein mediated cross presentation of exogenous antigens, chaperone-rich cell lysates (CRCLs) were prepared from various tumor cells and tested. Vaccination with CRCL resulted in a significant reduction in tumor size and longer survival in mice with fibrosarcoma. Peptide-embedded CRCL generated potent immunity against pre-established murine leukemia. Administration of CRCL triggered anti-HER2/neu antibody production and delayed the progression of established breast cancer in mice. Moreover, the unique ability of dendritic cells (DC) to pick up antigens and to activate naive and memory CD4~+ and CD8~+ T cells spawned the studies on developing TCL loaded DC tumor vaccines. In mouse model, the tumor cell lysate loaded dendritic cells appeared to be effective in generating potent T cell-mediated immune responses against T cell lymphoma, glioma, mesothelioma, acute myeloid leukemia, melanoma and fibrosarcomas. In clinical trials, the autologous TCL loaded DCs achieved immunologic and objective clinical responses in patients with cutaneous T-cell lymphoma, melonoma, colorectal cancer, renal carcinoma, malignant glioma and hepatocellular carcinoma. Alternatively, vaccination with GL26 glioma cell lysate pulsed DC engineered to express IL-12 induced anti-tumor immunity in both the subcutaneous and intracranial tumor models. Vaccination with IL-18 gene modified DCs pulsed by TCL induced effective anti-tumor immunity to pancreatic carcinoma in mice respectively. Interestingly, CRCL-activated DC resisted Treg suppression and might endow DC with the capacity to overcome tumor-induced Treg-inhibitory effects. Immunization with DCs loaded with leukemia-derived CRCL prolonged the survival of mice challenged with leukemia cells. Taken together, the tumor lysate based vaccines could generate enhanced anti-tumor immunity in animals as well as a subset of patients. However, so far, no clinical benefit of using the vaccines has been demonstrated in a properly controlled phase III study, calling for more efficient CTL based tumor vaccines. In recent years, unmethylated CpG containing oligodeoxynucleotides (CpG ODNs) have been demonstrated highly effective as adjuvants through TLR9 activating, thereby enhancing a predominant Th1 response, NK cell activation and CTL generation, which are hypothetically desired for improving the efficacy of TCL. Based on sequence as well as functions, CpG ODNs are grouped into three types. Type A CpGODN potently induces IFN-αand thus indirectly activates NK cells. Type B CpGODN preferentially activates B cells but moderately induces IFN-α. Type C CpGODN combines features of both type A and type B. Previously, type B CpG ODN was tested as adjuvant to develop TCL vaccine, showing that TCL plus CpG ODN was immunotherapeutic in mice inoculated with human papillomavirus immortalized tumor cells and SC administering CpG ODN mixed with tumor lysate could evoke an effective T-cell-mediated response against glioma.In the present study, using a poorly immunogenic melanoma B16, we show that prophylactic vaccination with whole tumor cell lysate plus a C class CpG ODN we developed could significantly inhibit tumor growth and prolong the survival of tumor bearing mice. The anti-tumor effect displayed is likely to be through the generation of tumor specific CTL and memory cells. The contents of this paper are focused on the following parts:1. The activation of human immune cells by BW008To develop an adjuvant to tumor vaccines, we have extensively screened a series of CpG ODNs designed by us for their immunostimulatory properties. The first round screening was conducted by conventional lymphoproliferative assays of human PBMC, using 2216, an A-type CpG ODN, 2006, a B-type CpG ODN and C274, a C-type CpG ODN as controls. Through the screening, a candidate CpG ODN designated as BW008 was found and studied in detail. As shown in Fig 2A, BW008 induced significant proliferation of human PBMC (p<0.05, versus PBS). The proliferation magnitude induced was similar to those induced by 2006 and C274 (p>0.05) but much higher than that induced by 2216 (p<0.05) (Fig 2A). To test type I interferon inducing potential of BW008, human PBMCs were incubated with BW008 ODN. After 3 days, type I IFN production was measured in the supernatant by vesicular stomatitis virus (VSV) bioassay. As shown in Fig 2B, BW008 induced significant level of type I IFN compared with medium control (p<0.05). The activity was weaker than that induced by 2216 (p>0.05), superior to that induced by 2006 (p>0.05) and similar to that induced by CpG-C274 (p>0.05). To assess the effect of BW008 on antigen presentation, we further tested whether BW008 could up-regulate co-stimulatory molecules and MHC class I. Human PBMCs were incubated with BW008 or 2216 or 2006 or C274 for 48-hour and then were examined for CD80, CD86, HLA-A2 expression by FACS analysis. The result (Fig 2C) showed that stimulation by BW008 resulted in an enhanced up-regulation of CD80 and CD86 as compared with medium control (p<0.05). Comparison of the effect of BW008, 2216, 2006 and C274 did not reveal a significant difference in CD80 and CD86 expression (p>0.05). Noticeably, HLA-A2 expression induced by BW008 was 1.6 times and 1.2 times superior, respectively, to those induced by 2216 and 2006, and close to C274. To identify the cells involved activation by BW008, human PBMCs were cultured with BW008 or 2216 or 2006 or C274, and then were double stained with FITC-anti-CD69 and PE-anti-CD19 or PE-anti-CD56. The results of a FACS analysis showed that BW008 could activate both B cells and NK cells. Overall, these results indicate that BW008 acts like a C type CpG ODN that could be an optimal candidate adjuvant for developing tumor vaccine of human use.2. The proliferation of mouse splenocytes stimulated with BW008To evaluate whether the BW008 defined for human was stimulatory in murine system, murine spleen cells were cultured with BW008 and assayed for its effect on proliferation of murine immune cells. Mouse splenocytes were cultured with BW008, 2006 and 1826 (a B-type CpG ODN for mouse) for 2 days, and [3H] TdR incorporation assay was used to evaluate the proliferation. BW008 could significantly stimulate mouse splenocytes proliferation. Its potent of stimulating the splenocytes proliferation was similar with 2006 and 1826 (p>0.05)(Fig 3). This result demonstrates that CpG 685 is less species specific, both human and mouse are sensitive for it. The adjuvant capacity of BW008 could be evaluated in mouse model.3. Tumor growth and survival of mice injected with B16 melanoma cell lysate plus BW008 prophylactively and therapeutically in s.c. tumor modelTo observe the prophylactic effect of tumor lysate plus BW008, C57BL/6 mice were injected subcutaneously (s.c.) at inguinal groove with B16 cell lysate lysate plus BW008 (BW008 enhanced B16 melanoma cell lysate, BEMCEL) for three times with a week interval. The control mice were injected with PBS or tumor lysate or BW008. Two days after the third immunization mice were inoculated with 2×10~5 B16 cells subcutaneously in the right hind leg. 8 days after tumor cell inoculation the mice showed the first signs of tumor growth. Tumor growth was monitored every 2 days using a caliper. As shown in Fig 11A, BEMCEL showed profound growth-inhibitory effects on B16 tumor growth compared with PBS (p=0.048) and B16 cell lysate (p=0.010). Notably, BW008 alone seemed to affect the tumor growth but the inhibition was not statistically significant. After tumor challenge, the mice were also monitored for survival. Mice received PBS became moribund between days 20 and 27. The survival of BEMCEL recipients was significantly increased compared with recipients of PBS (p=0.049) and B16 cell lysates (p=0.001) (Fig 11B). To observe the therapeutic effect of BEMCEL, C57BL/6 mice were subcutaneously (s.c.) inoculated with 2×10~5 B16 cells. On day 2 after the inoculation, the mice were injected subcutaneously (s.c.) at inguinal groove with BEMCEL for three times with a week interval. Mice received PBS or tumor lysate or BW008 were used as controls. The result showed that the therapeutic immunization with BEMCEL failed to induce the tumor inhibition and prolong the survival of the mice (Fig 12). These results reveal that BW008 is effective in facilitating the generation of anti-tumor effect induced by tumor lysate when used prophylactically, rather than therapeutically.4. Tumor growth and survival of mice injected with BEMCEL prophylactively and therapeutically in i.p. tumor modelTo further determine the efficacy of BEMCEL, mice were inoculated with B16 cells peritoneally (i.p) and treated prophylactically or therapeutically. In a prophylactic model, C57BL/6 mice were immunized with BEMCEL as described above. Mice received PBS or tumor lysate or BW008 were used as controls. On day 2 after the third immunization, the mice were inoculated i.p. with 7.5×10~4B16 cells. As shown in Fig 13A, all of mice received BEMCEL survived beyond 60 days (p=0.000 compared with the controls). Mice received PBS became moribund between days 14 and 27. There was no statistically significant difference among the survival of mice receiving tumor cell lysate, BW008, PBS (p>0.05). In a therapeutic model, C57BL/6 mice were inoculated i.p. with B16 cells on day 0. On day 2, 9 and 16, the mice were immunized with BEMCEL for three times. Mice received PBS, tumor lysate and BW008 were used as controls. The result showed that the survival of mice receiving BEMCEL was longer but not statistically significant from the survival of mice received PBS and tumor lysate (p>0.05). Conspicuously, life span of mice treated by BEMCEL was significantly longer than mice treated by BW008 alone (p=0.011) (Fig 14). These results are in agreement with those obtained in subcutaneous model, indicating that prophylactic not therapeutic administration with BEMCEL could induce the sufficient immunity against B16 cells. To check the abdominal cavity, an additional cohort of mice was sacrifice on day 16 after the tumor challenge in prophylactic model. Pathological examinations revealed complete disappearance of the tumor mass in their abdominal cavities of the mice received with BEMCEL. In contrast, tumor mass was found in the greater omentum, mesentery, diaphragm of mice received PBS (Fig 13B).5. The dose effect of BW008Next, dose effect of BW008 on immunogenicity of tumor lysate was observed in the prophylactic model. The three doses selected for testing were 5μg, 25μg and 50μg. The result showed that 5μg of BW008 in tumor lysate was sufficient to prolong the life span of mice inoculated i.p. with B16 cells (p<0.01). Although, the mice received tumor lysate plus 25μg or 50μg BW008 survived longer than those of 5μg group, there was no statistically significant difference between 5μg and 25μg group or 50μg group (p>0.05) (Fig 15). The result indicates that the efficient dose range of BW008 was wide, which may mean it will need more practice to find the ideal dose in a specific study.6. The immune activity of different fractions from B16 cell lysate with or without BW008Previous study showed that tumor cell lysate could be prepared as whole tumor lysate, tumor lysate supernatant and tumor lysate precipitate. Obviously, the immunogenicity of the three forms of tumor lysate may be various. To verify this, tumor lysate was separated into supernatant (sup) and precipitate (prec) by centrifugation, and then used in combination with BW008 to immunize mice in prophylactic model. Mice were injected with whole tumor lysate or sup or prec with or without BW008 (50μg) into inguinal groove subcutaneously for three times in a week interval. On day 2 after the third immunization, the mice were inoculated with 7.5×104 B16 cells intraperitonealy. The survival of mice in each group was observed. The result showed that life span of mice received whole tumor lysate or sup or prec with BW008 was significantly longer than that in PBS group (p<0.05). Comparatively, mice received whole tumor lysate plus BW008 and lysate sup plus BW008 live longer than mice received lysate sup or lysate prec (p<0.05). In addition, mice immunized with whole lysate plus BW008 or supernatant of lysate plus BW008 seemed to have longer life time than that immunized with lysate prec plus BW008 (Fig 16). This result may suggest that supernatant of tumor lysate contains more effective tumor antigens for inducing anti-tumor immunity.7. The induction of CTL by BEMCEL To assess the CTL-mediated immunological memory induced with BEMCEL, mice were injected with BEMCEL or tumor lysate or BW008 or PBS for three times at a week interval. After the third immunization, the spleen cells were isolated from the mice and then co-cultured with mitomycin C treated B16 tumor cells for 5 days. After removing B16 cells by Ficoll Hypaque centrifugation, the resultant cells were used as effector cells. As shown in Figure 21, significant killing of B16 cells by the spleen cells from the mice immunized with BEMCEL was observed (p<0.05, compared with each other group). When immunized with either tumor lysate, only weak killing of B16 cells was observed. In contrast, BW008 couldn't induce the generation of BW008 specific CTL. The results indicate that the BEMCEL induced specific CTL against B 16 cells, demonstrating that BW008 can facilitate tumor lysate to induce tumor antigen-specific CTL response.8. The immune memory induced by BEMCELBy using the prophylactic intraperitoneal inoculation model, we observed that B16 cell injected mice received tumor lysate or BW008 all developed tumors and died, whereas those received BEMCEL rejected the B16 cell showed survived. To investigate the in vivo consequence of lysate or BW008 immunization, the survived mice were tested for their resistance to the second challenge of B 16 cells. On day 60 after the first B16 cell challenge, the survived mice (n=6) were inoculated 7.5×10~4 B16 tumor cells/mouse intraperitoneally. B16 cell naive mice (n=6) were used as control. After the second challenge, the mice were observed for another 100 days. As shown in Fig 23, on the day 34 after the first tumor challenge, all of the 6 mice were succumbed. In contrast, on day 100 after the second tumor challenge, all of the 6 mice that survived from the first tumor challenge still survived without the sign of tumor development. To assess the Th-mediated immunological memory induced with BEMCEL, the survived mice from the first B16 cell challenge were observed for their delayed type hypersensitivity (DTH) to B16 cell lystate. On day 100 after B16 cell inoculation, the survived mice (n=3) were injected with tumor lysate (prepared from 1 x 106 tumor cell) at left hind foot pad. Naive mice injected with or without tumor lysate were used as negative controls. Thick of pad were measured by a slide caliper 48h after challenge. The result showed that BEMCEL induced B16 specific immunological memory (p <0.05) (Fig 24).To sum up, prophylactic administration with BEMCEL could inhibit the tumor growth and prolong the survival of mice in s.c. or i.p. tumor model; CTL cytotoxicity assay, immune memory response and DTH response demonstrated that CD4~+ T and CD8~+ T cell mediated the anti tumor effect by BEMCEL, and BEMCEL also induced the immune memory cells with the activity of preventing the mice from B16 rechallenge. This results indicated that C type CpG ODN has the potential to be powerful vaccine for tumor vaccine; The BW008 enhanced B16 melanoma cell lysate (BEMCEL) made from BW008 and tumor cell lysate cell lysate have the potential to be a new anti-tumor agent.