Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax®
© Mansour et al; licensee BioMed Central Ltd. 2007
Received: 18 February 2007
Accepted: 23 April 2007
Published: 23 April 2007
Melanoma tumors are known to express antigens that usually induce weak immune responses of short duration. Expression of both tumor-associated antigens p53 and TRP2 by melanoma cells raises the possibility of simultaneously targeting more than one antigen in a therapeutic vaccine. In this report, we show that VacciMax® (VM), a novel liposome-based vaccine delivery platform, can increase the immunogenicity of melanoma associated antigens, resulting in tumor elimination.
C57BL/6 mice bearing B16-F10 melanoma tumors were vaccinated subcutaneously 6 days post tumor implantation with a mixture of synthetic peptides (modified p53: 232–240, TRP-2: 181–188 and PADRE) and CpG. Tumor growth was monitored and antigen-specific splenocyte responses were assayed by ELISPOT.
Vaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-γ producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-γ producing splenocytes to one CTL epitopes (TRP2:180–188), suggesting that VM overcomes antigen dominance and enhances immunogenicity of multiple epitopes. Vaccination of mice bearing 6-day old B16-F10 tumors with both TRP2 and p53-peptides formulated in VM successfully eradicated tumors in all mice. A control vaccine which contained all ingredients except liposomes resulted in eradication of tumors in no more than 20% of mice.
A single administration of VM is capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors.
Previous studies using two mouse models have demonstrated that peptide-based vaccines delivered using VacciMax® (VM) can eradicate human papillomavirus (HPV) 16 induced tumors [1, 2]. The aim of the present study is to use VM in a therapeutic vaccine to treat C57BL/6 mice bearing B16-F10 melanoma tumors. Eradication of both HPV 16 and B16-F10 melanoma tumors would suggest that VM could enhance various therapeutic cancer vaccines. The identification of tumor-associated antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL) has led to the development of specific anti-tumor immunotherapies. Vaccine approaches for the treatment of cancer rely on the expansion and differentiation of tumor-specific CTL's, with the objective of rejecting established tumor. Peptide vaccines elicit a poor immune response and often require the use of potent adjuvants. Previous studies in our laboratory have shown that formulation of peptides in a vaccine enhancing platform, VM, can greatly enhance peptide-specific immune responses. VM is a liposome-based antigen delivery platform which we have shown to be effective in both preventing and eradicating established HPV 16-induced tumors in mouse models [1, 2]. The strong and specific CTL responses generated by VM against a variety of peptide antigens suggest that VM can be used to develop therapeutic vaccines against a variety of cancers. This report examines the use of VM in a melanoma mouse model.
A number of melanoma-associated antigens (MAA) have been identified as potential targets but most induce weak immune responses of limited duration. MAAs, expressed by both normal and malignant melanocytes, are self antigens that are difficult to target in a vaccine. MAAs include melanoma-specific antigens such as MART-1, gp100 and TRP2 , as well as antigens such as mutated p53 found in a variety of cancers [4–11]. The melanoma tumor cell line B16-F10 used in this study expresses two MAAs of interest, namely TRP-2 and mutated p53.
In this study, VM containing TRP2 and p53 CTL epitopes was used as a therapeutic vaccine to treat C57BL/6 mice bearing B16-F10 melanoma tumors. TRP2 was selected because clinical tumor regression has been associated with TRP2-specific T cells [12–17]. p53, on the other hand, was selected because it is expressed in a variety of cancers including melanoma [5, 18–23], and the ability to induce effective immune responses against p53 in a melanoma model may have implications for the treatment of other cancers.
Mice and cell lines
Pathogen-free C57BL/6 female mice, 6–8 weeks of age, were obtained from Charles River Laboratories (Wilmington, MA) and were housed under filter top conditions with water and food ad libitum. The B16-F10 melanoma cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA). The melanoma cell line was cultured in Dulbecco's Modified Dulbecco's Medium (DMDM, Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (Hyclone), 2 mM L-glutamine (Gibco), 5 mM 2-mercaptoethanol (Gibco), penicillin and streptomycin (100 μg/ml;Gibco). Cells were incubated at 37°C/5% CO2.
Peptides and oligonucleotide (ODN)
The tyrosinase-related protein peptides TRP2: 180–188 (SVYDFFVWL) and TRP2: 181–188 (VYDFFVWL), as well as the modified p53 peptide (p53: 232–240; KYICNSSCM) fused to PADRE (AKXVAAWTLKAAA, where X is cyclohexylalanine) were purchased from Dalton Chemical Laboratories Inc. (Toronto, ON, Canada). These peptides are presented by murine MHC-class I H-2K. The TRP2 and p53 peptides were stored as a 1 mg/ml stock solution containing DMSO to maintain solubility. Further dilutions for vaccine production were made using PBS.
All formulations of the vaccines contained either PADRE (AKXVAAWTLKAAA-OH; 25 μg/dose) and CpG ODN 1826 (5'-TCCATGACGTT CCTGACGTT-3'; Coley Pharmaceutical, Wellesley, MA; 50 μg/dose). The amino acid sequence of the irrelevant peptide used in ELISPOT was EGSRNQDWL (Dalton Chemical Laboratories Inc.).
B16-F10 cells (104 cells/mouse) were implanted subcutaneously in the left flank of C57BL/6 mice which were 6–8 weeks of age at time of challenge. Tumor were measured with calipers every 2–5 days. Data was reported as a percentage of tumor-bearing mice.
Four to six days after implantation of melanoma cells, mice (5 mice/group) received a single subcutaneous (s.c.) injection of VM containing TRP2: 180–188, TRP2: 181–188, or modified p53: 232–240 (25 μg of peptide/mouse). In addition, mice were injected with vaccine containing mixtures of two peptides (25 μg of each peptide/mouse). The mixtures contained either TRP2:180–188 and modified p53 or TRP2 181–188 and modified p53. VM formulations contained PADRE (25 μg/mouse) and CpG (50 μg/mouse) as adjuvants. The peptides and adjuvants were encapsulated in VM as previously described . In brief, lecithin and cholesterol in a ratio of 10:1 (0.2 g lecithin and 0.02 g cholesterol/dose) were dissolved in chloroform/methanol (1:1;v/v) and the solution was filter-sterilized using a PTFE 0.2 μm filter. Chloroform and methanol were removed under reduced pressure using a rotary evaporator and traces of the solvents were removed from the resulting thin lipid layer in vacuo. For liposome encapsulation, peptides with CpG and PADRE were dissolved in sterile PBS and the resulting solution added to the thin lipid layer with mixing to form liposomes. The resulting suspension of liposomes was emulsified in Montanide ISA51 (Seppic, France) by adding the liposome/PBS suspension to ISA51 to form a water-in-oil emulsion (PBS:ISA51; 1:1, v/v; 100 μl/dose). Control mice were injected with either vaccines that contained all components of test vaccines except liposomes or phosphate buffered saline (PBS, 100 μL/injection).
Ex vivo analysis of antigen-specific T cells by ELISPOT
Activated antigen-specific splenocytes harvested from immunized C57BL/6 mice were detected using the BD ELISPOT kit following the manufacturer's instructions (BD Bioscience, San Diego, CA). Briefly, on day 8 post-immunization, a 96-well nitrocellulose plate was coated overnight at 4°C with capture antibody (anti-mouse IFN-γ) and then blocked with complete media. Splenocytes were added to wells at an initial concentration of 1 million cells/well in a volume of 100 μl. Cells in a dilution series were either non-stimulated or stimulated with relevant or irrelevant peptides (10 μg/ml).
PMA (5 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma), served as positive controls and irrelevant peptide and media alone served as negative controls. The plate was incubated overnight at 37°C/5% CO2. The plate was then incubated with detection antibody (a biotinylated anti-mouse IFN-γ antibody), for 2 hours at room temperature. Unbound detection antibody was removed by washing and the enzyme conjugate (Streptavidin-HRP) was added. Following 1 hour incubation at room temperature, unbound enzyme conjugate was removed by washing and the plate was stained with an AEC substrate solution for 20 minutes. The plate was washed, allowed to air dry overnight, and foci of staining were counted using a magnifying lens.
CD4+ T cell responses to PADRE
The CD4+ T-cell helper response to PADRE was measured by ELISPOT. Eight days post-immunization, splenocytes were recovered from immunized mice and stimulated with PADRE (10 μg/ml). Production of IFN-γ was measured as described above.
Data are expressed as the mean ± SD and statistical significance was determined using paired two-tailed Student t-test (p < 0.05).
Eradication of B16-F10 melanoma tumors
Immunization against TRP2:180–188 alone plus CpG in VM eradicated tumors in 40% of the mice by 16 days post-immunization. These mice remained tumor-free for the entire monitoring period. Immunization against TRP2:181–188 alone also suppressed tumors in 40% of mice by 16 days post-immunization, but tumors regenerated in these mice by day 30 post-immunization (data not shown).
Immunization of mice against modified p53:232–240 alone delayed tumor development in one mouse but by day 21 post-immunization all mice were bearing tumors. Twenty percent of mice became tumor-free in the interval between day 25 and 27 post-immunization. All control mice injected with PBS developed tumors by day 14 post-immunization.
Induction of cytotoxic T cells
CD4+ T cell responses to PADRE
There is an unmet need for new vaccines that are cost effective, safe and yet mount robust, effective and durable immune responses . Liposomes have been described as safe vaccine adjuvants but their use has been limited to aqueous carriers, such as saline, phosphate buffered saline or oil-in-water emulsions. In contrast, liposomes stabilized to remain intact in hydrophobic carriers or water-in-oil emulsions (VM) significantly enhance both humoral and CTL immune responses [1, 2, 39]. To enhance immunity, the VM platform requires both liposomes and a hydrophobic carrier. This combination allows excellent antigen delivery and presentation to the immune system resulting in a durable immune response following a single vaccination. Most vaccines studied using the same mouse model as employed in our studies, are only useful as prophylactic vaccines. Clearance of established tumors by immunization against peptide antigens using VM have also been demonstrated in two independent HPV-cervical cancer models [1, 2]. This strategy is particularly effective when treating virally induced cancers. Self-antigens are tightly guarded by tolerance mechanisms and consequently, tumors presenting "self" antigens are more difficult to treat. Therefore, an effective therapeutic cancer vaccine platform with widespread applications must have the ability to induce immune responses against tumor-associated self antigens. Consequently, a therapeutic vaccine against melanoma must activate and expand T cell clonotypes that have escaped thymus clonal deletion but nevertheless are capable of targeting self epitopes on the surface of tumors.
The B16-F10 melanoma tumor model in C57BL/6 mice has been used in many pre-clinical melanoma studies [3, 24–34]. Consequently, this model was chosen to test VM-based melanoma vaccines that employ "self" antigens such as TRP2 and p53. Although p53 is rarely used as antigen for melanoma immunotherapy, an increased content of p53 in melanoma cell lines and induction of p53-specific CTL responses have been reported by others [4, 5, 10, 20, 23]. Cell-mediated immune responses to TRP epitopes correlated positively with tumor regression [12, 16, 40, 41]. Immune responses against these antigens are difficult to achieve because of immune tolerance mechanisms . Various approaches have been adopted for the generation of CTL responses against B16, including the use of antigen-pulsed DCs or transfection of B16 cells with the gene for GM-CSF [40, 43, 44]. Recently, Jerome and colleagues demonstrated high levels of CTL responses upon vaccination with TRP2 and CpG in liposome .
Surprisingly, preliminary studies employing VM detected an ex vivo CTL response against TRP2:180–188 in the absence of adjuvants (Fig. 2). Addition of CpG adjuvant however significantly increased the number of TRP2:180–188-specific IFN-γ producing splenocytes. To increase the number of IFN-γ producing splenocytes that were specific for modified p53:232–240, the presence of both PADRE and CpG were required. Since studies aimed at developing a therapeutic HPV vaccine demonstrated that multiple peptide antigens result in a better outcome than a single peptide antigen , a mixture of TRP2:180–188 and modified p53:232–240 in VM was evaluated to determine if T cell activation would be directed against one or both peptides. The TRP2:180–188-, modified p53:232–240- and PADRE-specific IFN-γ producing splenocytes were expanded in mice immunized with the mixture of three epitopes in VM (Figs 4 and 5). Without VM, only the number of TRP2:180–188-specific IFN-γ producing splenocytes was above background suggesting immuno-dominancy favoring TRP2-specific responses. Although a T helper response was mounted when all three epitopes plus CpG were used without VM, such helper response only supported CTL responses to one of the peptides (TRP2:180–188) and resulted in tumor suppression until day 25–30 in 20–40% of treated mice.
As a realistic model for melanoma immunotherapy, we used peptide antigens derived from the naturally expressed tumor-associated antigens TRP2 and p53 in the B16-F10 melanoma model. Poor immunogenicity of peptides and the fact that B16-F10 cells express very low amounts of MHC class 1 molecules make the B16-F10 model a challenging model for T-cell based immunotherapy . Immunization with liposome-encapsulated TRP2 peptide which were then mixed with CpG ODN caused complete tumor rejection in only 20% of vaccinated mice ; VM, on the other hand, caused tumor rejection in all treated mice when two antigens were targeted. The ability to co-deliver antigens and CpG adjuvant by encapsulation in liposomes is a significant advantage for the VM platform; co-encapsulation of antigen and adjuvant within the same liposome is superior to encapsulation of each in separate liposomes . Our study demonstrates that co-encapsulation of adjuvant with peptide antigens derived from two tumor-associated antigens in VM is superior to use of a single peptide antigen. For example, TRP2:180–188 and modified p53:232–240 encapsulated in VM with PADRE and CpG ODN adjuvants caused complete tumor eradication in 100% of vaccinated mice. The same treatment with only one CTL epitope (TRP2:180–188) caused complete tumor suppression for at least 20 days in 60% of vaccinated mice. VM-based vaccines containing more than two peptide antigens may improve the outcome of therapeutic melanoma vaccines as a result of VM's ability to overcome immuno-dominancy.
In summary, the VM vaccine platform simultaneously induces a robust CTL response to more than one peptide antigen contained therein. Immunization against two CTL epitopes in VM formulated with a T helper peptide and CpG adjuvant eradicated tumors in all treated mice. Although regeneration of melanoma tumors occurred in a significant number of mice that had been rendered tumor-free by vaccination, the strong CTL responses to multiple peptides from self-antigens by a single immunization and the rejection of aggressive 6-day old B16-F10 tumors to our knowledge have never been reported. Targeting more than 2 tumor-associated antigens in VM may improve the efficacy of the vaccine and reduce melanoma tumor regeneration.
VacciMax®, a liposome/Water-in-oil based vaccine delivery system, enhances the immunogenicity of melanoma-associated CTL epitopes simultaneously. A single administration of such vaccine elevated specific CTL responses and resulted in the rejection of 6-day old B16-F10 tumors.
cytotoxic T lymphocyte
enzyme-linked immunosorbent spot
pan DR epitope
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