Therapy of established B16-F10 melanoma tumors by a single vaccination of CTL/T helper peptides in VacciMax

Abstract

Background: 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.

Methods: 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.

Results: Vaccine formulated in VM increased the number of both TRP2- and p53-specific IFN-gamma producing splenocytes following a single vaccination. Vaccine formulated without VM resulted only in enhanced IFN-gamma 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.

Conclusion: 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.

Figure 1

All mice that received PBS (crosses) developed tumors by day 14 post-immunization. A single immunization against a mixture of TRP2:181–188 and modified p53:232–240 in VM (triangles) rendered all mice tumor-free by 21 days post-immunization. A single administration of TRP2:181–188 alone alone (diamonds) in VM rendered 2/5 mice tumor-free by day 16 post-immunization. A single administration of modified p53:232–240 in VM (squares) delayed tumor development in one mouse, but by day 21 post-immunization, all mice in this group were tumor bearing. In the interval between 25 and 27 days post-immunization, two mice in this group became tumor-free.

Figure 2

Ex-vivo detection of modified TRP2:180–188-specific IFN-γ producing splenocytes (spot forming cells, SFC) in mice 8 days following a single immunization against TRP2:180–188 in VM. Mice immunized against TRP2:180–188 in VM with CpG adjuvant produced the largest number of cytotoxic T cells. Omission of CpG from the vaccine produced approximately one-half as many SFC. TRP2:180–188 alone and an irrelevant peptide with CpG in VM produced background numbers of SFC.

Figure 3

Ex-vivo detection of modified p53:232–240-specific IFN-γ producing splenocytes (spot forming cells, SFC) in mice 8 days following a single immunization against modified p53:232–240 in VM. Mice immunized against modified p53:232–240 peptide in VM with CpG and PADRE adjuvants produced the largest number of cytotoxic T cells. Without VM or omission of CpG from the VM formulation produced low numbers of modified p53:232–240-specific IFN-γ producing splenocytes. Vaccination against an irrelevant peptide (IrrPep) also produced low numbers of IFN-γ producing splenocytes.

Figure 4

Spleens from mice immunized against a mixture of TRP2:180–188 and modified p53:232–240 in VM contained high numbers of TRP2:180–188-specific (right bar) and modified p53:232–240-specific (left bar) IFN-γ producing cells. In contrast, spleens from mice immunized against a mixture of TRP2:180–188 and modified p53:232–240 without VM contained high numbers of TRP2:180–188-specific IFN-γ producing cells but low numbers of modified p53:232–240-specific IFN-γ producing cells. The spleens of mice immunized with VM-formulated vaccine without CpG ODN had low numbers of both TRP2:180–188- and modified p53:232–240-specific IFN-γ producing cells as did the spleens of mice from mice immunized without VM.

Figure 5

Ex-vivo detection of PADRE-specific IFN-γ producing splenocytes (spot forming cells, SFC) in mice (N = 5) 8 days following a single immunization against a mixture of TRP2:180–188 and modified p53:232–240 with and without VM.