Therapeutic anticancer neoepitope vaccine
A new epitope, therapeutic technology, applied in the field of producing the vaccine, can solve the problem of difficult to control protein immune response and the like
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Embodiment 1
[0340] Example 1: Construction and expression of vaccines
[0341] Design the gene sequence according to the following structure:
[0342]
[0343] Exome sequencing and RNA-sequencing of the previously described mouse melanoma cancer cell line B16-F10 and mouse colon cancer cell line CT26 revealed hundreds to thousands of tumor-specific nonsynonymous mutations (Castle et al. 2012, Castle et al. 2014 and Kreiter et al. 2015). An in silico based approach was used to identify potentially immunogenic neo-epitopes. Mice were immunized with peptides encoding mutated epitopes, and their immunogenicity was observed as a specific T cell immune response (Elispot assay). Furthermore, vaccination of mice with the most immunogenic epitopes selected from Elispot conferred strong antitumor activity (Castle et al. 2012 and Kreiter et al. 2015).
[0344] Each neo-epitope is a peptide comprising 27 amino acids separated by flexible GGGGS linkers. Short peptides (20 amino acids) are selec...
Embodiment 2
[0352] Example 2: Comparison of vaccibodies comprising 3 or 10 neo-epitopes
[0353] Comparison of vaccibody vaccines containing 3 or 10 neoepitopes. In the 10 neoepitope vaccibody DNA construct, the position and order of the first 3 (N-terminal) peptides were similar to those in the 3 neoepitope vaccibody DNA construct. This was done in order to be able to compare the immunogenicity of the 3 neo-epitopes in the case with 3 epitopes and in the case with 7 more epitopes.
[0354] VB4001(VB10.NEO CT26-X), VB4002(VB10.NEO CT26-III), VB4003(VB10.NEOB16-X) and VB4004(VB10.NEO B16-III) were selected as vaccine candidates. A schematic diagram of the vaccine body is shown in figure 1 middle.
[0355] The novel epitopes for vaccines VB4001-VB4021 are shown below. For example, VB4015 contains three neoepitopes, B16pepM1+pepM8+pepM3, which are separated by a 5 amino acid linker. VB4018 contains 2 copies of the following 10 neoepitopes, B16 pepM1+pepM2+pepM3+pepM4+pepM11+pepM6+pepM7+...
Embodiment 3
[0374] Example 3: Comparison of Immunogenicity of VaccineBody DNA Vaccines and Corresponding Peptide Plus Adjuvanted Vaccines
[0375] Before the VB10.NEO construct was used in mouse vaccination studies, as previously reported in figure 2 As described in detail in the text, vaccibody protein expression and secretion in HEK293 cells was verified using a sandwich ELISA assay. The order of the neo-epitopes can have an effect on the expression and secretion of a functional vaccibody. exist Figure 5 In the upper panel of , slightly improved expression and secretion of functional vaccibody protein was observed for VB10.NEO B16-X construct VB4014 compared to VB10.NEO B16-X construct VB4003. The 10 neoepitopes in VB4014 were similar compared to VB4003, however, the order of the neoepitopes was changed and the most hydrophobic neoepitopes were located in the core of the neoepitope antigenic module. To test the immunogenicity of Vaccinator DNA vaccines VB4003 and VB4014 compared to...
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