A microbial-based cancer vaccine for induction of EGFRvIII-specific CD8+ T cells and anti-tumor immunity

Abstract

Dysregulated signaling via the epidermal growth factor receptor (EGFR)-family is believed to contribute to the progression of a diverse array of cancers. The most common variant of EGFR is EGFRvIII, which results from a consistent and tumor-specific in-frame deletion of exons 2-7 of the EGFR gene. This deletion generates a novel glycine at the junction and leads to constitutive ligand-independent activity. This junction forms a novel shared tumor neo-antigen with demonstrated immunogenicity in both mice and humans. A 21-amino acid peptide spanning the junctional region was selected, and then one or five copies of this 21-AA neo-peptide were incorporated into live-attenuated Listeria monocytogenes-based vaccine vector. These vaccine candidates demonstrated efficient secretion of the recombinant protein and potent induction of EGFRvIII-specific CD8+ T cells, which prevented growth of an EGFRvIII-expressing squamous cell carcinoma. These data demonstrate the potency of a novel cancer-specific vaccine candidate that can elicit EGFRvIII-specific cellular immunity, for the purpose of targeting EGFRvIII positive cancers that are resistant to conventional therapies.

Fig 1. Construction and confirmation of Lm-EGFRvIII.

a) Deletion of a 267 amino acid section of EGFR results in formation of EGFRvIII where amino acids from the amino terminus (blue) separated from the remainder of the EGFR molecule (green) by a novel glycine (red) at the junction. The novel EGFRvIII_20-40 sequence incorporates a characterized human HLA A0201 binding neoepitope. b) One or 5 copies of EGFRvIII_20-40, together with a single copy of the ovalbumin epitope SIINFEKL, were cloned in frame with the amino terminus of ActA and under control of the actA promoter in the parental LADD Lm vector. c) DC2.4s were infected with Lm-EGFRvIII x1, Lm-EGFRvIII x5 or controls and western blotted with antibodies specific for the mature amino terminus of the ActA fusion partner.

Fig 2. In vivo vaccination with Lm-EGFRvIII results in antigen specific T cell responses.

C57BL/6 mice were treated with Lm-EGFRvIII x1 and Lm-EGFRvIII x5 and seven days later splenocytes were tested for their response to i) DMSO vehicle, or ii) SIINFEKL peptide by intracellular cytokine staining. iii) Percent of CD3+CD8+ T cells and iv) absolute number of SIINFEKL-specific splenocytes. Each symbol represents one animal. NS = not significant. (Student’s T-test).

Fig 3. Identification of a novel murine class I epitope from EGFRvIII.

a) BALB/c, C57BL/6, C3H and SJL mice were vaccinated with Lm-EGFRvIII and 7 days later tested by ICS for EGFRvIII-specific T cells using a library of overlapping peptides from EGFRvIII_20-40, or a DMSO vehicle control. Spleens from three mice per strain were used for restimulation. The percentage of IFN-γ+ events within the CD3+CD8+ T cell gate are shown for each peptide; b) Splenocytes from C3H mice primed and boosted with Lm-EGFRvIII were stimulated using a library of overlapping peptides from EGFRvIII_20-40, or SIINFEKL as a negative control. A subgroup of peptides within the previously identified 15-mer were used to define the optimal 9-mer peptide by ICS. c) H2K^k binding of EGFRvIII or control peptides was assessed by stabilization of surface MHC I using flow cytometry. d) Identification of EGFRvIII-specific T cells in the spleen following prime-boost vaccination with Lm-EGFRvIII using H2K^k-tetramers folded with the defined peptide EEKKGNYV.

Fig 4. Immunogenicity of EGFRvIII vaccine candidates.

a) EGFRvIII_26-33 -specific CD8+ T cell responses in C3H mice vaccinated with Lm-EGFRvIIIx1 or Lm-EGFRvIIIx5, determined by IFN-γ ICS on day 7. b) Primary immunogenicity of PEPvIII and Lm-EGFRvIIIx5 in C3H mice, represented as the frequency of K^k-EGFRvIII_26-33 -tetramer+ cells within the CD3+CD8+ T cell population. c) 21 days after immunization with Lm-EGFRvIIIx5 or a 3 dose regimen of PEPvIII, the frequency of K^k-EGFRvIII_26-33 -tetramer+ cells within the CD3+CD8+ T cell population was determined. d) C3H mice were vaccinated x3 with PEPvIII, or a single dose of Lm-EGFRvIIIx5 as indicated. 21 days after the last vaccination, mice were boosted with PEPvIII or Lm-EGFRvIIIx5. Five days later, the frequency of K^k-EGFRvIII_26-33 -tetramer+ cells within the CD3+CD8+ T cell population was determined. Each symbol represents one animal. Bars represent mean ± SEM of groups containing 4–5 animals. Each figure represents a single experiment from a minimum of two replicates. (ANOVA).

Fig 5. Control of EGFRvIII-expressing tumors by Lm-EGFRvIII.

a) C3H mice were left untreated or vaccinated with Lm-EGFRvIIIx5 twice separated by 14 days. 7 days following the last vaccine, mice were challenged with SCCVII-control or SCCVII-EGFRvIII. Ii) 7 days following tumor challenge, EGFRvIII-specific CD8 T cells in the spleen were quantified by IFN-γ ICS. b) i) C3H mice were left untreated or vaccinated with Lm-EGFRvIII twice separated by 14 days. 7 days following the last vaccine, mice were challenged with SCCVII-control on one flank and SCCVII-EGFRvIII on the opposite flank. ii) Size of tumors in unvaccinated animals (left two columns) or vaccinated animals (right two columns) d10 following tumor challenge. c) C3H mice were vaccinated with Lm-OVA as a vector control or Lm-EGFRvIII twice separated by 14 days. 7 days following the last vaccine, mice were challenged with SCCVII-control or SCCVII-EGFRvIII and followed for ii) survival of vaccinated animals. d) C3H mice were implanted with SCCVII-EGFRvIII and left untreated or vaccinated with a single dose of Lm-EGFRvIII on d3 following tumor challenge. Graphs show average tumor growth of treatment groups. Key: * = p<0.05; ** = p< 0.01; *** = p<0.001; **** = p<0.0001 (a- ANOVA; b- T-test; c,d Log rank).