Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer

Abstract

The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch-repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as TGF beta RII and BAX. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in TGF beta RII, in TIL and peripheral blood lymphocytes from patients with MSI(+) tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI(+) colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC.

Figures

Figure 1

Frameshift mutations in TGFβRII. The human gene coding for TGFβRII contains a poly(A) sequence (A10) from base no. 709 to base no. 718. Frameshift mutations in this A10 repeat have been observed, both as A9 (−1A) and A11 (+1A). These mutations result in mutant peptide sequences with the first amino acid of the altered sequence in either position 128 or 129 as compared with the normal TGFβRII protein. We have synthesized several of these mutant peptides, among them peptide 538. From the SYFPEITHI–MHC Database (http.//www.medizin.uni-tuebingen.de/sfb510/index.html) we have predicted possible peptide motifs from peptide 538 for three HLA class II molecules.

Figure 2

Specificity of T cells from donor 6947 for p538. (a) Peptide-specific proliferation of bulk culture 6947 against autologous PBMCs pulsed with TGFβRII frameshift peptides. (b) Peptide-specific proliferation of the TLC 6947-22 against the p538 peptide. Autologous B-LCLs (6947 EB) were used as APCs. (c) Blocking of the TGFβRII specific response by anti-HLA-DP mAb. (d) Panel studies using homozygous B-LCLs corresponding to DPB1 alleles of donor 6947. Solid bars, APC + p538; open bars, APC − p538. The HLA-DPB1 type of the different B-LCLs was 6947; EB: DPB1*0301, 9097: DPB1*0301/0401, 9031: DPB1*0401, 9009: DPB1*0401/1401.

Figure 3

Proliferative responses of bulk cultures generated from colorectal cancer patients. Bulk cultures generated from six patients with colorectal cancer localized to the proximal colon were tested for proliferation to TGFβRII and Bax frameshift peptides. Autologous B-LCLs or PBMCs were used as APCs. Patients' mutation status is indicated.

Figure 4

Proliferative responses of bulk cultures generated from three HNPCC patients. PBMCs from HNPCC patients were stimulated with frameshift TGFβRII peptides and tested for peptide-specific response in a proliferation assay. Autologous PBMCs were used as APCs. The amino acid sequences of peptides p538, p540, and p541 are given, and the common/overlapping motif is underlined.

Figure 5

Specificity of TLC IMT8 and IMT9 for the TGFβRII frameshift peptide p538. (a) Peptide-specific proliferation against autologous B-LCLs loaded with mutated TGFβRII peptides. (b) Dose–response curves of TLC IMT8 and IMT9 obtained by pulsing the cell line 9054 (HLA-DRB1*1401) with increasing amounts of the p538 peptide. (c) Blocking of the peptide-specific response by anti-HLA class II mAbs. The cell line 9054 was used as APC. Filled squares and bars, TLC IMT8; open squares and bars, TLC IMT 9.

Figure 6

Photomicrographs of stained sections of the tumor biopsy obtained from donor IMT. Immunostaining with mAbs against HLA-DR (a) and same area magnified in b, CD4 (c) and CD8 (d).

Figure 7

ACDCE electropherogram and sequencing results of mutation analysis in the (10A) tract of TGFβRII in patient IMT. (Lower) The mutant and wild-type peaks of a heterozygous one base deletion in TGFβRII polyadenine tract. To verify the results the sample was sequenced and the result is illustrated Upper. Note that the sequence is out of frame after the polyadenine tract because of a heterozygous one base deletion in the sequence.