RhoC a new target for therapeutic vaccination against metastatic cancer

Abstract

Most cancer deaths are due to the development of metastases. Increased expression of RhoC is linked to enhanced metastatic potential in multiple cancers. Consequently, the RhoC protein is an attractive target for drug design. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The over-expression of RhoC in cancer and the fact that immune escape by down regulation or loss of expression of this protein would reduce the morbidity and mortality of cancer makes RhoC a very attractive target for anti-cancer immunotherapy. Herein, we describe an HLA-A3 restricted epitope from RhoC, which is recognized by cytotoxic T cells. Moreover, RhoC-specific T cells show cytotoxic potential against HLA-matched cancer cells of different origin. Thus, RhoC may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies.

Fig. 1

HLA-A3 restricted T-cell responses against Rho1L2 as measured by IFN-γ ELISPOT The average number of peptide specific IFN-γ spots formed in response to Rho1L2 among 5 × 10⁵ in vitro stimulated PBMC from five HLA-A3+ healthy donors (HD), PBL from ten renal cell carcinoma patients (RCC), and ten melanoma patients (MM). Measurements were made in triplicates

Fig. 2

T cell antigen specificity and cross reactivity. a Cytotoxicity by ⁵¹Cr-release assay of a bulk culture stimulated with Rho1L2-loaded autologous DC/autologous PBL. Specific lysis of T2-A3 cells with no peptide or pulsed with Rho1L2 (10 μM) or Rho1 (10 μM). E:T ratio = 60:1, 30:1. b Specificity of a T cell clone (clone 9) assayed by ⁵¹Cr-release assay. Lysis of T2-A3 cells with no peptide, pulsed with Rho1L2 (10 μM), Rho1 (10 μM) and of Rho1L2-pulsed T2–A3 cells with addition of the HLA-class I specific antibody W6/32 or HLA-A3 specific antibody GAP A3 at different E:T ratios (9:1; 3:1; 1:1; 0,3:1). All measurements were made in duplicates. Error bars indicate the standard deviation

Fig. 3

PCR products electrophoresed in agarose gel and stained with EtBr. The image represents five different cancer cell lines, all being positive for RhoC. For each cancer cell line the left band represents GAPDH and the right band represents RhoC

Fig. 4

Functional capacity of RhoC-specific T cells. a Cytotoxicity of a bulk culture stimulated with Rho1L2-loaded autologous DC/autologous PBL. Specific lysis of the HLA-A3+ melanoma cell line FM3 without and with the addition of the HLA-class I specific antibody W6/32. E:T ratio = 60:1, 40:1. b Lysis by a Rho1L2-specific clone (clone 9) of the HLA-A3+ melanoma cell line FM3, cell lysis with addition of unlabeled T2-A3 cells pulsed with Rho1L2 (10 μM) or no peptide (inhibitor to target ratio = 20:1), and cell lysis of the HLA-A3+ melanoma cell line FM9 and the HLA-A3- melanoma cell line FM82. Measurements were made in duplicates for all E:T ratios. c Repetition of the experiments shown in b) (without inclusion of FM9). d Lysis by a Rho1L2-specific clone of the HLA-A3+ breast cancer cell line BT-20, colon cancer cell line HT-29 and head and neck cancer cell line CRL-2095. All measurements were made in duplicates. Error bars indicate the standard deviation