RESUMEN
OBJECTIVE: Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies. DESIGN: We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. RESULTS: Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+ T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo. CONCLUSIONS: These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , ADN Complementario/análisis , Epítopos de Linfocito T/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteínas de Ciclo Celular/genética , Fosfatidilinositol 3-Quinasa Clase I , Análisis Mutacional de ADN , Epítopos de Linfocito T/inmunología , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Expresión Génica , Antígenos HLA/genética , Antígenos HLA/inmunología , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Madre Neoplásicas/inmunología , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína Smad4/genética , Proteína Smad4/inmunología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Clinical activity was observed in metastatic melanoma (MM) patients treated with ipilimumab (IPI) combined with fotemustine (FTM) in the phase II NIBIT-M1 study. Peripheral blood mononuclear cells (PBMCs) and serum were collected from MM patients at pre- and at weeks 12 and 24 post-treatment. A comprehensive phenotypic and functional immunomonitoring of circulating T cells, and the detection of soluble immunoregulatory molecules was carried out and correlated with clinical outcome. The frequency at baseline and along the treatment of circulating T central memory cells expressing activation/differentiation markers, such as CD3+CD4+CD45RO+BTLA+, CD3+CD4+4-1BB or Th17 lymphocytes correlated with the clinical outcome of MM patients. Moreover, either the absence or the presence of soluble NKG2D ligands (ULBP-1 or -2) at baseline in the serum of MM patients enabled to discriminate subjects with long-term survival (median overall survival, (OS) = 33.6 mo for ULBP-1 and -2) from poor survivors (OS = 9.8 or 6.6 mo, respectively). Conversely, no significant association between the levels of soluble MICA, MICB and ULBP-3 and the clinical outcome of patients was observed. An inverse correlation between circulating levels of these molecules at baseline and frequency of either CD3+CD4+CD45RO+BTLA+ or Th17 or CD3+CD4+4-1BB+ T cells occurred in patients with a favorable clinical outcome. The simultaneous monitoring of different immune parameters, though validation in a large cohort of patients is needed, allowed to identify an association between phenotypic and soluble markers representing a possible predictive immunological signature for the clinical activity of IPI plus FTM.
RESUMEN
Administration of NGR-TNF, a tumor vessel-targeting and tumor necrosis factor α TNFα) peptide conjugate, with immunotherapy has been shown to inhibit tumor growth in mice. Thus, we planned a Phase I pilot clinical trial to assess safety, immune and clinical response of this combination treatment for advanced melanoma. NA17.A2 and MAGE-3.A1 peptides were used as vaccine. HLA-A*0201 or HLA-A*01 metastatic melanoma patients received human NGR-hTNF i.v. alternating with s.c. weekly injections of either of the peptides emulsified in Montanide. The T-cell response was assessed ex-vivo using peripheral blood mononuclear cells (PBMCs) before, during and after therapy. The serum level of chromogranin A (CgA), soluble TNF receptors (sTNFR1/2), vascular endothelial growth factor (VEGF), and MIP-1ß and MCP-1 chemokines, was determined. In 3 subjects, pre- and post-treatment tumor lesions were examined by immunohistochemistry. Clinically, chills were observed in 4 patients during NGR-hTNF infusion and erythema at vaccination site was seen in 7 patients. T-cell response against the vaccine or against other melanoma-associated antigens was detectable after treatment in 6 out of 7 tested patients. Low level or reduction of CgA and sTNFR and increase of MIP-1ß and MCP-1 were found in patients sera. In the lesions examined the immune infiltrate was scanty but macrophage number increased in post-therapy lesions. From a clinical standpoint, a long term survival (>4 months) was found in 6 out of 8 evaluable patients (4, 4, 7, 11, 23+, 25+, 25+, 29+ months). The combination of NGR-hTNF and vaccine in metastatic melanoma patients was well tolerated, often associated with an ex-vivo T cell response and long-term overall survival. These findings warrant confirmation in a larger group of patients.