Natural Killer and NK-Like T-Cell Activation in Colorectal Carcinoma Patients Treated with Autologous Tumor-Derived Heat Shock Protein 96

Heat shock proteins (HSPs) are involved in the activation of both adaptive and innate immune systems. Here, we report that vaccination with autologous tumor-derived HSP96 of colorectal cancer patients, radically resected for liver metastases, induced a significant boost of natural killer (NK) activity detected as cytokine secretion and cytotoxicity in the presence of NK-sensitive targets. Increased NK activity was associated with a raise in CD3−CD56+ NK and/or CD3+CD56+ NK-like T cells, displaying enhanced expression of NKG2D and/or NKp46 receptors. Up-regulated expression of CD83 and CD40 and increased interleukin-12 release on stimulation were observed in CD14+ cells from post-HSP96 peripheral blood mononuclear cells, suggesting an indirect pathway of NK stimulation by HSP96-activated monocytes. Additionally, CD3−CD56+ and CD3+CD56+ lymphocytes were found to undergo functional and phenotypic activation on in vitro exposure to HSP96 even in the absence of monocytes, supporting a potential direct activity of HSP96 on these cell subsets. This evidence was confirmed by the specific binding of FITC-conjugated HSP96 to a subset of both CD3−CD56+ and CD3+CD56+ cells in peripheral blood mononuclear cells from colorectal cancer patients. Altogether, these findings identify the activation of the NK compartment as an additional immunologic effect of autologous tumor-derived HSP96 administration in cancer patients

Induction of Lymphocyte Apoptosis by Tumor Cell Secretion of FasL-bearing Microvesicles

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity

Cancer-Initiating Cells from Colorectal cancer Patients Escape from T Cell-Mediated Immunosurveillance In Vitro through Membrane-Bound IL-4

Cancer-initiating cells (CICs) that are responsible for tumor initiation, propagation, and resistance to standard therapies have been isolated from human solid tumors, including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display “tumor-initiating/stemness” properties, including the expression of CIC-associated markers (e.g., CD44, CD24, ALDH-1, EpCAM, Lgr5), multipotency, and tumorigenicity following injection in immunodeficient mice. The immune profile of these cells was assessed by phenotype analysis and by in vitro stimulation of PBMCs with CICs as a source of Ags. CICs, compared with non-CIC counterparts, showed weak immunogenicity. This feature correlated with the expression of high levels of immu- nomodulatory molecules, such as IL-4, and with CIC-mediated inhibitory activity for anti-tumor T cell responses. CIC-associated IL-4 was found to be responsible for this negative function, which requires cell-to-cell contact with T lymphocytes and which is impaired by blocking IL-4 signaling. In addition, the CRC-associated Ag COA-1 was found to be expressed by CICs and to represent, in an autologous setting, a target molecule for anti-tumor T cells. Our study provides relevant information that may contribute to designing new immunotherapy protocols to target CICs in CRC patient

Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells

Abstract Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8+ T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8+ T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-γ and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A27–35 in three of five HLA-A*0201 melanoma patients, and of CEA571–579 and EpCAM263–271 in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients

The Italian Network for Tumor Biotherapy (NIBIT): Getting together to push the field forward

As for a consolidated tradition, the 5th annual meeting of the Italian Network for Cancer Biotherapy took place in the Certosa of Pontignano, a Tuscan monastery, on September 20–22, 2007. The congress gathered more than 40 Italian leading groups representing academia, biotechnology and pharmaceutical industry. Aim of the meeting was to share new advances in cancer bio-immunotherapy and to promote their swift translation from pre-clinical research to clinical applications. Several topics were covered including: a) molecular and cellular mechanisms of tumor escape; b) therapeutic antibodies and recombinant constructs; c) clinical trials up-date and new programs; d) National Cooperative Networks and their potential interactions; e) old and new times in cancer immunology, an "amarcord". Here, we report the main issues discussed during the meeting

An Expanded Peripheral T Cell Population to a Cytotoxic T Lymphocyte (Ctl)-Defined, Melanocyte-Specific Antigen in Metastatic Melanoma Patients Impacts on Generation of Peptide-Specific Ctls but Does Not Overcome Tumor Escape from Immune Surveillance in Metastatic Lesions

It is not known if immune response to T cell–defined human histocompatibility leukocyte antigen (HLA) class I–restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA–peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-127–35–specific CTL precursors (CTLp) were ≥1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO+ memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA+ naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201–Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1–specific T cells. Furthermore, frequent lack of a “brisk” or “nonbrisk” CD3+CD8+ T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell–mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance

Defining the Critical Hurdles in Cancer Immunotherapy

ABSTRACT: Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators, others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet be overcome to improve outcomes of patients with cancer

Future perspectives in melanoma research: meeting report from the "Melanoma Bridge";: Napoli, December 3rd-6th 2014.

The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma