Targeting multiple inhibitory receptors to reverse melanoma-induced T cell dysfunction

It is now clearly established that dysfunctional/exhausted TA-specific T cells present in peripheral blood and at tumor sites co-express multiple inhibitory receptors. The implications of this important finding are two-fold. First, multiple subsets of TA-specific T cells can be identified in patients with advanced melanoma that exhibit variable levels of T cell dysfunction. Second, this observation supports the implementation of combinatorial therapies aiming at blocking multiple inhibitory pathways to enhance TA-specific immune responses and reverse tumor-induced T cell dysfunction. We have shown that a subset of highly dysfunctional TA-specific CD8+ T cells isolated from patients with advanced melanoma upregulate both PD-1 and Tim-3. PD-1 and Tim-3 blockades strongly enhance TA-specific CD8+ T cell expansion and function. Accordingly, targeting PD-1 and Tim-3 in vivo induces melanoma regression in mice. Therefore, the combination of PD-1 and Tim-3 blockade either alone or in combination with cancer vaccines appears to be a promising potent approach to reverse melanoma-induced T cell dysfunction and promote tumor regression in patients with advanced melanoma

SCIB2, an antibody DNA vaccine encoding NY-ESO-1 epitopes, induces potent antitumor immunity which is further enhanced by checkpoint blockade

Checkpoint blockade has demonstrated promising antitumor responses in approximately 10-40% of patients. However, the majority of patients do not make a productive immune response to their tumors and do not respond to checkpoint blockade. These patients may benefit from an effective vaccine that stimulates high-avidity T cell responses in combination with checkpoint blockade. We have previously shown that incorporating TRP-2 and gp100 epitopes into the CDR regions of a human IgG1 DNA (ImmunoBody®: IB) results in significant tumor regression both in animal models and patients. This vaccination strategy is superior to others as it targets antigen to antigen-presenting cells and stimulates high-avidity T cell responses. To broaden the application of this vaccination strategy, 16 NY-ESO-1 epitopes, covering over 80% of HLA phenotypes, were incorporated into the IB (SCIB2). They produced higher frequency and avidity T cell responses than peptide vaccination. These T cells were of sufficient avidity to kill NY-ESO-1-expressing tumor cells, and in vivo controlled the growth of established B16-NY-ESO-1 tumors, resulting in long-term survival (35%). When SCIB2 was given in combination with Treg depletion, CTLA-4 blockade or PD-1 blockade, long-term survival from established tumors was significantly enhanced to 56, 67 and 100%, respectively. Translating these responses into the clinic by using a combination of SCIB2 vaccination and checkpoint blockade can only further improve clinical responses

Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

BACKGROUND:Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered. PRINCIPAL FINDINGS:Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with 'MHC-loading enhancer' (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE. CONCLUSION:MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy

Human dendritic cells adenovirally-engineered to express three defined tumor antigens promote broad adaptive and innate immunity

Dendritic cell (DC) immunotherapy has shown a promising ability to promote anti-tumor immunity in vitro and in vivo. Many trials have tested single epitopes and single antigens to activate single T cell specificities, and often CD8+ T cells only. We previously found that determinant spreading and breadth of antitumor immunity correlates with improved clinical response. Therefore, to promote activation and expansion of polyclonal, multiple antigen-specific CD8+ T cells, as well as provide cognate help from antigen-specific CD4+ T cells, we have created an adenovirus encoding three full length melanoma tumor antigens (tyrosinase, MART-1 and MAGE-A6, “AdVTMM”). We previously showed that adenovirus (AdV)-mediated antigen engineering of human DC is superior to peptide pulsing for T cell activation, and has positive biological effects on the DC, allowing for efficient activation of not only antigen-specific CD8+ and CD4+ T cells, but also NK cells. Here we describe the cloning and testing of “AdVTMM2,” an E1/E3-deleted AdV encoding the three melanoma antigens. This novel three-antigen virus expresses mRNA and protein for all antigens, and AdVTMM-transduced DC activate both CD8+ and CD4+ T cells which recognize melanoma tumor cells more efficiently than single antigen AdV. Addition of physiological levels of interferon-α (IFNα) further amplifies melanoma antigen-specific T cell activation. NK cells are also activated, and show cytotoxic activity. Vaccination with multi-antigen engineered DC may provide for superior adaptive and innate immunity and ultimately, improved antitumor responses

Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4+ clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-γ in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-γ after melanoma stimulation. No CD4+ clones responded to stimulation with recipient haemopoietic cells. The majority of CD8+ clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4+ clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma

Selective identification of HLA-DP4 binding T cell epitopes encoded by the MAGE-A gene family

Because of the high frequency of HLA-DP4 in the Caucasian population, we have selectively delineated HLA-DP4 restricted T cell epitopes in the MAGE-A tumor antigens. We identified 12 good binders to HLA-DP4 and investigated the capacity of the seven best binders to induce in vitro specific CD4+ T cell lines from HLA-DP4 healthy donors. We found that the MAGE-A1 90-104 peptide exhibited a high and constant frequency of CD4+ T cell precursors in all the six tested donors. The MAGE-A1 268-282 peptide was found immunogenic in only two donors but with a high precursor frequency. The MAGE-A12 127-141 peptide was T cell stimulating in six different donors and induced fewer T cell lines. The peptide-specific T cell lines were stimulated by DC loaded with the lysates of cells transfected with MAGE-A1 or MAGE-A12, or loaded with the recombinant protein. We also show that the immunoreactivity of CD4+ T cell epitopes restricted to the same HLA II molecule may vary from one individual to another, as a result of inter-individual variations in the CD4+ T cell repertoire