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NY-ESO-1 is a \"cancer-testis\" antigen expressed in many cancers. ISCOMATRIX is a saponin-based adjuvant that induces antibody and T cell responses. We performed a placebo-controlled clinical trial evaluating the safety and immunogenicity of recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant. Forty-six evaluable patients with resected NY-ESO-1-positive tumors received three doses of vaccine intramuscularly at monthly intervals. The vaccine was well tolerated. We observed high-titer antibody responses, strong delayed-type hypersensitivity reactions, and circulating CD8+ and CD4+ T cells specific for a broad range of NY-ESO-1 epitopes, including known and previously unknown epitopes. In an unplanned analysis, vaccinated patients appeared to have superior clinical outcomes to those treated with placebo or protein alone. The vaccine is safe and highly potent immunologically.

There is increasing evidence showing the involvement of CD4+T cells in initiating and maintaining antitumor immune responses. NY-ESO-1 is expressed by various tumors but not normal tissues except testis. We conducted a cancer clinical trial by using full-length NY-ESO-1 protein formulated with ISCOMATRIX adjuvant and injected into patients intramuscularly. Autologous dendritic cells pulsed with NY-ESO-1 ISCOMATRIX in combination with overlapping synthetic peptides were used to identify immunodominant T cells from a vaccinated patient. We show here the identification and characterization of two novel CD4+T cell epitopes. T cells specific to these epitopes not only recognized autologous dendritic cells loaded with NY-ESO-1 but also NY-ESO-1-expressing tumor cell lines treated with IFN-γ. One of the two responses identified was greater than the previously identified immunodominant HLA-DPA-restricted response and correlated with NY-ESO-1-specific CD8+T cell induction after vaccination. This T cell response was vaccinated in most patients who expressed HLA-DR2. This study has systematically surveyed patients vaccinated with full-length tumor antigen for a vaccinated CD4 helper T cell response.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Clinical outcomes from cancer vaccine trials in patients with advanced melanoma have so far been disappointing. This appears at least partially due to a state of immunosuppression in these patients induced by an expansion of regulatory cell populations including regulatory T cells (Tregs). We have previously demonstrated potent immunogenicity of the NY-ESO-1/ISCOMATRIX™ vaccine in patients with resected melanoma (study LUD99-08); however, the same vaccine induced only a few vaccine antigen-specific immune responses in patients with advanced disease (study LUD2002-013). Pre-clinical models suggest that the alkylating agent cyclophosphamide can enhance immune responses by depleting Tregs. Therefore, we have enrolled a second cohort of patients with advanced melanoma in the clinical trial LUD2002-013 to investigate whether pre-treatment with cyclophosphamide could improve the immunogenicity of the NY-ESO-1/ISCOMATRIX™ vaccine. The combination treatment led to a significant increase in vaccine-induced NY-ESO-1-specific CD4 + T cell responses compared with the first trial cohort treated with vaccine alone. We could not detect a significant decline in regulatory T cells in peripheral blood of patients 14 days after cyclophosphamide administration, although a decline at an earlier time point cannot be excluded. Our observations support the inclusion of cyclophosphamide in combination trials with vaccines and other immune-modulatory agents.

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

Background NY-ESO-1 protein formulated in ISCOMATRIX™ results in CD4+, CD8+ T cell and antibody-mediated immunity. We evaluated persistence of immunity, relapse-free survival and tumour antigen expression upon relapse in patients vaccinated in an earlier trial. Methods Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252–1,155 days (median = 681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX™ adjuvant at three protein doses 10 μg, 30 μg or 100 μg ( n  = 14); 100 μg NY-ESO-1 protein ( n  = 8) or placebo ( n  = 6), together with 1 μg of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2 days after DTH skin testing with NY-ESO-1 protein (1 μg) or peptides (10 μg). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17. Results Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX™ adjuvant. In contrast, immunity only persisted in 3/14 who received 100 μg un-adjuvanted NY-ESO-1 protein (3/8) or 2 μg DTH protein (0/6) P  = 0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred. Conclusion Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX™. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.

Clinical outcomes from cancer vaccine trials in patients with advanced melanoma have so far been disappointing. This appears at least partially due to a state of immunosuppression in these patients induced by an expansion of regulatory cell populations including regulatory T cells (Tregs). We have previously demonstrated potent immunogenicity of the NY-ESO-1/ISCOMATRIX(TM) vaccine in patients with resected melanoma (study LUD99-08); however, the same vaccine induced only a few vaccine antigen-specific immune responses in patients with advanced disease (study LUD2002-013). Pre-clinical models suggest that the alkylating agent cyclophosphamide can enhance immune responses by depleting Tregs. Therefore, we have enrolled a second cohort of patients with advanced melanoma in the clinical trial LUD2002-013 to investigate whether pre-treatment with cyclophosphamide could improve the immunogenicity of the NY-ESO-1/ISCOMATRIX(TM) vaccine. The combination treatment led to a significant increase in vaccine-induced NY-ESO-1-specific CD4 super(+) T cell responses compared with the first trial cohort treated with vaccine alone. We could not detect a significant decline in regulatory T cells in peripheral blood of patients 14 days after cyclophosphamide administration, although a decline at an earlier time point cannot be excluded. Our observations support the inclusion of cyclophosphamide in combination trials with vaccines and other immune-modulatory agents.

IntroductionCombination therapy with BRAF and MEK inhibition is currently in clinical development for the treatment of BRAF mutated malignant melanoma. BRAF inhibitors are associated with enhanced antigen-specific T-lymphocyte recognition in vivo. Consequently BRAF inhibition has been proposed as pro-immunogenic and there has been considerable enthusiasm for combining BRAF inhibition with immunotherapy. MEK inhibitors inhibit ERK phosphorylation regardless of BRAF mutational status and have been reported to impair T-lymphocyte and dendritic cell function. In this study we investigate the effects on isolated T-lymphocytes and monocyte-derived dendritic cells (moDCs) of a MEK and BRAF inhibitor combination currently being evaluated in a randomized controlled clinical trial.Experimental designThe effects of a BRAF inhibitor and a MEK inhibitor, alone and in combination were studied on isolated normal T-lymphocyte and moDCs. Lymphocyte viability, together with functional assays including proliferation, cytokine production and antigen-specific expansion, were assessed. moDC phenotype in response to lipo-polysaccharide stimulation was evaluated by flow-cytometry, as were effects on antigen crosspresentation.ResultsBRAF inhibition did not have an impact on T-lymphocytes or moDCs, whereas MEK inhibition alone or in combination with BRAF inhibition suppressed T-lymphocyte proliferation, cytokine production and antigen-specific expansion. Similarly, moDC cross-presentation was suppressed in association with enhanced maturation following combined inhibition of MEK and BRAF. However no significant decrease in CD4+ or CD8+ T-lymphocyte viability was observed following kinase inhibition.ConclusionsMEK inhibition, alone or in combination with BRAF inhibition can suppress immune cell function and further studies in vivo will be required to evaluate the potential clinical impact of these findings.