Qiyuan Chen

NY-ESO-1 Protein Formulated in ISCOMATRIX Adjuvant Is a Potent Anticancer Vaccine Inducing Both Humoral and CD8+ T-Cell-Mediated Immunity and Protection against NY-ESO-1+ Tumors

NY-ESO-1 is a 180 amino-acid human tumor antigen expressed by many different tumor types and belongs to the family of “cancer-testis” antigens. In humans, NY-ESO-1 is one of the most immunogenic tumor antigens and NY-ESO-1 peptides have been shown to induce NY-ESO-1-specific CD8+ CTLs capable of altering the natural course of NY-ESO-1-expressing tumors in cancer patients. Here we describe the preclinical immunogenicity and efficacy of NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant (NY-ESO-1 vaccine). In vitro, the NY-ESO-1 vaccine was readily taken up by human monocyte-derived dendritic cells, and on maturation, these human monocyte-derived dendritic cells efficiently cross-presented HLA-A2-restricted epitopes to NY-ESO-1-specific CD8+ T cells. In addition, epitopes of NY-ESO-1 protein were also presented on MHC class II molecules to NY-ESO-1-specific CD4+ T cells. The NY-ESO-1 vaccine induced strong NY-ESO-1-specific IFN-γ and IgG2a responses in C57BL/6 mice. Furthermore, the NY-ESO-1 vaccine induced NY-ESO-1-specific CD8+ CTLs in HLA-A2 transgenic mice that were capable of lysing human HLA-A2+ NY-ESO-1+ tumor cells. Finally, C57BL/6 mice, immunized with the NY-ESO-1 vaccine, were protected against challenge with a B16 melanoma cell line expressing NY-ESO-1. These data illustrate that the NY-ESO-1 vaccine represents a potent therapeutic anticancer vaccine.

CD8+ T cell responses against a dominant cryptic HLA-A2 epitope after NY-ESO-1 peptide immunization of cancer patients

NY-ESO-1 is a germ cell antigen aberrantly expressed in different tumor types that elicits strong humoral and cellular immune responses in cancer patients. Monitoring spontaneous CD8+ T cell responses against NY-ESO-1 peptides 157–165 (S9C) and 157–167 (S11L) in a series of HLA-A2+ cancer patients showed that these two peptides had overlapping antigenic profiles and were equally immunogenic. However, discrepancies between S9C and S11L reactivities were observed upon vaccination with both peptides to generate or boost T cell responses to NY-ESO-1 in cancer patients. We here analyze the fine specificity of these responses and describe an HLA-A2-restricted epitope, NY-ESO-1 peptide 159–167 (L9L), which is strongly recognized by CD8+ T cells as a result of peptide vaccination of cancer patients. Responses to L9L were stimulated by S11L and appeared early in the course of vaccination, independently of S9C responses. However, L9L-specific CD8+ T cells failed to recognize tumor cells naturally expressing NY-ESO-1 or B lymphoblastoid cells transduced with NY-ESO-1. Processing of L9L could be rescued after IFN-γ treatment of tumor cells or by dendritic cells pulsed with NY-ESO-1 protein/antibody immune complexes. The present results demonstrate a dual specificity within peptide S11L, with S9C as the natural antigenic tumor epitope, and L9L as a cryptic epitope with dominant immunogenicity upon vaccination that diverts the immune response from tumor recognition. These unanticipated findings raise questions about the use of S11L in the clinic and emphasize the importance of analyzing the fine specificity of vaccine-induced T cell responses in patients as a basis for constructing effective cancer vaccines.

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients.

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10–specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.

Exogenous Peptides Presented by Transporter Associated with Antigen Processing (TAP)-Deficient and TAP-Competent Cells: Intracellular Loading and Kinetics of Presentation

This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous β2-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.

Striking Immunodominance Hierarchy of Naturally Occurring CD8+ and CD4+ T Cell Responses to Tumor Antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient’s immune system, especially the “cancer-testis” tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Spontaneous T cell responses to melanoma differentiation antigens from melanoma patients and healthy subjects

Abstract The spontaneous cytotoxic T cell responses to melanoma differentiation antigens and influenza matrix peptide were compared in 20 HLA-A2+ melanoma patients and 17 healthy A2+ individuals. Cytotoxic T lymphocyte (CTL) responses were determined by mixed lymphocyte peptide culture (MLPC) involving two stimulations of unfractionated peripheral blood lymphocytes (PBLs) with peptide in vitro. CTL responses to Melan-A 9-mer (amino acids 27–35, AAGIGILTV) peptide were detected in 4 out of 16 normal individuals, but in none of the melanoma patients. CTL specific for influenza matrix peptide were frequently found in both normal individuals and melanoma patients, suggesting that generalized immuno-suppression was not responsible for this difference. No significant responses were observed in either normal individuals or melanoma patients to Melan-A 10-mer (26–35, EAAGIGILTV), two gp100 epitopes (280–288, YLEPGPVTA; 457–466, LLDGTATLRL) and two tyrosinase epitopes (1–9, MLLAVLYCL; 368–376, YMDGMSQV). Melan-A (27–35)-specific CTL cells generated by normal individuals and melanoma patients recognized both synthetic peptide-pulsed T2 cells and two HLA-A2+, Melan-A+ melanoma cell lines (ME272, LAR1) in an antigen-specific, MHC class I restricted manner. T cells generated against Melan-A 9-mer were also able to recognize Melan-A 10-mer-pulsed target cells. Spontaneous CTL responses to Melan-A 9-mer from three known responder normal individuals were further evaluated over a prolonged time course (6–11 months). All 3 subjects demonstrated specific Melan-A 9-mer responses throughout the study period, although lytic activity fluctuated over time for a given individual. We found the MLPC assay to be reliable and easy to perform for monitoring T cell responses, although it may still not be sufficiently sensitive to detect low numbers of precursor T cells.

Effects of oncostatin M and tamoxifen on human melanoma cells

New agents are required in the treatment of malignant melanoma, to be used alone or in combination with established therapies. Oncostatin M (OSM), a member of the gp130 family of cytokines, has previously been shown to inhibit the growth of melanoma cell lines. Tamoxifen (TAM) is widely used in the treatment of melanoma, typically in combination with chemo- and/or immunotherapy. A component of the antitumour activity of TAM is via modulation of transforming growth factor-β (TGFβ) which has previously been shown to be synergistic with OSM in vitro. To further investigate the clinical potential of OSM, alone and in combination with TAM, set concentrations of each were added to nine fresh and two well-established melanoma cell lines. The proliferation of seven of the 11 cell lines was inhibited by OSM, while two were unresponsive at the dose range tested. Of particular interest was the finding that the growth of two of the cell lines was significantly stimulated at low doses of OSM. The combination of OSM with TAM produced widely divergent results, most frequently resembling the effects of OSM alone. No synergism between the two was evident in any of the cell lines tested. Our results indicate that a combination of OSM and TAM in clinical trials needs further evaluation before it can be recommended. Furthermore, while OSM alone may be useful in the treatment of melanoma, this may be complicated by the possibility of stimulating tumour growth in some instances.

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor α) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1+ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing “immunodominant” alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.

A direct comparison of cytolytic T-lymphocyte responses to Melan-A peptides in vitro: differential immunogenicity of Melan-A27-35 and Melan-A26-35.

&NA; In this study we directly compared the in vitro responses of T‐cells from normal donors and melanoma patients to Melan‐A27‐35 and Melan‐A26‐35. These peptides have been previously used in peptide‐based vaccination studies. Following three stimulations with peptide‐pulsed antigenpresenting cells in vitro, Melan‐A‐specific cytolytic T‐lym‐phocytes (CTLs) were generated from seven of 20 subjects; two of the seven subjects responded reproducibly to both Melan‐A27‐35 and Melan‐A26‐35, three to only Melan‐A27‐35 and two to only Melan‐A26‐35. However, CTLs generated with either Melan‐A27‐35 or Melan‐A26‐35 showed cross recognition, and both types of CTL could recognize naturally processed antigen displayed on HLA‐A2+ tumour cells. Furthermore, Melan‐A‐specific CTLs could also be generated by stimulating peripheral blood mononuclear cells with autologous melanoma cells. Our results suggest that some subjects may have a bias in their CTL repertoire which favours the generation of Melan‐A27‐35 specific CTLs, while others may favour Melan‐A26‐35 specific CTLs. It is also likely that CTL precursors capable of detecting both peptides may have different affinities to the two Melan‐A peptides. Since it is difficult to predict the CTL responses to Melan‐A peptide in a given individual, we suggest vaccinating with both Melan‐A27‐35 and Melan‐A26‐35 peptides in clinical trials.