Search

PDF file - 41K, Table S1. Characteristics of patients enrolled in the clinical trial.

The cancer–testis/cancer germline antigen, NY-ESO-1, is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Preclinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1–specific CTL-mediated responses. On the basis of these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T-cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in six of ten evaluable patients. On the basis of these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted. Cancer Immunol Res; 2(1); 37–49. ©2014 AACR.

Vaccination of patients with ovarian cancer with overlapping long peptides (OLP) from cancer-testis antigen NY-ESO-1 and poly-ICLC in Montanide-ISA-51 (Montanide) was found to consistently induce integrated immune responses (antibody, CD4+, and CD8+ T cells). Using detailed methods, we investigated the respective effects of poly-ICLC and Montanide adjuvant on pre- and postvaccine NY-ESO-1–specific CD4+ T cells, because of their central function for induction and maintenance of both antibody and CD8+ T cells. Polyclonal NY-ESO-1–specific CD4+ T-cell lines were generated from 12 patients using CD154-based selection of precursors before and after vaccination with (i) OLP alone, (ii) OLP in Montanide, or (iii) OLP and poly-ICLC in Montanide. Kinetics, quantification, fine specificity, avidity, and cytokine-producing pattern were analyzed in depth and compared between vaccine cohorts. Vaccination with OLP alone did not elicit CD4+ T-cell responses; it suppressed high-avidity CD4+ T-cell precursors that recognized naturally processed NY-ESO-1 protein before vaccination. Emulsification of OLP in Montanide was required for the expansion of high-avidity NY-ESO-1–specific CD4+ T-cell precursors. Poly-ICLC significantly enhanced CD4+ Th1 responses while suppressing the induction of interleukin (IL)-4–producing Th2 and IL-9–producing Th9 cells. In summary, Montanide and poly-ICLC had distinct and cooperative effects for the induction of NY-ESO-1–specific Th1 cells and integrated immune responses by OLP vaccination. These results support the use of admixing poly-ICLC in Montanide adjuvant to rapidly induce antitumor type I immune responses by OLP from self/tumor antigens in human cancer vaccines. Cancer Immunol Res; 1(5); 340–50. ©2013 AACR.

PDF file - 13027K, Comparison of apparent avidities of NY-ESO-1-specific CD4+ T cells before and after vaccination. Apparent avidity (EC50) for recognition of the NY-ESO-1 assay OLP was determined for NY-ESO-1-specific CD4+ T cell lines before and after the vaccination. (A) Cohort 1 (OLP alone); (B) Cohort 2 (OLP in Montanide); and (C) Cohort 3 (OLP and poly-ICLC in Montanide).

PDF file - 59K

PDF file - 51K, Figure S1. NY-ESO-1-specific CD8+ T cell response to a melanoma cell line.

PDF file - 3271K, Comparison of NY-ESO-1-specific cytokine production between 3 cohorts before vaccination. Normalized cytokine production from NY-ESO-1-specific CD4+ T cells before vaccination is shown for statistical consideration. The shape of symbols indicates a single patient as shown in Fig. 1B. Bars indicate mean plus-minus SD. *: p=0.04 by Student's t-test. All other pairs show no statistically significant difference.

PDF file - 45K, Table S2. Adverse events observed in the clinical trial.

PDF file - 62K

PDF file - 54K, Figure S2. Recognition of naturally processed NY-ESO-1 antigen by NY-ESO-1 specific T cells, pre- and post-therapy.

PDF file, 64K, In-vitro immunostimulatory activity of MBV on PBMC.

PDF file 90K, Supplementary Figure Legends

Conflict of Interest from NY-ESO-1 DNA Vaccine Induces T-Cell Responses That Are Suppressed by Regulatory T Cells

PDF file - 1810K, Detection of NY-ESO-1-specific CD4+ T cells by intracellular cytokine staining. CD4+ T cells were stimulated once with T-cell depleted PBMC pulsed with NY-ESO-1 assay OLP and after 22-23 day culture, NY-ESO-1-specific IFN-γ and IL-5 production was evaluated by intracellular cytokine staining. Peptide-pulsed or unpulsed autologous EBV-B cells pulsed or unpulsed with 20-mer NY-ESO-1 peptides covering areas indicated were used as target cells. Responses shown at week 16 after vaccination in two patients (M09 and M19) representative of Cohort 2 and 3, respectively.

PDF file, 39K, Reactivation of cytokine response by further MBV administration after a MBV treatment-free period.

PDF file - 68K

PDF file 79K, GAG-HERV-K ch22q11.23 features. Endogenous retrovirus K from chromosome 22 is located on the q11.23 region, nearby the BCR, ZDHHC8P, IGLL1 and RGL4 genes. HERV-K ch22q11.23 has GAG-PRO, POL and ENV genes flanked by LTR sequences. The GAG gene has an ORF encoding a 715aa protein that contains P10, P24 and Zinc finger domains. {delta}: deletions; I: early stop codons; --> primer binding regions; ABS: antibody binding site for monoclonal antibody TI-35 (see Supplementary Fig. S3C)

PDF file, 82K, Trial design schema.

PDF file - 2015K, Injection site reaction in patient M21

PDF file, 62K.

Gene specific PCR primers

Purpose: Different vaccination strategies against the NY-ESO-1 antigen have been employed in an attempt to induce antitumor immune responses. Antigen-specific effector T-cell responses have been reported in a subset of vaccinated patients; however, these responses have not consistently correlated with disease regression. Here, we report for the first time clinical and immune responses generated by the NY-ESO-1 DNA vaccine administered by particle-mediated epidermal delivery to cancer patients.Experimental Design: Eligible patients received treatment with the NY-ESO-1 DNA vaccine. Clinical outcomes and immune responses were assessed.Results: The NY-ESO-1 DNA vaccine was safely administered and induced both antigen-specific effector CD4 and/or CD8 T-cell responses in 93% (14 of 15) of patients who did not have detectable pre-vaccine immune responses. Despite the induction of antigen-specific T-cell responses, clinical outcomes consisted predominantly of progressive disease. Detectable effector T-cell responses were inconsistent and did not persist in all patients after completion of the scheduled vaccinations. However, high-avidity CD4 T-cell responses that were either undetectable pre-vaccine or found to be diminished at a later time during the clinical trial were detected in certain patients' samples after in vitro depletion of regulatory T cells.Conclusions: Regulatory T cells play a role in diminishing vaccine-induced antigen-specific effector T-cell responses in cancer patients. The NY-ESO-1 DNA vaccine represents a feasible immunotherapeutic strategy to induce antigen-specific T-cell responses. Counteracting regulatory T-cell activity before vaccination may lead to prolonged effector T-cell responses and possibly antitumor responses in cancer patients.

PDF file - 70K, Incidence of treatment-related adverse events by maximum grade

PDF file 204K, Alignment of GAG proteins derived from human endogenous retroviruses and respective epitopes recognized by prostate cancer patient sera. A, Detail alignment from several GAG-HERV-K family members and the TI-35 binding site (red) highlighting the amino-acid differences present in GAG-HERV-K ch22q11.23 (blue). B, Western-blot analysis of immunoprecipitation of GAG-HERV-K from VCaP and LNCaP prostate cancer cell lines. WFP prostate cancer patient serum (WFP) was incubated with VCaP or LNCaP protein extract for 2 hours at 4{degrees}C. Protein-antibody complexes were pulled down using anti-human IgG sepharose beads. The pull-down pellet was submitted to SDS-PAGE and Western-blot analysis. Reactivity to GAG-HERV-K protein was developed by incubating the membrane with TI-35 monoclonal antibody. A pool of healthy donor sera was used as control (Con). C, Summary of ELISA results from epitope mapping using 20mers peptides overlapping by 10aa (71 peptides tested in total from JPT Innovative Peptide Solution, Berlin, Germany) spanning GAG-HERV-K and tested individually for recognition by monoclonal antibody TI-35 and different prostate cancer patient sera seropositive for GAG-HERV-K protein. The square symbol represents a positive reaction to the corresponding peptide (labeled by aminoacid start and end position). A pool of healthy donor sera was used as control (Con pool). Plates were coated with 5 uM peptide and the reaction was performed as previously described in methods

Purpose: Cancer-testis genes mapping to the X chromosome have common expression patterns and show similar responses to modulators of epigenetic mechanisms. We asked whether cancer-testis gene expression occurred coordinately, and whether it correlated with variables of disease and clinical outcome of non–small cell lung cancer (NSCLC).Experimental Design: Tumors from 523 NSCLC patients undergoing surgery were evaluated for the expression of nine cancer-testis genes (NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7/MAGE-C1, SSX2, and SSX4) by semiquantitative PCR. Clinical data available for 447 patients were used to correlate cancer-testis expression to variables of disease and clinical outcome.Results: At least one cancer-testis gene was expressed by 90% of squamous carcinoma, 62% of bronchioloalveolar cancer, and 67% of adenocarcinoma samples. Statistically significant coexpression was observed for 34 of the 36 possible cancer-testis combinations. Cancer-testis gene expression, either cumulatively or individually, showed significant associations with male sex, smoking history, advanced tumor, nodal and pathologic stages, pleural invasion, and the absence of ground glass opacity. Cox regression analysis revealed the expression of NY-ESO-1 and MAGE-A3 as markers of poor prognosis, independent of confounding variables for adenocarcinoma of the lung.Conclusions: Cancer-testis genes are coordinately expressed in NSCLC, and their expression is associated with advanced disease and poor outcome.

Purpose: Long peptides are efficiently presented to both CD4+ and CD8+ T cells after intracellular processing by antigen-presenting cells. To investigate the safety and in vivo immunogenicity of synthetic overlapping long peptides (OLP) from a human tumor self-antigen, we conducted a phase I clinical trial with OLP from cancer-testis antigen NY-ESO-1 in various adjuvant combinations.Experimental Design: Twenty-eight patients with advanced ovarian cancer in second or third remission were enrolled sequentially in three cohorts and received at least one vaccination. Patients in Cohort 1 (n = 4) received 1.0 mg OLP, Cohort 2 (n = 13) received OLP in Montanide-ISA-51, and Cohort 3 (n = 11) received OLP + 1.4 mg Poly-ICLC in Montanide-ISA-51 on weeks 1, 4, 7, 10, and 13. Humoral and cellular responses were evaluated by standardized immunomonitoring techniques (ELISA, ELISPOT assay, intracellular cytokine staining, and tetramer staining).Results: The vaccine was generally well tolerated with injection site reactions and fatigue that resolved. NY-ESO-1–specific antibody and CD8+ T cells were undetectable after vaccination with OLP alone, but were found in 6 of 13 (46%) and 8 of 13 (62%) patients, respectively, after vaccination with OLP+Montanide, and in 10 of 11 (91%) and 10 of 11 (91%) patients, respectively, after vaccination with OLP+Montanide+Poly-ICLC. NY-ESO-1–specific CD4+ T cells were detected in all patients with greater frequency and polyclonality when Montanide-ISA-51 was used for vaccination. Inclusion of Poly-ICLC as an adjuvant further accelerated the induction of NY-ESO-1–specific immune responses.Conclusions: The current study shows that NY-ESO-1 OLP vaccine is safe and rapidly induces consistent integrated immune responses (antibody, CD8+ and CD4+) in nearly all vaccinated patients when given with appropriate adjuvants. Clin Cancer Res; 18(23); 6497–508. ©2012 AACR.

PDF file - 196K, Humoral immune responses to NY-ESO-1 protein and peptides in individual patients throughout vaccination with NY-ESO-1 OLP. A) Reciprocal titers of plasma IgG antibodies were measured throughout vaccination by ELISA against NY-ESO-1 recombinant protein. Each patient was tested for all available time points, from prestudy (pre) up to week (w) 16. B) IgG plasma antibody responses were analyzed using 20-mer OLP in ELISA from samples at the time points where maximal titers had been achieved against NY-ESO-1 protein following vaccination. Peaks represent titers against 20-mer individual peptides centered around amino-acids from NY-ESO-1 shown along the X-axis. Reciprocal titers were considered significant if > 100. Assays representative of at least 2 repeats.

Supplementary Figure S1; Supplementary Table S1.

Purpose: Mixed bacterial vaccine (MBV, Coley's toxins) is a historical, vaguely defined preparation of heat-inactivated Streptococcus pyogenes and Serratia marcescens used as nonspecific immunotherapy in the treatment of cancer. The mechanism of action is suspected to have an immunologic basis, yet it is poorly defined up to now. We developed a new, biochemically well defined and current good manufacturing practice–compliant MBV preparation, which has been investigated in patients with NY-ESO-1 expressing cancers.Experimental Design: Patients received MBV subcutaneously at a starting dose of 250 EU (endotoxin units) twice a week. The MBV dose was escalated in each patient until a body temperature of 38°C to 39.5°C was induced or up to the maximum dose of 547.000 EU. Changes in serum cytokine levels were determined and immune responses to NY-ESO-1 were evaluated. Tumor response was assessed according to RECIST.Results: Twelve patients were enrolled and 11 of them developed fever after the administration of MBV. Ten of 12 patients showed a consistent increase in serum IL-6 levels with the highest levels coinciding with the highest body temperature. A subgroup of patients showed increasing levels of TNF-α, IFN-γ, and IL1-β. A patient with metastatic bladder cancer showed a partial tumor response strongly correlated with MBV-induced fever and highly elevated levels of several cytokines.Conclusions: MBV at fever-inducing dose levels can lead to a massive induction of immunoregulatory cytokines that may be involved in inducing tumor regressions. We propose to further explore the role of MBV as a potent immune modulator at higher dose levels and in conjunction with antigen-specific cancer vaccines. Clin Cancer Res; 18(19); 5449–59. ©2012 AACR.

PDF file - 65K, Immunohistochemical analysis of NY-ESO-1 expression (mAb E978) in tumor tissue and vaccine regimen

CT-X expression frequency and clinical characteristics

Purpose: Human endogenous retroviruses (HERV) encode 8% of the human genome. While HERVs may play a role in autoimmune and neoplastic disease, no mechanistic association has yet been established. We studied the expression and immunogenicity of a HERV-K GAG protein encoded on chromosome 22q11.23 in relation to the clinical course of prostate cancer.Experimental Design:In vitro expression of GAG-HERV-K was analyzed in panels of normal and malignant tissues, microarrays, and cell lines, and effects of demethylation and androgen stimulation were evaluated. Patient sera were analyzed for seroreactivity to GAG-HERV-K and other self-antigens by ELISA and seromics (protein array profiling).Results: GAG-HERV-K expression was most frequent in prostate tissues and regulated both by demethylation of the promoter region and by androgen stimulation. Serum screening revealed that antibodies to GAG-HERV-K are found in a subset of patients with prostate cancer (33 of 483, 6.8%) but rarely in male healthy donors (1 of 55, 1.8%). Autoantibodies to GAG-HERV-K occurred more frequently in patients with advanced prostate cancer (29 of 191 in stage III–IV, 21.0%) than in early prostate cancer (4 of 292 in stages I–II, 1.4%). Presence of GAG-HERV-K serum antibody was correlated with worse survival of patients with prostate cancer, with a trend for faster biochemical recurrence in patients with antibodies to GAG-HERV-K.Conclusions: Preferential expression of GAG-HERV-K ch22q11.23 in prostate cancer tissue and increased frequency of autoantibodies observed in patients with advanced prostate cancer make this protein one of the first bona fide retroviral cancer antigens in humans, with potential as a biomarker for progression and biochemical recurrence rate of prostate cancer. Clin Cancer Res; 19(22); 6112–25. ©2013 AACR.

PDF file 63K, Correlation between antibodies against GAG-HERV-K and group risk or Gleason score in cancer patients. Prostate cancer patients were split (A) in 3 groups according to Gleason score and classified from group risk 1 to 3 and compared to healthy donors (HD), or (B) in 3 groups according to Gleason score as indicated. Antibodies anti-GAG-HERV-K were detected and analyzed as in Fig. 4. Asterisk denotes significantly higher frequency of seropositive patients (titer > 100) in risk group 3 and Gleason {greater than or equal to} 8 (Chi-square test, p

PDF file 341K, qPCR gene expression analysis of chromosome 22q11.23 region in cancer tissues. Total RNA from cancer tissues was submitted to DNAse treatment and reverse transcriptase reaction for cDNA synthesis. The resulting cDNA was used on qPCR to detect the expression of GAG-HERV-K and the neighbor genes BCR, IGGL1, RGL4 and ZDHHC8P. The results are expressed as relative expression to GAG-HERV-K levels in normal prostate. GAG-HERV-K expression is relative to GAPDH. BCR, IGGL1, RGL4 and ZDHHC8P expression is relative to TFRC

PDF file, 86K, In-vitro activity of MBV on DC.

Supplementary Data from NY-ESO-1 DNA Vaccine Induces T-Cell Responses That Are Suppressed by Regulatory T Cells

Purpose: NY-ESO-1, one of the most immunogenic tumor antigens, is expressed in 15% to 25% of metastatic prostate cancers. The immunological and clinical effects of vaccination with recombinant NY-ESO-1 protein combined with CpG as adjuvant were evaluated.Experimental Design: In a phase I clinical study, patients with advanced prostate cancer were vaccinated with recombinant NY-ESO-1 protein (100 μg) mixed with CpG 7909 (2.5 mg) every 3 weeks intradermally for 4 doses. Objectives of the study were the safety of the vaccine and changes of specific humoral and cellular immunological responses to NY-ESO-1 in relation to detectable NY-ESO-1 expression in the individual tumor.Results: All 12 baseline sero-negative patients developed high-titer NY-ESO-1 antibody responses. B-cell epitope mapping identified NY-ESO-1 p91–110 to be recognized most frequently by vaccine-induced antibodies. Two patients developed significant antibody titers against the adjuvant CpG. NY-ESO-1-specific CD4+ and/or CD8+ T-cell responses were induced in 9 patients (69%). Five of these 9 patients did not express NY-ESO-1 in the autologous tumor. Postvaccine CD8+ T-cell clones recognized and lyzed HLA-matched tumor cell lines in an antigen-specific manner.Conclusion: Our data provide clear evidence for the capacity of NY-ESO-1 protein/CpG vaccine to induce integrated antigen-specific immune responses in vivo and to efficiently prime CD8+ T-cell responses in NY-ESO-1 antigen-negative patients. Our results may also support further clinical vaccination protocols with NY-ESO-1 protein not only focused on the treatment of existing cancer, but also to prevent further development of NY-ESO-1 positive cancers in vivo. Clin Cancer Res; 17(4); 1–10. ©2010 AACR.

PDF file - 1MB