Supplementary Figure 1 from The Regulatory T Cell–Associated Transcription Factor FoxP3 Is Expressed by Tumor Cells

BACKGROUND:To compare the clinical efficacy of New York Esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccine with ISCOMATRIX adjuvant versus ISCOMATRIX alone in a randomized, double-blind phase II study in participants with fully resected melanoma at high risk of recurrence. METHODS:Participants with resected stage IIc, IIIb, IIIc and IV melanoma expressing NY-ESO-1 were randomized to treatment with three doses of NY-ESO-1/ISCOMATRIX or ISCOMATRIX adjuvant administered intramuscularly at 4-week intervals, followed by a further dose at 6 months. Primary endpoint was the proportion free of relapse at 18 months in the intention-to-treat (ITT) population and two per-protocol populations. Secondary endpoints included relapse-free survival (RFS) and overall survival (OS), safety and NY-ESO-1 immunity. RESULTS:The ITT population comprised 110 participants, with 56 randomized to NY-ESO-1/ISCOMATRIX and 54 to ISCOMATRIX alone. No significant toxicities were observed. There were no differences between the study arms in relapses at 18 months or for median time to relapse; 139 vs 176 days (p=0.296), or relapse rate, 27 (48.2%) vs 26 (48.1%) (HR 0.913; 95% CI 0.402 to 2.231), respectively. RFS and OS were similar between the study arms. Vaccine recipients developed strong positive antibody responses to NY-ESO-1 (p≤0.0001) and NY-ESO-1-specific CD4+ and CD8+ responses. Biopsies following relapse did not demonstrate differences in NY-ESO-1 expression between the study populations although an exploratory study demonstrated reduced (NY-ESO-1)+/Human Leukocyte Antigen (HLA) class I+ double-positive cells in biopsies from vaccine recipients performed on relapse in 19 participants. CONCLUSIONS:The vaccine was well tolerated, however, despite inducing antigen-specific immunity, it did not affect survival endpoints. Immune escape through the downregulation of NY-ESO-1 and/or HLA class I molecules on tumor may have contributed to relapse.

Aim: Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. Patients & methods: Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+B cells followed by positive selection of CD1c+PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. Results: The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. Conclusion: This method was feasible and safe but was minimally immunogenic.

The ability of recombinant antibodies to adequately penetrate into tumours is a key factor in achieving therapeutic effect; however, the behaviour of antibodies at a cellular level in tumours is poorly understood. The purpose of this study was to investigate those factors that influence the macroscopic and microscopic intratumoural distribution of an IgG1-humanized antibody, huA33, in colorectal tumours.

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.

To determine the effect of Cancer-Testis Antigen (CTAg) expression on the natural history of primary cutaneous melanoma we compared its impact on prognosis with that of known prognostic factors and its relationship with other clinicopathologic characteristics. The immunohistochemical expression of three CTAgs (MAGE-A1, MAGE-A4 and NY-ESO-1) in 348 cases of stage I and stage II primary cutaneous melanoma was analysed and correlated with clinicopathologic characteristics, relapse free survival (RFS) and overall survival (OS). A Cox proportional hazards regression model was used to analyse factors which independently predicted RFS. All three CTAgs were significantly co-expressed with each other (p < 0.001). The median RFS for patients with CTAg-negative tumours and CTAg-positive tumours was 72 months and 45 months, respectively, (P = 0.008). Univariate analysis demonstrated that the impact of CTAg expression on RFS was comparable in magnitude to that of Breslow thickness, ulceration and tumour mitotic rate. Multivariate Cox regression analysis indicated that CTAg expression was a powerful independent predictor of RFS (risk ratio (RR) = 1.715, 95% confidence interval (CI) = 0.430-0.902, P = 0.010). In contrast, CTAg expression was demonstrated to have no prognostic impact on overall survival. This study demonstrates that CTAg expression in primary cutaneous melanoma is a strong independent predictor of RFS and it is comparable to other known important prognostic factors. CTAg expression has no relationship with overall survival, suggesting anti-melanoma immunity directed towards CTAg expression may contribute to the natural history of the disease. In view of these results, further investigation of the function of CTAgs and their potential use in therapeutic targeting is warranted.

Professional antigen-presenting cells (APCs) are sentinel cells of the immune system that present antigen to T lymphocytes and mediate an appropriate immune response. It is therefore surprising that knowledge of the professional APCs in human lymph nodes is limited. Using 3-color immunohistochemistry, we have identified APCs in human lymph nodes, excluding plasmacytoid APCs, that fall into 2 nonoverlapping classes: (1) CD209+ APCs, coexpressing combinations of CD206, CD14, and CD68, that occupied the medullary cords, lined the capsule and trabeculae and were also scattered throughout the diffuse T-lymphocyte areas of the paracortex; and (2) APCs expressing combinations of CD1a, CD207, and CD208, that were always restricted to the paracortex. Surprisingly, this second class of APCs was almost entirely absent from many lymph nodes. Our data suggest that most CD208+ cells, often referred to as "interdigitating cells," derive from migratory APCs, and that the major APC subset consistently resident in the paracortex of human lymph nodes is the CD209+ subset. All APC subsets were demonstrated to be in close contact with the fibroreticular network. The identification of 2 distinct APC populations in the paracortex of human lymph nodes has important implications for understanding T-lymphocyte responses and optimizing vaccine design.

Abstract FoxP3 is a member of the forkhead family of transcription factors critically involved in the development and function of CD25+ regulatory T cells (Treg). Until recently, FoxP3 expression was thought to be restricted to the T-cell lineage. However, using immunohistochemistry and flow cytometric analysis of human melanoma tissue, we detected FoxP3 expression not only in the tumor infiltrating Treg but also in the melanoma cells themselves. FoxP3 is also widely expressed by established human melanoma cell lines (as determined by flow cytometry, PCR, and Western blot), as well as cell lines derived from other solid tumors. Normal B cells do not express FoxP3; however, expression could be induced after transformation with EBV in vitro and in vivo, suggesting that malignant transformation of healthy cells can induce FoxP3. In addition, a FOXP3 mRNA variant lacking exons 3 and 4 was identified in tumor cell lines but was absent from Treg. Interestingly, this alternative splicing event introduces a translation frame-shift that is predicted to encode a novel protein. Together, our results show that FoxP3, a key regulator of immune suppression, is not only expressed by Treg but also by melanoma cells, EBV-transformed B cells, and a wide variety of tumor cell lines. [Cancer Res 2008;68(8):3001–9]

MEDICAL SCIENCES. For the article ‘‘A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors,’’ by Andrew M. Scott, Fook-Thean Lee, Niall Tebbutt, Rebecca Herbertson, Sanjeev S. Gill, Zhanqi Liu, Effie Skrinos, Carmel Murone, Timothy H. Saunder, Bridget Chappell, Anthony T. Papenfuss, Aurora M. T. Poon, Wendie Hopkins, Fiona E. Smyth, Duncan MacGregor, Lawrence M. Cher, Achim A. Jungbluth, Martin W. Brechbiel, Roger Murphy, Antony W. Burgess, Eric W. Hoffman, Terrance G. Johns, and Lloyd J. Old, which appeared in issue 10, March 6, 2007, of Proc Natl Acad Sci USA (104:4071– 4076; first published February 28, 2007; 10.1073 pnas.0611693104), the authors request that Gerd Ritter be added to the author list, between Achim A. Jungbluth and Martin W. Brechbiel, and be credited with contributing new reagents/ analytic tools. The online version has been corrected. The corrected author and affiliation lines, author contributions, and related footnote appear below.

An array of cell-surface antigens expressed by human cancers have been identified as targets for antibody-based therapies. The great majority of these antibodies do not have specificity for cancer but recognize antigens expressed on a range of normal cell types (differentiation antigens). Over the past two decades, our group has analyzed thousands of mouse monoclonal antibodies for cancer specificity and identified a battery of antibodies with limited representation on normal human cells. The most tumor-specific of these antibodies is 806, an antibody that detects a unique epitope on the epidermal growth factor receptor (EGFR) that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor in cancer. In vitro immunohistochemical specificity analysis shows little or no detectable 806 reactivity with normal tissues, even those with high levels of wild-type (wt)EGFR expression. Preclinical studies have demonstrated that 806 specifically targets a subset of EGFR expressed on tumor cells, and has significant anti-tumor effects on human tumor xenografts, primarily through abrogation of signaling pathways. The present clinical study was designed to examine the in vivo specificity of a chimeric form of mAb 806 (ch806) in a tumor targeting/biodistribution/pharmacokinetic analysis in patients with diverse tumor types. ch806 showed excellent targeting of tumor sites in all patients, no evidence of normal tissue uptake, and no significant toxicity. These in vitro and in vivo characteristics of ch806 distinguish it from all other antibodies targeting EGFR.

Desmoplastic melanoma is a diagnostic and therapeutic challenge. Immunohistochemical analysis with antibodies to melanoma antigens can complement morphologic evaluation. Although staining for S100 protein is generally positive, staining for other melanoma differentiation antigens, particularly gp100, Melan-A/MART1 and tyrosinase, is often negative despite being commonly positive in other melanoma types. A high clinical index of suspicion and better diagnostic techniques are essential as atypical features and incorrect diagnosis can lead to poor clinical outcomes. Antigens associated with melanoma, such as the melanocyte differentiation and cancer testis antigen, may become important targets for immune therapies. We characterized the patterns of antigen expression of desmoplastic melanoma from 32 patients, including gp100, Melan-A/MART-1, tyrosinase, MAGE-A1, MAGE-A4 and NY-ESO-1. Consistent positive staining with S100 was observed. Differentiation antigens were expressed more frequently than cancer testis antigens regardless of the histological subtype of desmoplastic melanoma. When present, cancer testis antigen expression correlated to positive staining with differentiation antigens. The diagnostic yield of desmoplastic melanoma did not increase with the addition of cancer testis antigen typing. Low levels of expression of cancer testis antigen may indicate that they are suboptimal targets for vaccine development in desmoplastic melanoma.

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.

Purpose: We report a first-in-man trial of a humanized antibody (hu3S193) against the Ley antigen. Experimental Design: Patients with advanced Ley-positive cancers received four infusions of hu3S193 at weekly intervals, with four dose levels (5, 10, 20, and 40 mg/m2). The first infusion of hu3S193 was trace labeled with Indium-111, and biodistribution, pharmacokinetics, tumor uptake, and immune response were evaluated in all patients. Results: A total of 15 patients (7 male/8 female; age range, 42-76 years; 6 breast, 8 colorectal cancer, and 1 non–small-cell lung cancer) were entered into the study. Transient grade 1 to 2 nausea and vomiting was observed following infusion of hu3S193 at the 40mg/m2 dose level only. There was one episode of dose-limiting toxicity with self-limiting Common Toxicity Criteria grade 3 elevated alkaline phosphatase observed in one patient with extensive liver metastases. The biodistribution of 111In-hu3S193 showed no evidence of any consistent normal tissue uptake, and 111In-hu3S193 uptake was observed in cutaneous, lymph node, and hepatic metastases. Hu3S193 displayed a long serum half-life (T1/2β = 189.63 ± 62.17 h). Clinical responses consisted of 4 patients with stable disease and 11 patients with progressive disease, although one patient experienced a 89% decrease in a lymph node mass, and one patient experienced inflammatory symptoms in cutaneous metastases, suggestive of a biological effect of hu3S193. No immune responses (human anti-human antibody) to hu3S193 were observed. Conclusion: Hu3S193 is well tolerated and selectively targets tumors, and the long half-life and biological function in vivo of this antibody makes it an attractive potential therapy for patients with Ley-expressing cancers.

Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 microg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines.

We conducted an open label dose-escalation phase I trial of chimeric anti-GD3 mAb KM871 in patients with metastatic melanoma. Patients were entered into one of five dose levels (1, 5, 10, 20, and 40 mg/m2) and received three infusions of KM871 at 2-wk intervals. A metastatic melanoma site was biopsied at day 7-10. Pharmacokinetics, immune function, and mechanism of action of KM871 were analysed. A total of 17 patients were entered into the trial; 15 were evaluable. KM871 had a serum half-life (T1/2-beta) based on ELISA of 10.39 +/- 1.12 d (mean +/- SD). Trough levels >1.0 microg/mL KM871 at 2 wk postinfusion were seen with the 10 mg/m2 and higher dose levels. There were no significant changes in white blood cell subsets or serum complement levels during KM871 treatment. KM871 was stable in vivo and maintained binding affinity and complement-dependent cytotoxicity (CDC) function up to 2 wk postinfusion. No significant trends in CDC or antibody-dependent cellular-cytotoxicity (ADCC) activity in patients were observed during treatment. Analysis of tumour biopsies demonstrated a significant increase in CD4+ T cell infiltrates compared to control patient tumours (P = 0.010), and in patients with either stable disease (2 patients) or a clinical partial response (1 patient) at restaging, a significant increase in CD3 and CD4 infiltrates in tumour over nonresponding patients was observed. The favourable immune properties of KM871, combined with this preliminary clinical data, indicate that KM871 has potential for the treatment of metastatic melanoma.

Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 (rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.