Frederic Lehmann

Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate.

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20xa0months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10xa0months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation.

Transient expansion of peptide‐specific lymphocytes producing IFN‐γ after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage‐A1/A3‐positive tumors

Assessment of T‐cell activation is pivotal for evaluation of cancerimmunotherapy. We initiated a clinical trial in patients with MAGE‐A1and/or ‐A3 tumors using autologous DC pulsed with MAGE peptides aimedat analyzing T‐cell‐derived, IFN‐γ secretion by cytokine flowcytometry and ELISPOT. We also tested whether further KLH additioncould influence this response favorably. Monocyte‐derived DC weregenerated from leukapheresis products. They were pulsed with therelevant MAGE peptide(s) alone in group A (n=10 pts) andadditionally with KLH in group B (n=16 pts). A specific buttransient increase in the number of peripheral blood T lymphocytessecreting IFN‐γ in response to the vaccine peptide(s) was observed in6/8 patients of group A and in 6/16 patients of group B. We concludethat anti‐tumor vaccination using DC pulsed with MAGE peptides inducesa potent but transient anti‐MAGE, IFN‐γ secretion that is notinfluenced by the additional delivery of a nonspecific, T‐cellhelp.

Phase I clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion

BackgroundPatients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib.MethodsWe immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured.ResultsThis regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%).ConclusionsdHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway.Trial registryClinicalTrials.gov NCT00952692

IL-2 Production by Virus- and Tumor-Specific Human CD8 T Cells Is Determined by Their Fine Specificity

Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-γ, whereas effector memory CD8 cells (CD45RAdullCCR7−) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-γ. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.

Future perspectives in melanoma research. Meeting report from the "Melanoma Research: a bridge Naples-USA. Naples, December 6th-7 th2010"

Progress in understanding the molecular basis of melanoma has made possible the identification of molecular targets with important implications in clinical practice. In fact, new therapeutic approaches are emerging from basic science and it will be important to implement their rapid translation into clinical practice by active clinical investigation.The first meeting of Melanoma Research: a bridge Naples-USA, organized by Paolo A. Ascierto (INT, Naples, Italy) and Francesco Marincola (NIH, Bethesda, USA) took place in Naples, on 6-7 December 2010.This international congress gathered more than 30 international and Italian faculty members and was focused on recent advances in melanoma molecular biology, immunology and therapy, and created an interactive discussion across Institutions belonging to Government, Academy and Pharmaceutical Industry, in order to stimulate new approaches in basic, translational and clinical research. Four topics of discussion were identified: New pathways in Melanoma, Biomarkers, Clinical Trials and New Molecules and Strategies.

Safety and immunogenicity of the PRAME cancer immunotherapeutic in metastatic melanoma: results of a phase I dose escalation study

Purpose The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment. Patients and methods Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays. Results 66 patients were treated (20, 24 and 22 in the respective cohorts). AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected. Conclusions The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500u2005µg PRAME immunotherapeutic dose. Trial registration number NCT01149343, Results.

Correction: phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion

Corrections In our original manuscript [1], the corresponding author, Michael A Morse, was missed from the authors’ list. Therefore the correct author list should be: Erika Hamilton, Kimberly Blackwell, Amy C Hobeika, Timothy M Clay, Gloria Broadwater, Xiu-Rong Ren, Wei Chen, Henry Castro, Frederic Lehmann, Neil Spector, Junping Wei, Takuya Osada, H Kim Lyerly and Michael A Morse. Also the title of our original manuscript ‘Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion’ is also incorrect. The title should read:‘Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition’. Journal of Translational Medicine regret any inconvenience that this inaccuracy might have caused.

Seventh annual meeting of the Italian Network for Tumor Biotherapy (NIBIT), Siena, 1–3 October 2009

Abbreviations ACT Adoptive cell therapy APC Antigen-presenting cells AS Adjuvant system CIK Cytokine-induced killer COA Colon antigen CRC Colorectal cancer CSC Cancer stem cells CTC Circulating tumor cells CTL Cytotoxic T lymphocytes CTLA-4 Cytotoxic T lymphocyte-associated antigen-4 CTX Cyclophosphamide DC Dendritic cells DXR Doxorubicin HDI High-dose interferon-alpha-2b IDO Indoleamine 2,3-dioxygenase IFN Interferon IL Interleukin mAb Monoclonal antibody MM Metastatic melanoma NB Neuroblastoma NGR Asn-Gly-Arg NHL Non-Hodgkin lymphoma NK Natural killer PC Pancreatic cancer

Abstract A37: Identification of a gene expression signature predictive of clinical activity following MAGE‐A3 ASCI treatment

Clinical data today highlight the encouraging potential of the MAGE‐A3 Antigen‐Specific Cancer Immunotherapeutic (ASCI) for cancer treatment. The MAGE‐A3 gene encodes a tumor‐specific antigen expressed on tumor cells only. When used as a recombinant protein and combined with an immunological Adjuvant System designed to enhance the immune response to the MAGE‐A3 antigen, it was shown to induce specific T‐cell responses and long‐lasting clinical objective responses in metastatic melanoma (Phase II NCT00086866 trial) (Kruit et al. JCO 2008 26:9065), and demonstrated proof‐of‐concept in Non‐Small Cell Lung Cancer (NSCLC; Phase II NCT00290355 trial) (Vansteenkiste et al. JCO 2007 25:7554). In these 2 trials, gene expression profiling by microarrays was used to identify biomarkers predictive of the clinical activity of the MAGE‐A3 ASCI. A first analysis carried out on samples from melanoma patients using supervised hierarchical clustering of 2 objective responders and 2 non‐responders identified 2 gene clusters based on differential gene expression. This immune gene expression signature was then confirmed in an analysis of 22‐patient samples, and independently validated on an additional 30‐patient testing set; furthermore these analyses confirmed the association of clinical benefit and the molecular signature. Most of the identified genes are immune‐related, defining a particular biological context present in the tumor environment before immunization. This was confirmed by selecting genes using all patients eligible for gene expression profiling. Upon crossvalidation, median overall survival was improved significantly in the population of patients whose tumor presented the gene signature (GS): 28 months in the GS+ population, 16.2 months in the GS−. The predictive value of the melanoma signature was tested in NSCLC. A subset of 49 genes discovered in the melanoma Phase II trial was assayed by qPCR on biopsies taken prior to any ASCI treatment from 137 patients of the lung Phase II trial. Applied to NSCLC patients, this biomarker showed that the relative reduction in the risk of recurrence upon MAGE‐A3 ASCI treatment is increased by about 2 fold in the patients with the predictive gene signature as compared to the overall population: from 25% relative improvement in the overall population to 53% in patients whose tumor presents the predictive gene signature. In conclusion, two clinical proof‐of‐concepts have been obtained with the MAGE‐A3 ASCI in 2 different types of tumors, NSCLC and melanoma. More importantly, we have identified a gene expression signature predictive of clinical activity of the MAGE‐A3 ASCI treatment. The initiation of Phase III studies in NSCLC (MAGRIT) and melanoma (DERMA) is a unique opportunity to validate prospectively these biomarkers with the ultimate goal to select patients that are the most likely to benefit from the MAGE‐A3 ASCI therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A37.