Peptide-specific Immune Responses Research Articles

BCR-ABL Derived Peptide Vaccine in Chronic Myeloid Leukemia Patients with Molecular Minimal Residual Disease During Imatinib: Interim Analysis of a Phase 2 Multicenter GIMEMA CML Working Party Trial.

Abstract 648 Introduction:Imatinib (IM) 400mg daily is the standard treatment for chronic myeloid leukemia (CML) patients and a complete cytogenetic response (CCyR) is achieved in the majority of patients within one year of treatment. In addition, a considerable number of patients reach a major molecular response (i.e BCR-ABL/ABL ratio <0.1%) but BCR-ABL transcript is still measurable in most of treated patients revealing the persistence of a minimal residual disease (MRD). In a previous small pilot study, vaccinations with p210 b3a2-derived fusion peptides in IM treated CML patients appeared to induce both a peptide specific immune response and a reduction of residual disease surviving IM. Methods: To investigate the efficacy of this immune based targeted approach in a larger cohort of patients we designed a phase 2 multicenter study (GIMEMA CML0206) employing 5 p210 b3a2-derived peptides (CMLVAX100 vaccine) in CML patients with at least 18 months of IM treatment and persistence of molecular residual disease. Each vaccination consisted of CMLVAX100 plus 2 doses of GM-CSF as immunological adjuvant. Treatment schedule included 6 biweekly vaccinations (immunization phase) followed by 3 monthly boosts (reinforcement phase) and 2 tri-monthly boosts (maintenance phase). The primary endpoint of the trial was to assess the rate of response (patients showing a reduction by at least 50% of peripheral blood BCR-ABL/ABL ratio compared to the individual prevaccine level) evaluated after immunization and reinforcement boosts (evaluation after 6 months, ) and persisting at the 9th month (after 10th vaccination). Secondary endpoints included the rate of undetectable transcript at any time after immunization and the rate of peptide-specific immune response induced by the vaccinations. Patients population: At present 57/69 planned patients have been enrolled and 43/57 are evaluable for response. Twentyseven are males and 16 are females with a median age of 56.5ys (range 29-78). At diagnosis, 25/43 (58%) patients presented with low, 15/43 (35%) with intermediate and 3/43(7%) with a high Sokal risk. Twenty-one out of 43 patients (49%) started standard IM treatment while in late chronic phase (CP) after a median time from diagnosis of 29 months during which they mainly received alpha interferon therapy. On the contrary 22/43(51%) patients started IM immediately after diagnosis. All patients entered the vaccination protocol after at least 18 months of IM treatment and the median time of exposure to this tyrosin kynase inhibitor before peptide vaccination was 54 months (range 23-100). All patients had obtained a CCyR before entering the study (after a median time of 6 months of IM treatment) and were still in CCyR at enrollment with a median duration of CCyR of 47 months. All patients started vaccination with persisting measurable molecular disease in peripheral blood (any level of BCR/ABL transcript). Results:Current interim analysis shows that vaccinations (a total of over 400 shots) were very well tolerated, with CMLVAX100-GMCSF toxicity consisting exclusively of some redness and itching at the site of injection and with only 4/43 patients (9%) experiencing a mild fever. Regarding immune response induced by vaccination, 29/43 patients (67%) showed a significant in vitro b3a2-peptide-specific CD4+ T cell proliferation. With respect to MRD response, we observed a reduction of at least 50% of pre-vaccine BCR-ABL/ABL values after 6 months of treatment (i.e. after 9 vaccinations) in 22/43 (51%) patients and the reduction was confirmed in 14/29 (48%) patients who reached the 9th month evaluation (i.e. after 10 vaccinations); 14/43(32%) patients had at least one documented undetectable transcript during this time period. In 2/43 patients we observed a significant raise of BCR-ABL transcript level, after 3 and 18 months from starting vaccinations with subsequent loss of CCyR. Conclusions: CMLVAX100 vaccine appears to induce a reduction of long lasting molecular MRD surviving IM in about half of vaccinated CML patients, thus confirming preliminary results. If this BCR-ABL-specific immune control of MRD will have a substantial impact on the rate of BCR-ABL mutations, disease evolution and ultimately survival needs longer observation time to be determined. Disclosures:Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Peptide-Vaccine Treatment Associated with TKI Therapy in Patients with CML Is Able to Induce Immunologic, Cytogenetic and Molecular Responses: a Single Center Experience with Long-Term Follow up.

Abstract 2185Poster Board II-162 Introduction:The main objective in the intent to “cure” chronic myeloid leukemia (CML) is to obtain complete cytogenetic remission (CCR) and molecular remission. Tyrosin kinase inhibitors (TKI) treated patients (pts) achieve CCR, but BCR-ABL transcripts can still be detectable, and complete molecular remission (CMR), intended as undetectable transcript, are rare. Moreover about 10% of up-front treated patients show resistance to Imatinib, that reaches 30% in late chronic phase, loss of response during treatment is not negligible, and treatment cannot be stopped. Thus the eradication of residual disease still appears a difficult goal for a TKI alone. An alternative approach to target the residual disease is an active specific immunotherapy. We associated TKI therapy and immunogenic peptides derived from the p210 b3a2 and b2a2 fusion protein (developed by M. Bocchia et al. University of Siena) in pts with chronic phase (CP) CML with stable disease in trying to obtain a specific immunologic activation able to induce a measurable clinical response. Patients population and methods:17 pts (11 M:6 F) with CP CML, median age 55,5 (range 30-68) treated with Imatinib (16) or Dasatinib (1) were enrolled in the studies. All patients but one were in late chronic phase and had been treated with 2 (2 pts), 3 (11 pts) or >3 (4 pts) lines of therapies. Median time from diagnosis was 64.1(16-143) months, and patients were treated for a median of 30.8 months with TKI before peptide vaccination. 15 pts had b3a2 and 2 a b2a2 transcript. Pts presented with stable, measurable disease at cytogenetic or molecular level from at last 6 months. Vaccination included GM-CSF pre treatment and administration of 5 p210 b3a2 (CMLVAX100) or 1 b2a2 (CMLVAX25) derived peptides. Treatment plan consisted of an induction plan of 6 vaccinations every two weeks, followed by additional boosts every 3-6 months for responding patients. During vaccination, patients continued their conventional treatment with Imatinib/Dasatinib. Prior to vaccine all patients were tested with an intradermal injection of peptides (DTH) to evaluate their sensitivity to the CML antigens, and all of them resulted negative. Cytogenetics, FISH and molecular biology, peptide-specific immune responses (DTH, CD4+ proliferation, immunophenotype) were analyzed before and during treatment. Results:15/17 pts are evaluable (2 patients had just completed the first 3 months and were not considered for their short follow up), and all patients but one showed a variable degree of response. All patients presented with some degree of DTH indicating the “recognition” of peptides by effector T cells (biologic response). 5/9 pts with positive cytogenetic (2-66% Ph+) reached CCR, and 3 also CMR, while 1 patient did not respond (the one with high tumor burden, 66% Ph+). 3/6 pts in CCR at time of vaccination reached CMR. The majority of responses were rapidly reached (after the induction) and were long lasting. After 69 month follow up 6/15 patients are still treated. Patients suspended vaccination due to: no response (1), lost CCR (5), progression (1), 2nd neoplasm (1), allergic reaction (1). One patient that reached CCR and MCR after vaccination stopped imatinib and was closely monitored thereafter. She is now treated with only vaccine boosts twice/year and still in CMR after 28 month. Specific immune response will be described. Conclusions:These data suggest that addition of b3a2 or b2a2-specific vaccine may have synergistc effect with TKI favouring reduction of residual disease and increasing the number of patients that reach CMR. A multicentric trial is ongoing through the GIMEMA CML study group, and a pilot study to stop TKI in long lasting CMR is in preparation. Disclosures:No relevant conflicts of interest to declare.

Universal influenza DNA vaccine encoding conserved CD4 + T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid™ in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4 + T cell responses to protection against influenza infection.

Multipeptide vaccination in cancer patients

Since the identification of tumor associated antigens (TAA) in different tumor histotypes, many vaccination strategies have been investigated, including peptide-based vaccines. Results from the first decade of clinical experimentation, though demonstrating the feasibility and the good toxicity profile of this approach, provided evidence of clinical activity only in a minority of patients, despite inducing immunization in up to 50% of them. In this review, we discuss the different approaches recently developed in order to induce stronger peptide-induced immune-mediated tumor growth control, possibly translating into improved clinical response rates, with specific focus on multipeptide-based anti-cancer vaccines. This strategy offers many advantages, such as the possibility of bypassing tumor heterogeneity and selection of antigen (Ag)-negative clones escaping peptide-specific immune responses, or combining HLA class I- and class II-restricted epitopes, thus eliciting both CD4- and CD8-mediated immune recognition. Notably, advances in Ag discovery technologies permit further optimization of peptide selection, in terms of identification of tumor-specific and unique TAA as well as Ags derived from different tumor microenvironment cell components. With the ultimate goal of combining peptide selection with patient-specific immunogenic profile, peptide based anti-cancer vaccines remain a promising treatment for cancer patients, as attested by of pre-clinical and clinical studies.

Vaccination with human papillomavirus type 16-derived peptides using a tattoo device

Tattooing has been shown to be very efficient at inducing immunity by vaccination with DNA vaccines. In this study, we examined the usability of tattooing for delivery of peptide vaccines. We compared tattooing with subcutaneous (s.c.) needle injection using peptides derived from human papillomavirus type 16 (HPV16) proteins. We observed that higher peptide-specific immune responses were elicited after vaccination with the simple peptides (E7 44–62 and E7 49–57) and keyhole limpet hemocyanin-(KLH)-conjugated peptides (E7 49–57, L2 18–38 and L2 108–120) with a tattoo device compared to s.c. inoculation. The administration of the synthetic oligonucleotide containing immunostimulatory CpG motifs (ODN1826) enhanced the immune responses developed after s.c. injection of some peptides (E7 44–62, KLH-conjugated L2 18–38 and L2 108–120) to levels close to or even comparable to those after tattoo delivery of identical peptides with ODN1826. The highest efficacy of tattooing was observed in combination with ODN1826 for the vaccination with the less immunogenic E6 48–57 peptide and KLH-conjugated and non-conjugated E7 49–57 peptides which form the visible aggregates that could negatively influence the development of immune responses after s.c. injection but probably not after tattooing. In summary, we first evidenced that tattoo administration of peptide vaccines that might be useful in some cases efficiently induced both humoral and cell-mediated immune responses.

Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge in vitro and in vivo (129.16)

Abstract The release of Bacillus anthracis spores can cause significant morbidity and mortality and an effective vaccine as well as directed immunotherapeutics are needed. The currently licensed vaccine requires six primary vaccinations, annual boosters, and has been associated with serious adverse events. Thus there is increasing interest in developing an improved vaccination strategy which can generate lasting protective immunity with reduced vaccinations. This study seeks to identify the major humoral targets of B. anthracis that provide protection. To this end, plasma was obtained from two hundred vaccinated individuals and thirty unvaccinated controls. Ninety-five percent of all vaccinated individuals had anti-protective antigen antibodies and these antibodies correlated with in vitro protection against lethal toxin. Utilizing overlapping decapeptides, we identified common antigenic regions within those individuals with high toxin neutralizing activity. Epitope-specific antibodies directed against three of these regions were isolated and were able to mediate protection in vitro and in a mouse model of in vivo lethal toxin challenge. Identification of common, protective, peptide-specific humoral immune responses provide key information for further immunotherapeutic and vaccine development. NIAID: 5U19AI062629

The effects of age on CD4 upregulation and Treg distribution in a TAChR peptide-specific immune response (47.1)

Abstract Studies of age-related defects in T cell function have been complicated by the difficulty of identifying antigen-specific T cells early in the activation process. Thus, peptide-responsive T cells were identified and characterized by the upregulation of CD4, an early event following T cell receptor ligation. We focused on the Torpedo californica acetylcholine receptor (TAChR) peptide, p146-162, since responses to this peptide have been well defined in young mice. In old mice, the fine specificity of the TAChR response was preserved with age; p146-162 remained immunodominant. Yet, in vitro proliferative responses to p146-162 were diminished in a dose-dependent manner in lymph node cells (LNC) derived from old peptide-primed mice. When p146-162-specific Vβ6+CD4high T cells derived from young (2-5 months) and old (20-27 months) mice were examined, the upregulation of CD4 was maintained in old age but the frequency of Vβ6+CD4high T cells was often lower; reflecting fewer CD4+ LNC, changes in TCR repertoire selection and/or altered T cell regulation. Interestingly, Foxp3+CD25+CD4+ T regulatory cell (Treg) frequencies were elevated in the old peptide-responding populations. The Vβ6-CD4normal Treg phenotype predominated in both age groups, yet a unique "activated" CD4high Treg subpopulation was also identified. Future studies may help elucidate the potential role of these CD4high Tregs in promoting age-related immune dysfunction. Support: Myasthenia Gravis Foundation, Nathan Shock Aging Center, NIH R03 AG 14557 and San Antonio Area Foundation.

Antitumoral Immune Response by Recruitment and Expansion of Dendritic Cells in Tumors Infected with Telomerase-Dependent Oncolytic Viruses

Virotherapy can potentially be used to induce tumor-specific immune responses and to overcome tumor-mediated tolerance mechanisms because apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor-specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide-specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T-cell responses against intracellular tumor antigens. Triggering of DC and T-cell infiltration to the oncolytic tumors by macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Although immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1alpha/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1alpha/FLT3L virotherapy, we observed systemic antitumoral effects by MIP-1alpha/FLT3L virotherapy on uninfected lung metastasis in immunocompetent mice but not in nude mice. Furthermore, MIP-1alpha/FLT3L virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long-term survival of 50% of treated animals. In summary, improvement of cross-presentation of tumor antigens by triggering of DC and T-cell infiltration during virotherapy enhances antitumoral immune response that facilitates an effective viroimmunotherapy of primary tumors and established metastases.

Antitumoral immune response by recruitment and expansion of dendritic cells in tumors infected with telomerase-dependent oncolytic viruses

Virotherapy can potentially be used to induce tumor specific immune responses and to overcome tumor-mediated tolerance mechanisms, since apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T cell responses against intracellular tumor antigens. Triggering of DC and T cell infiltration to the oncolytic tumors by Macrophage Inflammatory Protein 1a (MIP-1a, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Though immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1a/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1a/FLT3L-virotherapy, we observed systemic antitumoral effects by MIP-1a/FLT3L-virotherapy on uninfected lung metastasis in immunocompetent mice, but not in nude mice. Furthermore, MIP-1a/FLT3L-virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long term survival of 50% of treated animals.

Clinical and immunologic response of patients with advanced solid tumors vaccinated with an optimized cryptic hTERT peptide (Vx-001)

3030 Background: The clinical and immunologic efficacy of the optimized peptide TERT572Y (Vx-001) presented by HLA- A*0201 in patients with advanced malignancies was investigated. Methods: In the context of an expanded phase I-II study, 71 patients with advanced solid tumors (breast cancer n=10; NSCLC n=11; prostate cancer n=10; mCRC n=2; RCC n=6; pancreatic cancer/cholangiocarcinoma n=14; melanoma n=8; HCC n=3; others n=7) who had been previously treated with standard chemotherapy (disease status at enrollment: stable disease n=21 and progressive disease n=50) received two subcutaneous injections of 2 mg of the optimized TERT572Y peptide followed by four injections of 2 mg of the native TERT572 peptide given every three weeks. The peptide-specific immune responses were assessed by interferon-γ Elispot at baseline, before the 3rd (early response) and after the 6th (late response) vaccination. Clinical outcome was evaluated after the 6th vaccination (based on RECIST criteria) and every three months thereafter for patients who did not progress. Results: Thirty-seven (52%) out of 71 patients completed the vaccination program. The main toxicity was grade 1 local skin reactions. An early and late immunologic response was detected in 29 of 56 (51.8%) and 25 of 30 (83%) evaluable patients, respectively. There were three (4.2%) objective clinical responses (HCC n=1; NSCLC n=2) and 22 (31%) disease stabilizations. All disease stabilizations occurred in early immunologically responding patients. Among the patients with PD before vaccination the median overall survival was 23.5 versus 7 months (p=0.056) in early immune responders versus non-responders, respectively, and was significantly higher in patients with late immunologic response (not reached) than in non-responding patients (9.5 months) (p=0.007). Conclusions: Vx-001 is a strongly immunogenic vaccine capable of inducing clinical responses in immunologically responding patients with progressive advanced solid tumors. Detailed and updated results will be presented at the meeting. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Vaxon Biotech

A multicenter phase I/II study of gemcitabine and personalized peptide vaccination combination therapy for metastatic pancreatic cancer patients

4633 Background: The present multicenter phase I/II study was conducted to confirm the efficacy and toxicity of immunochemotherapy for metastatic pancreatic cancer. Methods: HLA-A2 or A24 positive patients with histologically or cytologically proven pancreatic adenocarcinoma with at least one measurable metastatic lesion were eligible for the study. Other eligibility criteria included: age of 20–80 years, ECOG performance status (PS) of 0 or 1, and adequate organ function. Gemcitabine was given intravenously at a dose of 1,000 mg/m2 over 30 min once a week for three weeks, followed by 1 week of rest, and three or four peptides that had been positive for pre-existing peptide-specific immune responses in the circulation (personalized peptide vaccine) were injected subcutaneously in the femoral area once a week. The objective response rate was assessed according to RECIST. Results: Twenty patients from 2 institutions were enrolled in this study. The median age was 63 years (range: 41–80 years). The PS was 0 in 10 patients (50%) and 1 in 10 patients (50%). The efficacy and toxicity were analyzed in 20 patients who received at least one course (8 weeks) of immunochemotherapy. Although no complete response was seen, a partial response was achieved in 5 patients, resulting in an overall response rate of 25%. Eleven patients (55%) had stable disease. The median overall survival was 8.5 months with a 1-year survival rate of 33%. The major grade 3–4 toxicities using CTCAE criteria were neutropenia (25%), anemia (20%), thrombocytopenia (20%) and anorexia (5%). All 17 dermatologic reactions at the vaccination site were scored as grade 1 or 2. No treatment-related deaths occurred during the study. Augmentation of peptide-specific CTL responses in the post-vaccination peripheral blood mononuclear cells was observed in 72%, while increased titer of peptide-specific IgG antibodies was observed in the post-vaccination plasma in 78%. Conclusions: This study could be recommended for further stages of clinical trials because of safety, boosting of immune responses, and potential clinical benefits. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GreenPeptide GreenPeptide GreenPeptide Grant-in-Aid for Scientific Research from Research Center of Innovative Cancer Therapy of the 21st Century COE Program for Medical Science of Japan, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan

Contribution of influenza immunity and virosomal-formulated synthetic peptide to cellular immune responses in a phase I subunit malaria vaccine trial

We have demonstrated recently in a phase Ia clinical trial that synthetic malaria peptides delivered by immuno-potentiating reconstituted influenza virosomes (IRIV) induced long-lived peptide-specific antibody responses in all volunteers. In the current ancillary study to this clinical trial we have investigated the cellular immune responses specific for IRIV and the surface bound synthetic malaria peptides tested. After vaccination, in 50% (8/16) of the volunteers at least one positive lymphoproliferative response specific for the 49mer peptide derived from the Plasmodium falciparum apical membrane antigen-1 (AMA-1) was observed with stimulation indices ranging from 2 to 4.5. All volunteers showed pre-existing IRIV specific cellular immunity assessed by ex vivo IFN-γ ELISpot analysis and lymphoproliferation. The pre-existing influenza specific T cell responses did not interfere negatively with the induction of malaria peptide-specific humoral and cellular immune responses. Our results support the view that IRIV constitute a safe antigen delivery system for induction of peptide-specific immune responses in human populations.

Fine Specificity of Neonatal Lymphocytes to an Abundant Malaria Blood-Stage Antigen: Epitope Mapping ofPlasmodium falciparumMSP133

Cord blood T cells have been reported to respond to a variety of exogenous Ags, including environmental allergens and various viruses and parasites, as demonstrated by enhanced proliferation and cytokine secretion. This finding is evidence that Ags in the maternal environment transplacentally prime and result in fetal development of memory T cells. Some studies suggest these neonatal T cell responses may arise by nonspecific activation of T cells that express TCRs with low binding affinity, thus lacking fine lymphocyte specificity. To address this question, we examined malaria Ag stimulation of human cord and adult blood mononuclear cells in samples from residents of a malaria endemic area in Kenya. We constructed overlapping 18-mer peptides derived from sequences contained in dimorphic alleles of the C-terminal 33-kDa fragment of Plasmodium falciparum merozoite protein 1. This study identified a dominant T cell epitope for one MSP1(33) allele (MAD20) and two T cell epitopes for the second allele (K1); these epitopes were nonoverlapping and allele specific. In a given donor, peptide-specific proliferation and IFN-gamma secretion were highly concordant. However, IL-10 and IL-13 secretion were not correlated. Importantly, the fine specificity of lymphocyte proliferation and cytokine secretion in cord and adult blood mononuclear cells was similar. Cord blood cells obtained from malaria-infected pregnant women were 4-fold more likely to acquire a peptide-specific immune response. We conclude that the fetal malaria response functions in a fully adaptive manner and that this response may serve to help protect the infant from severe malaria during infancy.

Quantification and Phenotypic Characterization of Circulating Tumor Cells for Monitoring Response to a Preventive HER2/neu Vaccine-Based Immunotherapy for Breast Cancer: A Pilot Study

Ongoing cancer vaccine trials are limited by the inability of immunologic assays to monitor clinically relevant surrogates of response. Recent advances in the ability to quantify and phenotype circulating tumor cells (CTCs) in breast cancer patients may lead to a role for CTCs in monitoring response to vaccine-based immunotherapy. The CellSearch System (Veridex-LLC, Warren, NJ) was used to enumerate total and HER2/neu+ CTCs in 20 mL of blood from all 16 node-positive (NP) breast cancer patients active in our NP HER2/neu E75 peptide vaccine trial at the initiation of this pilot study. These patients were vaccinated with E75 (1000 microg)/GM-CSF (250 microg) monthly x 6 after completion of multimodality therapy. Mean (+/-SEM) number of CTCs and HER2/neu+ CTCs were compared in unmatched (n = 16) and matched (n = 9) prevaccination and postvaccination cases. CTCs were detected in 14 of 16 (88%) patients (mean: 3.4 +/- 0.2 CTC/20 mL). After vaccination, a reduction in CTC/20 mL (prevaccination 3.9 +/- 1.5 vs postvaccination 0.7 +/- 0.4, P = .077) and HER2/neu+ CTC/20 mL (prevaccination 2.8 +/- 1.0 vs postvaccination 0.5 +/- 0.2, P = .048) was demonstrated. A significant delayed-type hypersensitivity (DTH) response suggesting that vaccination was effective in eliciting a peptide-specific immune response was confirmed (22.3 +/- 4.1 vs 3.0 +/- 2.2 [controls] mm, P < .01). All nine patients followed throughout the vaccination series also showed significant reduction in CTCs (4.8 +/- 1.5 vs 0.3 +/- 0.2, P < .01) and HER2/neu+ CTCs (3.0 +/- 0.9 vs 0.4 +/- 0.2, P = .013). CTCs are readily demonstrated in posttreatment, clinically disease-free NP breast cancer patients. E75+GM-CSF vaccination appears to reduce the number of CTCs. These data suggest a potential role for this clinically validated CTC assay in assessing response to preventive vaccine-based immunotherapy, and further validation studies are underway.

Vaccination of Patients With Advanced Non–Small-Cell Lung Cancer With an Optimized Cryptic Human Telomerase Reverse Transcriptase Peptide

To evaluate the immunological and clinical response as well as the safety of the optimized peptide telomerase reverse transcriptase p572Y (TERT572Y) presented by HLA-A*0201 in patients with advanced non-small-cell lung cancer (NSCLC). Twenty-two patients with advanced NSCLC and residual (n = 8) or progressive disease (PD; n = 14) following chemotherapy and/or radiotherapy received two subcutaneous injections of the optimized TERT572Y peptide followed by four injections of the native TERT572 peptide administered every 3 weeks. Peptide-specific immune responses were monitored by enzyme-linked immunosorbent spot assay and/or TERT572Y pentamer staining. Twelve (54.5%) of 22 patients completed the vaccination program. Toxicity consisted primarily of local skin reactions. TERT572-specific CD8+ cells were detected in 16 (76.2%) of 21 patients after the second vaccination, and 10 (90.9%) of 11 patients after the sixth vaccination. Stable disease (SD) occurred in eight (36.4%) of 22 vaccinated patients, with three (13.6%) having had PD before entering the study. The median duration of SD was 11.2 months. After a median follow-up of 10.0 months, patients with early developed immunological response (n = 16) had a significantly longer time to progression and overall survival (OS) than nonresponders (n = 5; log-rank tests P = .046 and P = .012, respectively). The estimated median OS was 30.0 months (range, 2.8 to 40.0 months) and 4.1 months (range, 2.4 to 10.9 months) for responders and nonresponders, respectively. TERT572Y peptide vaccine is well tolerated and effective in eliciting a specific T cell immunity. Immunological response is associated with prolonged survival. These results are encouraging and warrant further evaluation in a randomized study.

Vaccination of patients with advanced non-small cell lung cancer with an optimized cryptic hTERT peptide (Vx-001)

7642 Background: To evaluate the immunological and clinical efficacy of the optimized peptide TERT572Y (Vx-001) presented by HLA-A*0201 in patients with advanced non-small cell lung cancer (NSCLC). Methods: Twenty-two patients with residual (n=8) or progressive (n=14) advanced NSCLC following chemotherapy and/or radiotherapy received two subcutaneous injections of the optimized TERT572Y peptide followed by four injections of the native TERT572 peptide given every 3 weeks. Peptide-specific immune responses were monitored by Elispot assay and/or TERT572Y pentamer staining. Clinical outcome was compared with that of 22 case-matched historical control patients. Results: Thirteen (59%) out of 22 patients completed the vaccination program. Toxicity consisted primarily of local skin reactions. TERT572-specific CD8+ cells were detected in 16 (76.2%) out of 21 patients after the 2nd vaccination and 10 (90.9%) out of 11 patients after the 6th vaccination. Stable disease occurred in 8 (36.4%) patients with a median duration of 11.2 months. Patients with an early immunological response (n=16) had a significantly longer time to disease progression and overall survival than non-responders (n=5) (log-rank tests p=0.046 and p=0.012, respectively). The estimated median overall survival was 30.0 (range, 2.8–40.0) and 4.1 (range, 2.4–10.9) months for immunological responders and non-responders, respectively. Moreover, median overall survival was 30.6 months (95% CI:10.9–48.9) and 6.1 months (95% CI:4.4–7.8) for the vaccinated and case-matched historical control patients, respectively (p=0.074). Conclusions: TERT572Y peptide vaccine is well tolerated and effective in eliciting a specific T-cell immunity which seems to be associated with prolonged patients’ survival. No significant financial relationships to disclose.

The combination of GM-CSF and IL-2 as local adjuvant shows synergy in enhancing peptide vaccines and provides long term tumor protection

Many strategies have been used to enhance the peptide vaccine immune response and to establish therapeutic benefits. This includes the utilization of cytokines to improve antigen presentation or enhance T cell response. Here, we have tested the combination of GM-CSF and IL-2 as locally administered adjuvant to enhance the immune response to the HPV16 E7 peptide. Female C57BL/6 mice were immunized intradermally with a 9-mer HPV16 E7 peptide (aa: 49-57) alone, or in combination with GM-CSF, IL-2, or both cytokines. Specific immune responses were measured by ELISA and Chromium-Release Assays. Furthermore, therapeutic effects of these vaccines and long term tumor protection were assessed in mice bearing established tumors. We showed that GM-CSF and IL-2, when co-administered locally in an emulsion with peptide, exert a synergistic effect in enhancing the immune response to the antigen. This combination induced higher CTL and cytokine release responses and did not increase the T(reg) population. Therapeutic intervention with this synergistic combination led to a complete response of established tumors. Furthermore, this combination induced a memory response which protected mice against subsequent additional tumor challenge. We identified a new vaccine adjuvant, a local combination of GM-CSF and IL-2, which is synergistic in enhancing peptide specific immune response through local effect without increasing T(reg) cells. This immune response was found to be long lasting and protective in tumor bearing mice.

Induction of Peptide-Specific Immune Response in Patients with Primary Malignant Melanoma of the Esophagus after Immunotherapy Using Dendritic Cells Pulsed with MAGE Peptides

Primary malignant melanoma of the esophagus (PMME) is a very rare disease with an extremely poor prognosis. Surgery is currently considered its best treatment, while any other measures are ineffective. We studied the effect of active specific immunotherapy using monocyte-derived dendritic cells (DCs) pulsed with the epitope peptides of melanoma-associated antigens (MAGE-1, MAGE-3) in patients with PMME after surgery, for the first time. The patient received passive immunotherapy with lymphokine-activated killer cells concomitantly. Two HLA-A24-positive patients with PMME were treated. Both patients initially received radical esophagectomy with regional lymphadenectomy, followed by adjuvant chemotherapy with dacarbazine, nimustine, vincristine and interferon-alpha. In the case 1 patient, active specific immunotherapy was used to treat a large abdominal lymph node metastasis that became obvious 21 months after surgery. The disease remained stable for 5 months, and the patient survived for 12 months after the initiation of immunotherapy. In the case 2 patient, immunotherapy was tried as post-operative adjuvant treatment after adjuvant chemotherapy. There was no tumor recurrence for 16 months after the immunotherapy. As of 49 months after esophagectomy, the patient is still alive. In both patients, the ability of peripheral lymphocytes to produce IFN-gamma in vitro in response to peptide stimulation was significantly enhanced and delayed-type hypersensitivity skin test response to MAGE-3 peptide was turned positive after immunotherapy. In conclusion, active specific immunotherapy for PMME with the use of DCs and MAGE peptides was safe and capable of inducing peptide-specific immune responses. This case report warrants further clinical evaluation of this immunotherapy for PMME.

Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells

BackgroundCervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.ResultsIn this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors.ConclusionWe show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Dendritic Cell Vaccination Induces HCMV Specific T-Cell Responses in Allogeneic Stem Cell Recipients.

Human cytomegalovirus (HCMV) infection remains a major and life threatening infectious complication after allogeneic stem cells transplantation (SCT). We performed a Dendritic cell (DC) vaccination trial by utilizing HCMV peptide loaded mature DC to boost HCMV specific T-cell responses which have been demonstrated to be protective against the development of HCMV disease. DCs were pulsed with nonamer peptides from the HCMV proteins pp65 and pp150, restricted by the HLA-class I elements A1,A2,A3,A11,A68 and B7. We enrolled 24 allogeneic SCT recipients, 6 patients received prophylactic vaccination in view of high risk for HCMV disease. 18 patients were vaccinated therapeutically after HCMV reactivation failed to respond to 4 weeks course of antiviral chemotherapy. Our primary objectives were safety and feasibility of DC vaccination after allogeneic SCT. As all patients with active HCMV infections already received antiviral chemotherapy at the time of DC vaccination, viral load was not a suitable efficacy parameter. Thus, evaluation of efficacy as a secondary objective was based on reconstitution of HCMV-specific CTL responses and long term control of HCMV infection. The study protocol was approved by the local ethical committee and all patients gave written informed consent. DC were generated under GMP conditions and displayed typical surface markers of mature DC (CD1a+/CD14−/CD83+). No local or systemic acute side effects occurred during the first week post vaccination. An observation period of 3 months was determined to evaluate long term side effects and control of HCMV infection. Six patients died during this observation period from other causes and one had no follow-up blood samples, so, 17 patients (5 received prophylactic and 12 received therapeutic vaccination) were evaluable. Only one patient developed Graft-Versus-Host-Disease (GVHD) grade III of the skin and gut. Due to a time lag of 2 months between vaccination and the onset of GVHD, a causative relationship seems to be unlikely. Four of the five patients receiving prophylactic vaccination never showed HCMV reactivation. 10 patients from the therapeutic group cleared their HCMV infection after a mean of 50 days post vaccination. Therefore, 15 of the 17 evaluable patients demonstrated control of HCMV infection after DC vaccination. Among these 15 patients, 10 had detectable specific T-cell response against the vaccine peptides after a mean of 23 days post vaccination. Only 2 from these 10 patients developed a further HCMV reactivation after high dose steroid therapy. Our results show that no relevant side effect was observed in this first DC vaccination trial among allogeneic SCT patients. Additionally, DC are able to induce an efficient peptide specific immune response among allogeneic SCT patients which is capable to protect against HCMV reactivation. In the future, further investigations should be performed to evaluate the feasibility of DC vaccination not only against other infectious complications but also against tumour associated antigens to induce specific T-cell response effectively targeting the particular tumour cell.