DTH Skin Test Research Articles

First step results from a phase II study of a dendritic cell vaccine in glioblastoma patients (CombiG-vax).

Glioblastoma (GBM) is a poor prognosis grade 4 glioma. After surgical resection, the standard therapy consists of concurrent radiotherapy (RT) and temozolomide (TMZ) followed by TMZ alone. Our previous data on melanoma patients showed that Dendritic Cell vaccination (DCvax) could increase the amount of intratumoral-activated cytotoxic T lymphocytes. This is a single-arm, monocentric, phase II trial in two steps according to Simon's design. The trial aims to evaluate progression-free survival (PFS) at three months and the safety of a DCvax integrated with standard therapy in resected GBM patients. DCvax administration begins after completion of RT-CTwith weekly administrations for 4 weeks, then is alternated monthly with TMZ cycles. The primary endpoints are PFS at three months and safety. One of the secondary objectives is to evaluate the immune response both in vitro and in vivo (DTH skin test). By December 2022, the first pre-planned step of the study was concluded with the enrollment, treatment and follow up of 9 evaluable patients. Two patients had progressed within three months after leukapheresis, but none had experienced DCvax-related G3-4 toxicities Five patients experienced a positive DTH test towards KLH and one of these also towards autologous tumor homogenate. The median PFS from leukapheresis was 11.3 months and 12.2 months from surgery. This combination therapy is well-tolerated, and the two endpoints required for the first step have been achieved. Therefore, the study will proceed to enroll the remaining 19 patients. (Eudract number: 2020-003755-15 https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-003755-15/IT).

143 Follow up of Cell-Immune Response using CovidCELL DTH skin test in Kidney Transplanted Patients

143 Follow up of Cell-Immune Response using CovidCELL DTH skin test in Kidney Transplanted Patients

Results from the first step of a phase II study of vaccination with autologous dendritic cells loaded with autologous tumour homogenate in glioblastoma.

e14025 Background: Glioblastoma (GBM) is a poor prognosis malignant grade IV glioma. After surgical resection, standard therapy consists of concomitant radiotherapy (RT) and temozolomide (TMZ) followed by TMZ alone. Multiple phase I/II trials and at least 3 meta-analysis showed improved survival (OS) and progression free survival (PFS) with DC vaccination in High-grade gliomas (HGGs) patients (pts). In pts developing antitumor immunity, DC vaccination increases the amount of intratumoral activated cytotoxic T lymphocytes and decreases the number of FoxP3 positive regulatory T cells. Based on these data we have developed a phase II protocol with DC vaccine concomitant to standard RT-CT in pts who have undergone radical surgery for GBM. Methods: This is a single-arm, monocentric, phase II trial of a DC vaccination integrated to standard therapy in resected GBM pts. All pts receive a dendritic cell vaccine loaded with autologous tumor homogenate for up to one year. The vaccine administration starts at the end of the RT-CT (Induction Phase) and then is alternated to TMZ cycles (Maintenance Phase). Primary end points are PFS and safety, among secondary end points there are the in vitro (Elispot, Plasma Cytokines, Tumor tissue analysis) and in vivo (DTH skin test) Immune response evaluations. A Simon's two-stage design has been used for the sample size calculation. In the first stage, 9 patients have been accrued and a total of 28 patients will be enrolled at the end of the second step. Results: From July 2021, the 9 pts of the first step has been enrolled, 4 females and 5 males with median age of 58 years. Five pts had no MGMT mutation. All 9 pts concluded the induction phase. To November 2022 four pts have progressed with a median PFS, from the date of leukapheresis, of 6.5 months (3.2-NE). DTH test became positive in 5 out of 9 evaluable pts. Median OS from the date of surgery was 11 months. No gr 3-4 vaccine related toxicities have been observed and gr 1-2 toxicities were mostly due to local skin reactions. Conclusions: On the basis of the results described above and the vaccine favorable toxicity profile the Study Team decided to proceed with the second step. Clinical trial information: EUCTR220-003755-15 . [Table: see text]

Pilot study on the camel brucellosis and paratuberculosis in Western India

Brucellosis, a major zoonosis, has attracted attention due to its reemergence. Similarly, paratuberculosis is one of the major health and production problems of animals with zoonotic concerns. Brucellosis and paratuberculosis have been reported from all parts of country with multiple species involvement. The Rose Bengal plate agglutination test (RBPT) and indirect ELISA (iELISA) are most commonly used tests for diagnosing brucellosis. Similarly, plate ELISA and DTH skin tests are popular tests for paratuberculosis. The recently developed penside test is suitable for door step diagnosis of brucellosis. In the present study plate ELISA and penside test were used to investigate brucellosis, and plate ELISA was used to investigate paratuberculosis in camels. Both the plate ELISA and penside tests revealed 4 (11%) animals to be positive for anti-Brucella antibodies. Penside test revealed results at par to plate ELISA test and showed promising results to be adopted for doorstep Brucella sp. testing. Out of 36 camels suspected for paratuberculosis infection, 12 (33.3%) were positive for the paratuberculosis antibodies in plate ELISA. All brucellosis positive animals were negative for paratuberculosis.

OA13.06 Autologous Dendritic Cells Loaded with Allogeneic Tumor Cell Lysate (Pheralys®) in Patients with Mesothelioma: Final Results of a Phase I Study

Mesothelioma is an aggressive malignancy without curative treatment options. We have previously shown promising activity of dendritic cell (DC) immunotherapy loaded with autologous tumor cell lysate (Hegmans 2013, Cornelissen 2016). Because of quality and quantity issues (availability, standardization etc) with the autologous lysate, we have developed an off-the-shelf allogenic tumor cell lysate from human mesothelioma cell lines (Pheralys®). Patients (pts) with advanced mesothelioma, either treatment naive, or non-progressing after chemotherapy, were included. Leucapheresis was performed to obtain an enriched monocyte fraction from which immature DC were generated which were loaded with the allogenic lysate. The DC were matured, frozen and stored. In subsequent cohorts of 3 pts 10, 25, or 50 × 106 DC were administered IV and intradermally, 3 times at a bi-weekly interval and after 3 and 6 months. Primary endpoint was toxicity occurring within 8 weeks after the first vaccination. Secondary endpoints were response rate (RR), progression free survival (PFS) and overall survival (OS). PFS and OS were determined from time of registration in the trial. Immunological read-outs were performed (DTH skin testing, peripheral blood testing). Nine pts (median age 69yrs, 8 male, 1 female) were included. All patients developed transient grade 1-fever and a grade 1-2 injection site reaction. No dose limiting toxicities or autoimmunity signs were observed. In 2 pts (22%), both treated with 25 ×106 cells, a partial response (PR) was observed, the other 7 pts had stable disease as best overall response. All patients are alive with a median follow up of 11.9 months after trial inclusion(range 7.6-16.5 months). Median PFS was 8.3 months (95% confidence interval (CI) 3.7-not yet reached), PFS at 12 months was 33% (95% CI 8%-62%). Data on immunological read outs are pending. DC immunotherapy with allogenic tumor cell lysate is safe and clinically active. Data on PFS and OS are promising and still maturing. The recommended dose for future studies will be 25 * 106 cells based on the responses and logistic reasons (the number of monocytes obtained during leucapheresis to generate 5 vaccinations). A randomized trial comparing DC therapy with Pheralys versus best supportive care as maintenance treatment after chemotherapy is planned to start in Q1 2017.

Fine mapping under linkage peaks for symptomatic or asymptomatic outcomes of Leishmania infantum infection in Brazil

Infection with the protozoan Leishmania infantum can lead to asymptomatic infection and protective immunity, or to the progressive and potentially fatal disease visceral leishmaniasis (VL). Published studies show host genetic background determines in part whether infected individuals will develop a symptomatic or asymptomatic outcome. The purpose of the current study was to fine map chromosome regions previously linked with risk for symptomatic (chromosome 9) or asymptomatic (chromosomes 15 and 19) manifestations of L. infantum infection. We conducted a family-based genetic study of VL and asymptomatic infection (detected by a DTH skin test) with a final post quality control sample of 961 individuals with full genotype and phenotype information from highly endemic neighborhoods of northeast Brazil. A total of 5485 SNPs under the linkage peaks on chromosomes 9, 15 and 19 were genotyped. No strong SNP associations were observed for the DTH phenotype. The most significant associations with the VL phenotype were with SNP rs1470217 (p=5.9e−05; pcorrected=0.057) on chromosome 9, and with SNP rs8107014 (p=1.4e−05; pcorrected=0.013) on chromosome 19. SNP rs1470217 is situated in a 180kb intergenic region between TMEM215 (Transmembrane protein 215) and APTX (Aprataxin). SNP rs8107014 lies in the intron between exons 26 and 27 of a 34 exon transcript (ENST00000204005) of LTBP4, (Latent transforming growth factor-beta-binding protein 4a). The latter supports growing evidence that the transforming growth factor-beta pathway is important in the immunopathogenesis of VL.

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25+ cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25+ cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

Intranasal administration of a therapeutic HIV vaccine (Vacc-4x) induces dose-dependent systemic and mucosal immune responses in a randomized controlled trial.

BackgroundVacc-4x, a Gag p24-based therapeutic HIV vaccine, has been shown to reduce viral load set-points after intradermal administration. In this randomized controlled pilot study we investigate intranasal administration of Vacc-4x with Endocine as adjuvant.MethodsSafety and immunogenicity were tested in patients on effective ART. They were randomized to low, medium or high dose Vacc-4x or adjuvant alone, administered four times at weekly intervals with no booster. Vacc-4x-specific T cell responses were measured in vitro by proliferation and in vivo by a single DTH skin test at the end of study. Nasal and rectal mucosal secretions were analyzed for Vacc-4x-specific antibodies by ELISA. Immune regulation induced by Vacc-4x was assessed by functional blockade of the regulatory cytokines IL-10 and TGF-β.ResultsVacc-4x proliferative T cell responses increased only among the vaccinated (p≤0.031). The low dose group showed the greatest increase in Vacc-4x CD8+T cell responses (p = 0.037) and developed larger DTH (p = 0.005) than the adjuvant group. Rectal (distal) Vacc-4x IgA and IgG antibodies also increased (p = 0.043) in this group. In contrast, the high dose generated higher nasal (local) Vacc-4x IgA (p = 0.028) and serum IgG (p = 0.030) antibodies than the adjuvant. Irrespective of dose, increased Vacc-4x CD4+T cell responses were associated with low proliferation (r = −0.82, p<0.001) and high regulation (r = 0.61, p = 0.010) at baseline.ConclusionIntranasal administration of Vacc-4x with Endocine was safe and induced dose-dependent vaccine-specific T cell responses and both mucosal and systemic humoral responses. The clinical significance of dose, immune regulation and mucosal immunity warrants further investigation.Trial RegistrationClinicalTrials.gov NCT01473810

Vaccination with PSA146-154 peptide for advanced prostate cancer: A 10-year follow-up.

e14024 Background: Prostate cancer is the second leading cause of cancer-related death among men in the United States. Although localized disease may be definitively treated with prostatectomy or radiotherapy, advanced metastatic disease can only be managed transiently with androgen-deprivation therapy (ADT) and other systemic therapies. Given the prevalence and lack of curative options for advanced disease, novel treatments warrant investigation. Methods: A study begun in 2002 at UIC examined whether vaccination against PSA146-154 peptide elicited specific T cell immunity in 28 patients with advanced disease. In order to measure immune responses, three distinct readouts were used: induction of responses to DTH skin testing for PSA146-154 peptide, detection of CD8+ PSA146-154 peptide-tetramer +T cells, and via PSA146-154 peptide-induced release of IFN-γ in pre versus post-vaccine peripheral blood mononuclear cell samples. All three assays were completed by 26 and 52 weeks following vaccination. The current study examines the long-term effects of the vaccine ten years post immunization and determines survival differences between immune responders and non-responders. Results: Individuals who demonstrated a DTH response against PSA146-154 peptide have improved survival compared to individuals who did not develop a DTH response against the peptide (80.154 months ± 7.849 v. 72.69 months ± 10.358, p = .12). By contrast, specific responses to the PSA peptide detected by IFN gamma production or tetramer staining did not correlate with improved survival. Analysis of mRNA from peripheral blood mononuclear cells of DTH responders revealed increased expression of genes involved in plasmacytoid dendritic cell function (7 genes, with .5 fold to 4.25 fold increased expression in DTH responders). Conclusions: Together, these findings suggest that vaccination against the PSA146-154 peptide may lead to improved survival in patients who effectively mount a peptide-specific DTH response. Furthermore, the RNA expression profile of responders implicates an antitumor role for plasmacytoid dendritic cells.

Adoptive Transfer of Autologous CD25-Depleted, CD3/CD28-Costimulated T Cells After Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma: Long-Term Follow up

Abstract 1631 Introduction:Fludarabine-based chemotherapy combinations are highly effective in patients with low-grade follicular lymphoma but cause severe and long-lasting immunosuppression related to depletion of normal T cells, especially CD4 T cells. Aside from increasing the risk of serious infections, this toxicity may limit the ability of the immune system to eliminate minimal residual disease. To determine whether adoptive transfer of autologous CD25-depleted, CD3/CD28-costimulated T cells expanded ex vivo (ACTC) improves immune reconstitution following cyclophosphamide - fludarabine chemotherapy, we conducted a phase I study in patients with relapsed/refractory follicular lymphoma. Patients and Methods:Eligible patients had previously treated, purine analog naïve, relapsed or refractory follicular lymphoma (grade 1 or 2). After leukapheresis, patients received 4 cycles of fludarabine (25 mg/m2) and cyclophosphamide (250 mg/m2) on days 1 – 3 of a 28 – 42 day cycle. Four weeks after last chemotherapy, responding patients [complete response (CR), complete response unconfirmed (CRu), and partial response (PR)] received escalating doses of ACTC prepared from autologous T cells collected prior to chemotherapy. Results:Fifteen patients were enrolled. Median age was 49 years (range: 32 – 68), median number of prior therapies was 2 (range: 1 – 3), and 14 patients (93%) were rituximab-refractory. Five patients were withdrawn from the study, 2 due to chemotherapy-related hematologic toxicity and 3 for progressive disease during chemotherapy. For 10 evaluable patients completing chemotherapy and ACTC infusion, 8 patients were in CR and 2 patients in PR at the time of ACTC infusion; 5 patients received 1 – 5 × 109 ACTC and 5 patients received 5 – 10 × 109 ACTC. There were no adverse events related to T cell infusions. Following chemotherapy and before ACTC infusion, median CD4 and CD8 counts were 91/μL (range: 15 –169) and 64/μL (range: 19 – 273), respectively; four weeks after ACTC, median CD4 and CD8 counts were 502/μL (range: 156 – 1053) and 509/μL (range: 158 – 1860). Median %CD4+FOXP3+ blood cells before chemotherapy was 15.8% (n = 8; range: 5.07 – 37.1); after chemotherapy / before ACTC, 21.4% (n = 7; range: 6.15 – 56.3); on day 60 after ACTC, 3.3% (n = 8; range: 1.5 – 20.4) (p = 0.01 for both pre ACTC and post ACTC comparisons). Before receiving chemotherapy and ACTC, all 10 patients were anergic to candida antigen by DTH skin testing; 60 days after ACTC, 5 patients developed a positive DTH response to candida antigen (p = 0.03). From the day of ACTC infusion, median progression-free survival (PFS) is 14.1 months. Most significantly, no patient relapsed or had progression of lymphoma after 14.1 months with follow-up from 16.5 – 101.5 months. In addition, PFS is significantly longer than time to progression from prior therapy (p = 0.01; see figure below). Overall survival after ACTC was 60% with median follow-up of 68 months. Conclusions:Adoptive transfer of ACTC after fludarabine - cyclophosphamide chemotherapy enhances recovery of CD4 and CD8 lymphocyte counts compared to historical observations, results in a relative reduction in peripheral blood FOXP3+ regulatory T cells, enhances recovery of lymphocyte function, and can result in prolonged remission. Our findings suggest that reversal of immunosuppression by adoptive transfer of autologous co-stimulated T-cells may significantly improve therapeutic outcome after cytotoxic therapy for patients with follicular lymphoma. [Display omitted] Disclosures:Levine:US Patent: Financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight., Financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight. Patents & Royalties. June:Life Technologies / Invitrogen: cell culture technology patents owned by U.S. Government and licensed to Life Technology / Invitrogen, cell culture technology patents owned by U.S. Government and licensed to Life Technology / Invitrogen Patents & Royalties.

Advax™, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses

Advax™ adjuvant is derived from inulin, a natural plant-derived polysaccharide that when crystallized in the delta polymorphic form, becomes immunologically active. This study was performed to assess the ability of Advax™ adjuvant to enhance influenza vaccine immunogenicity and protection. Mice were immunized with influenza vaccine alone or combined with Advax™ adjuvant. Immuno-phenotyping of the anti-influenza response was performed including antibody isotypes, B-cell ELISPOT, CD4 and CD8 T-cell proliferation, influenza-stimulated cytokine secretion, DTH skin tests and challenge with live influenza virus. Advax™ adjuvant increased neutralizing antibody and memory B-cell responses to influenza. It similarly enhanced CD4 and CD8 T-cell proliferation and increased influenza-stimulated IL-2, IFN-γ, IL-5, IL-6, and GM-CSF responses. This translated into enhanced protection against mortality and morbidity in mice. Advax™ adjuvant provided significant antigen dose-sparing compared to influenza antigen alone. Protection could be transferred from mice that had received Advax™-adjuvanted vaccine to naïve mice by immune serum. Enhanced humoral and T-cell responses induced by Advax™-formulated vaccine were sustained 12months post-immunization. Advax™ adjuvant had low reactogenicity and no adverse events were identified. This suggests Advax™ adjuvant could be a useful influenza vaccine adjuvant.

Serum CEACAM1 Correlates with Disease Progression and Survival in Malignant Melanoma Patients

The search for melanoma biomarkers is crucial, as the incidence of melanoma continues to rise. We have previously demonstrated that serum CEACAM1 (sCEACAM1) is secreted from melanoma cells and correlates with disease progression in metastatic melanoma patients. Here, we have used a different cohort of melanoma patients with regional or metastatic disease (N = 49), treated with autologous vaccination. By monitoring sCEACAM1 in serum samples obtained prior to and after vaccination, we show that sCEACAM1 correlates with disease state, overall survival, and S100B. The trend of change in sCEACAM1 following vaccination (increase/decrease) inversely correlates with overall survival. DTH skin test is used to evaluate patients' anti-melanoma immune response and to predict response to vaccination. Importantly, sCEACAM1 had a stronger prognostic value than that of DTH, and when sCEACAM1 decreased following treatment, this was the dominant predictor of increased survival. Collectively, our results point out the relevance of sCEACAM1 in monitoring melanoma patients.

Efficacy and safety of Id-protein-loaded dendritic cell vaccine in patients with multiple myeloma – Phase II study results

In a phase II clinical study, pretreated multiple myeloma patients with relapsing or stable disease received autologous anticancer vaccine containing dendritic cells loaded with Id-protein. Patients received a total of 6 vaccine doses intradermally in monthly intervals. No clinical responses were observed. During the follow-up with a median of 33.1 months (range: 11-43 months), the disease remained stable in 7/11 (64%) of patients. Immune responses measured by ELISpot were noted in 3/11 (27%) and DTH skin test for Id-protein was positive in 8/11 (73%) of patients; out of those, 1/11 (9%) and 5/11 (46%), respectively, had preexisting immune response to Id-protein before the vaccination began. Outcomes were compared to those of a control group of 13 patients. A trend to lower cumulative incidence of progression in the vaccinated group was observed at 12 months from the first vaccination (p= 0.099). More patients from the control group compared to vaccinated patients required active anticancer therapy [4/11 (36%) vs. 8/13 (62%)]. Vaccines based on dendritic cells loaded with Id-protein are safe and induce specific immune response in multiple myeloma patients. Our results suggest that the vaccination could stabilize the disease in approximately two-thirds of patients. dendritic cells, immunotherapy, anticancer vaccines, Id-protein, multiple myeloma.

Immunoediting and persistence of antigen-specific immunity in patients who have previously been vaccinated with NY-ESO-1 protein formulated in ISCOMATRIX™.

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. Immunity was measured in 28 patients with resected NY-ESO-1-expressing tumours (melanoma 25, breast 3) 252-1,155days (median=681) after vaccination. In the earlier vaccination, trial patients received NY-ESO-1 with ISCOMATRIX™ adjuvant at three protein doses 10μg, 30μg or 100μg (n=14); 100μg NY-ESO-1 protein (n=8) or placebo (n=6), together with 1μg of intradermal (ID) NY-ESO-1 protein twice for DTH skin testing. Immune responses assessed in the current study included antibody titres, circulating NY-ESO-1-specific T cells and DTH reactivity 2days after DTH skin testing with NY-ESO-1 protein (1μg) or peptides (10μg). Relapse-free survival was determined for 42 melanoma patients. On relapse NY-ESO-1 and HLA, class I was assessed by immunohistochemistry in 17. Persisting anti-NY-ESO-1 immunity was detected in 10/14 recipients who had previously received vaccine with ISCOMATRIX™ adjuvant. In contrast, immunity only persisted in 3/14 who received 100μg un-adjuvanted NY-ESO-1 protein (3/8) or 2μg DTH protein (0/6) P=0.02. Hence, persisting NY-ESO-1 immunity was associated with prior adjuvant. Tumour NY-ESO-1 or HLA class I was downregulated in participants who relapsed suggesting immunoediting had occurred. Immunoediting suggests that a signal of anti-tumour activity was observed in high-risk resected melanoma patients vaccinated with NY-ESO-1/ISCOMATRIX™. This was associated with measurable persisting immunity in the majority of vaccinated subjects tested. A prospective randomised trial has been undertaken to confirm these results.

Increased humoral and cellular immunity in patients (pts) with advanced melanoma treated with ipilimumab

3031 Background: Ipilimumab is an anti-CTLA-4 monoclonal antibody (Ab) that overcomes T-cell suppression. In this phase II study (CA184004), humoral and cellular responses were assessed in ipilimumab-treated pts with unresectable stage III/IV melanoma. Methods: Ipilimumab 3 or 10 mg/kg was given every 3 weeks x 4. Tetanus boosters were given ≤10 days pre-treatment. Influenza and pneumococcal vaccines were given 5 days after first ipilimumab dose. Tetanus, anti-influenza, and anti-pneumococcal Ab levels were assessed at pre-dose and Wk 7. Humoral response to 5 tumor antigens (Ag) and a control Ag (DHFR) were examined at baseline (BL) and at Wks 4, 8–9, and 12. DTH skin tests were given at pre-dose and Wk 4, with responses recorded 15 minutes (min) and 24–72 hours (hrs) post-test. Peripheral T-cell populations were evaluated through flow cytometry at BL, Wk 4, and Wk 12. Results: Pts received ipilimumab 3 (n = 40) or 10 mg/kg (n = 42). Increases from BL in humoral responses to pneumococcal (40–50/78 pts, depending on Ab) and tetanus (58/78 pts) vaccines were noted, even in pts who did not receive on-study pneumococcal (4–9 pts) or tetanus (7 pts) vaccines. Increased humoral response to influenza only occurred in pts receiving the influenza vaccine. Maximum increase from BL of ≥ 5-fold titer (clinically meaningful threshold) in humoral response to tumor Ag MELANA (23.2% of pts), SSX2 (20.3%), NYES01 (18.8%), MAGEA4 (10.1%), and P53 (4.3%) (DHFR, 4.3%) was noted without tumor vaccines. Tumor Ag response was not associated with clinical activity (complete or partial response, or stable disease ≥ 24 wks). Increased DTH reactions were noted for tetanus, 24–72 hrs (3 mg/kg: 5/7 pts; 10 mg/kg: 3/6 pts); tuberculin, 15 min (3 mg/kg: 7/15 pts; 10 mg/kg: 4/15 pts); Candida, 15 min (3 mg/kg: 4/6 pts; 10 mg/kg: 2/7 pts); and Trichophyton, 15 min (3 mg/kg: 3/4 pts; 10 mg/kg: 2/5 pts). Significant increases from BL in percents of HLA-DR+ CD4+ (p = 9.3x10-7), HLA-DR+ CD8+ (p = 0.018), and ICOS+ CD4+ (p = 0.0027) effector T cells were noted. Conclusions: Humoral immunity in ipilimumab-treated pts increased (± vaccination) in an Ag-dependent manner and cellular immunity was enhanced. Change in tumor Ag response was not associated with clinical activity. [Table: see text]

Vaccination therapy of pancreatic carcinoma patients with autologous, tumor-lysate pulsed dendritic cells: Results of a phase II study

3069 Background: Vaccine therapy using dendritic cells can induce antitumor immunity. We report about the final results of a phase II-study using autologous, tumor-lysate pulsed dendritic cells (DC) for the treatment of patients with advanced pancreatic carcinoma. As all patients received concomitant chemotherapy, we investigated systematically the interplay between DC therapy and gemcitabine chemotherapy in a murine pancreatic carcinoma model, correlating the results of ex vivo immuno monitoring tools with clinical efficacy. Methods: Pancreatic carcinoma patients receiving abdominal surgery were recruited to the study. DC therapy was started after recurrence of pancreatic carcinoma or in a primarily palliative situation. Immune response was monitored by ELISPOT and proliferation assays. A pancreatic carcinoma vaccination model was established by transfecting murine pancreatic carcinoma cell line Panc02 with an OVA vector. Mice were vaccinated with bone marrow derived DC loaded with apoptotic Panc02OVA cells, OVA protein or SIINFEKL peptide. Intracellular IFN- γ staining and streptamer staining were used to examine the induction of antigen specific T cells. For tumor induction, Panc02OVA cells were orthotopically injected into C57BL/6 mice. Results: Twelve patients received DC vaccination. Four cases of at least intermittent partial remission or stable disease were seen. These patients had developed strong immunological antitumor responses as demonstrated by DTH skin testing, T cell proliferation assays and ELISPOT assays. Data in the murine model indicate that there is a synergistic clinical efficacy of gemcitabine and DC vaccination therapy despite reduction of specific cytotoxic T cells by concomitant gemcitabine therapy. Results of an in vitro cytotox assay using SIINFEKL-specific, transgene T-cells demonstrate that gemcitabine sensitizes Panc02OVA cells towards T cell mediated lysis. Conclusions: DC vaccination therapy can be of clinical benefit in the setting of advanced pancreatic carcinoma. DC therapy led to immunological responses despite concomitant chemotherapy. Data obtained in a murine model of pancreatic cancer support the concept of a combination of vaccination and chemotherapy. No significant financial relationships to disclose.

Humoral shift and increased early recurrence with GM-CSF + vaccine versus vaccine alone

3013 Background: Vaccines are currently under evaluation in melanoma, and many vaccine strategies use GM-CSF as an immune adjuvant. There are few randomized data yet available to determine the clinical impact of the addition of GM-CSF to vaccination. We conducted a randomized, prospective trial of an allogeneic melanoma vaccine with or without GM-CSF to determine the immune and clinical effects. Methods: 95 patients were stratified by stage and randomized to Canvaxin vaccine (with bacille Calmette-Guerin [BCG]) or Canvaxin (+BCG) and GM-CSF (200 mcg/m2/d) injected at the vaccine sites after vaccination for 5 days. Antibody titers against a urinary tumor-associated melanoma antigen were measured by ELISA, and DTH skin testing was done using PPD and unadulterated vaccine cells. Results: There was a significant early rise in both IgG and IgM titers in the GM-CSF arm with an early high plateau at about 8 weeks for both. The rise in antibody titer was slower in the non-GM-CSF arm with IgG rising throughout the study period and IgM peaking at 20 weeks. Conversely there was a delay in PPD responsiveness in the GM-CSF arm. PPD conversion percentage surpassed 90% in the non-GM-CSF arm at 6 weeks and peaked at 100% at 12 weeks. In the GM-CSF arm, only 76% converted at 6 weeks and only 87% were ever sensitized. A trend toward diminished DTH response was noted in the GM-CSF arm as early as 4 weeks and became significant at 16 weeks (p=0.01) and when considered throughout the trial. There was a marked increase in early recurrences in the GM-CSF arm leading to a significant survival difference when examined at 2 years (96% versus 72%; log-rank p=0.0017). Conclusions: When given at the site of vaccination, GM-CSF appears to accelerate the onset of a humor immune response while delaying cellular responses. In addition there appears to be evidence of adverse clinical effects in terms of early recurrence and possibly survival. Recent reports of increased myeloid suppressor cells with GM-CSF treatment may provide an immune mechanism for our findings. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration CancerVax

Immunotherapy of patients with pancreatic adenocarcinoma: Influence of adjuvants

15542 Background: Because peptide vaccines are poorly immunogenic, combination with immunological adjuvants such as GM-CSF, have been given in previous phase I/II studies of the hTERT peptide vaccine GV1001. The objective of the present study was to compare the immunological response after GV1001 treatment, combined with different immunological adjuvants. Methods: The study was an open phase II study of patients with irresectable adenocarcinoma of the pancreas, 18–75 years old. All patients received GV1001 as an intradermal injection eight times over a period of 10 weeks using imiquimod as an adjuvant in treatment group 1 and imiquimod and GM- CSF as adjuvants in treatment group 2 and 3. In addition, patients in group 2 received palliative irradiation (3Gy × 10) prior to immunotherapy while patients in group 3 received one dose of cyclophosphamide three days prior to start of immunotherapy. Primary endpoint was immune response, measured by DTH skin test reaction and specific T-cell response. Results: All together 15, 10 and 14 patients were included in groups 1–3 respectively. For the evaluable patients the immune response in treatment group 1, 2 and 3 was 39%, 50% and 64%, respectively. A total of 221 adverse events (AEs) were reported by 39 patients, 86 in treatment group 1, 41 in treatment group 2 and 94 in treatment group 3. The incidence rate (number of AEs per patient per month) of adverse events was lower in treatment group 2 (0.67 AEs/patient/months) compared to treatment groups 1 and 3 with incidence rates of 1.13 and 1.27, respectively.Most AEs were scored as unlikely related to study treatment and were of mild to moderate severity. Conclusions: The study demonstrated that immune response to GV1001 can be obtained using different treatment regimens, but none of these adjuvant regimens generated higher response levels than GM-CSF alone. It can further be concluded that GV1001 is well tolerated, both in combination with palliative irradiation and with cyclophosphamide. No significant financial relationships to disclose.

Immunological and clinical response after vaccination therapy of pancreatic carcinoma patients with autologous, tumor-lysate pulsed dendritic cells: Results of a phase II-study

4579 Background: Multiple studies in the experimental and in the clinical setting have shown that vaccine therapy using dendritic cells can induce antitumor immunity. Here, we report about the results of a phase II-study using autologous, tumor-lysate pulsed dendritic cells for the treatment of patients with advanced pancreatic carcinoma. Methods: Pancreatic carcinoma patients receiving abdominal surgery were included into to the study protocol. Tumor-lysate was derived by freeze-taw-cycles from surgically derived tissue specimens. Patients were eligible for DC vaccination after recurrence of pancreatic carcinoma or in a primarily palliative situation. DC were derived from PBMC according to a six-day protocol, loaded with tumor lysate and stimulated with TNF-a and PgE2. DC were applicated intracutaneously into the groin region every other week for three cycles, then monthly. All patients received standard chemotherapy with gemcitabine concomitantly. Immune response was controlled by DTH skin testing. Samples of non adherent cells were frozen for MLR and ELISPOT assays to monitor immune response ex vivo. Main study end point was partial or complete remission. Results: Ten patients have received dendritic cell vaccination so far. Of these, one patient developed a partial remission after a four-months course of vaccination therapy. Another patient showed stable disease after having received five vaccinations. Both patients showed immunological response. The patient with stable disease had a mean of 56 IFN-γ positive spots per 50E3 DC-stimulated non adherent cells prior to vaccination. After vaccination, this number increased to 191 spots per 50E3 cells (negative control: 5; positive control: 315). Both patients are alive 13 and 7 months after the start of vaccination therapy, respectively. Conclusions: Vaccination therapy with dendritic cells can be of clinical benefit in the setting of advanced pancreatic carcinoma. Clinical responses were associated with the induction of a stable immunological response. No significant financial relationships to disclose.

The immunology of bovine tuberculosis and progression toward improved disease control strategies

Failure to remove cattle diseased with Mycobacterium bovis has immense financial implications for disease control, animal health and agricultural trade as well as the zoonotic risk to human health. Current disease control strategies based on DTH skin testing fail to detect all diseased cattle and additional measures are urgently needed to improve detection of disease and to prevent naïve animals becoming exposed to infection. Experimental models of bovine TB traditionally based on intra-nasal instillation, intra-tracheal inoculation or placed in-contact with infected cattle, have been further developed using aerosolised bacteria delivered to the respiratory tract, allowing field-like bovine TB to be recreated under controlled, experimental conditions. Experimental infection models have already been used to improve diagnostic tests. Specificity of DTH skin testing can be improved under experimental conditions, using recombinant ESAT-6, while laboratory assays such as IFN-gamma release have benefited from the use of defined proteins to improve assay specificity. In combination, antigen cocktails may also improve test sensitivity. There is a concerted international effort to evaluate vaccines for use in cattle populations and to define vaccination strategies which will eliminate disease from infected herds. DNA, protein and genetically modified vaccines inoculated in a single dose, given as prime-boost or injected concurrently, will elicit significant protection against challenge with M. bovis under controlled conditions. However, vaccines and vaccination strategies require evaluation under field conditions. Furthermore, complementary strategies are under development to differentiate immune responses that follow vaccination from those following disease. This paper describes those recent advances which may lead to the introduction of improved disease control strategies.