Positive Delayed-type Hypersensitivity Response Research Articles

Abstract P4-07-23: Results of a Phase 2 Trial of Combination Immunotherapy with Concurrent Nelipepimut-S + GM-CSF and Trastuzumab in High-risk HER2+ Breast Cancer Patients

Abstract INTRODUCTION: The HER2-targeted peptide vaccine nelipepimut-S + GM-CSF (NeuVax) has been shown to be safe, immunogenic, and potentially synergistic with trastuzumab. Here we present the results of a randomized phase 2 trial assessing the ability of nelipepimut-S/GM-CSF versus GM-CSF alone, added to the standard adjuvant Trastuzumab, to prevent recurrences in high-risk HER2-positive breast cancer patients. METHODS: The study was a multi-center, prospective, randomized, controlled, single-blinded, phase 2 trial. Enrolled patients had high risk HER2+ breast cancer defined by the presence of residual disease post neoadjuvant therapy or by the presence of positive lymph nodes after upfront surgery. Eligible patients had completed an approved trastuzumab-chemotherapy containing regimen and they were receiving adjuvant Trastuzumab monotherapy. Enrollment was limited to patients with HLA-A2, A3, A24, and/or A26 alleles. Patients received intradermal injections of nelipepimut-S + GM-CSF or placebo + GM-CSF every three weeks for six total vaccinations with concurrent, standard monotherapy with iv trastuzumab. After completion of the primary vaccine series, booster inoculations were administered every six months for four doses. The primary outcome measure was invasive disease-free survival (iDFS) at 36 months. Secondary outcome measures were distant recurrence-free survival (DRFS), toxicity assessment, and evaluation of immune response. RESULTS: 100 patients were enrolled and randomized 1:1 to nelipepimut-S/GM-CSF or GM-CSF alone. There were no significant clinicopathologic differences between the groups. There was no difference in related local (p=0.49) or systemic toxicities (p=0.41). Kaplan-Meier estimates of iDFS at 36 months were 79% in the nelipepimut-S arm and 92% in the placebo arm (log rank, p=0.11). DRFS at 36 months was estimated to be 90% in the nelipepimut-S arm and 95% in the placebo arm (log rank, p=0.40). Delayed type hypersensitivity (DTH) response to nelipepimut-S was measured and considered positive if there was more than 5 mm induration after 48 hours. DTH response converted from negative to positive in 11% of patients in the vaccine group versus 5% of patients in the placebo group (p=0.36). In both groups, iDFS at 36 months was 100% for those with a positive DTH response post-inoculation and 88% for those with a negative DTH response post-inoculation (log rank, p=0.29). CONCLUSION: Combination immunotherapy with concurrent nelipepimut-S + GM-CSF and trastuzumab is safe, however there was no difference in iDFS or DRFS among high-risk HER2+ breast cancer patients who received nelipepimut-S + GM-CSF compared to GM-CSF alone. We observed a trend towards improved iDFS in patients with a positive DTH response to nelipepimut-S, though it was not statistically significant. Citation Format: Ankur Tiwari, Guy Clifton, Carmen Calfa, Gheath Alatrash, Jarrod Holmes, Isabelle Bedrosian, George Peoples, Elizabeth A. Mittendorf. Results of a Phase 2 Trial of Combination Immunotherapy with Concurrent Nelipepimut-S + GM-CSF and Trastuzumab in High-risk HER2+ Breast Cancer Patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-23.

Abstract P2-13-29: Analysis of GP2 immune response and relationship to recurrence in a prospective, randomized, placebo-controlled, single-blinded, multicenter, phase IIb study evaluating the reduction of recurrences using HER2/neu peptide GP2 (GLSI-100) vs. GM-CSF alone after adjuvant trastuzumab in HER2 positive women with operable breast cancer

Abstract Background: Delayed type hypersensitivity (DTH) skin tests in the randomized, active-controlled, single-blinded, multicenter Phase IIb trial investigating GLSI-100 (GP2+GM-CSF) administered in the adjuvant setting to node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 (immuno-histochemistry [IHC] 1-3+) (NCT00524277) have been analyzed. The trial enrolled HLA-A*02 patients randomized to receive GLSI-100 versus GM-CSF alone. The trial's primary objective was to determine if treatment with GLSI-100, a HER2-derived peptide, reduces recurrence rates. Analyses for this trial showing GLSI-100 to be efficacious, safe and immunogenic have been previously reported by Patel et al. and Mittendorf et al.. Methods: Consented patients were randomized and scheduled to receive GLSI-100 (500 mcg GP2: 125 mcg GM-CSF) or control (GM-CSF only) via 6 intradermal injections every 3-4 weeks as part of the Primary Immunization Series (PIS) for the first 6 months and 4 booster intradermal injections every 6 months thereafter. Boosters were introduced during the trial, thus some patients did not receive all 4 boosters. DTH skin tests were assessed at baseline and after the 6th dose with the orthogonal mean of each skin reaction measured 48-72 hours after injection using the sensitive ballpoint-pen method.. Results: The study enrolled 180 patients across 16 clinical sites with both HER2 3+ positive and low HER2 expressors (1-2+). After 5 years of follow-up, the Kaplan-Meier estimated 5-year DFS rate in the 46 HER2 3+ patients treated with GLSI-100, if the patient completed the PIS, was 100% versus 89.4% (95% CI:76.2, 95.5%) in the 50 placebo patients treated with GM-CSF (p = 0.0338). GLSI-100 was shown to be well tolerated with no SAEs deemed related to study medication and elicited a potent immune response measured by local skin tests and immunological assays. Injection site reactions were common, occurring in almost 100% of patients treated with either GLSI-100 or GM-CSF alone. Previous publications have reported the increase in DTH response reported among patients after treatment with GLSI-100. However, it was of interest to understand the positive DTH responses to GP2 noted at baseline. 22.8% of patients reacted to GP2 at baseline with induration of 5mm or greater. In the subgroup of patients who later experienced a breast cancer recurrence, 36.4% (8/22) had such a baseline response. Analysis of the time to recurrence among those recurring found that the median time to recurrence was 0.6 years for those with a baseline response while those that did not have a positive baseline DTH response to GP2 took 1.2 years to recur.. Conclusions: This study demonstrated that GLSI-100 safely elicited a potent immune response as evidenced by increased DTH skin responses with treatment paired with improved disease-free survival. It is theorized that a positive baseline DTH skin test to GP2 may be evidence of an existing immune response to GP2 associated with residual disease, impending recurrence, or prior treatments. Further studies assessing if GP2 immune response is an important prognosticator of cancer disease state or recurrence are planned. Citation Format: Snehal S Patel, David B McWilliams, Mira S Patel, Christine T Fischette, Jaye Thompson, F. Joseph Daugherty. Analysis of GP2 immune response and relationship to recurrence in a prospective, randomized, placebo-controlled, single-blinded, multicenter, phase IIb study evaluating the reduction of recurrences using HER2/neu peptide GP2 (GLSI-100) vs. GM-CSF alone after adjuvant trastuzumab in HER2 positive women with operable breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-29.

Abstract 1699: Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine

Abstract Introduction: Immune checkpoint blockade by anti-CTLA-4 antibody and anti-PD-1 antibody led to clinical breakthrough for the treatment of patients with solid tumors. Moreover, recent reports have shown that neoantigens are important functional targets mediating response to immune checkpoint inhibitors. Therefore, sequential combination of personalized cancer vaccines and immune checkpoint blockade has emerged as a potential therapeutic strategy. However, existing drawbacks is that it is difficult to identify robust biomarkers for patient selection in vaccination with cancer vaccine. In this study, we examined whether the development of delayed-type hypersensitivity (DTH) response, measured after vaccination with autologous formalin-fixed tumor vaccine (AFTV), correlates to parameters of immune function such as whole blood cytokine levels and peripheral regulatory T cells (Treg) in patients with solid tumors. Methods: Subjects comprised 52 patients with solid malignancy. AFTV treatment consisted of 3 courses of vaccination performed at intervals of 2 weeks. DTH test was performed before and after vaccination. A positive DTH response was defined as a ≥ 10mm diameter erythema/induration. We evaluated whole blood cytokine production after phytohemagglutinin (PHA) stimulation using the bioplex array system. We also assessed the number of peripheral Tregs as well as routine hematological and biochemical parameters. Results: DTH positive patients and DTH negative patients did not differ with respect to the number of peripheral Tregs, leukocytes, lymphocytes or granulocyte/lymphocyte ratio. Whole blood IFN-γ production levels after phytohemagglutinin (PHA) stimulation at baseline in DTH positive patients were significantly higher than those in DTH negative patients (p=0.0397). Conclusion: These data indicate that whole blood IFN-γ production at baseline is likely to be useful to assess immunologic competence in vaccinated patients with AFTV. Consequently, clinical utility of whole blood IFN-γ production, as immunomonitoring method for patient selection in vaccination with cancer vaccine, is required to investigate in larger clinical studies in the future. Citation Format: Naoyuki Sakamoto, Takeshi Ishikawa, Tetsuya Okayama, Tomoyo Yasuda, Toshifumi Doi, Mari Tanigawa, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1699. doi:10.1158/1538-7445.AM2017-1699

Recombinant proteins encoded by genes present in Mycobacterium tuberculosis-specific regions of difference induce delayed-type hypersensitivity skin responses (43.31)

Abstract The world-wide control of tuberculosis (TB) requires affordable and easy to apply test(s), which could differentiate TB from BCG vaccination and exposure to environmental mycobacteria. In this study, the proteins encoded by Mycobacterium tuberculosis-specific regions of difference (RD) genes, which are absent in BCG and most environmental mycobacteria, were studied for delayed-type hypersensitivity (DTH) skin responses in guinea pigs. The genes of RD1 (CFP10 and ESAT-6), RD7 (Rv2346 and Rv2347) and RD9 (Rv3619 and Rv3620) proteins were cloned in plasmid vectors and expressed in Escherichia coli. The expressed recombinant proteins were purified to homogeneity using affinity columns. Guinea pigs were injected with 1x1010 heat-killed M. tuberculosis, and phosphate buffered saline (PBS), as a placebo control. After four weeks of injection, all the animals were tested for DTH skin responses by intradermal injection with PBS (negative control), purified protein derivative (PPD) and sonicates of M. tuberculosis (positive control), and purified recombinant proteins. The results showed that PBS failed to induced DTH responses in any group, whereas PPD, M. tuberculosis sonicates and the recombinant proteins induced positive DTH responses in animals injected with heat-killed M. tuberculosis, but not in PBS-injected animals. These results suggest that purified RD proteins are active in inducing DTH responses in vivo, and may be useful in specific diagnosis of TB.

A Pilot Trial of WT1 Peptide-Loaded Allogeneic Dendritic Cell Vaccine and Donor Lymphocyte Infusion for WT1-Expressing Hematologic Malignancies and Post-Transplant Relapse

Abstract 3055 Background:Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains challenging. The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies with limited expression in normal tissues. This pilot trial incorporates antigen-specific immunotherapy and allogeneic adoptive cell transfer for pediatric and adult patients with relapsed hematologic malignancies after alloHSCT. The primary objectives are to assess safety and feasibility of a peptide-loaded donor-derived dendritic cell (DC) vaccine and donor lymphocyte infusion (DLI) designed to enhance the graft-vs -leukemia effect. Secondary objectives are to determine if immunologic and clinical responses to WT1 specific peptides can be generated by this novel vaccine strategy after alloHSCT. Design:HLA-A2+ patients with WT1-expressing hematologic malignancies that have relapsed after alloHSCT are eligible. Donor-derived DC vaccines are given every 2 weeks for 6 doses and DLI every 4 weeks for 3 doses. Peripheral blood monocyte-derived DCs are loaded with a combination of three HLA-A2 binding WT1 peptides (WT1 37–45; WT1 126–134; WT1 187–195) linked to the 11-mer HIV TAT protein transduction domain peptide (47–57) designed to enhance antigen presentation. The DCs are generated from donor peripheral blood monocytes that are separated from DLI collected by apheresis. Monocytes are incubated with GM-CSF and IL-4 followed by maturation with LPS and IFN-g. WT1 expression is assessed using immunohistochemistry and/or quantative RT-PCR. Study endpoints included toxicity, feasibility, and antigen-specific immune and clinical responses. Results:4 patients, aged 9–19 years have been treated to date, 3 with acute lymphoblastic leukemia (ALL) and one with Hodgkin lymphoma (HL). Donors were matched siblings in 3 cases and a 10/10 HLA matched father in one case. Vaccines were successfully produced from all donors. All patients tolerated vaccine and DLI administrations well. The most common adverse events were mild, reversible pain and pruritus at vaccine administration and delayed type hypersensitivity (DTH) skin test sites. One patient developed Grade I skin GVHD that did not require treatment. Immune responses were observed in all 3 patients with ALL. ELISPOT was considered positive when it was at least two times greater than and had at least 10 spots more than background. 3 of 4 (75%) patients had positive ELISPOT responses to WT1 peptides. 3 of 4 (75%) patients had positive DTH responses to the keyhole limpet hemocyanin (KLH) control and 2 of 4 (50%) to the WT1 peptides. Median overall survival was 12 months. 1 patient remains in remission 12 months after initiation of therapy and 3 have died of disease. All donors tolerated apheresis well and there were no complications from donor procedures. Conclusions:This novel allogeneic immunotherapy regimen is feasible, well-tolerated and can induce immune responses in the allogeneic setting. Accrual is ongoing.Table:Summary of DTH and ELISPOT ResponsesPatient #DiagnosisKLH DTH ResponseWT1 DTH ResponseWT1 ELISPOT Response1ALL+++2HL---3ALL+-+4ALL+++ Disclosures:Off Label Use: This is a pilot clinical trial of an investigational cancer vaccine.

Humoral and In Vivo Cellular Immunity against the Raw Insect-Derived Recombinant Leishmania infantum Antigens KMPII, TRYP, LACK, and papLe22 in Dogs from an Endemic Area

Leishmania infantum causes visceral leishmaniasis, a severe zoonotic and systemic disease that is fatal if left untreated. Identification of the antigens involved in Leishmania-specific protective immune response is a research priority for the development of effective control measures. For this purpose, we evaluated, in 27 dogs from an enzootic zone, specific humoral and cellular immune response by delayed-type hypersensitivity (DTH) skin test both against total L. infantum antigen and the raw Trichoplusia ni insect-derived kinetoplastid membrane protein-11 (rKMPII), tryparedoxin peroxidase (rTRYP), Leishmania homologue of receptors for activated C kinase (rLACK), and 22-kDa potentially aggravating protein of Leishmania (rpapLe22) antigens from this parasite. rTRYP induced the highest number of positive DTH responses (55% of leishmanin skin test [LST]-positive dogs), showing that TRYP antigen is an important T cell immunogen, and it could be a promising vaccine candidate against this disease. When TRYP-DTH and KMPII-DTH tests were evaluated in parallel, 82% of LST-positive dogs were detected, suggesting that both antigens could be considered as components of a standardized DTH immunodiagnostic tool for dogs.

Immunization responses in rheumatoid arthritis patients treated with rituximab: Results from a controlled clinical trial

To examine immunization responses in patients with rheumatoid arthritis (RA) treated with rituximab and to investigate the effects of rituximab-induced CD20+ B cell depletion on immune responses to tetanus toxoid (T cell-dependent antigen), pneumococcal polysaccharide (T cell-independent antigen), and keyhole limpet hemocyanin (KLH) (neoantigen) and on delayed-type hypersensitivity (DTH). In a controlled trial, we enrolled 103 patients with active RA receiving a stable dose of methotrexate (MTX). Tetanus toxoid, pneumococcal polysaccharide, and KLH vaccines as well as a Candida albicans skin test were administered to 1 group of patients receiving rituximab plus MTX (called rituximab-treated patients) for 36 weeks and to 1 group of patients receiving MTX alone for 12 weeks. The primary end point was the proportion of patients with a >or=4-fold rise in antitetanus IgG levels. Antitetanus, antipneumococcal, and anti-KLH serum IgG levels were measured prior to and 4 weeks following vaccine administration. The DTH response to C albicans was measured 2-3 days following placement. Responses to tetanus toxoid vaccine (>or=4-fold rise) were similar in both groups (39.1% of rituximab-treated patients and 42.3% of patients treated with MTX alone). The ability to maintain a positive DTH response to the C albicans skin test was comparable in both groups (77.4% of rituximab-treated patients and 70% of patients treated with MTX alone), showing no effect of rituximab treatment. Rituximab-treated patients had decreased responses to pneumococcal polysaccharide vaccine (57% of patients had a 2-fold rise in titer in response to >or=1 serotype, compared with 82% of patients treated with MTX alone) and to KLH vaccine (47% of patients had detectable anti-KLH IgG, compared with 93% of patients treated with MTX alone). Recall responses to the T cell-dependent protein antigen tetanus toxoid as well as DTH responses were preserved in rituximab-treated RA patients 24 weeks after treatment. Responses to neoantigen (KLH) and T cell-independent responses to pneumococcal vaccine were decreased, but many patients were able to mount responses. These data suggest that polysaccharide and primary immunizations should be administered prior to rituximab infusions to maximize responses.

Mapping TB resistance

The first genetic resistance factor for tuberculosis (TB) infection is now reported by Cobat et al. The group has identified one locus that determines whether an individual will respond to the TB skin test and a second that controls the extent of that response. The results suggest that one major genetic locus controls innate resistance to the pathogen in humans. Two-thirds of the global population is infected with Mycobacterium tuberculosis, the agent responsible for TB. Yet the disease only manifests in about 10% of infected individuals. To find genetic variants controlling susceptibility to infection, the authors studied 128 families in Cape Town, South Africa, where TB is highly endemic. Although most subjects were likely to have been exposed to M. tuberculosis, about 40% did not show delayed type hypersensitivity (DTH) in a skin antigen test. This strong resistance mapped to a 6-Mbp chromosome region, 11p14. This locus may unveil cellular mechanisms that might one day be manipulated to prevent TB—an important goal given the recent rise in drug-resistant strains. The second locus, in the 2.9-Mbp 5p15 region, segregated with differing extents of positive DTH responses. These genetic factors might contribute to whether an infected individual keeps the bacterium dormant or develops the disease. The same chromosomal region is associated with susceptibility to sarcoidosis, an immune disorder of persistent inflammation that is often associated with TB. A candidate 5p15 gene is a solute carrier (SLC) family member. A human SLC member called NRAMP1 is known to influence granuloma responses to mycobacteria, and loss of the mouse SLC6A3 protein reduces DTH response to ovalbumin. NL

Humoral and cellular immune response to a crude exo-antigen and purified keratinase of Microsporum canis in experimentally infected guinea pigs

In order to understand better the host–parasite relationship and to compare with previous observations in Microsporum canis naturally infected cats, the humoral and cellular immune responses to both a crude exo-antigen and a 31·5 kDa purified keratinase were evaluated in 12 M. canis experimentally infected guinea pigs. Humoral and cellular responses were assessed by ELISA from days 0 to 56 postinfection (PI) and by measurement of delayed-type hypersensitivity (DTH) responses on days 14 and 57 PI, respectively. Additionally, immunohistochemical staining was performed and demonstrated that the keratinase was produced in infected guinea pig skin, as previously reported in cats. Despite a marked interindividual variation, all the guinea pigs produced specific IgG to the crude exo-antigen from day 21 PI onwards, but no anti-keratinase IgG was detected. Strongly positive DTH responses to the exo-antigen were observed on both dates, whereas the keratinase elicited no and weak DTH on days 14 and 57 PI, respectively. These results are in agreement with those previously described for naturally infected cats, and indicate that the 31·5 kDa keratinase is not a major antigen in M. canis infection.

Phase II trial of combination therapy of tumor lysate-pulsed dendritic cells and adoptive transfer of anti-CD3 activated T cells (DC/VAC) to lower postsurgical recurrence rates of cholangiocellular carcinoma (CCC)

3068 Background: Postsurgical recurrence of CCC is frequent and fatal. This phase II study investigated the efficacy and safety of DC/VAC in patients (pts) who underwent curative resection for CCC to lower postsurgical recurrence rates. Methods: Patients (pts) with CCC who underwent curative resection received DC/VAC. DC/VAC consisted of dendritic cells with autologous lymphocytes activated with interleukin-2 and antibody to CD3 at 14-day intervals for 3 months. The immune responses to tumor antigens (Tgs) were monitored by delayed- type hypersensitivity (DTH). The primary endpoint was relapse-free survival (RFS). Secondary endpoints were overall survival (OS), the monitoring of immune responses to Tgs, and toxicity. Results: Between September 2000 and August 2005, 26 pts were included, with a median age of 61.4 years, a median tumor size of 5.8 cm. Twenty-five pts (96 %) underwent segmentectomy. On the pathological findings, vascular invasions, intrahepatic metastases (IM), and lymph node involvements (LNM) were 77 %, 27 %, and 50 %, respectively. The median RFS was 7.4 months. Eight pts showed positive DTH responses to Tgs. Among these pts, the median PFS was 22.5 months. Conclusions: This result suggests that DC/VAC is very effective in pts with positive DTH responses to Tgs who underwent curative resection for CCC to lower postsurgical recurrence rates. No significant financial relationships to disclose.

Adoptive Immunotherapy with Autologous CD25-Depleted and CD3/CD28-Costimulated T-Cells (ACTC) Enhances Numeric and Functional Lymphocyte Recovery after Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma.

Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal 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. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) may enhance immune reconstitution and improve disease control. We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1 – 3 and cyclophosphamide (250 mg/m2) days 1 – 3. Four weeks after last chemotherapy, responding patients (CR, CRu, PR) receive escalating doses of ACTC prepared from autologous T-cells collected prior to chemotherapy. Thirteen pts have been enrolled to date. Median age is 49 y (range: 32 – 68). Median number of prior therapies is 2 (range: 1 – 3). Two pts were withdrawn from the study due to hematologic toxicity related to chemotherapy, one patient was withdrawn for progressive disease during chemotherapy, and one patient has not completed first response assessment after ACTC infusion. For 9 assessable pts completing chemotherapy and ACTC infusion, 7 pts achieved a CR and 2 pts achieved a PR; 5 pts received 1 – 5 x 109 and 4 patients received 5 – 10 x 109 CD3+ ACTC. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 20 months (range: 2 – 42 months). CD4 counts increased in all patients by 1 month after T-cell infusion, with a median increase of 3.8 fold (n = 8; range: 1.5 – 71). For patients at dose level 1, the median increase was 2.2 fold (n = 4; range: 1.5 – 21); at dose level 2, it was 4.2 fold (n = 4; range: 3.8 – 71). CD8 counts also increased, with a median increase of 8.1 fold (range: 1.0 – 30). All 9 pts receiving ACTC were anergic to candida antigen by delayed type hypersensitivity (DTH) skin testing before chemotherapy; 5 pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. From the start of therapy for patients receiving T-cells, median follow-up is 24 months (range: 6 – 47) with median progression-free survival of 18 months, which is significantly longer than the time to progression from last therapy (median 11 months) (p = 0.01, log-rank test). Thus, ACTC results in more rapid CD4+ lymphocyte recovery in pts after cyclophosphamide-fludarabine chemotherapy compared to historical controls and results in reconstitution of recall immunity. These encouraging clinical responses provide the rationale for clinical trials to test ACTC as a way to enhance cellular immunity to tumor-specific antigens after chemotherapy.

Adaptive Immunotherapy with Autologous CD25-Depleted and CD3/CD28-Costimulated T-Cells Enhances Lymphocyte Recovery after Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma.

BACKGROUND: Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal 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. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells (ACTC) expanded ex vivo may enhance immune reconstitution and improve disease control.METHODS: We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after the last cycle, responding patients (CR/CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cellss collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28.RESULTS: Eleven pts have been enrolled to date. Median age is 49 y (range: 33–64y). Median number of prior therapies is 2 (range: 1 – 3). Two pts were withdrawn from the study due to hematologic toxicity related to chemotherapy, one patient was withdrawn for progressive disease during chemotherapy, and one patient has not yet completed chemotherapy. Of the 7 pts completing chemotherapy and proceeding to T-cell infusion, 5 pts achieved a CR/CRu and 2 pts achieved a PR; 5 pts received 1 – 5 x 109 CD3+ cells and 2 patients received 5 – 10 x 109 CD3+ cells. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 19 mos (range: 3 – 26 mos). CD4+ counts increased in all patients by 1 month after T-cell infusion, with a median increase of 3.2 fold (range: 1.5 – 70, p= .028)(see figure). For patients at dose level 1, the median increase was 2.2 fold (n=4; range: 1.5 – 3.3); at dose level 2 it was 37 fold (n=2; range: 3.8 – 70). CD8+ counts also increased, with a median increase of 8.1 (range: 1.0 – 30, p= .046)(see figure). All 7 pts receiving ACTC were anergic to candida antigen by delayed type hypersensitivity (DTH) skin testing before chemotherapy. Four pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. For patients receiving ACTC, median follow-up is 24 mos (range: 7 – 31 mos) with a median progression-free survival of 18 months which is significantly longer than the time to progression from last therapy (p= .024).CONCLUSIONS: ACTC infusion results in significant CD4+ and CD8+ numerical and functional lymphocyte recovery after cyclophosphamide-fludarabine chemotherapy in pts with low-grade FL. This compares very favorably to historical controls treated with fludarabine-based regimens. T-cell dose escalation is ongoing. [Display omitted]

Objective Clinical Regression of Metastatic Breast Cancer in Disparate Sites after Use of Whole-Cell Vaccine Genetically Modified to Release Sargramostim

A patient with recurrent breast cancer metastases following initial response to chemotherapy and hormonal maintenance was treated with a whole-cell tumor vaccine, resulting in a prompt objective complete remission of a lung lesion on computed tomography (CT) scans and near-complete regression of multiple breast lesions on magnetic resonance imaging (MRI). Three months after completion of the protocol, metastases were again found in the breast and lung, with new lesions in the brain and liver. Reinstitution of vaccine inoculation resulted in major regression of the brain and breast lesions, improvement in all other areas, and no indication of new lesions. Therapy consisted of inoculation of 20 x 10(6) SV-BR-1-GM cells, a unique breast cancer cell line transfected to release sargramostim (granulocyte macrophage colony-stimulating factor [GM-CSF]). Following lethal irradiation to 200 cGy, vaccine was injected intradermally in four divided doses to the back and thighs, every 2 weeks x 3, then every month x 3. Each treatment was preceded 48 hours earlier with low-dose cyclophosphamide 300 mg/m2 to abrogate regulatory T-cell activity. Interferon (IFN)-alpha, 20,000 IU, was injected into each inoculation site at 48 and 96 hours postinoculation to provide an additional "danger signal." The patient developed positive delayed-type hypersensitivity responses and also antibody reactivity to the vaccine cells.

Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate‐pulsed mature, monocyte‐derived dendritic cells

We performed a clinical trial to assess the feasibility and efficacy of immunotherapy with peptides, tumor lysate or both peptides and tumor lysate-pulsed mature, monocyte-derived dendritic cells (DC) for advanced malignant melanoma patients that are resistant to conventional therapies. Sixteen patients were enrolled in this trial. All patients received DC vaccines i.d. in the proximal thigh, close to the inguinal lymph nodes, one treatment per week or 2 weeks. Several factors such as clinical findings, computed tomography (CT) images, delayed type hypersensitivity (DTH) response, enzyme-linked immunosorbent spot (ELISPOT) assay, and immunohistochemistry in primary, metastatic lesions and the DTH site were evaluated. Clinical results through DC vaccination were as follows: in 11 evaluable cases, three stable disease, six progression of disease and two disease-free from the time of study entry to the completion of one vaccination course. One patient showed reduction of the tumors in the metastases on chest CT during the first and second course of DC vaccination. Ten out of 14 evaluable cases showed positive DTH responses to more than one treatment with melanoma peptides or tumor lysate. Eight out of 13 evaluable cases showed positive immunological responses to more than one treatment with melanoma peptides or tumor lysate in an ELISPOT assay. As for the experiences with toxicity and adverse reactions, autosensitization dermatitis-like eruptions appeared in five cases during DC vaccination. No severe adverse effects were seen in any of the patients. In our study, the clinical efficacy in prolongation of the patients' survival was confirmed. At the same time, cancer immunoediting of the tumor was also found. It will be necessary to improve the tumor-specificity of this therapeutic approach and to analyze the mechanism(s) of tumor escape from immunosurveillance in melanoma.

Control of Residual Disease in Imatinib Treated Chronic Myeloid Leukemia Patients with Peptide Vaccinations: 2 Years Follow up of CMLVAX100 Trial.

In the past five years, the body of data concerning imatinib mesylate had certainly defined the role of this drug as very effective first line debulking therapy for CML patients. However, both the persistence of molecular disease in most of patients together with the evidence that discontinuation of imatinib inevitably exerts on a rapid loss of response, suggest that with imatinib alone the cure of CML is unlikely. CMLVAX100 is a peptide-based vaccine that specifically targets p210-b3a2 in CML cells through the induction of a peptide-specific T cell response in b3a2-CML vaccinated patients. We recently published the preliminary results of a pilot study testing the immunological response and antitumor activity of vaccinations with CMLVAX100 in 10 patients showing persistent residual disease during treatment with imatinib. We here report the results on a larger cohort of patients and with a longer follow up. Up to date 21 patients were enrolled in this study. After a median time of 24 months (range 12–50) of imatinib treatment, patients started vaccinations with CMLVAX100 showing different degrees of persistent residual disease: 8/21 patients presented with molecular disease, 10/21 showed residual cytogenetic disease (range 2–45% of Ph+ metaphases), while 3/21 presented only an hematological response (100% of Ph+ metaphases). No improvement of their residual disease was documented for a median of 12 months (range 6–24) before starting vaccinations. Vaccine treatment plan included 6 vaccinations at 2 weeks interval (immunization) and for responder patients additional boosts of vaccine every 4–6 months. So far 18/21 patients completed the immunization and are evaluable for response; in addition 8/18 received 4 further boosts of vaccine. After immunization, CMLVAX100 induced a prompt immunological response in most of the patients as 17/18 patients showed peptide specific T cell response in vitro and 12/18 developed a positive delayed type hypersensitivity response in vivo. After 6 vaccinations 6/10 patients with persisting cytogenetic disease reached a complete cytogenetic response (CCR), with 3 of them achieving an undetectable level of bcr-abl transcript. In addition, 3/5 evaluable patients starting vaccinations with persistent molecular disease, further reduced their bcr-abl level, wth one reaching molecular negativity. None of the 3 patients vaccinated in hematological remission had cytogenetic response. Of the 8 patients who underwent 4 additional boosts of vaccine, one reached a complete molecular response, 5 maintained the response obtained after immunization, while 2 patients who previously achieved an undetectable level of bcr-abl transcript, lost the complete molecular response, while maintaining CCR, thus suggesting that a 6 months interval between boosts could be too long to maintain an efficient immune control on residual leukemic cells. In conclusion, we confirm that CMLVAX100 has a synergistic antitumor effect with imatinib in CML patients with persistent minimal residual disease. However, follow-up data recommend that closer boosts of vaccine are necessary in order to maintain an optimal level of response.

Adoptive Immunotherapy with Autologous CD25-Depleted and CD3/CD28-Costimulated T-Cells (ACTC) Enhances Numeric and Functional Lymphocyte Recovery after Cyclophosphamide - Fludarabine Chemotherapy in Patients with Low-Grade Follicular Lymphoma.

Fludarabine-based chemotherapy combinations are highly effective in patients (pts) with low-grade follicular lymphoma (FL), but cause severe and long-lasting immunosuppression due to depletion of normal 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. Adoptive immunotherapy using autologous CD25-depleted, CD3/CD28-costimulated T-cells expanded ex vivo (ACTC) may enhance immune reconstitution and improve disease control. We initiated a phase I study in pts with purine analog-naive relapsed/refractory FL (grades 1 and 2). After leukapheresis, pts receive 4 cycles of fludarabine (25 mg/m2) days 1–3 and cyclophosphamide (250 mg/m2) days 1–3. Four weeks after last chemotherapy, responding patients (CR, CRu, PR) receive escalating doses of ACTC prepared ex vivo from autologous T-cells collected prior to chemotherapy and depleted of regulatory CD4+/CD25+ cells, then expanded and activated using anti-CD3 and anti-CD28. Eight pts have been enrolled to date. Median age is 40.5 y (range: 32–64). Median number of prior therapies is 2 (range: 1–3). Two pts were withdrawn from the study due to hematologic toxicity related to the chemotherapy; one patient has not completed chemotherapy. Of the 5 pts completing chemotherapy, 3 pts achieved a CR and 2 pts achieved a PR; 4 pts received 5 x 109 and 1 patient received 1 x 1010 CD3+ ACTC. There have been no adverse events related to T-cell infusions. Median follow-up after ACTC infusion is 9 months (range: 2–15 months). Median time to CD4 count >200 /uL was 29 days following T-cell infusion (range 28–127 days). CD4 counts increased in all patients by 1 month after T-cell infusion, with a median increase of 126% from baseline (range 50 to 484%). CD8 counts also increased, with a median increase of 82% (range −4 to 976%). All 5 pts receiving ACTC were anergic to candida antigen by Delayed Type Hypersensitivity (DTH) skin testing before chemotherapy. Three pts developed a positive DTH response to candida antigen 60 days after ACTC infusion. From the start of therapy for patients receiving T-cells, median follow-up is 14 months (range 7–20 months) with median progression-free survival not reached; 4 pts remain in remission and 1 patient had progression of disease. ACTC results in significant CD4+ lymphocyte recovery in previously treated pts receiving cyclophosphamide-fludarabine chemotherapy and compares very favorably with historical controls. T-cell dose escalation is ongoing.

An emerging peri-urban pattern of infection with Leishmania chagasi, the protozoan causing visceral leishmaniasis in northeast Brazil.

Peri-urban visceral leishmaniasis (VL) caused by Leishmania chagasi is emerging in a new epidemiologic pattern in Brazilian cities. We studied peri-urban VL in endemic neighborhoods surrounding Natal, Brazil, identified through hospitalized individuals with VL. Clinical and environmental information obtained for 1106 members of 216 families living in endemic neighborhoods enabled us to identify 4 groups: VL: individuals with current or prior symptomatic visceral leishmaniasis (n = 135); DTH+: individuals with positive delayed-type hypersensitivity response with no history of VL (n = 390); Ab +: individuals with negative DTH response and seropositive (n = 21); DTH -: individuals with negative DTH and seronegative (n = 560). The mean +/-SD age of VL was 9.3+/-12.3 y. The gender distribution was nearly equal below age 5, but skewed toward males at higher ages. Acutely infected VL subjects had significantly lower hematocrits, neutrophils, and eosinophils than other categories. AB+ subjects also had lower eosinophil counts than others, a possible immune marker of early infection. VL was not associated with ownership of dogs or other animals, raising the question whether the reservoir differs in peri-urban settings. This new pattern of L. chagasi infection enables us to identify epidemiological and host factors underlying this emerging infectious disease.

Immunopharmacologic Analysis of an Autologous, Hapten-Modified Human Melanoma Vaccine

We have previously reported a clinical trial of a human cancer vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP), in patients with clinical stage III melanoma. Here we present a follow-up report expanded to 214 patients with 5-year follow-up. Two hundred fourteen patients with clinical stage III melanoma (117 patients with stage IIIC and 97 patients with stage IIIB) who were melanoma-free after standard lymphadenectomy were treated with multiple intradermal injections of autologous, DNP-modified vaccine mixed with bacille Calmette-Guérin. Four vaccine dosage schedules were tested sequentially, all of which included low-dose cyclophosphamide. Patients were tested for delayed-type hypersensitivity (DTH) to autologous melanoma cells, both DNP-modified and unmodified, and to control materials. The 5-year overall survival (OS) rate of the 214 patients was 44%. DTH responses to unmodified autologous melanoma were induced in 47% of patients. The OS of this DTH-positive group was double that of DTH-negative patients (59.3% v 29.3%; P <.001). In contrast, positive DTH responses to DNP-modified autologous melanoma cells and to purified protein derivative developed in almost all patients but did not affect OS. Surprisingly, the OS after relapse was also significantly longer in patients who developed positive DTH to unmodified tumor cells (25.2% v 12.3%; P <.001). Finally, the development of DTH was dependent on the schedule of administration of the vaccine, specifically, the timing of an induction dose administered at the beginning of the treatment program. This study underscores the importance of the immunopharmacology of the autologous, DNP-modified vaccine and may be relevant to other cancer vaccine technologies.

Delayed type hypersensitivity response to recall antigens does not accurately reflect immune competence in advanced stage breast cancer patients.

The development of delayed-type hypersensitivity (DTH) response to recall antigens has long been utilized as a measure of immune competence. It is assumed that because patients with advanced stage cancers exhibit multiple immune system defects they may not be responsive to immunization. We pre-selected patients with advanced HER-2/neu (HER2) overexpressing breast and ovarian cancers for enrolment into a phase I trial designed to evaluate the immunogenicity of a HER2 peptide vaccine based on the patient's immune competence as assessed by DTH skin testing to common recall antigens (Multitest CMI, Institut Merieux, Lyon, France). At the time of a positive DTH response to tetanus toxoid (tt) peripheral blood was obtained to measure T cell responses to tt. Of 53 patients evaluated, 38 (72%) were not anergic. Among the 15 (28%) who were, seven patients with advanced stage breast cancer were re-tested a median of 26 days (range 12-150 days) after receiving a tt bopster vaccination. Five of the seven had positive DTH responses when re-challenged with tt and six had peripheral blood tetanus specific T cell response with stimulation index >2.0. Thus, the majority of patients studied with advanced stage breast or ovarian cancer were able to mount a DTH response to common recall antigens. Moreover, a negative response by DTH testing to a battery of common recall antigens was not a reflection of the breast cancer patient's ability to mount a cell-mediated immune response to a vaccinated antigen, tt.

Clinical and immuno-virologic characterization of the efficacy of stavudine, lamivudine, and indinavir in human immunodeficiency virus infection

Clinical, virologic, and immunologic outcomes were analyzed in children with vertically transmitted human immunodeficiency virus (HIV) infection (n = 25) and clinical symptoms and evidence of immunosuppression to establish the efficacy of 18 months’ treatment with stavudine, lamivudine, and indinavir. Children were naive for treatment with protease inhibitors and lamivudine and had minimal exposure to stavudine. At 1, 6, 12, and 18 months, the proportions of patients with HIV-RNA <400 copies/mL were 79%, 100%, 94%, 87% in Centers for Disease Control and Prevention (CDC) immunologic class 2 and 50%, 67%, 67%, 72% in CDC immunologic class 3. At 12 months, the median CD4 + count and percent increased significantly in both CDC immunologic class groups, but to a greater extent in the class 3 group. In the 12- to 18-month period, there were no significant changes within the groups. In both groups there was a steady increase in the proportion and number of children with positive skin test responses. Children in class 2 were more likely to have a positive delayed-type hypersensitivity response and a greater number of positive responses. Lymphocyte proliferative response to recall antigens improved significantly in all patients. The rate of increase in positive test results was faster in children in class 2 than in those in class 3. Only minor clinical events occurred during 18 months of therapy. Potent antiretroviral therapy achieves a sustained benefit in HIV-infected children, but immune reconstitution is more likely achieved in children with less advanced disease. (J Pediatr 1999;135:675-82)