Endogenous Immunity Research Articles

Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

BackgroundRecombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.Methodology/Principal FindingsPeptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.Conclusions/SignificanceAP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.

Atrial Natriuretic Peptide Receptor Signaling Plays a Critical Role in Induction of Tolerogenic Dendritic Cells

RATIONALE: Atrial natriuretic peptide (ANP) is an important endogenous hormone, controlling inflammation and immunity by acting through dendritic cells (DCs), however, the mechanisms remain unclear. We sought to determine whether ANP receptor, NPRA, signaling modulates DC function and analyze the downstream signaling events. METHODS: The role of NPRA signaling in modulating DC function was examined using NPRA-signaling inhibiting peptide NP73-102. Immunoblotting and immunoprecipitation assays of human monocyte-derived DC (hmDC) were used to define the potential downstream molecules and the protein interactions among these molecules involved in this signaling pathway. Regulatory T cell phenotype and function were analyzed using flow cytometry and BrdU proliferation ELISA assay. Adoptive transfer studies in mice involved cytokine assays and immunohistochemical analyses. RESULTS: Attenuation of NPRA signaling in DC generates more Treg in our in vitro T-DC co-culture system. Analysis of DC surface marker expression and in vitro phagocytosis suggested that DC maturation was unaffected significantly by modulated NPRA signaling. NP73-102-mediated increases in levels of IL-10 and TGF-β, but not of IL-12 and IFN-γ, compared to those in cells given ANP or vector alone. DC adoptive transfer studies in mice indicated that NPRA signaling in DCs primes DCs to induce Treg cells and attenuated lung inflammation. NPRA signaling is associated with TLR-2, SOCS3 and STAT3 and affects induction of IL-6, IL-10 and TGF-β but not IL-12. CONCLUSIONS: TLR2 and SOCS3 are integral to NPRA signaling in DCs leading to immunity or tolerance induction.

Harnessing the effect of adoptively transferred tumor-reactive T cells on endogenous (host-derived) antitumor immunity.

Adoptive T cell transfer therapy, the ex vivo activation, expansion, and subsequent administration of tumor-reactive T cells, is already the most effective therapy against certain types of cancer. However, recent evidence in animal models and clinical trials suggests that host conditioning interventions tailored for some of the most aggressive and frequent epithelial cancers will be needed to maximize the benefit of this approach. Similarly, the subsets, stage of differentiation, and ex vivo expansion procedure of tumor-reactive T cells to be adoptively transferred influence their in vivo effectiveness and may need to be adapted for different types of cancer and host conditioning interventions. The effects of adoptively transferred tumor-reactive T cells on the mechanisms of endogenous (host-derived) antitumor immunity, and how to maximize their combined effects, are further discussed.

Phase I-II Study of Denileukin Diftitox (ONTAK®) in Patients with Advanced Refractory Breast Cancer.

Abstract Background: CD4+CD25+Foxp3+ regulatory T cells (Tregs) are potent suppressors of CD4+ and CD8+ T cells, produce the immunosuppressive cytokine TGF-β; and as such, may down-regulate immune responses to tumor antigens. Additionally, Tregs are increased in the peripheral blood (PB) and tumors of breast cancer patients; and are associated with poor prognosis. Depletion of PB and tumor-associated Tregs may induce anti-tumor immunity by augmenting anti-tumor effector T cells and enhancing endogenous tumor specific immunity. ONTAK®, a diphtheria/IL-2R fusion protein depletes PB Tregs when given intravenously (IV) and selectively targets tumor cells that overexpress IL-2R. Breast tumors have been shown to overexpress IL-2R which is associated with their malignant potential. We hypothesized that ONTAK® could (1) have direct anti-tumor activity in breast cancers that overexpress IL-2R, and (2) deplete Tregs resulting in generation of functional immune effector cells and enhanced anti-tumor immunity. A phase I-II study was conducted to evaluate the safety of IV ONTAK® and assess its effect on Tregs and endogenous immunity in patients with advanced refractory breast cancer.Materials and Methods: 15 patients with progressive stage IV breast cancer following standard therapy were sequentially enrolled and received IV ONTAK® 18 mcg/kg/day on Days1-5 every 21 days for a total of 6 cycles and/or maximal tumor response. Toxicity was evaluated on Days 1 8, and 14 of each cycle per CTEP CTCAE v3.0. Tumor response was evaluated per RECIST at baseline, and after cycles 3 and 6. PB was collected at baseline and after cycles 2, 4, and 6 for evaluation of Tregs, sIL-2R, and endogenous tumor-antigen specific T cell immunity to HER-2/neu (HER2), CEA, and MAGE-3 via RT-PCR, LUMINEX and IFN-γ ELISPOT assay, respectively. Expression of IL-2R in patient paraffin embedded tumor samples was analyzed by IHC analysis.Results: 15 subjects have been enrolled and 14/15 have completed treatment; median age is 58 years (range, 32-69) and median salvage regimens is 3 (range, 2-8). 7/14 subjects had triple negative tumors. 7 subjects completed 1-2 and 7 completed 3-6 ONTAK® cycles, respectively. 4 subjects who completed 6 cycles of ONTAK® had SD or PR per RECIST. ONTAK®-related toxicities have been primarily grade I and II fatigue, nausea, and headache; and transient grade 3 hypoalbuminemia and lymphopenia. Preliminary data in 2 subjects shows enhanced tumor-antigen specific T cell immunity defined as mean tumor antigen-specific T cell precursors:PBMC to CEA (pre- ONTAK® 1:250,000; post- ONTAK® 1:15,000) and HER2 (pre- ONTAK® 1:63,000; post- ONTAK® 1:6,312). Immunologic analyses are ongoing and will be presented along with clinical data on all patients.Conclusions: ONTAK® is well-tolerated when used as a salvage regimen in heavily pretreated breast cancer patients. Additionally, ONTAK® treatment can enhance endogenous immunity to known breast cancer antigens and potentially lead to more effective eradication of tumor. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4130.

CCL5-Mediated Endogenous Antitumor Immunity Elicited by Adoptively Transferred Lymphocytes and Dendritic Cell Depletion

Adoptive transfer of antitumor T cells is a promisingly effective therapy for various cancers, but its effect on endogenous antitumor immune mechanisms remains largely unknown. Here, we show that the administration of naive T cells de novo primed for only 7 days against tumor antigens resulted in the durable rejection of otherwise lethal ovarian cancers when coupled with the depletion of tumor-associated immunosuppressive dendritic cells (DC). Therapeutic activity required tumor antigen specificity and perforin expression by the adoptively transferred T cells, but not IFN-gamma production. Importantly, these shortly primed T cells secreted large amounts of CCL5, which was required for their therapeutic benefit. Accordingly, transferred T cells recruited CCR5(+) DCs into the tumor, where they showed distinct immunostimulatory attributes. Activated CCR5(+) host T cells with antitumor activity also accumulated at tumor locations, and endogenous tumor-specific memory T cells remained elevated after the disappearance of transferred lymphocytes. Therefore, persistent, long-lived antitumor immunity was triggered by the administration of ex vivo activated T cells, but was directly mediated by immune cells of host origin. Our data unveil a CCL5-dependent mechanism of awakening endogenous antitumor immunity triggered by ex vivo expanded T cells, which is augmented by tumor-specific targeting of the cancer microenvironment.

Phase II experience with MDX-1106 (Ono-4538), an anti-PD-1 monoclonal antibody, in patients with selected refractory or relapsed malignancies

3018 Background: Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, may suppress antitumor immunity. This expansion cohort in a phase I/II trial of MDX-1106, a fully human IgG4 antibody blocking PD-1, sought to further evaluate the safety and activity of the 10 mg/kg dose. Other objectives included evaluation of pharmacokinetics (PK) and immunological effects. Methods: Patients (pts) with treatment refractory metastatic non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), colon cancer (CC), melanoma (MEL), or prostate cancer (CRPC), and no history of autoimmune disease received a single infusion of MDX-1106 at 10 mg/kg. Disease status was evaluated at week (wk) 8 by RECIST criteria. Pts with stable disease or lesional responses could receive additional MDX-1106 at wks 12 and 16. Those with PR/CR were observed without retreatment. Results: 21 pts (5 CC, 2 NSCLC, 8 MEL, 5 HRPC, 1 RCC) were treated from 10/07 to present, and 6 were retreated. No MDX-1106 related SAEs occurred. One pt developed arthritic symptoms requiring treatment, and 2 pts had asymptomatic TSH elevation. One pt with RCC had a PR after 3 doses, lasting 5+ months (mo). Lesional regressions (“mixed response”) were seen in 2 MEL pts; to date, one has received 7 doses of MDX-1106 over 15 mo without serious toxicity. Biopsy of a regressing MEL lymph node metastasis showed a moderately increased and selective CD8+ T cell infiltrate post treatment. The median serum t 1/2 of MDX-1106 was 20.6 days, which was roughly 50% longer than observed for lower doses. There was no evidence of immunogenicity based on HAHA development. Interestingly, in contrast to PK results, flow cytometric analysis demonstrated sustained occupancy of 60–80% PD-1 molecules on T cells for at least 3 mo following a single dose. Analyses of circulating lymphocyte subsets and tumor B7-H1 expression are in progress. Conclusions: Intermittent dosing of MDX-1106 at 10 mg/kg demonstrated clinical activity against RCC and MEL without serious toxicity. Therapy with MDX-1106 to enhance endogenous antitumor immunity, either alone or combined with other immunological therapies, warrants further study. [Table: see text]

Potentiating Endogenous Antitumor Immunity to Prostate Cancer through Combination Immunotherapy with CTLA4 Blockade and GM-CSF

CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment antitumor immunity in animal models and is being developed as a treatment for cancer patients. As has been seen in preclinical models, combining CTLA4 blockade and granulocyte macrophage colony-stimulating factor (GM-CSF)-based immunotherapies can enhance the antitumor efficacy of this approach. We therefore examined whether CTLA4 blockade could be combined with GM-CSF administration. We treated 24 patients with metastatic, castration-resistant prostate cancer in a phase I trial where sequential cohorts were treated with increasing doses of ipilimumab, a fully human anti-CTLA4 antibody. Study subjects also received s.c. injections of GM-CSF at a fixed dose. Of the six patients treated at the highest dose level, three had confirmed PSA declines of >50%, including one patient that had a partial response in visceral metastases. Expansion of activated, circulating CD25(+) CD69(+) CD8(+) T cells occurred more frequently at higher doses of treatment and was greater in magnitude than was seen in patients who received the same doses of either ipilimumab or GM-CSF alone. By screening sera with protein arrays, we showed that our treatment can induce antibody responses to NY-ESO-1. These results show that this combination immunotherapy can induce the expansion not only of activated effector CD8 T cells in vivo but also of T cells that are specific for known tumor-associated antigens from the endogenous immune repertoire.

Modulators of arginine metabolism support cancer immunosurveillance

BackgroundTumor-associated accrual of myeloid derived suppressor cells (MDSC) in the blood, lymphoid organs and tumor tissues may lead to perturbation of the arginine metabolism and impairment of the endogenous antitumor immunity. The objective of this study was to evaluate whether accumulation of MDSC occurred in Th2 prone BALB/c and Th1 biased C57BL/6 mice bearing the C26GM colon carcinoma and RMA T lymphoma, respectively, and to investigate whether N(G) nitro-L-arginine methyl ester (L-NAME) and sildenafil, both modulators of the arginine metabolism, restored antitumor immunity.ResultsWe report here that MDSC accumulate in the spleen and blood of mice irrespective of the mouse and tumor model used. Treatment of tumor-bearing mice with either the phosphodiesterase-5 inhibitor sildenafil or the nitric-oxide synthase (NOS) inhibitor L-NAME significantly restrained tumor growth and expanded the tumor-specific immune response.ConclusionOur data emphasize the role of MDSC in modulating the endogenous tumor-specific immune response and underline the anti-neoplastic therapeutic potential of arginine metabolism modulators.

Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines

Cancers express tumor associated antigens that should elicit immune attack, but spontaneous immune rejection of established cancer is rare. Recent data demonstrate that specific and active tumor-mediated mechanisms hinder host anti-tumor immunity. CD4+CD25+ T regulatory cells (Tregs) are important mediators of active immune evasion in cancer. Disrupting tumor-mediated mechanisms hindering host immunity is a novel approach to tumor immunotherapy. Treg depletion improves endogenous anti-tumor immunity and the efficacy of active immunotherapy in animal models for cancer, suggesting that inhibiting Treg function could also improve the limited successes of human cancer immunotherapy. We have identified five strategies to block Treg activity: depletion, interference with trafficking, inhibition of differentiation, blockade of function or raising the effector T cell threshold for suppression. Discovery of additional regulatory cell populations expands the potential targets for these approaches. The fusion toxin denileukin diftitox (Ontak) reduces Treg numbers and function in the blood of some patients with cancer. We discuss specific strategies to block Treg activity and present some of our preliminary data in this area. Combining Treg depletion with active vaccination and other approaches poses additional challenges that are discussed.

CD4+Foxp3+ Regulatory T Cells Persist in High Numbers in Peripheral Blood after Induction of Clinical Remission with Standard Chemotherapy and Suppress Tumor-Specific Effector T Cells in Patients with Follicular Lymphoma

Follicular lymphoma (FL) is one of the most immune responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of endogenous immunity as well as therapeutic vaccines and adoptive T-cell therapy. CD4+CD25+CD127loFoxp3+ regulatory T cells (Tregs) are one of the most potent suppressors of effector T cells and were recently shown to be increased in peripheral blood and tumors of patients with various malignancies. Here, we determined the percentage of Tregs, in the tumors and the peripheral blood mononuclear cells (PBMC) of patients with FL (n = 13) at the time of initial diagnosis and after induction of clinical remission with a uniform cyclophosphamide and doxorubicin containing chemotherapy regimen, PACE (Modified ProMACE without Methotrexate; Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide). We observed that Tregs are markedly increased in number both in the tumors (median 19.45%, range 13.4%–44.5%) and PBMC (median 17.7%, range 7.9%–64.5%) of patients with FL at the time of initial diagnosis as compared with PBMC from normal donors (median 3.33%, range 1.88%–6.3%). Unexpectedly, Tregs persisted in high numbers in peripheral blood after induction of clinical remission with a cyclophosphamide and doxorubicin containing chemotherapy regimen (median 19.95%, range 7.6%–67.2%). To determine the immunosuppressive effects of Tregs, we assessed the proliferation of CFSE-labeled effector T cells following polyclonal stimulation with anti-CD3 and anti-CD28 antibodies. The presence of high numbers of Tregs was associated with significantly decreased proliferation of peripheral blood CD4+ T cells in patients with FL both at baseline (median 24.9%, range 3.7%–40.7%) and after chemotherapy induced clinical remission (median 15.8%, range 1.5%–23.5%) as compared with normal donors (median 38%, range 11.7%–58.8%). A similar decrease in proliferation was also noted in CD8+ T cells in the presence of high numbers of Tregs both at baseline and after induction of clinical remission in patients with FL as compared with normal donors. More importantly, intratumoral and peripheral blood Tregs significantly inhibited effector T cells proliferating in response to autologous tumor cell stimulation. In conclusion, these results suggest that CD4+CD25+CD127loFoxp3+ Tregs that inhibit the function of tumor-specific effector T cells are markedly increased in number in both tumor and peripheral blood of patients with FL and persist in high numbers despite induction of clinical remission with standard cyclophosphamide and doxorubicin containing chemotherapy. This observation may potentially explain the failure of two recent Phase III therapeutic vaccine trials in patients with FL. Furthermore, these results suggest that a state of peripheral tolerance continues to be present after induction of clinical remission and strategies that deplete or block function of Tregs may be necessary to enhance the efficacy of therapeutic vaccines and adoptive T-cell therapy in patients with follicular and possibly other non-Hodgkin's lymphomas.

Maternal transfer of antibodies in vertebrates: trans-generational effects on offspring immunity.

Maternal effects by which females provide their offspring with non-genetic factors such as hormones, nutrients and antibodies can have an important impact on offspring fitness. In vertebrates, maternal antibodies (matAb) are transferred from the mother, via the placenta, egg yolk or milk during lactation to offspring until they are 2 weeks (birds), 4-10 weeks (rodents) and 9 months (humans) old, respectively. matAb transfer can have direct effects on offspring growth rate in birds and rodents, probably by passively protecting the newborn from common pathogens before their endogenous immune system has matured. Indirect long-term effects of matAb transfer on the offspring's own immunity can be synergistic, if matAb act as antigen templates of the accumulated immunological experience of the mother and educate the newborn's immune system. However, it may also be suppressive if matAb reduce antigen presentation to the newborn resulting in antigen-specific blocking of offspring endogenous immunity. Our aim is to review the mechanisms and direct effects of matAb transfer in vertebrates with an emphasis on birds, outline a framework for research on the long-term effects of matAb on the endogenous immune system of the mature offspring and encourage ecological and evolutionary studies of matAb transfer in non-domesticated animals.

Potent Tumor-Specific Protection Ignited by Adoptively Transferred CD4+ T Cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.

Safety and activity of MDX-1106 (ONO-4538), an anti-PD-1 monoclonal antibody, in patients with selected refractory or relapsed malignancies

3006 Background: Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, may suppress antitumor immunity. Objectives of this first in human phase I trial of MDX-1106 (ONO-4538), a fully human IgG4 anti-PD-1 blocking antibody, are to evaluate safety and maximum tolerated dose (MTD). Other objectives include evaluation of pharmacokinetics (PK) and immunological effects. Methods: Patients (pts) had treatment refractory metastatic non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), colon cancer (CC), melanoma (MEL), or prostate cancer (HRPC), and no history of autoimmune disease. Sequential cohorts of 6 pts received a single IV dose of MDX-1106 at 0.3, 1, 3, or 10 mg/kg. Disease status was evaluated at wk 8 by RECIST criteria. Pts with SD or lesional responses could receive additional MDX-1106 at wks 12 and 16, at the original dose. Results: 24 pts, 6 at each dose level (9 CC, 5 NSCLC, 6 MEL, 4 HRPC), were enrolled through 11/07. 23 pts have been assessed for tumor response at 8 wks. All doses were well tolerated and no MDX-1106 related SAEs occurred. Two pts (treated at 3 and 10 mg/kg) developed arthritic symptoms requiring treatment, and 2 pts had an asymptomatic TSH elevation. Objective tumor responses (CR or PR) were not observed within 8 wks. Three pts had significant lesional regressions (1 NSCLC pt at 1 mg/kg, 2 MEL pts at 10 mg/kg). Five pts with SD or lesional regressions received retreatment. Biopsy of a regressing MEL lymph node metastasis showed a moderately increased CD8+ T cell infiltrate post treatment. PK and immunological evaluations are underway. Conclusions: The targeted maximum dose of 10 mg/kg of MDX-1106 was achieved without observing dose-limiting toxicities, and lesional tumor regressions were observed in 3 of 23 pts evaluated. An expansion cohort at 10 mg/kg is enrolling 15 additional pts. Therapy with MDX-1106 to enhance endogenous antitumor immunity, either alone or in combination with other therapeutic modalities, warrants further study. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Amplimmune, Inc., Medarex Amplimmune, Inc., Bristol-Myers Squibb, Medarex, Inc. Amplimmune, Inc., Medarex Cell Genesys, Inc., Medarex

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Endogenous Cytotoxic T-Cell Response Contributes to the Long-Term Antiretroviral Protection Induced by a Short Period of Antibody-Based Immunotherapy of Neonatally Infected Mice

Neutralizing monoclonal antibodies (MAbs) are increasingly being considered for blunting human viral infections. However, whether they can also exert indirect effects on endogenous antiviral immune responses has been essentially overlooked. We have recently shown that a short (several-day) period of immunotherapy with the neutralizing 667 MAb of mouse neonates shortly after infection with the lethal FrCas(E) retrovirus not only has an immediate effect on the viral load but also permits an endogenous antiviral immunity to emerge. Even though passive immunotherapy was administered during the particular period of immunocompetence acquisition, the endogenous response eventually arising was protective and persisted long (>1 year) after the MAb has disappeared. As very high levels of anti-FrCas(E) antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype and showing strong neutralization activity, were found in the sera of MAb-treated mice, it was necessary to address whether this humoral immunity was sufficient on its own to confer full protection against FrCas(E) or whether a cytotoxic T-lymphocyte (CTL) response was also necessary. Using a variety of in vivo assays in young and adult animals previously infected by FrCas(E) and treated by 667, we show here that transient 667 immunotherapy is associated with the emergence of a CTL response against virus-infected cells. This cytotoxic activity is indispensable for long-term antiviral protective immunity, as high neutralizing antibody titers, even enhanced in in vivo CD8(+) cell depletion experiments, cannot prevent the FrCas(E)-induced death of infected/treated mice. Our work may have important therapeutic consequences, as it indicates that a short period of MAb-based immunotherapy conducted at a stage where the immune system is still developing can be associated with the mounting of a functional Th1-type immune response characterized by both CTL and IgG2a-type humoral contributions, the cooperation of which is known to be essential for the containment of chronic infections by a variety of viruses.

Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect againstBacillus anthracisInfection and Enhance Endogenous Immunity to Anthrax

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-mug dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, tadalafil, and vardenafil) are agents currently in clinical use for nonmalignant conditions. We report the use of PDE5 inhibitors as modulators of the antitumor immune response. In several mouse tumor models, PDE5 inhibition reverses tumor-induced immunosuppressive mechanisms and enables a measurable antitumor immune response to be generated that substantially delays tumor progression. In particular, sildenafil, down-regulates arginase 1 and nitric oxide synthase–2 expression, thereby reducing the suppressive machinery of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) recruited by growing tumors. By removing these tumor escape mechanisms, sildenafil enhances intratumoral T cell infiltration and activation, reduces tumor outgrowth, and improves the antitumor efficacy of adoptive T cell therapy. Sildenafil also restores in vitro T cell proliferation of peripheral blood mononuclear cells from multiple myeloma and head and neck cancer patients. In light of the recent data that enzymes mediating MDSC-dependent immunosuppression in mice are active also in humans, these findings demonstrate a potentially novel use of PDE5 inhibitors as adjuncts to tumor-specific immune therapy.

Antioxidant Micronutrients in Major Trauma and Burns: Evidence and Practice

There has been a growing interest in micronutrients as a result of their essential role in endogenous antioxidant defense mechanisms and immunity. Critically ill burn and trauma patients are characterized by an increased free radical production, which is proportional to the severity of the injury. In addition, they are at high risk of negative trace element balances, which contribute to the imbalance in endogenous antioxidant capacity and the extension of primary lesions. Although selenium, zinc, and vitamin C and E status are altered in all injured patients, patients with major burns are unique for having copper deficiency. In major burns, high-dose ascorbic acid for 24 hours achieves reduction of resuscitation fluid requirements by endothelial antioxidant mechanisms both in animal models and in 1 human trial. Supplementation trials in trauma and burns including selenium and zinc have shown that early provision of micronutrients improves recovery. Vitamin supplementation trials without selenium have not achieved definitive effects. The human studies show that reinforcing antioxidant defenses early in the course of major injury is rational and that substituting the large initial micronutrient losses of selenium and zinc is safe in trauma, as is the addition of copper in burns. The IV route seems the only way to deliver the doses required to obtain a clinical effect.

The effect of immunotherapy with extracted tumor antigens on sinecomitant immunity.

The fate of hosts after tumor resection depends upon the level of their sinecomitant immunity, which can destroy residual neoplastic cells. In addition, active specific immunotherapy can stimulate the resistance of mice incompletely resected of large tumors and therefore destined to develop recurrent disease. The studies presented herein, assessing host resistance by in vivo tumor challenge and an in vitro growth inhibition assay (GIA) measuring the effect of splenic lymphocytes on 3H-thymidine incorporation into monolayers of sarcoma cells, revealed an antagonistic relationship between sinecomitant immunity and tumor antigen immunotherapy. Treatment of hosts bearing high levels of endogenous sinecomitant immunity with Fraction 15 pI 5.95 partially purified from 3 M KCl extracts by preparative isoelectric focusing, decreased their resistance to tumor challenge and the capacity of their spleen cells to perform in the in vitro GIA. When sinecomitant immunity was not demonstrable, therapy with tumor antigen induced host resistance. If therapy was delayed until sinecomitant immunity had naturally waned, hosts displayed improved resistance toward secondary challenge. These findings suggest that not only does antigen therapy effect tumor resistance by a mechanism independent of sinecomitant immunity, but also these two mechanisms are mutually antagonistic. Therefore, the design of active specific immunotherapy protocols utilizing tumor antigens may depend on the level of the host endogenous sinecomitant resistance.

Patients with breast and ovarian cancer generate immune responses to the folate receptor alpha

20043 Background: Studies have demonstrated that the generation of endogenous immunity to specific tumor antigens is associated with improved prognosis for many cancers. Thus, there has been increased efforts to identify tumor-associated antigens to which there is immunity, as these may be tumor rejection antigens. A candidate antigen is the folate receptor alpha (FRα) which is overexpressed in malignancies, especially breast and ovarian cancer. Prior studies involving ovarian and breast cancer patients have demonstrated the presence of FRα-specific cytotoxic T cells in the tumor-associated lymphocyte population. Methods: Using the RANKPEP CD4 T cell epitope prediction algorithm, we predicted promiscuous immunogenic regions of FRα, and tested for immunity in 30 breast or ovarian cancer patients and 18 healthy volunteer donors in order to attain a better understanding of the levels and extent of the endogenous FRα immune response. Immunity was examined using IFN-γ ELIspot analysis and IgG ELISAs. Results: Fourteen peptides were predicted as potential epitopes to which T cells may respond, seven each from the carboxy- and amino-terminus halves of the protein. Greater than 70% of patients demonstrated T cell immunity to at least one of the fourteen peptides. Patients responded to an average of 3 ± 0.7 peptides while healthy donors responded to only 1 ± 0.5 peptides (p = 0.02). Five peptides were recognized by >25% of patients. Responses to three of these peptides were higher (p > 0.05) in the patients than in healthy donors, suggesting that patients generated immunity upon cancer development. Compared to healthy individuals, patients developed higher immunity to the amino-terminus half of the receptor, suggesting natural epitope spreading (p = 0.03). There was no difference between patients and healthy donors in the immune responses to either non-specific stimuli (p = 0.2) or viral peptides (p = 0.5). Patients had detectable FRα-specific IgG antibody consistent with active FRα-specific helper T cell immunity. Conclusions: These findings demonstrate that the FRα is a target of the immune system in patients with breast or ovarian cancer. Understanding the antigens that are naturally targeted by the immune system may be important for diagnosis, prognosis, and immune-based therapies. No significant financial relationships to disclose.