Break Immune Tolerance Research Articles

Immunogenicity and therapeutic efficacy of a dual-component genetic cancer vaccine cotargeting carcinoembryonic antigen and HER2/neu in preclinical models.

Several cancer vaccine efforts have been directed to simultaneously cotarget multiple tumor antigens, with the intent to achieve broader immune responses and more effective control of cancer growth. Genetic cancer vaccines based on in vivo muscle electro-gene-transfer of plasmid DNA (DNA-EGT) and adenoviral vectors represent promising modalities to elicit powerful immune responses against tumor-associated antigens (TAAs) such as carcinoembryonic antigen (CEA) and human epidermal growth factor receptor-2 (HER2)/neu. Combinations of these modalities of immunization (heterologous prime-boost) can induce superior immune reactions as compared with single-modality vaccines. We have generated a dual component-dual target genetic cancer vaccine consisting of a DNA moiety containing equal amounts of two plasmids, one encoding the extracellular and transmembrane domains of HER2 (ECD.TM) and the other encoding CEA fused to the B subunit of Escherichia coli heat-labile toxin (LTB), and of an adenoviral subtype 6 dicistronic vector carrying the same two tumor antigens gene constructs. The CEA/HER2 vaccine was tested in two different CEA/HER2 double-transgenic mouse models and in NOD/scid-DR1 mice engrafted with the human immune system. The immune response was measured by enzyme-linked immunospot assay, flow cytometry, and ELISA. The CEA/HER2 vaccine was able to break immune tolerance against both antigens. Induction of a T cell and antibody immune response was detected in immune-tolerant mice. Most importantly, the vaccine was able to slow the growth of HER2/neu⁺ and CEA⁺ tumors. A significant T cell response was measured in NOD/scid-DR1 mice engrafted with human cord blood cells. In conclusion, the CEA/HER2 genetic vaccine was immunogenic and able to confer significant therapeutic effects. These data warrant the evaluation of this vaccination strategy in human clinical trials.

Immunotherapy of tumor with vaccine based on basic fibroblast growth factor-activated fibroblasts

Cancer-associated fibroblasts play a key role in tumor progression. It is conceivable that the breaking of immune tolerance of "self-antigens" associated with tumor cells and tumor stromal is an attractive approach for tumor immunotherapy. To test this concept, we used basic fibroblast growth factor (bFGF) to activate normal fibroblasts and used these activated fibroblasts as one vaccine against tumor. Normal fibroblasts were treated with bFGF; their expressions of a-SMA and FAP were assessed by Western blot. We immunized mice with bFGF-activated fibroblasts. Auto-antibodies were assessed by flow cytometric and Western blot analysis. The deposition of auto-antibodies within the tumor tissues was assessed. The inhibition of proliferation of tumor cells and fibroblasts by purified immunoglobulins was investigated. The anti-tumor effects of purified immunoglobulins and lymphocytes of immunized mice were assessed. The bFGF-activated fibroblasts were effective in affording protection from tumor onset, growth, and prolonging survival of tumor-bearing mice. The immunized sera exhibited positive staining for fibroblasts and tumor cells in FCAS and Western blot analysis. The purified immunoglobulins of immunized serum could inhibit the proliferation of tumor cells and fibroblasts in vitro and had the anti-tumor activity in vivo. There was the deposition of auto-antibodies within the tumor tissues. Adoptive transfer of lymphocytes of immunized mice revealed that cellular immune response is also involved. The anti-tumor activity could be abrogated by the depletion of CD4(+), CD8(+) T lymphocytes and NK cells. In summary, bFGF-activated fibroblasts could induce an autoimmune response which was simultaneously against both cancer-associated fibroblasts and tumor cells in a cross-reaction.

Combining oncolytic HSV-1 with immunogenic cell death-inducing drug mitoxantrone breaks cancer immune tolerance and improves therapeutic efficacy.

Although antitumor activity of herpes simplex virus 1 (HSV-1) ICP0 null oncolytic vectors has been validated in murine breast cancer models, oncolytic virus treatment alone is insufficient to break immune tolerance. Thus, we investigated enhancing efficacy through combination therapy with the immunogenic cell death-inducing chemotherapeutic drug, mitoxantrone. Despite a lack of enhanced cytotoxicity in vitro, HSV-1 ICP0 null oncolytic virus KM100 with 5 μmol/L mitoxantrone provided significant survival benefit to BALB/c mice bearing Her2/neu TUBO-derived tumors. This protection was mediated by increased intratumoral infiltration of neutrophils and tumor antigen-specific CD8(+) T cells. Depletion studies verified that CD8-, CD4-, and Ly6G-expressing cells are essential for enhanced efficacy of the combination therapy. Moreover, the addition of mitoxantrone to KM100 oncolytic virus treatment broke immune tolerance in BALB-neuT mice bearing TUBO-derived tumors. This study suggests that oncolytic viruses in combination with immunogenic cell death-inducing chemotherapeutics enhance the immunogenicity of the tumor-associated antigens, breaking immunologic tolerance established toward these antigens.

Successful clinical translation of preclinical combinations of radiation and immunotherapy

Ionising radiation (IR) can induce immunogenic cell death in tumours, an effect likely to contribute to the success associated with radiotherapy (RT) for cancer. Recent studies suggest that radiotherapy can be applied as a powerful adjuvant to immunotherapy, and can even contribute to converting the irradiated tumour into an in situ vaccine, resulting in specific immunity against metastases [1]. Importantly, preclinical models of syngeneic tumours have reliably predicted clinical success in several distinct tumour settings and with several different immunotherapy/radiation combinations. As a proof-of-principle trial, we have translated the preclinical evidence of a successful combination with Flt3 ligand and RT to a protocol of granulocyte–monocyte colony-stimulating factor (GM-CSF) and IR, and demonstrated out-of-field objective responses in 27% of patients with multiple metastases of solid tumours, defined as an abscopal effect [2] In-parallel mechanistic studies in the laboratory were conducted in the syngeneic mouse models of metastatic breast cancer. Results showed that radiation was synergistic with anti-CTLA-4 treatment, a strategy to break immune tolerance to the tumor. Multiple mechanisms contributed to thus synergy, including the RT-induced upregulation of a chemokine that promoted homing of effector T cells to the tumor. Intravital microscopy demonstrated that while both IR and CTLA-4 blockade given as monotherapy enhanced the motility of activated CD8 T cells infiltrating 4T1 tumours, IR with anti-CTLA-4 increased the arrest of T cells in contact with tumour cells, promoting the formation of an immune synapse between cytotoxic T cells and their targets. The latter required interaction between NKG2D on CD8+ T cells and its ligand, retinoic acid early inducible-1 (Rae-1), on the tumour cells, which was up-regulated by IR. Blocking NKG2D–Rae-1 interactions markedly increased the motility of anti-CTLA-4 treated T cells within irradiated tumours, inhibiting their contact with tumour cells and also abrogated immune-mediated tumour rejection [4]. The preclinical success of the combination of anti-CTLA-4 antibody and IR was mirrored by abscopal responses seen in metastatic melanoma and non-small-cell lung cancer (NSCLC) patients irradiated in one lesion during ipilimumab therapy. These clinical observations strongly suggest that the effects of IR identified in experimental models are relevant to patients. IR can stimulate the anti-tumor immune response, increasing the proportion of patients who respond to checkpoint blockade treatment, an hypothesis currently being tested in clinical trials. The same pattern of abscopal responses has been observed in preclinical models was also demonstrated in clinical cases of lymphomas and breast cancers treated by combinations of RT and toll-like receptor agonists. Finally, blocking tumour growth factor beta (TGF-beta) during RT preclinically has demonstrated abscopal effects that have been confirmed in one patient accrued to a trial of anti-human TGFb antibody fresolimumab and RT. While promising, this evidence remains preliminary and warrants more research to define the optimal combinations of immunotherapy and RT, to best exploit this novel role of ionising radiation [3].

Development of a Transgenic Mouse Model with Immune Tolerance for Human Coagulation Factor VIIa

Human factor VIIa (FVIIa) is commonly used as bypassing therapy to treat bleeding episodes in hemophilia patients with neutralizing antibodies to factors VIII (FVIII) or IX (FIX). There is a need for a suitable animal model to assess the immunogenicity of new FVIIa products during preclinical development. The aim of this study was the design of a novel transgenic mouse model with immune tolerance to human FVIIa. The model was generated by transgenic expression of human F7 cDNA. FVIIa-specific immune responses after treatment with human FVIIa were assessed by analyzing circulating antibodies, antibody producing plasma cells and CD4(+) T cells. In contrast to wild-type mice, human FVII transgenic mice did not develop antibodies when treated with human FVIIa. The immune tolerance was specific and could be broken by application of human FVIIa together with a strong stimulus of the innate immune system. Break of tolerance was associated with increased numbers of pro-inflammatory FVIIa-specific CD4(+) T cells. The new mouse model is suitable to study the influence of the innate immune system on maintenance and break of immune tolerance against FVIIa and could be used to assess the immunogenicity of new FVIIa products during pre-clinical development.

The antigen-binding site of an N-propionylated polysialic acid-specific antibody protective against group B meningococci is consistent with extended epitopes†

Monoclonal antibodies 13D9 and 6B9 are both specific for N-propionylated polysialic acid (NPrPSA); however, while 13D9 is protective against meningitis caused by group B meningococci and Escherichia coli capsular type K1 infection, 6B9 is not. The crystal structures of the Fabs from the two antibodies determined at 2.06 and 2.45 Å resolutions, respectively, reveal markedly different combining sites, where only the surface of 13D9 is consistent with the recognition of extended helical epitopes known to exist in the capsular polysaccharides of etiological agents of meningitis. Interestingly, complementarity determining region H2 on 13D9 lies in a non-canonical conformation that docking studies show is a critical feature in the generation of negative free energy of binding. Finally, the model of extended NPrPSA decasaccharide bound to 13D9 derived from docking studies is consistent with saturation transfer difference nuclear magnetic resonance experiments. Together, these results provide further evidence that extended epitopes have the ability to break immune tolerance associated with the polysialic acid capsule of these pathogens.

Results of a phase III clinical trial with an HBsAg-HBIG immunogenic complex therapeutic vaccine for chronic hepatitis B patients: Experiences and findings

Even though various experimental therapeutic approaches for chronic hepatitis B infection have been reported, few of them have been verified by clinical trials. We have developed an antigen-antibody (HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as adjuvant (YIC), aimed at breaking immune tolerance to HBV by modulating viral antigen processing and presentation. A double-blind, placebo-controlled, phase II B clinical trial of YIC has been reported previously, and herein we present the results of the phase III clinical trial of 450 patients. Twelve doses of either YIC or alum alone as placebo were administered randomly to 450 CHB patients and they were followed for 24weeks after the completion of immunization. The primary end point was HBeAg seroconversion, and the secondary end points were decrease in viral load, improvement of liver function, and histology. In contrast to the previous phase II B trial using six doses of YIC and alum as placebo, six more injections of YIC or alum resulted in a decrease of the HBeAg seroconversion rate from 21.8% to 14.0% in the YIC group, but an increase from 9% to 21.9% in the alum group. Decrease in serum HBV DNA and normalization of liver function were similar in both groups (p>0.05). Overstimulation with YIC did not increase but decreased its efficacy due to immune fatigue in hosts. An appropriate immunization protocol should be explored and is crucial for therapeutic vaccination. Multiple injections of alum alone could have stimulated potent inflammatory and innate immune responses contributing to its therapeutic efficacy, and needs further investigation.

Endoglin as a target of antitumor therapy

Blood vascular supply significantly affects progression of tumor growth. Inhibition of endothelial cell proliferation by antiangiogenic drugs should lead to growth arrest of both primary tumors and metastases. During the course of lengthy therapy, endothelial cells may, however, become refractory to the action of antiangiogenic agents. Novel approaches to anticancer treatment should explore the issue of drug resistance shown by endothelial cells. One possible therapeutic solution might be tumor immunotherapy directed against antigens expressed on the surface of endothelial cells which co-form tumor blood vasculature. Such therapy is supposed to break immune tolerance to own antigens and to eliminate tumor blood vessel endothelial cells by activating cytotoxic T lymphocytes. This kind of response can be obtained against endoglin (CD105). Endoglin is overexpressed in proliferating endothelial cells which line tumor blood vessels. Presence of endoglin in solid tumor blood vessels has prognostic value in cancer treatment. CD105 is also expressed by certain cancer cells (prostate, melanoma and Ewing sarcoma). It appears that therapeutic strategies directed against endoglin allow several mechanisms of resistance to antiangiogenic drugs to be omitted. The therapeutic approach that we propose, i.e. a tumor blood vessel-destroying strategy combined with immunotherapy, may become an effective therapeutic tool.

Xenogeneic Human p53 DNA Vaccination by Electroporation Breaks Immune Tolerance to Control Murine Tumors Expressing Mouse p53

The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA). Immunization against a self-antigen is challenging because an antigen-specific immune response likely generates only low affinity antigen-specific CD8+ T-cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous TAAs expressed by tumors. The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine. The vaccine employs a xenogeneic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Mice immunized with xenogeneic full length hp53 DNA plasmid intramuscularly followed by electroporation were protected against challenge with murine colon cancer MC38 while those immunized with mp53 DNA were not. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53 DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8+ T-cell precursors compared to vaccination with mp53 DNA. Antibody depletion experiments also demonstrated that CD8+ T-cells play crucial roles in the antitumor effects. This study showed intramuscular vaccination with xenogeneic p53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against tumors expressing mutated p53 through CD8+ T cells.

Innovative DNA Vaccine to Break Immune Tolerance Against Tumor Self-Antigen

Vaccination is, in theory, a safe and effective approach for controlling disseminated or metastatic cancer due to the specificity of the mammalian immune system, yet its success in the clinic has been hampered thus far by the problem of immune tolerance to tumor self-antigen. Here we describe a DNA vaccination strategy that is able to control cancer by overcoming immune tolerance to tumor self-antigen. We engineered a DNA construct encoding a dimeric form of a secreted single-chain trimer of major histocompatibility complex class I heavy chain, β2-microglobulin, and peptide antigen linked to immunoglobulin G (SCT-Ag/IgG). The chimeric protein was able to bind to antigen-specific CD8(+) T cells with nearly 100% efficiency and strongly induce their activation and proliferation. In addition, the chimeric protein was able to coat professional antigen-presenting cells through the F(c) receptor to activate antigen-specific CD8(+) T cells. Furthermore, intradermal vaccination with DNA-encoding SCT-Ag/IgG could generate significant numbers of cytotoxic effector T cells against tumor self-antigen and leads to successful therapeutic outcomes in a preclinical model of metastatic melanoma. Our data suggest that the DNA vaccine strategy described in the current study is able to break immune tolerance against endogenous antigen from melanoma and result in potent therapeutic antitumor effects. Such strategy may be used in other antigenic systems for the control of infections and/or cancers.

Epitope-optimization creates highly immunogenic alpha fetoprotein antigen to break immune tolerance and potently activates CD8 T cells to prevents autochthonous hepatocellular carcinoma

Meeting abstracts In this study, we investigated whether mouse alpha fetoprotein (mAFP), the shared self/tumor antigen of hepatocellular carcinoma (HCC), could be rationally engineered to create effective vaccine to break tolerance and potently activate CD8 T cells to prevent clinically-relevant

Abstract A17: Cancer immunotherapy using naked DNA vaccine targeting p53.

Abstract Background: The pivotal role of p53 as a tumor suppressor protein is illustrated by the fact that this protein is found mutated in more than 50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein in normal tissue and cause it to accumulate in the cytoplasm of tumors. The overexpression of mutated p53 in tumor cells makes p53 a potentially desirable target for the development of cancer immunotherapy. However, p53 protein represents an endogenous tumor-associated antigen (TAA), and immunization against self-antigen is challenging because antigen-specific immune response likely only generate low affinity antigen-specific CD8+ T cells. This represents a bottleneck of tumor immunotherapy when targeting endogenous antigens expressed by tumors. Purpose: The objective of the current study is to develop a safe cancer immunotherapy using a naked DNA vaccine employing xenogenic p53 gene to break immune tolerance resulting in a potent therapeutic antitumor effect against tumors expressing mutated p53. Our study assessed the therapeutic antitumor effect after immunization with DNA encoding human p53 (hp53) or mouse p53 (mp53). Results: C57BL/6 mice immunized with xenogenic full length of hp53 DNA plasmid intramuscularly followed by electroporation were capable of protecting against challenge with murine colon cancer MC38. In contrast, mice immunized with mp53 DNA were incapable of protecting mice against challenge with MC38 tumors. In a therapeutic model, established MC38 tumors were also well controlled by treatment with hp53DNA therapy in tumor bearing mice compared to mp53 DNA. Mice vaccinated with hp53 DNA plasmid also exhibited an increase in mp53-specific CD8+ T cell precursors compared to vaccination with mp53 DNA. In antibody depletion experiments, we also demonstrated that CD8+ T cells play crucial roles in the antitumor effects. Conclusion: This study showed intramuscular vaccination with xenogenic hp53 DNA vaccine followed by electroporation is capable of inducing potent antitumor effects against mutated p53 expressing tumors through CD8+ T cells. Citation Format: Rueyshyang Soong, Chien-Fu Hung, T.-C. Wu. Cancer immunotherapy using naked DNA vaccine targeting p53. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A17.

Immunosurveillance against cancer-associated hyperploidy

Immunosurveillance against cancer-associated hyperploidy

A Novel Model of Immune-Mediated Disease Equilibrium in Acute Lymphoblastic Leukemia

AbstractAbstract 3540Acute lymphoblastic leukemia (ALL) is a challenging target for immunotherapy due to its poor antigen presentation, lack of costimulatory molecule expression, and resistance to cytotoxic killing by immune effector cells. In order to develop new strategies to generate autologous immune-mediated protection against ALL progression, we sought to understand more about the immune requirements that must be met in order to achieve disease control. To do this, we have transfected B cell precursor ALL cell lines derived from leukemic Eμ-ret transgenic BALB/c mice with a lentiviral construct encoding green fluorescent protein (GFP) and firefly luciferase (luc) in tandem. The luciferase protein serves a dual purpose as a potent neoantigen capable of stimulating leukemia-specific immune responses and as a bioluminescent reporter for highly sensitive detection of minimal residual disease (MRD).Intravenous injection of 106 non-transfected Eμ-ret-derived leukemia cells is consistently lethal in wild type BALB/c mice within 3–5 weeks. However, adoptive transfer of 106 GFP/luc-expressing leukemia cells into wild type BALB/c mice does not lead to morbidity or mortality (p=0.025, logrank for survival). These mice show detectable disease in bone marrow by bioluminescent imaging below the limit of detection by peripheral blood flow cytometry that increases until day 7–10, followed by a spontaneous decrease in leukemia burden to undetectable levels by imaging. BALB/c mice lacking B and T cells (RAG1−/−) or B, T and NK cells (gamma common−/−) injected with GFP/luc ALL cells develop leukemia that is lethal by day 25 (p=0.037, logrank). Early T cell-depletion in wild type BALB/c mice on days 0, 3, 6, and 9 with CD4- and/or CD8-depleting antibodies also results in complete breakthrough of disease, while NK cell-depletion with asialo-GM1 on the same days does not significantly alter the timeframe of leukemia clearance (p<0.0001, 2-way ANOVA; Figure 1). These results demonstrate that the presence of a neoantigen is sufficiently immunogenic to generate effective T cell-mediated control of the disease.We have followed immunocompetent mice that received GFP/luc ALL for up to 12 months. While most mice have long-term remissions both by imaging and peripheral blood sampling, a subset have evidence of persistent MRD by bioluminescent imaging that waxes and wanes in luminosity but may not break through to a fulminant relapse. Relapses in these mice have occurred up to 11 months from injection. Late T cell-depletion with CD4 and CD8 antibodies (weekly x12, beginning at day 28) leads to an increased rate of leukemia breakthroughs and occurs more frequently in mice who had cleared their leukemia in the setting of early NK cell depletion (p<0.001, 2-way ANOVA), suggesting early crosstalk between NK and T cells to develop a sustained immune response. In addition, many mice relapse with GFP/luc-expressing leukemia, while a proportion of mice have late relapses with GFP/luc-negative leukemia, suggesting antigen loss is one of several potential mechanisms of immune evasion in ALL.Finally, BALB/c mice transgenic for GFP and luciferase do not generate T cell protection against outgrowth of injected GFP/luc ALL cells and succumb to disease by day 20 (p = 0.027 versus wild type mice, logrank). This result suggests that tolerance mechanisms, rather than antigen presentation deficiencies, may undermine host immune responses against ALL.We present a novel model of immune equilibrium in ALL, made evaluable by exquisitely sensitive bioluminescent imaging. Importantly, despite its well-documented lack of immunogenicity, we have shown ALL can be a sensitive target for long-term leukemia-specific T cell-mediated immune control. We are currently investigating additional cellular mechanisms of immune protection, the role of antigen spreading in control of ALL, and the mechanisms allowing leukemia to escape from immune control. Our approach will enable the development of strategies to break immune tolerance towards leukemia antigens and generate control of MRD in ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

In vivo electroporation improves therapeutic potency of a DNA vaccine targeting hepadnaviral proteins

This preclinical study investigated the therapeutic efficacy of electroporation (EP)-based delivery of plasmid DNA (pDNA) encoding viral proteins (envelope, core) and IFN-γ in the duck model of chronic hepatitis B virus (DHBV) infection. Importantly, only DNA EP-therapy resulted in a significant decrease in mean viremia titers and in intrahepatic covalently closed circular DNA (cccDNA) levels in chronic DHBV-carrier animals, compared with standard needle pDNA injection (SI). In addition, DNA EP-therapy stimulated in all virus-carriers a humoral response to DHBV preS protein, recognizing a broader range of major antigenic regions, including neutralizing epitopes, compared with SI. DNA EP-therapy led also to significant higher intrahepatic IFN-γ RNA levels in DHBV-carriers compared to other groups, in the absence of adverse effects. We provide the first evidence on DNA EP-therapy benefit in terms of hepadnaviral infection clearance and break of immune tolerance in virus-carriers, supporting its clinical application for chronic hepatitis B.

Abstract 5241: Development of a single Listeria monocytogene-based vaccine simultaneously targeting two antigens

Abstract Previously published studies reported that recombinant Listeria monocytogenes (Lm-LLO)-based vaccines expressing either tumor associated antigens (TAA) or angiogenesis associated antigens fused to an immunogenic fragment of listeriolysin O (LLO) have demonstrated efficacy in mouse models with several types of tumors such as lung, breast, prostate or melanoma. Therefore, we hypothesized that an Lm-LLO vaccine capable of delivering two disparate antigens with two different therapeutic mechanisms would likely have a synergistic effect on decreasing tumor growth by 1) targeting blood vessels, which may enhance effector T cell infiltration into the tumor and 2) improving the therapeutic efficacy of the vaccine. In addition, a dual vaccine construct creates a flexible platform for future use. The Lm-LLO-cHER2 vaccine was developed using a chimeric HER2/neu (cHER2) construct, and was found to regress tumors, elicit a strong T cell immune response and break immune tolerance towards the HER2/neu self-antigen in experimental animals. The Lm-LLO-VEGR2 vaccine has been shown to eradicate established breast tumors, reduce microvascular density, protect against tumor rechallange and metastases, and induce epitope spreading to various regions of the TAA such as HER2/neu. A dual Lm-LLO vaccine was developed that expresses and secretes both the VEGFR2 and cHER2 antigens as LLO-based proteins and is based on a highly attenuated Listeria monocytogenes backbone Lm Δ dal dat actA, which is cleared 48 hours post-injection in interferon gamma knock-out mice. Initial characterization of the dual vaccine indicates that the two antigens cHER2 and VEGFR2 are stably expressed and secreted after two in vivo mouse passages. Currently, we are evaluating the anti-tumor effects and antigen specific immune responses generated by the dual vaccine in mouse models. If successful, this dual vaccine may offer a new immunotherapy for the treatment of cancer in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5241. doi:1538-7445.AM2012-5241

Inflammation-Associated Nitrotyrosination Affects TCR Recognition through Reduced Stability and Alteration of the Molecular Surface of the MHC Complex

Nitrotyrosination of proteins, a hallmark of inflammation, may result in the production of MHC-restricted neoantigens that can be recognized by T cells and bypass the constraints of immunological self-tolerance. Here we biochemically and structurally assessed how nitrotyrosination of the lymphocytic choriomeningitis virus (LCMV)-associated immunodominant MHC class I-restricted epitopes gp33 and gp34 alters T cell recognition in the context of both H-2Db and H-2Kb. Comparative analysis of the crystal structures of H-2Kb/gp34 and H-2Kb/NY-gp34 demonstrated that nitrotyrosination of p3Y in gp34 abrogates a hydrogen bond interaction formed with the H-2Kb residue E152. As a consequence the conformation of the TCR-interacting E152 was profoundly altered in H-2Kb/NY-gp34 when compared to H-2Kb/gp34, thereby modifying the surface of the nitrotyrosinated MHC complex. Furthermore, nitrotyrosination of gp34 resulted in structural over-packing, straining the overall conformation and considerably reducing the stability of the H-2Kb/NY-gp34 MHC complex when compared to H-2Kb/gp34. Our structural analysis also indicates that nitrotyrosination of the main TCR-interacting residue p4Y in gp33 abrogates recognition of H-2Db/gp33-NY complexes by H-2Db/gp33-specific T cells through sterical hindrance. In conclusion, this study provides the first structural and biochemical evidence for how MHC class I-restricted nitrotyrosinated neoantigens may enable viral escape and break immune tolerance.

Immune Responses in Wild-type Mice Against Prion Proteins Induced Using a DNA Prime-Protein Boost Strategy

Objective To break immune tolerance to prion (PrP) proteins using DNA vaccines. Methods Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests. Results Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response. Conclusion Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.

Deciphering of CD4+ T Cell Subsets Involved in Maintenance and Break of Immune Tolerance in a Humanized Mouse Model That Is Immune Tolerant to Human FVII

Abstract 218825%-30% of patients with hemophilia A develop neutralizing antibodies following replacement therapy with factor VIII (FVIII). These patients can be treated with factor VIIa (FVIIa) which triggers the extrinsic pathway of coagulation and thereby bypasses the requirement for FVIII. We developed a new mouse model that is transgenic for human FVII and expresses specific immune tolerance to native human FVIIa. We aim to investigate the immunological impact of modified FVIIa product candidates and to characterize their immunogenicity by analyzing emerging FVIIa-specific T cell responses.The new mouse model offers a unique opportunity to study central and peripheral immune regulatory mechanisms and the generation of immune responses by pro-inflammatory antigen-specific effector T cells (Teff). We hypothesized that FVIIa-specific Teff having escaped clonal deletion are present in the periphery and may be actively suppressed by FVIIa-specific regulatory T cells (Tregs). To study this hypothesis, we immunized mice with recombinant FVIIa (rFVIIa) with or without LPS, a well-described “danger signal” being able to break immune tolerance by stimulating the innate immune system. Intravenous or subcutaneous administration of rFVIIa alone did not elicit antibody responses and thus immune tolerance to rFVIIa was not broken. However, co-administration of rFVIIa and LPS resulted in a specific antibody response that was not isotypically restricted. To further analyze the mechanisms behind this break of specific immune tolerance, we characterized rFVIIa-specific T cells by the expression of CD154, a marker of antigen-specific T cells. Cytokine production and CD154 expression were assessed upon re-stimulation with rFVIIa. In contrast to mice that were immunized with rFVIIa only, we found increased numbers of rFVIIa-specific T cells in rFVIIa-LPS-treated mice displaying a stable, highly pro-inflammatory (IL-2+/IFN-g+) memory phenotype.These data could suggest that rFVIIa-specific Teff that escaped clonal deletion during induction of central immune tolerance, are present in the periphery of human FVII-transgenic mice. This would imply that rFVIIa-specific Teff could be actively suppressed by Tregs. This suppression could be overcome by danger signals like LPS. We currently study the regulatory mechanisms that maintain tolerance upon administration of FVIIa without LPS. We are approaching this question by correlating the characteristics of FVIIa-specific Teff and Treg responses under both tolerant and non-tolerant conditions.Ultimately, we aim to understand which danger signals have to be provided to break immune tolerance and how tolerance is regulated. Understanding these regulatory mechanisms will enable us to develop new therapeutic strategies and prevent conditions that lead to the induction of antibodies against drug products in patients. Disclosures:Lenk:Baxter BioScience: Employment. Pasztorek:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment. de la Rosa:Baxter BioScience: Employment.

Induction and Break of Immune Tolerance to Human FVIII in a HLA-DRB1*1501 Humanized Hemophilic Mouse Model

Abstract 2280Development of neutralizing antibodies against FVIII is the major complication in the treatment of patients with hemophilia A. Although several genetic and environmental risk factors have been identified, it remains unclear why some patients develop antibodies while others do not. Understanding the underlying mechanisms that drive the decision of the immune system whether or not to make antibodies against FVIII would help to design novel therapeutics.We used a new humanized hemophilic mouse model that expresses the human MHC-class II molecule HLA-DRB1*1501 on the background of a complete knock out of all murine MHC-class II genes. Initial studies had indicated that only a fraction of these mice developed antibodies when intravenously (i.v.) treated with human FVIII. These findings which resemble the situation in patients with severe hemophilia A, evoked the question if the lack of antibody development in non-responder mice reflects the induction of specific immune tolerance after i.v. application of FVIII or represent non-responsiveness for other reasons. We addressed this question by choosing another application route (subcutaneous, s.c.) and by combining i.v. application with a concomitant activation of the innate immune system applying LPS, a well characterized ligand for toll-like receptor 4, together with FVIII. Both strategies resulted in the development of antibodies in all mice included in the study what suggested that non-responsiveness against i.v. FVIII does not reflect an inability to develop antibodies against FVIII. Next, we asked if i.v. FVIII does induce immune tolerance in non-responder mice. We pretreated mice with i.v. FVIII, selected non-responder mice and challenged them with s.c. FVIII. None of the mice developed antibodies what indicated that i.v. pretreatment had induced immune tolerance in non-responder mice. Currently, we test the hypothesis that immune tolerance after i.v. application is induced and maintained by FVIII-specific regulatory T cells.The differences in responder rates after i.v. and s.c. application of FVIII raised the question if there are differences in FVIII T-cell epitopes involved in the initial activation of FVIII-specific CD4+ T cells. We obtained spleen cells from mice treated with either i.v. or s.c. FVIII and generated CD4+ T-cell hybridoma libraries that were tested for peptide specificities. For this purpose we used a FVIII peptide library containing 15 mers with an offset of 3 amino acids. Our results indicate that the pattern of FVIII-specific T-cell epitopes involved in the activation of FVIII-specific CD4+ T cells after i.v. and s.c. application of FVIII is almost identical and represents a small set of FVIII peptides distributed over the A1, A2, B, A3 and C1 domains.Based on our results we conclude that the new HLA-DRB1*1501 hemophilic mouse model represents an interesting opportunity to uncover the mechanisms that drive the decision of the immune system whether or not to develop antibodies against FVIII. Disclosures:Steinitz:Baxter BioScience: Employment. Binder:Baxter BioScience: Employment. Lubich:Baxter BioScience: Employment. Ahmad:Baxter BioScience: Employment. Weiller:Baxter BioScience: Employment. de la Rosa:Baxter BioScience: Employment. Schwarz:Baxter BioScience: Employment. Scheiflinger:Baxter BioScience: Employment. Reipert:Baxter Innovations GmbH: Employment.