Antigen-specific Immune Therapy Research Articles

Antigen-specific immune therapy (CNP-106) for treatment of generalised myasthenia gravis: rationale and design of first-in-human randomised controlled trial

IntroductionMyasthenia gravis (MG) is a T cell-dependent B cell-mediated autoimmune disease with pathogenic antibodies directed against components of the acetylcholine receptor (AChR). Current therapies do not address the root cause...

T-cell subsets in allergy and tolerance induction.

Antigen-specific T lymphocytes are the central regulators of tolerance versus immune pathology against otherwise innocuous antigens and key targets of antigen-specific immune therapy. Recent advances in the understanding of T cells in tolerance and allergy resulted from improved technologies to directly characterize allergen-specific T cells by multiparameter flow cytometry or single-cell sequencing. This unravelled phenotypically and functionally distinct populations, such as Type 2a T helper cells (Th2a), follicular Th cells (Tfh), regulatory T cells (Treg),Type 1 regulatory T cells (Tr1), and follicular T regulatory cells. Here we will discuss the role of the different Th-cell subsets in the healthy state, during sensitization and development of allergy, and in tolerance induction by allergen immunotherapy (AIT). To date, the mechanisms of AIT as the only causal treatment of allergy are not completely understood. The analyses of allergen-specific T cells directly ex vivo during AIT support the concept of specific-Th2(a) cell deletion rather than an expansion of allergen-specific Tr1 or Treg cells as underlying mechanism.

Therapeutic regulation of autoreactive B cells using bispecific molecules

Abstract Type 1 diabetes (T1D) occurs when autoreactive T cells destroy insulin-producing beta cells. Anti-insulin B cells promote this disease, acting as essential antigen-presenting cells. Rituximab, which globally depletes B cells, provided temporary preservation of insulin production in early onset T1D, but increases susceptibility to infection. Antigen-specific immune therapy (ASIT) may be a viable option for treating T1D by targeting anti-insulin B cells for removal from the B cell repertoire. In this study we investigate the efficacy of soluble antigen arrays containing insulin (SAgAins) developed by the Berkland lab. These constructs consist of a polyethylene glycol (PEG) backbone with four linker arms for bioconjugation. We conducted in vitro experiments to investigate the effects of constructs containing insulin and a trisaccharide ligand (CD22L) for inhibitory B cell receptor CD22 on transgenic anti-insulin B cells. We found that incubating insulin-PEG with anti-insulin B cells stimulates proliferation and CD86 upregulation. However, when CD22L is added (CD22L-PEG-insulin) the expression of CD86 is dampened. This suggests that this novel construct harnesses the regulatory power of CD22 to dampen activation of B cells in an autoantigen-specific manner.

Induced pluripotent stem cell-derived myeloid cells expressing OX40 ligand amplify antigen-specific T cells in advanced melanoma.

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune-related adverse events have been reported. Therefore, more effective and antigen-specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS-ML). In this study, we generated OX40L-overexpressing iPS-ML (iPS-ML-Zsgreen-OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS-ML-Zsgreen-OX40L suppressed the progression of B16-BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)-expressing B16 melanoma (MO4). The number of antigen-specific CD8+ T cells was higher in spleen cells treated with OVA peptide-pulsed iPS-ML-Zsgreen-OX40L than in those without OX40L. The OVA peptide-pulsed iPS-ML-Zsgreen-OX40L significantly increased the number of tumor-infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS-ML in the clinical applications, iPS-ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS-ML-Zsgreen-OX40L therapy might be a new method for antigen-specific cancer immunotherapy.

Grave effects of a specific immune therapy.

Antigen-specific immune therapy unexpectedly worsens Graves’ disease in a spontaneous animal model.

Restoring Immune Tolerance in Atherosclerosis: Role of Regulatory Immune Response in Atheroprotection.

Cardiovascular diseases remain the most significant cause of global mortality despite advances in medicine and new drug development. Atherosclerotic lesions start developing in childhood, progress over decades and manifest as coronary artery disease, stroke or peripheral arterial disease later in life. Chronic inflammation in the arterial wall mediated by altered immune response is vital during the development of atherosclerosis. Antigen specific immune therapy is an elegant approach to target disease related antigens. Immune tolerance to atherogenic self antigens has gained importance as an efficient therapy to control atherosclerosis in recent years. This review discusses the recent development and our understanding of immune tolerance in atherosclerosis and the role of regulatory immune response in protection against the disease.

T-cell Responses to Oncogenic Merkel Cell Polyomavirus Proteins Distinguish Patients with Merkel Cell Carcinoma from Healthy Donors

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with strong evidence of viral carcinogenesis. The association of MCC with the Merkel cell polyomavirus (MCPyV) may explain the explicit immunogenicity of MCC. Indeed, MCPyV-encoded proteins are likely targets for cytotoxic immune responses to MCC as they are both foreign to the host and necessary to maintain the oncogenic phenotype. However, to date only a single MCPyV-derived CD8 T-cell epitope has been described, thus impeding specific monitoring of T-cell responses to MCC. To overcome this limitation, we scanned the MCPyV oncoprotein large T and small T antigens and the virus capsid protein VP1 for potential T-cell epitopes, and tested for MHC class I affinity. We confirmed the relevance of these epitopes using a high-throughput platform for T-cell enrichment and combinatorial encoding of MHC class I multimers. In peripheral blood from 38 patients with MCC and 30 healthy donors, we identified 53 MCPyV-directed CD8 T-cell responses against 35 different peptide sequences. Strikingly, T-cell responses against oncoproteins were exclusively present in patients with MCC, but not in healthy donors. We further demonstrate both the processing and presentation of the oncoprotein-derived epitopes, as well as the lytic activity of oncoprotein-specific T cells toward MHC-matched MCC cells. Demonstrating the presence of oncoprotein-specific T cells among tumor-infiltrating lymphocytes further substantiated the relevance of the identified epitopes. These T-cell epitopes represent ideal targets for antigen-specific immune therapy of MCC, and enable tracking and characterization of MCPyV-specific immune responses.

Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

Pretransplant donor lymphocyte infusion (DLI) has been shown to enhance donor-specific allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored donor cell subsets after DLI in a murine MHC class I Ld-mismatched skin transplantation model. We found that donor B cells, but not DCs, are the major surviving donor APCs in recipients following DLI. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Furthermore, mice that had received donor B cells showed higher expression of Ly6A and CD62L on antigen-specific TCRαβ+CD3+CD4−CD8−NK1.1− double negative (DN) regulatory T cells (Tregs). B cells presented alloantigen to DN Tregs and primed their proliferation in an antigen-specific fashion. Importantly, DN Tregs, activated by donor B cells, showed increased cytotoxicity toward anti-donor CD8+ T cells. These data demonstrate that donor B cells can enhance skin allograft survival, at least partially, by increasing recipient DN Treg-mediated killing of anti-donor CD8+ T cells. These findings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specific immune therapy to enhance allograft survival.

2011 Update: antigen-specific therapy in type 1 diabetes.

To update on the clinical trials using antigen-specific therapies in autoimmune diabetes. Type 1 diabetes is now a predictable disease with the measurement of islet autoantibodies, and the incidence is increasing dramatically. Well tolerated and effective interventions are needed to stop the underlying autoimmune destruction of insulin-producing beta cells. Beta-cell antigens, insulin and glutamic acid decarboxylase, are being used to preserve endogenous insulin production in individuals with new-onset diabetes and to prevent diabetes. The results of antigen-specific immune intervention trials are reviewed and consideration is given to future directions for inducing tolerance in type 1 diabetes. Antigen-specific immune therapies act by enhancing regulatory T cell function, in animal models often locally and selectively in islets or pancreatic lymph nodes while inhibiting effector T cells. This therapeutic pathway provides a safe treatment to preserve beta cell function in new-onset diabetic individuals with the GAD-Alum vaccine being the most extensively studied therapy. Insulin is being used in many forms to prevent diabetes and stop the underlying autoimmune process. For the future, combination immune therapies targeting different pathways in the immune system will be needed to effectively induce sustained tolerance in type 1 diabetes.

Abstract 4793: MUC1 immunogenicity in a triple transgenic mouse model for epithelial ovarian cancer

Abstract Background: Ovarian cancer continues to be associated with a very high mortality rate. Improvements on the development of new therapeutic approaches, including antigen-specific immune therapies, depend in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 mucin is a tumor associated antigen and a potential target for cancer immunotherapy. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models with de novo lesions expressing the human antigen are needed. Objective: To identify MUC1 antigen-specific immune regulation in mice with de novo malignant ovarian lesions. Methods: In order to generate a human MUC1 antigen expressing ovarian cancer mouse model, we crossed the MUC1 transgenic mice which express human MUC1 under the endogenous promoter with the previously described KrasG12D/+PtenloxP/loxP mice that develop orthotopic ovarian tumors. Results: The newly generated MUC1+/−KrasG12D/+ PtenloxP/loxP triple transgenic mice constitute a valuable Cre-loxP conditional in vivo model for MUC1 expressing ovarian tumors. Upon injection of Cre-encoding adenovirus under the ovarian bursa, the triple transgenic mice develop aggressive ovarian tumors with clear cell histology. The lesions express high levels of MUC1 and are estrogen receptor and cytokeratin 7-positive, markers classically used to diagnose malignant epithelial proliferation. The tumors are accompanied by ascites and numerous peritoneal implants, mimicking late stage disease clinically seen in women. We identified increased accumulation of Treg cells in the para-aortic (draining) lymph nodes and presence of Th17 cells in ascites, mimicking closely the immune phenotype of the human disease. In addition, using quantitative PCR T cell gene profiler arrays, we identified 5 significantly up- and 16 down-regulated genes in the spleen T cells of tumor-bearing mice. One of the upregulated genes was osteopontin (Spp1), recently validated as a marker in detecting human recurrent ovarian cancer. Among the down-regulated genes we identified, Cd4, Cd40ligand (Cd40lg), Interferon-g (Ifnγ), IL-23 receptor (IL-23ra) all suggestive of a shift in immune effectors, some of which may promote the chronic inflammatory background. Conclusions: Our results reveal important immune regulatory mechanisms in mice with de novo MUC1-expressing ovarian tumors and provide unique opportunities for future in vivo testing of MUC1 vaccines as ovarian cancer therapy and potentially immune prevention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4793.

New prospects for immunotherapy at diagnosis of type 1 diabetes

Immune intervention at diagnosis of type 1 diabetes (T1D) aims to prevent or reverse the disease by blocking autoimmunity, thereby preserving/restoring beta-cell mass and function. Recent clinical trials of non-specific and of antigen-specific immune therapies have demonstrated the feasibility of modulation of islet-specific autoimmunity in patients with partial prevention of loss of insulin secretion. In a series of review articles published in this issue of the journal, some of the most promising approaches of immune intervention in T1D are presented. Here we outline the rationale of such interventions and future prospects in this area.

Genetically Manipulated Human Embryonic Stem Cell-Derived Dendritic Cells with Immune Regulatory Function

Genetically manipulated dendritic cells (DC) are considered to be a promising means for antigen-specific immune therapy. This study reports the generation, characterization, and genetic modification of DC derived from human embryonic stem (ES) cells. The human ES cell-derived DC (ES-DC) expressed surface molecules typically expressed by DC and had the capacities to stimulate allogeneic T lymphocytes and to process and present protein antigen in the context of histocompatibility leukocyte antigen (HLA) class II molecule. Genetic modification of human ES-DC can be accomplished without the use of viral vectors, by the introduction of expression vector plasmids into undifferentiated ES cells by electroporation and subsequent induction of differentiation of the transfectant ES cell clones to ES-DC. ES-DC introduced with invariant chain-based antigen-presenting vectors by this procedure stimulated HLA-DR-restricted antigen-specific T cells in the absence of exogenous antigen. Forced expression of programmed death-1-ligand-1 in ES-DC resulted in the reduction of the proliferative response of allogeneic T cells cocultured with the ES-DC. Generation and genetic modification of ES-DC from nonhuman primate (cynomolgus monkey) ES cells was also achieved by the currently established method. ES-DC technology is therefore considered to be a novel means for immune therapy.

Experimental autoimmune diabetes is caused by IFN-γ+/CD8+ T cell immunity against a single insulin A-chain epitope

Insulin and its primary gene product, preproinsulin (PPI), are considered central autoantigens in human type 1 diabetes (T1D), with potential value for antigen-specific immune therapy. We have previously demonstrated (Diabetes 51:3237–44) that PPI can adversely induce hyperglycemia and experimental autoimmune diabetes (EAD). Here we identify a C-terminal insulin A-chain epitope as the cause of EAD. RIP-B7.1 transgenic mice were immunized by DNA vaccination using PPI-expressing plasmid vectors, resulting in CD4+/CD8+ insulitis and hyperglycemia in >95% animals after only 2–3 weeks. Adoptive spleen cell transfer shows that autoreactive PPI-specific CD8 T cell clones can be induced in both transgenic and wild type mice, but beta cell co-stimulation (B7.1) is required for diabetes manifestation. Using gene-targeted B7.1 mice we demonstrate that interferon-γ, but not perforin, is required for autoimmune beta cell damage in EAD. Using PPI minigene constructs and a PPI peptide library, we localize and fine-map an immunodominant CD8 T cell epitope to the C-terminus of the A chain of insulin. We identify its MHC restriction as class I Db by a stabilization experiments in TAP-deficient H2 cell lines. After i.m. injection as peptide-DNA complex, this peptide is sufficient to induce insulitis and hyperglycemia in mice. Our data show that experimental autoimmune diabetes results from CD8+ T cell immunity against a Db-restricted T cell epitope of insulin, located in the C-terminal moiety of the insulin A-chain.

Rationale for Designing of Antigen-Specific Immune Therapy Including Dendritic Cell-Based Therapy in Patients with Chronic Hepatitis B Virus Infection

Rationale for Designing of Antigen-Specific Immune Therapy Including Dendritic Cell-Based Therapy in Patients with Chronic Hepatitis B Virus Infection

Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health

Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index ≥2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-γ (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.

Self epitopes shared between human skeletal myosin and Streptococcus pyogenes M5 protein are targets of immune responses in active juvenile dermatomyositis.

To identify self T cell epitopes associated with proinflammatory immune responses and clinically active juvenile dermatomyositis (juvenile DM). The target of our search for relevant epitopes was represented by amino acid sequences shared between human skeletal myosin and Streptococcus pyogenes M5 protein. The long-term objective of the project is to identify suitable targets for immunotherapy of the disease. We used computerized algorithms to identify putative agretopes on both the human myosin and Streptococcus M5 proteins. Direct binding assays for homolog peptides were used to confirm such predictions. Antigenicity and functional cross-reactivity were evaluated by cytotoxicity assays and by measurement of cytokine levels. Specific T cells were isolated by T cell capture, and T cell receptor (TCR) V(beta) gene usage was identified by reverse transcriptase-polymerase chain reaction. We identified peptides that are targets of disease-specific cytotoxic T cell responses. T cell reactivity against the self peptides correlates with clinical signs of early, active myositis. Such reactivity is accompanied by production of proinflammatory cytokines, which may contribute to the damage. T cell cross-recognition of bacterial and human homologs was shown functionally as well as by sorting peptide-specific T cells and identifying oligoclonal and largely overlapping TCR V(beta) gene usage. These findings represent the first identification of a self epitope in juvenile DM, providing a potential candidate for antigen-specific immune therapy.

Mucosal modulation of immune responses to heat shock proteins in autoimmune arthritis.

Induction of oral tolerance to antigens that are targets of self-reactive immune responses is an attractive approach to antigen-specific immune therapy of autoimmune diseases. Oral tolerization has indeed proven to be safe and effective in amelioration of autoimmune diseases in animal models. In humans, results have been somewhat controversial. The emphasis given to clinical outcome rather than to immunomodulation, and the difficulty in identifying appropriate candidate antigens contribute to the controversy. Heat shock proteins are promising targets for immune intervention. Immune reactivity to heat shock proteins has indeed been correlated with autoimmune arthritis in animal models, and abnormal immune responses to heat shock proteins have been described in human arthritis as well. Despite significant recent progress, little is known at a molecular level regarding the mechanisms which are responsible for a switch from autoimmunity to tolerance in humans. This is particularly true with respect to sequential analysis of several molecular and immunologic markers during both the course and treatment of disease. Novel approaches are currently under way to fill the gaps. We will briefly detail here the experience gained to date, and identify some of the avenues which future research will explore.

Graves' ophthalmopathy: current concepts regarding pathogenesis and management.

Referring to the hyperadrenergic theory on the pathogenesis of Graves' ophthalmopathy, a 1934 JAMA editorial (513) stated "the mechanism of exophthalmos is well understood though the knowledge would seem to be poorly disseminated." Several subsequent theories on pathogenesis, stated equally emphatically, have experienced a similar fate to that prompting this remark, but not before negatively impacting upon the management of patients suffering from this disorder. Thus, it is with an element of caution that we conclude that the sequence of events contributing to the pathogenesis of Graves' ophthalmopathy has become progressively more lucid, due to the assiduous efforts of many investigators in this field. Demonstration of an inextricable link between the eye and the thyroid in Graves' ophthalmopathy seems limited only by our ability to detect subtle involvement of one or the other of these two organs in exceptional cases, although not all authors share this viewpoint (514). The factors modulating the degree of expression of the thyroid component, such as concurrent lymphocytic thyroiditis or qualitative differences in TRAb seem more tangible than those affecting the clinical expression of eye involvement. Environmental factors such as smoking are associated, to a degree that matches or surpasses that known for genetic predisposition to this disorder. Local anatomy, including such factors as vulnerability to obstruction of venous drainage, must play a role as evidenced by asymmetric eye involvement and rapid relief of inflammatory changes after orbital decompression surgery. The transition from palliative to curative measures in Graves' ophthalmopathy will require further advances in our understanding of the putative shared thyroid-eye antigens, demonstration that these antigens are etiologically important and concomitant advances in antigen-specific immune therapy.