Antigen-specific Immunotherapy Research Articles

Nanoparticles versus Dendritic Cells as Vehicles to Deliver mRNA Encoding Multiple Epitopes for Immunotherapy

The efficacy of antigen-specific immunotherapy relies heavily on efficient antigen delivery to antigen-presenting cells and engagement of as many disease-relevant T cells as possible in various lymphoid tissues, which are challenging to achieve. Here, we compared two approaches to deliver mRNA encoding multiple epitopes targeting both CD4+ and CD8+ T cells: a lipid-based nanoparticle platform to target endogenous antigen-presenting cells in vivo versus ex vivo mRNA-electroporated dendritic cells. After intraperitoneal injection, the nanoparticle platform facilitated efficient entry of mRNA into various endogenous antigen-presenting cells, including lymph node stromal cells, and elicited robust T cell responses within a wider network of lymphoid tissues compared with dendritic cells. Following intravenous injection, mRNA-electroporated dendritic cells and the nanoparticle platform localized primarily in lung and spleen, respectively. When administered locally via an intradermal route, both platforms resulted in mRNA expression at the injection site and in robust T cell responses in draining lymph nodes. This study indicates that multiple epitopes, customizable for specific patient populations and encoded by mRNA, can be targeted to different lymphoid tissues based on delivery vehicle and route, and constitute the groundwork for future studies using mRNA to reprogram exogenous or endogenous APCs for immunotherapy.

1151P - HLA-ligandome analysis reveals target antigens of oropharyngeal squamous cell carcinoma

BackgroundIn Germany, approximately 30-50% of Oropharyngeal squamous-cell carcinoma (OPSCC) are related to human papillomaviruses (HPV), of which 90% are caused by HPV-16. The analysis of cancer-associated antigens is needed to develop antigen-specific immunotherapy and to determine the level of personalization required for therapeutic cancer vaccines. MethodsHuman leucocyte antigen (HLA) ligands from fresh frozen OPSCC biopsies were analyzed. HLA molecules were extracted from tumor tissue using immunoaffinity chromatography. HLA-bound peptides were subjected to high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Tumor-exclusive antigens in OPSCC were identified by comparative profiling against an in-house benign and malignant database. Additionally, whole exome sequencing of RNA and DNA isolated from the respective cancer biopsies was performed to complement the database. ResultsTo date, HLA ligandomes from 28 OPSCC (15 HPV+; 13 HPV-) patients were analyzed. Compared to the benign samples database, 148 source proteins were tumor-exclusive for HLA class I and 200 source proteins for HLA class II. The mean number of tumor-exclusive proteins per sample was 6.0 (0-14) for HLA class I and 7.7 for HLA class II (0-41) respectively. Among the tumor exclusive proteins for HLA class I, 23 had not been detected in other malignant tumor ligandomes (n=703 samples) and 75 for HLA class II (n=433 samples). A principal component analysis of the HLA-ligandomes from HPV+ and HPV- revealed significant differences between the two groups. For HLA-class I, 85 tumor-exclusive proteins were only found in HPV+ and 55 in HPV-, whereas 8 proteins were found in both groups. Among the 200 HLA class II tumor-exclusive proteins, 96 were only found in HPV+ and 95 in HPV- respectively, whereas 9 were shared by HPV+ and HPV-. ConclusionsThe analysis of HLA-ligandomes from OPSCC revealed tumor-exclusive and newly discovered antigens. We found clear differences between the HLA ligandomes of HPV+ and HPV- patients. These differences need to be taken into account for therapeutic vaccination strategies. The level of personalization needed for such vaccines remains to be determined. Legal entity responsible for the studyThe authors. FundingUniversity of Ulm, Clinician Scientist Programme. DisclosureS. Laban: Honoraria (institution), Advisory / Consultancy, Travel / Accommodation / Expenses: Merck Sharp & Dohme (MSD); Honoraria (institution), Advisory / Consultancy, Travel / Accommodation / Expenses: Bristol-Myers Squibb; Honoraria (institution), Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (institution): Merck Serono. P.J. Schuler: Advisory / Consultancy: Bristol-Myers Squibb. T.K. Hoffmann: Honoraria (institution), Advisory / Consultancy: Merck Sharp & Dohme (MSD); Honoraria (institution): Bristol-Myers Squibb; Honoraria (institution): Merck Serono. H. Rammensee: Advisory / Consultancy, Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Immatics; Advisory / Consultancy, Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: CureVac; Advisory / Consultancy, Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: Synimmune. All other authors have declared no conflicts of interest.

A transgenic mouse that spontaneously develops pathogenic TSH receptor antibodies will facilitate study of antigen-specific immunotherapy for human Graves' disease.

Graves' hyperthyroidism can be treated but not cured. Antigen-specific immunotherapy would accomplish this goal, for which purpose an animal model is an invaluable tool. Two types of animal models are available. First, pathogenic TSHR antibodies (TSHRAb) can be induced by injecting mice with fibroblasts co-expressing the human TSHR (hTSHR) and MHC class II, or in mammals using plasmid or adenovirus vectors encoding the hTSHR or its A-subunit. Second, a mouse model that spontaneously develops pathogenic TSHRAb resembling those in human disease was recently described. This outcome was accomplished by transgenic intrathyroidal expression of the hTSHR A-subunit in NOD.H2h4 mice that are genetically predisposed to develop thyroiditis but, without the transgene, do not generate TSHRAb. Recently, novel approaches to antigen-specific immunotherapy have been tested, primarily in the induced model, by injecting TSHR A-subunit protein or cyclic TSHR peptides. T-cell tolerance has also been induced in "humanized" HLA-DR3 mice by injecting synthetic peptides predicted in silico to mimic naturally processed TSHR T-cell epitopes. Indeed, a phase 1 study based on the latter approach has been conducted in humans. In the spontaneous model (hTSHR/NOD.H2h mice), injection of soluble or nanoparticle-bearing hTSHR A-subunits had the unwanted effect of exacerbating pathogenic TSHRAb levels. A promising avenue for tolerance induction, successful in other conditions and yet to be tested with the TSHR, involves encapsulating the antigen. In conclusion, these studies provide insight into the potential outcome of immunotherapeutic approaches and emphasize the importance of a spontaneous model to test future novel, antigen-specific immunotherapies for Graves' disease.

Adjuvants as Delivery Systems in Antigen-Specific Immunotherapies

Combining autoantigens with immune-modulating drugs has emerged as an attractive approach to selectively reinstate tolerance in autoimmune diseases. The disparate properties of autoantigens and small-molecule immunosuppressants commonly used to treat autoimmune diseases can confound efforts to co-deliver these therapies. However, both components may be co-delivered with adjuvants which have been successful in delivering antigens to immune cells. We evaluated several common adjuvants as vehicles to co-deliver a model antigen and immunosuppressant, ovalbumin (OVA) and dexamethasone (DEX), respectively. Formulations were developed, and the release of DEX from adjuvants was investigated. Next, the effect of adjuvant, DEX, and OVA was tested in vitro using a DC line. A MF59-analog (MF59a) formulation was advanced to more sophisticated co-culture studies using OVA-primed bone marrow–derived dendritic cells and splenocytes or T-cells from OT-II mice. Most of these studies indicated MF59a-based antigen-specific immunotherapies could diminish the markers of inflammation associated with OVA recognition. We rationalized MF59a co-delivery of antigen and drug could reduce the risk of side effects typically associated with these drugs and reinstate immune tolerance, thus prompting continued investigation of emulsion adjuvants as delivery vehicles for antigen-specific immunotherapy of autoimmune diseases.

Is there RCT evidence showing the effects of antigen-specific active immunotherapy in women with ovarian cancer?

Is there RCT evidence showing the effects of antigen-specific active immunotherapy in women with ovarian cancer?

Abstract 3188: Transimmunization prevents recurrence and reprograms the immune milieu in a mouse model of ovarian cancer

Abstract Introduction: Targeting recurrent disease is vital in the improvement of survival in ovarian cancer patients. Transimmunization (TI) is a modification of extracorporeal photochemotherapy, which is a dendritic cell (DC)-based vaccination, currently used in cutaneous T cell lymphoma but has never been tested in gynecologic tumors. The objective of this study is to determine if TI can induce a tumor-specific immune response capable of preventing recurrence in ovarian cancer. Methods of study: Ovarian cancer cells expressing mCherry fluorescent protein were injected i.p. in C57bl/6 mice to mimic the establishment of recurrent disease. These cells (TKO cancer cells) were isolated from spontaneously formed ovarian tumors from p53LSL-R172H/+Dicerflox/floxPtenflox/flox Amhr2cre/+ mice. Tumor burden was quantified using mCherry fluorescence ROI area. TI vaccination was conducted as follows: (1) cancer cells were treated with 8-MOP and exposed to UVA to induce apoptosis; (2) PBMCs were collected from tumor-bearing mice, mixed with apoptotic cancer cells, and circulated through the TI chamber to induce functional DC from monocytes; (3) cells collected were incubated overnight to facilitate antigen uptake and presentation and injected i.p. Treatment groups were as follows: (1) PBS control, n = 20 ; (2) TI TKO, n =10 (i.e. using apoptotic TKO cells); and (3) TI YUMM, n = 10 (using apoptotic mouse melanoma YUMM1.7 cells as antigen-unrelated control). Immune milieu was characterized by IHC on tumors and FACS analysis using cells isolated from peritoneal lavage. Results: Eighty percent of mice in the TI TKO group were disease free compared to 35% in the PBS control group and 40% in the TI YUMM group. In addition, TI TKO vaccination (p < 0.0001), but not TI YUMM vaccination (p = 0.1079) induced a statistically significant decrease in tumor kinetics compared to PBS control. IHC analysis showed significantly higher levels of infiltrating CD8+ (p = 0.0037) and CD4+ T cells (p = 0.0079) in TI TKO group compared to PBS group. Similarly, mice in the TI TKO group have higher i.p. levels of CD8+/CD44+/CD62L+ central memory T cells (p = 0.004), higher levels of CD8+/CD44+/CD62L- effector memory T cells (p = 0.03), lower levels of CD8+/CD44-/CD62L+ naïve T cells (p = 0.02), and lower levels of CD11b+/Gr-1high myeloid-derived suppressor cells (p = 0.01) compared to PBS control group. Levels of these cells in TI YUMM group were not significantly different from the PBS control. Conclusion: We demonstrate for the first time that TI vaccination with autologous cancer cells can prevent recurrence and modify the immune phenotype in ovarian cancer. TI efficacy depends on the tumor antigen source, confirming TI as an antigen-specific DC-based immunotherapy. Our results highlight the value of TI in ovarian cancer and its potential application to other i.p. cancers. Citation Format: Ayesha B. Alvero, Douglas Hanlon, Mary Pitruzzello, Renata Filler, Eve Robinson, Olga Sobolev, Roslyn Tedja, Alessandra Ventura, Richard L. Edelson, Gil Mor. Transimmunization prevents recurrence and reprograms the immune milieu in a mouse model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3188.

Soluble Antigen Arrays Displaying Mimotopes Direct the Response of Diabetogenic T Cells.

Type 1 diabetes (T1D) is an autoimmune disorder which develops when insulin-producing, pancreatic beta cells are destroyed by an aberrant immune response. Current therapies for T1D either treat symptoms or cause global immunosuppression, which leave patients at risk of developing long-term complications or vulnerable to foreign pathogens. Antigen-specific immunotherapies have emerged as a selective approach for autoimmune diseases by inducing tolerance while mitigating global immunosuppression. We previously reported SAgAs with multiple copies of a multiple sclerosis (MS) autoantigen grafted onto hyaluronic acid (HA) as an efficacious therapy in experimental autoimmune encephalomyelitis. While the immune response of MS is distinct from that of T1D, the mechanism of SAgAs was hypothesized to be similar and via induction of immune tolerance to diabetes antigens. We synthesized SAgAs composed of HA polymer backbone conjugated with multiple copies of the T1D autoantigen mimotope p79 using aminooxy chemistry (SAgAp79) or using copper-catalyzed alkyne-azide cycloaddition (cSAgAp79) chemistry. SAgAs constructed using the hydrolyzable aminooxy linkage, thus capable of releasing p79, exhibited physicochemical properties similar to the triazole linkage. Both SAgAp79 versions showed high specificity and efficacy in stimulating epitope-specific T cells. SAgAs can be taken up by most immune cell populations but do not induce their maturation, and conventional dendritic cells are responsible for the brunt of antigen presentation within splenocytes. cSAgAp79 was more stimulatory than SAgAp79 both in vitro and in vivo, an effect that was ascribed to the peptide modification rather than the type of linkage. In summary, we provide here the first proof-of-principle that SAgA therapy could also be applicable to T1D.

Myeloid Derived Suppressor Cells: Key Drivers of Immunosuppression in Ovarian Cancer.

The presence of tumor infiltrating lymphocytes (TILs) is associated with a longer overall survival in advanced stage epithelial ovarian cancer. Despite the prognostic impact of TILs, response to checkpoint-inhibitors and antigen-specific active immunotherapy is limited in ovarian cancer. The goal of our study was to investigate the interaction between ovarian cancer and the innate and adaptive immune system in the ID8-fLuc syngeneic ovarian cancer mouse model. For the in vivo experiments C57BL/6, B6.129S7-Rag1tm1Mom/J, and B6.129P2(SJL)-Myd88tm1.1Defr/J mice were inoculated with ID8-fLuc. In vivo depletion experiments were performed using clodronate liposomes (CL), anti-CD8a, anti-GR1, anti-colony stimulating factor 1 (anti-CSF1), and TMβ1 (anti-CD122). Immune read out was performed by fluorescent activated cell sorting analysis for effector T cells, regulatory T cells, natural killer cells, B cells, macrophages, and myeloid derived suppressor cells (MDSC), immunohistochemistry for MDSC and tumor-associated macrophages (TAM) and immunofluorescence for M1 and M2 TAM in the vascular context. The effect of MDSC on T cell proliferation and phenotype were studied in vitro. We discovered that the absence of T and B cells did not influence tumor growth or survival of B6.129S7-Rag1tm1Mom/J mice compared to immunocompetent C57BL/6 mice. CL-induced macrophage depletion promoted tumor proliferation and shortened survival in C57BL/6 mice (p = 0.004) and in B6.129S7-Rag1tm1Mom/J mice (p = 0.0005). During CL treatment, we observed a clear increase of pro-inflammatory cytokines (p ≤ 0.02) and monocytic MDSC (p ≤ 0.01). Selective depletion of MDSC by anti-GR1 improved survival, certainly in comparison to mice treated with anti-CSF1 (p = 0.01—median survival 91 vs. 67.5 days). B6.129P2(SJL)-Myd88tm1.1Defr/J mice displayed to a longer median survival compared to C57BL/6 mice (90 vs. 76 days). MDSC activated by ID8-fLuc conditioned medium or ascites of tumor-bearing mice showed T cell suppressive functions in vitro. Based on these findings, we conclude that the adaptive immune system does not efficiently control tumor growth in the ID8-fLuc model. In addition, we discovered a prominent role for MDSC as the driver of immunosuppression in the ID8-fLuc ovarian cancer mouse model.

94-OR: Lipid Nanoparticle-Mediated Delivery of Enhanced Costimulation Blockade to Prevent Type 1 Diabetes

Type 1 diabetes (T1D) remains an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Because of its complex etiology, many immunotherapy strategies have been investigated, but with disappointing results. Costimulation blockade, blocking the CD28 pathway via administration of CTLA4-Ig, is a promising approach, but recent observations suggest its efficacy is antagonized by inflammatory factors. As antigen presenting cells malfunctions and aberrant accumulation of type 1 interferons are associated with T1D, we pose that inhibiting the signaling of inflammatory cytokines via Tofacitinib (Tofa), a JAK inhibitor, would enhance the efficacy of CTLA4-Ig to prevent T1D development. The objective of this study was to design the controlled and localized delivery of Tofa via implementation of biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess the immunomodulatory impact of this strategy. We identified a specific composition of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging using fluorescently-labeled NLC confirmed that these particles have the unique property of accumulating in lymphoid tissues. Moreover, when administrated via oral gavage, they bypassed first-pass metabolism and accumulated in spleen, pancreatic and mesenteric lymph nodes. Ex-vivo, Tofa- NLC rapidly delivered Tofa to mouse antigen presenting cells preventing their maturation and the release of inflammatory cytokines. Ongoing experiments show that short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in NOD promotes a significant reduction of T1D onset. Overall, Tofa-NLC represent a promising strategy to complement CTLA4-Ig (currently investigated) and we envision this combination strategy could enhance the efficacy of antigen specific immunotherapy. Disclosure Y. Zhang: None. J. Wang: None. X. Calderon-Colon: None. O. Tiburzi: None. M. Iglesias Lozano: None. J. Patrone: None. G. Raimondi: None.

Endoplasmic reticulum stress enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV vaccines

BackgroundEndoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodal treatment methods that utilize and combine conventional cancer therapies with antigen-specific immunotherapies have emerged as promising approaches for the treatment and control of cancer. However, it is not well known whether endoplasmic reticulum stress-inducing agents can influence the efficacy of tumor antigen-targeting vaccines.MethodsIn the past, we developed a therapeutic human papillomavirus (HPV) DNA vaccine that encodes for calreticulin (CRT) linked to the HPV16 E7 antigen (CRT/E7). In this study, we utilize the CRT/E7 and further encode for an endoplasmic reticulum (ER) stress-inducing agent, 3-bromopyruvate (3-BrPA), in a preclinical model, by harnessing its potential to enhance HPV16 E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors (TC-1 cells). E7-specific CD8+ T cells were added to evaluate the cytotoxicity of luciferase-expressing TC-1 tumor cells treated with 3-BrPA in vitro, as measured with an IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-BrPA-treated TC-1 cells. TC-1 tumor-bearing mice were treated with either 3-BrPA (10 mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30 μg/mouse).ResultsTreatment of E7-expressing TC-1 tumor cells with 3-BrPA induced significantly higher in vitro cytotoxicity and resulted in upregulation of endoplasmic reticulum stress markers (CHOP and GRP78). More importantly, combination treatment of 3-BrPA and the CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice.ConclusionsOur data indicate that 3-BrPA can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation and provide novel strategies for the treatment of HPV-associated diseases.

Effect of subcutaneous multipeptide active antigen-specific immunotherapy at lymph nodes and tumor sites on clinical outcomes in progressive tumors.

2636 Background: The tumor and lymph nodes (LN) microenvironment clinically speaking are mainly unexplored and there are limited clinical studies of immunotherapeutic approaches manipulating those in progressive cancer patients. The tumor and LN microenvironment offers an opportunity to treat locally with multi-peptide immunotherapy in challenging clinical situations where we can have still a potential safe and effective therapeutic approach using the concept treat locally treat systemically. Methods: N = 11 patients were enrolled after the local IRB ethic committee approved the pilot clinical study number CICS/SS/0035. Previous identified targets such as FAP, b-2-microglobulin, Fascin, RCAS1/EBAG9, Bcl-2, survivin, Sox2, Ape-1, Valosin containing-protein (VCP) and EGFR were analyzed by IHC, Th1 and CD8 long-peptides were predicted, immune assays using PBMCs including tumor microenvironment were performed to detect Granzyme B by ELISPOT, naturally processed epitopes by T-cell expansion and cytokines. Patients were treated subcutaneously (S.C.) eight times in the axillary and inguinal LN area one week apart. Afterwards we treated S.C. as well but now in the areas with tumor activity according with the CT or PET every week 10 times. Initial DTH and at the end of the treatment was performed. Pathological, clinical and immunological correlations were made using multivariate analysis. Results: Despite progressive disease 100% of the patients responded to the treatment. 80% had CR and 20% pseudo-progression and then CR. 100% of the patients increased the levels of Granzyme B against Bcl-2 (p = 0.001), VCP (p = 0.0001), Ape-1 (p = 0.005) and RCAS1 (P = 0.0001) and this correlated with the scans post-treatment. The patients showed more CD8 infiltration in the DTH test at the tumor site (p = 0.002) than in the non-tumor site (p = 0.01). The number of metastatic lesions in lungs (p = 0.004) and hepatic tissue (p = 0.001) disappeared and correlated with increased levels of IL-12 production in vivo. Conclusions: Treating immunologically the LN and the microenvironment of the areas with tumor activity is a feasible and safe approach to limit systemic toxicity and improve the clinical outcomes in patients with progressive cancer including bone sarcoma, epithelial ovarian cancer, PDAC, pediatric sarcomas and TNBC.

Current Status of Immunotherapies for Treating Pancreatic Cancer.

Despite all efforts, pancreatic ductal adenocarcinoma (PDAC) remains a disease that causes substantial morbidity and mortality, with a 5-year survival rate of 7%. Innovative paradigms for treating PDAC are urgently needed. We discuss the advances and difficulties in using immunotherapy and developing immunotherapeutic vaccines for PDAC. Current excitement about antigen-specific immunotherapy has been propelled by advances in multiple areas, such as next-generation sequencing to identify neoantigens and manufacturing to produce immunotherapeutic vaccines. Antigen-specific immunotherapy is being actively explored in clinical trials. As the field of immunotherapy matures and as our understanding of the complex interactions between tumor and host develops, we hope to identify new methods for treating and managing PDAC.

Antigen-Drug Conjugates as a Novel Therapeutic Class for the Treatment of Antigen-Specific Autoimmune Disorders.

Multiple sclerosis represents the world's most common cause of neurological disability in young people and is attributed to a loss of immune tolerance toward proteins of the myelin sheath. Typical treatment options for MS patients involve immunomodulatory drugs, which act nonspecifically, resulting in global immunosuppression. The study discussed herein aims to demonstrate the efficacy of antigen-specific immunotherapies involving the conjugation of disease causing autoantigen, PLP139-151, and a potent immunosuppressant, dexamethasone. Antigen-drug conjugates (AgDCs) were formed using copper-catalyzed azide-alkyne cycloaddition chemistry with the inclusion of a hydrolyzable linker to maintain the activity of released dexamethasone. Subcutaneous administration of this antigen-drug conjugates to SJL mice induced with experimental autoimmune encephalomyelitis, protected the mice from a symptom onset throughout the 25 day study, demonstrating enhanced efficacy in comparison to dexamethasone treatment. These results highlight the benefits of co-delivery of autoantigens with immunosuppressant drugs as AgDCs for the treatment of autoimmune diseases.

Combinatorial, Microparticle-Based Delivery of Immune Modulators Reprograms the Dendritic Cell Phenotype and Promotes Remission of Collagen-Induced Arthritis in Mice.

Safe, effective, antigen-specific therapy for rheumatoid arthritis (RA) remains an elusive clinical goal with a few lasting, viable options on the horizon. Existing therapeutic interventions are indiscriminate and inconsistently immunosuppressive, often leaving patients susceptible to infection. Herein, we investigate the use of a dual-sized, microparticle "regulatory vaccine" (REGvac) that passively targets dendritic cells for antigen-specific biomaterial-based immunotherapy of RA. This REGvac employs poly(d,l-lactic-co-glycolic-acid) (PLGA) microparticles (MPs) encapsulating (i) a dendritic cell chemoattractant, (ii) potent immunosuppressive molecules, (iii) and an RA-relevant autoantigen to provide a multifaceted approach for the treatment of collagen-induced arthritis (CIA), the primary mouse model of RA. Subcutaneous administrations of the REGvac after mice had developed moderate clinical symptoms markedly diminished overt inflammation in the paws, halted cartilage degradation, and restored gait parameters within 56 days after initial treatment. Positron emission tomography imaging corroborated reduction of inflammation in the paws of REGvac-treated mice. In-depth immunological assessments showed a decreased expression of CD80, CD86, and MHC II on CD11c+ dendritic cells in joint-associated lymph nodes. Further, we observed significant increases in conventional regulatory CD25+FOXP3+ T cells, as well as programmed cell death protein-1 (PD-1)-expressing CD4+ T cells in joint-proximal lymph nodes and the spleen. Real-time PCR analysis of joint tissues from treated mice revealed significant decreases in inflammatory cytokine expression (IL-6), while IL-10 mRNA levels were significantly increased. These observations strongly hint toward the induction of multiple tolerogenic mechanisms by administration of this MP regulatory vaccine. With regards to antigen specificity, ex vivo antigen recall assays revealed a lack of response to collagen by CD4+ T cells from the popliteal and inguinal lymph nodes of REGvac-treated mice, contrasting with the proliferative response of CD4+ T cells from CIA+ mice. Taken altogether, our results strongly support the application of this MP regulatory vaccine as a potent, biomaterial-based, antigen-specific therapy for RA.

T regulatory antigen-specific multi-peptide immunotherapy induce clinical responses in autoimmune diseases

Abstract Background The current treatment for several fatal autoimmune diseases is mainly palliative with minimal clinical benefit. By other hand the biological and clinically relevant potential targets to stop the auto immune response are mainly unknown, including the innate and adaptive auto-immune micro-environment. We performed a pilot trial with (n=5) progressive patients with myasthenia gravis (MG), multiple sclerosis (MS) and amyotrophic sclerosis lateral (ASL) using a multi-peptide T regulatory immunotherapy. Methods We performed a systematic review to found potential proteins involved in the auto-immune response in these three autoimmune diseases using the terms “misfolded proteins and autoimmune disease and prognosis and multivariate analysis”. initially N=25 repository samples from autoimmune disease patients were analyzed to test the predicted T regulatory peptides immunogenicity for ELISA, IL-10 ELISPOT and T –cell expansion. Results We found that predicted T regulatory peptides from Bcl-2, peptide C (p=0.001), survivi, peptide A (0.001) and VCP, peptide 8 = (P=0.005) were able to expand antigen-specific T regulatory cells and there was a clinical correlation in symptoms improvement in all the three diseases (mOS=28 months). Discussion The multi-peptide T regulatory peptide immunotherapy is feasible therapeutic approach to treat incurable autoimmune diseases and this data could be used as proof of principle to prepare a large clinical study in order to potentially improve clinical outcomes in combination with other approaches that may impact the clinical scenario in autoimmune diseases with limited or nor therapeutic options. No clinical toxicity was observed.

Lipid Nanoparticle-Mediated Delivery of Enhanced Costimulation Blockade to Prevent Type 1 Diabetes

Abstract Type 1 diabetes (T1D) remains an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Costimulation blockade, blocking the CD28 pathway via CTLA4-Ig, is one promising immunotherapeutic, but recent observations suggest its efficacy is antagonized by inflammatory factors. As antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons are associated with T1D, we pose that inhibiting the signaling of inflammatory cytokines via Tofacitinib (Tofa), a JAK inhibitor, would enhance the efficacy of CTLA4-Ig in preventing T1D development. The objective of this study was to design a controlled and localized delivery of Tofa via biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess the immunomodulatory impact of this strategy. We identified a specific composition of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have unique property of accumulating in lymphoid tissues, particularly in spleen, pancreatic and mesenteric lymph nodes when administrated via oral gavage. Ex-vivo, Tofa-NLC rapidly delivered Tofa to mouse APCs preventing their maturation and the release of inflammatory cytokines. Ongoing experiments show that short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in disease prone NOD mice promotes a significant reduction of T1D onset. Overall, Tofa-NLC represent a promising strategy to complement CTLA4-Ig and we envision this combined strategy could enhance the efficacy of antigen specific immunotherapy.

Acute B-Cell Inhibition by Soluble Antigen Arrays Is Valency-Dependent and Predicts Immunomodulation in Splenocytes.

Antigen valency plays a fundamental role in directing the nature of an immune response to be stimulatory or tolerogenic. Soluble antigen arrays (SAgAs) are an antigen-specific immunotherapy that combats autoimmunity through the multivalent display of autoantigen. Although mechanistic studies have shown SAgAs to induce T- and B-cell anergy, the effect of SAgA valency has never been experimentally tested. Here, SAgAs of discrete antigen valencies were synthesized by click chemistry and evaluated for acute B-cell signaling inhibition as well as downstream immunomodulatory effects in splenocytes. Initial studies using the Raji B-cell line demonstrated SAgA valency dictated the extent of calcium flux. Lower valency constructs elicited the largest reductions in B-cell activation. In splenocytes from mice with experimental autoimmune encephalomyelitis, the same valency-dependent effects were evident in the downregulation of the costimulatory marker CD86. The reduction of calcium flux observed in Raji B-cells correlated strongly with downregulation in splenocyte CD86 expression after 72 h. Here, a thorough analysis of SAgA antigenic valency illustrates that low, but not monovalent, presentation of autoantigen was ideal for eliciting the most potent immunomodulatory effects.

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (−40 to −60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.

Determining Antigen Specificity of Human Islet Infiltrating T Cells in Type 1 Diabetes.

Type 1 diabetes, the immune mediated form of diabetes, represents a prototypical organ specific autoimmune disease in that insulin producing pancreatic islets are specifically targeted by T cells. The disease is now predictable in humans with the measurement of type 1 diabetes associated autoantibodies (islet autoantibodies) in the peripheral blood which are directed against insulin and beta cell proteins. With an increasing incidence of disease, especially in young children, large well-controlled clinical prevention trials using antigen specific immunotherapy have been completed but with limited clinical benefit. To improve outcomes, it is critical to understand the antigen and T cell receptor repertoires of those cells that infiltrate the target organ, pancreatic islets, in human type 1 diabetes. With international networks to identify organ donors with type 1 diabetes, improved immunosequencing platforms, and the ability to reconstitute T cell receptors of interest into immortalized cell lines allows antigen discovery efforts for rare tissue specific T cells. Here we review the disease pathogenesis of type 1 diabetes with a focus on human islet infiltrating T cell antigen discovery efforts, which provides necessary knowledge to define biomarkers of disease activity and improve antigen specific immunotherapy approaches for disease prevention.

Efficacy and Safety of Antigen-specific Immunotherapy in the Treatment of Patients with Non-small-cell Lung Cancer: A Systematic Review and Meta-analysis.

We performed this systematic review and meta-analysis to assess the efficacy and safety of antigen-specific immunotherapy (Belagenpumatucel-L, MAGE-A3, L-BLP25, and TG4010) in the treatment of patients with non-small-cell lung cancer (NSCLC). A comprehensive literature search on PubMed, Embase, and Web of Science was conducted. Eligible studies were clinical trials of patients with NSCLC who received the antigenspecific immunotherapy. Pooled hazard ratios (HRs) with 95% confidence intervals (95%CIs) were calculated for overall survival (OS), progression-free survival (PFS). Pooled risk ratios (RRs) were calculated for overall response rate (ORR) and the incidence of adverse events. In total, six randomized controlled trials (RCTs) with 4,806 patients were included. Pooled results showed that, antigen-specific immunotherapy did not significantly prolong OS (HR=0.92, 95%CI: 0.83, 1.01; P=0.087) and PFS (HR=0.93, 95%CI: 0.85, 1.01; P=0.088), but improved ORR (RR=1.72, 95%CI: 1.11, 2.68; P=0.016). Subgroup analysis based on treatment agents showed that, tecemotide was associated with a significant improvement in OS (HR=0.85, 95%CI: 0.74, 0.99; P=0.03) and PFS (HR=0.70, 95%CI: 0.49, 0.99, P=0.044); TG4010 was associated with an improvement in PFS (HR=0.87, 95%CI: 0.75, 1.00, P=0.058). In addition, NSCLC patients who were treated with antigen-specific immunotherapy exhibited a significantly higher incidence of adverse events than those treated with other treatments (RR=1.11, 95%CI: 1.00, 1.24; P=0.046). Our study demonstrated the clinical survival benefits of tecemotide and TG4010 in the treatment of NSCLC. However, these evidence might be limited by potential biases. Therefore, further well-conducted, large-scale RCTs are needed to verify our findings.