Soluble antigen arrays provide increased efficacy and safety over free peptides for tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but clinical implementation has been challenging. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA used (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Over time, the peptides induced an IgE-independent anaphylactic reaction, the incidence of which was significantly delayed when peptides were in SAgA form rather than in free form. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Immunologic anaphylaxis occurred in NOD mice in a dose-dependent manner but not in C57BL/6 or BALB/c mice; however, its incidence did not correlate with the level of anti-peptide antibodies. We provide evidence that SAgAs significantly improve the efficacy of peptides to induce tolerance and prevent autoimmune diabetes while at the same time reducing their anaphylactogenic potential.

Soluble antigen arrays improve the efficacy and safety of peptide-based tolerogenic immunotherapy.

Autoantigen-specific immunotherapy using peptides offers a more targeted approach to treat autoimmune diseases, but the limited in vivo stability and uptake of peptides impedes clinical implementation. We previously showed that multivalent delivery of peptides as soluble antigen arrays (SAgAs) efficiently protects against spontaneous autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Here, we compared the efficacy, safety, and mechanisms of action of SAgAs versus free peptides. SAgAs, but not their corresponding free peptides at equivalent doses, efficiently prevented the development of diabetes. SAgAs increased the frequency of regulatory T cells among peptide-specific T cells or induce their anergy/exhaustion or deletion, depending on the type of SAgA (hydrolysable (hSAgA) and non-hydrolysable 'click' SAgA (cSAgA)) and duration of treatment, whereas their corresponding free peptides induced a more effector phenotype following delayed clonal expansion. Moreover, the N-terminal modification of peptides with aminooxy or alkyne linkers, which was needed for grafting onto hyaluronic acid to make hSAgA or cSAgA variants, respectively, influenced their stimulatory potency and safety, with alkyne-functionalized peptides being more potent and less anaphylactogenic than aminooxy-functionalized peptides. Both SAgA variants significantly delayed anaphylaxis compared to their respective free peptides. The anaphylaxis, which occurred in NOD mice but not in C57BL/6 mice, was dose-dependent but did not correlate with the production of IgG1 or IgE against the peptides. We provide evidence that SAgAs significantly improve the efficacy and safety of peptide-based immunotherapy.

Epitope-based precision immunotherapy of Type 1 diabetes.

Antigen-specific immunotherapies (ASITs) address important clinical needs in treating autoimmune diseases. However, Type 1 diabetes is a heterogeneous disease wherein patient characteristics influence responsiveness to ASITs. Targeting not only disease-relevant T cell populations, but also specific groups of patients using precision medicine is a new goal toward achieving effective treatment. HLA-restricted peptides provide advantages over protein as antigens, however, methods for profiling antigen-specific T cells need to improve in sensitivity, depth, and throughput to facilitate epitope selection. Delivery approaches are highly diverse, illustrating the many ways relevant antigen-presenting cell populations and anatomical locations can be targeted for tolerance induction. The role of persistence of antigen presentation in promoting durable antigen-specific tolerance requires further investigation. Based on the outcome of ASIT trials, the field is moving toward using patient-specific variations to improve efficacy, but challenges still lie on the path to delivering more effective and safer treatment to the T1D patient population.

Preclinical evaluation of a precision medicine approach to DNA vaccination in type 1 diabetes.

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject's MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes.

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address the limited clinical efficacy of ASIT. Soluble antigen arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using either hydrolysable linkers (hSAgAs) or stable click chemistry linkers (cSAgAs). They were evaluated for the ability to block spontaneous development of disease in a nonobese diabetic mouse model of type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgAs) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers, such as CD73, interleukin-10, programmed death-1, and KLRG-1. Anaphylaxis caused by free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely nonoverlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to free peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

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.

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.

Transgenic substitution with Greater Amberjack Seriola dumerili fish insulin 2 in NOD mice reduces beta cell immunogenicity.

Type I diabetes (T1D) is caused by immune-mediated destruction of pancreatic beta cells. This process is triggered, in part, by specific (aa 9-23) epitopes of the insulin Β chain. Previously, fish insulins were used clinically in patients allergic to bovine or porcine insulin. Fish and human insulin differ by two amino acids in the critical immunogenic region (aa 9-23) of the B chain. We hypothesized that ß cells synthesizing fish insulin would be less immunogenic in a mouse model of T1D. Transgenic NOD mice in which Greater Amberjack fish (Seriola dumerili) insulin was substituted for the insulin 2 gene were generated (mouse Ins1-/- mouse Ins2-/- fish Ins2+/+). In these mice, pancreatic islets remained free of autoimmune attack. To determine whether such reduction in immunogenicity is sufficient to protect ß cells from autoimmunity upon transplantation, we transplanted fish Ins2 transgenic (expressing solely Seriola dumerili Ins2), NOD, or B16:A-dKO islets under the kidney capsules of 5 weeks old female NOD wildtype mice. The B:Y16A Β chain substitution has been previously shown to be protective of T1D in NOD mice. NOD mice receiving Seriola dumerili transgenic islet transplants showed a significant (p = 0.004) prolongation of their euglycemic period (by 6 weeks; up to 18 weeks of age) compared to un-manipulated female NOD (diabetes onset at 12 weeks of age) and those receiving B16:A-dKO islet transplants (diabetes onset at 12 weeks of age). These data support the concept that specific amino acid sequence modifications can reduce insulin immunogenicity. Additionally, our study shows that alteration of a single epitope is not sufficient to halt an ongoing autoimmune response. Which, and how many, T cell epitopes are required and suffice to perpetuate autoimmunity is currently unknown. Such studies may be useful to achieve host tolerance to ß cells by inactivating key immunogenic epitopes of stem cell-derived ß cells intended for transplantation.

Efficient Presentation of Multiple Endogenous Epitopes to Both CD4+ and CD8+ Diabetogenic T Cells for Tolerance.

Antigen-specific immunotherapy of type 1 diabetes, typically via delivery of a single native ß cell antigen, has had little clinical benefit to date. With increasing evidence that diabetogenic T cells react against multiple ß cell antigens, including previously unappreciated neo-antigens that can be emulated by mimotopes, a shift from protein- to epitope-based therapy is warranted. To this end, we aimed to achieve efficient co-presentation of multiple major epitopes targeting both CD4+ and CD8+ diabetogenic T cells. We have compared native epitopes versus mimotopes as well as various targeting signals in an effort to optimize recognition by both types of T cells in vitro. Optimal engagement of all T cells was achieved with segregation of CD8 and CD4 epitopes, the latter containing mimotopes and driven by endosome-targeting signals, after delivery into either dendritic or stromal cells. The CD4+ T cell responses elicited by the endogenously delivered epitopes were comparable with high concentrations of soluble peptide and included functional regulatory T cells. This work has important implications for the improvement of antigen-specific therapies using an epitope-based approach to restore tolerance in type 1 diabetes and in a variety of other diseases requiring concomitant targeting of CD4+ and CD8+ T cells.