IntroductionThe etiology of Canine Diabetes Mellitus (CDM) is poorly understood but findings like increased CD3+CD4+CD40+ pathogenic effector T cells (Th40 cells), support an autoimmune contribution. Despite insulin supplementation and possible residual C-peptide in CDM, many dogs remain severely dysglycemic, with weight loss, cataracts, and chronic and recurrent infections. In human and murine autoimmune disease, CD40-CD154 acts as a prominent inflammation driver but targeting that interaction, and others, with antibodies has been plagued by complications such as thrombotic emboli or immunosuppression. We developed small peptides that target CD40 and that are not accompanied by the side effects attributed to antibodies. In mice, such a peptide prevented and reversed type 1 diabetes.MethodsWe utilized a CD40-targeting peptide, OPT501, to treat CDM dogs via an intravenous or subcutaneous route and followed their disease status and clinical outcomes as well as their inflammatory status.ResultsTreatment with OPT501 significantly decreased pathogenic Th40 cells, the systemic inflammatory index, and fructosamine (an analog to human HbA1c). This led to lowered insulin requirements while improving blood glucose regulation. OPT501 also significantly reduced cholesterol and alkaline phosphatase, and significantly increased plasma C-peptide, a measure of endogenous insulin production.ConclusionsThis pilot and proof-of-concept study demonstrates that targeting CD40 with a peptide is feasible and impacts the inflammatory status of the recipient CDM dogs, with improved disease management as a result. The C-peptide result is consistent with preservation of islet beta cell health and function. These data support translation of a CD40 targeting peptide approach to human type 1 diabetes.

Background: Type 1 diabetes (T1D) is a complex autoimmune disease demonstrating substantial heterogeneity in age of onset, residual C-peptide levels, clinical outcomes, and therapeutic response. Although the autoimmune classification of T1D has traditionally relied on detection of autoantibodies indicating B-cell involvement, studies targeting total CD3+ T cells have underscored the importance of T-cell regulation. Th40 cells, a pathogenic subset of CD3+ T cells, first identified in NOD mice, become significantly increased during diabetogenesis. Human subjects with T1D exhibit variable but significantly elevated Th40 levels in peripheral blood. Methods: To target pathogenic effector Th40 cells, we developed OPT101, a 15-mer peptide, and found that it interacts with CD40 in association with an activated integrin, identifying a novel inflammatory receptor complex. We conducted a phase 1b, double-blind, first-in-human clinical trial to evaluate OPT101 and met the primary objectives of safety and tolerability. Results: OPT101 generated only Grade 1 and 2 adverse events. Across eight doses, administered over six weeks, no product-related immune suppression was observed. Secondary objectives included immunologic outcomes and potential efficacy. Subjects with higher Th40 levels had low or undetectable C-peptide, higher (>7.0%) HbA1c, and elevated inflammatory cytokines. Th40 levels were significantly higher in subjects diagnosed before age eighteen. OPT101 treatment significantly reduced Th40 percentages without cell ablation, increased Treg levels, and decreased inflammatory cytokines. Serum blood glucose levels and HbA1c were significantly reduced by visit 8 in treated subjects. In two subjects, 11 and 13 years post-diagnosis, with undetectable C-peptide at screening, C-peptide became detectable post-treatment. Conclusions: OPT101 proved safe and effective in human T1D subjects with only mild and a few moderate adverse events. In this short-term study, OPT101 improved beta cell functions thus warranting further exploration.

Background/Objectives: One of the first-line disease-modifying treatments of multiple sclerosis (MS) is Glatiramer Acetate (GA), which requires daily or three-times-weekly subcutaneous injections. Disease progression, while slowed, still occurs with time. Increasing the impact of the treatment while decreasing the frequency of injections would be ideal. The mechanism of action of GA remains undefined. We developed an alternate approach, KGYY6, whose mechanism of action targets the CD40 receptor with promising results in an Experimental Autoimmune Encephalomyelitis (EAE) model. Methods: GA and a CD40-targeting peptide, KGYY6, were formulated as slow-release particles used to treat EAE in C57BL/6 mice. Results: Compared to liquid formulations, the particle formulations vastly improved drug efficacy in both cases, which would be advantageous in treating MS. GA is a combination of randomly generated peptides, in the size range of 5000–9000 Da, using the amino acids E, A, Y, and K. This approach introduces batch differences that impacts efficacy, a persistent problem with GA. KGYY6 is generated in a controlled process and has a motif, K-YY, which could be generated when manufacturing GA. When testing two different lots of GA or KGYY6, the latter performed equally well across lots, while GA did not. Conclusions: Slow-release formulations of both GA and KGYY6 vastly improve the efficacy of both, and KGYY6 is more consistent in efficacy across different lots.

IntroductionCanine diabetes mellitus (CDM) is a relatively common endocrine disease in dogs. Many CDM clinical features resemble human type 1 diabetes mellitus (T1DM), but lack of autoimmune biomarkers makes calling the disease autoimmune controversial. Autoimmune biomarkers linking CDM and T1DM would create an alternative model for drug development impacting both human and canine disease. MethodsWe examined peripheral blood of diagnosed CDM dog patients comparing it to healthy control (HC) dogs. Dogs were recruited to a study at the Colorado State University Veterinary Teaching Hospital and blood samples collected for blood chemistry panels, complete blood counts (CBC), and immunologic analysis. Markers of disease progression such as glycated albumin (fructosamine, the canine equivalent of human HbA1c) and c-peptide were addressed. ResultsSignificant differences in adaptive immune lymphocytes, innate immune macrophages/monocytes and neutrophils and differences in platelets were detected between CDM and HC based on CBC. Significant differences in serum glucose, cholesterol and the liver function enzyme alkaline phosphatase were also detected. A systemic immune inflammation index (SII) and chronic inflammation index (CII) as measures of dynamic changes in adaptive and innate cells between inflammatory and non-inflammatory conditions were created with highly significant differences between CDM and HC. Th40 cells (CD4+CD40+ T cells) that are demonstrably pathogenic in mouse T1DM and able to differentiate diabetic from non-diabetic subjects in human T1DM were significantly expanded in peripheral blood mononuclear cells.ConclusionsBased on each clinical finding, CDM can be categorized as an autoimmune condition. The association of significantly elevated Th40 cells in CDM when compared to HC or to osteoarthritis, a chronic but non-autoimmune disease, suggests peripheral blood Th40 cell numbers as a biomarker that reflects CDM chronic inflammation. The differences in SII and CII further underscore those findings.

CD40 signaling has long been a target in autoimmunity. Attempts to block signaling between CD40 and CD154 during clinical trials using monoclonal antibodies suffered severe adverse events. Previously, we developed a peptide, KGYY15, that targets CD40 and, in preclinical trials, prevents type 1 diabetes in >90% of cases and reverses new-onset hyperglycemia in 56% of cases. It did so by establishing normal effector T-cell levels rather than ablating the cells and causing immunosuppression. However, the relationship between KGYY15 and other elements of the complex signaling network of CD40 is not clear. Studying interactions between proteins from autoimmune and nonautoimmune mice, we demonstrate interactions between CD40 and integrin CD11a/CD18, which complicates the understanding of the inflammatory nexus and how to prevent autoinflammation. In addition to interacting with CD40, KGYY15 interacts with the integrins CD11a/CD18 and CD11b/CD18. We argue that modulation of CD40-CD154 signaling may be more advantageous than complete inhibition because it may preserve normal immunity to pathogens.

NOD mice are the accepted model for Type 1 Diabetes Mellitus (T1DM) Canine Diabetes (CD) demonstrates clinical outcomes identical to NOD and human T1DM. We described helper T cells that express the CD40 receptor, termed Th40 cells. In human T1DM Th40 cells expand significantly in number in peripheral blood prior to hyperglycemia; and respond to human islets. In NOD mice, Th40 cells increase first in the pancreatic lymph nodes, expand in number in the periphery prior to hyperglycemia, are the prominent T cell type detected in the pancreas during insulitis, and transfer diabetes to SCID recipients. Naturally occurring CD is insulin requiring and demonstrates the same clinical parameters as human disease. In a clinical pilot study using diabetic dogs we discovered significantly elevated Th40 cell numbers in PBMC compared to non-diabetic dogs (p < 0.0001) . Th40 cell numbers may help predict risk of development of T1DM as a subset of “at-risk” pre-T1DM human subjects who developed T1DM showed elevated Th40 numbers. Using a novel approach to target CD40-mediated inflammation we created a series of small peptides. The lead candidate, OPT101, has an approved IND and completed Phase 1a human clinical trials establishing human safety profiles. OPT501, a canine version caused a rapid reduction in Th40 cell numbers in diabetic dogs. Daily insulin requirements were reduced on average by 75% and in 2 cases by 90%. Fructosamine, the canine equivalent of HbA1c, was reduced 40.2% on average with 3 subjects achieving normal range. All participants had significantly reduced daily blood glucose averages, and 3 subjects maintained normal glucose levels and time-in-range (TIR) increased to greater than 50%, and in some cases greater than 90%, from a previous TIR of 10%. All participants demonstrated increased c-peptide levels, a measure of restored beta cell function, after 6 weeks of treatment. Equivalent clinical outcomes have not been reported for any other drug candidate in veterinary or human T1DM trials. Disclosure D.H.Wagner: Other Relationship; Op-T, LLC. G.M.Vaitaitis: None. Funding National Institutes of Health (AI131784; AI128592)

Treating MS has been difficult. One successful drug is Ocrelizumab (anti-CD20), used for the chronic relapsing MS (RMS) and the progressive MS (PMS) forms. TH40 cells are pathogenic effector T cells that increase in percentage and numbers during chronic inflammation. Here we show that in the earliest MS course, clinically isolated syndrome (CIS), TH40 cells expand in number. In PMS TH40 cell numbers remain expanded demonstrating sustained chronic inflammation. In RMS TH40 cells were found in CSF and express CD20. Ocrelizumab reduced TH40 cells to healthy control levels in patients. During treatment inflammatory cytokine producing TH40 cells were decreased.

Abstract Canine Diabetes (CD) is identical to human T1DM. Helper T cells expressing the CD40 receptor (TH40) are prominent in human T1DM. TH40 cell expansions occur regardless of HLA haplotype or auto-antibody status and remain elevated throughout disease. TH40 cells from T1DM respond to human islets, TH40 from healthy controls do not. At a Veterinary Hospital 6 female and 2 male dogs that met clinical criteria for T1DM were recruited. Physical exam, blood panels and CBC were done. Dogs were dosed with a OPT501 a drug targeting CD40 mediated inflammation by IV infusion. Doses were 2 and later 4 mg/kg, given on days 1, 4, 7, then weekly for 8 weeks. Diabetic dogs had significantly elevated TH40 numbers compared to non-diabetic dogs (p < 0. 0001). OPT501 reduced TH40 cell numbers to normal, following which insulin requirements reduced on average by 75% and up to 90%. The canine equivalent of HbA1c reduced 40.2% on average; 3 subjects were normal range. Daily blood glucose reduced significantly, 3 subjects maintained normal glucose levels. Time-in-range (TIR) increased to up to 90%, from a previous 10%. Increased c - peptide levels were universally detected after 8 weeks of treatment. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Type 1 diabetes (T1D) , a prototypic autoimmune disease, creates several serious complications of which atherosclerosis is the major co-morbid condition. Atherosclerosis is defined by arterial plaque deposition due to coagulation blood products, lipid deposition, and inflammation. The latter has been known for more than a decade but only recently have inflammatory mediators proven to be a beneficial target once traditional treatment has been exhausted. T-cell mediators associated with the CD40-CD154 inflammatory dyad are found in autoimmune diseases such as T1D and rheumatoid arthritis and may be a cause of the added atherosclerotic risk which these disease states maintain. This study seeks to explore the role of T cells in atherogenesis through the use of pro-atherogenic ApoE-/- mice bred to create T cell deficiency (ApoE-/- TCRα-/-) for analysis of the overall effect of T cells in atherogenesis. Mice are sacrificed at 8 months of age and dissected to obtain the aortas and hearts. The aorta is used for en-face Sudan IV staining analysis while serial aortic valve cross sections are used to characterize the lesion in terms of area and content. Analyses of aortic valve cross sections (A-D) and whole aortas (E-F) of this novel model not only demonstrated a significant reduction in overall plaque, but also revealed a change in plaque composition due to T cell deficiency. Future aims are to define the specific subset of pathogenic T cells. Disclosure A.J.Shepherd: None. M.Yussman: None. D.H.Wagner: Other Relationship; Op-T, LLC. G.M.Vaitaitis: None. Funding National Institutes of Health (4R42DK115296-02)

CONTEXT:The incidence of type 1 diabetes (T1D) is increasing worldwide. The quest to understand T1D etiology and how to predict diabetes is ongoing; and, in many ways, those goals intertwine. Although genetic components associate with T1D, not all individuals with T1D have those components, and T1D does not develop in all subjects with those components.OBJECTIVE:More robust methods for prediction of T1D are needed. We investigated if high CD4+CD40+ T-cell (Th40) levels can be used as a biomarker.METHODS:Th40 levels were assessed along with other parameters in blood collected from prediabetic subjects in TrialNet.RESULTS:In prediabetic subjects stratified according to Th40 cell level, patterns paralleled those seen between control subjects and those with T1D. Cytokine patterns were significantly different between those with high Th-40 levels (Th40-high) and those with low levels, and a CD4/CD8 double-positive population was more represented in Th40-high groups. Subjects experiencing impaired glucose tolerance had a significantly higher Th40 level than did control subjects. HLA DR4/DR4 and DQ8/DQ8 were more likely found among Th40-high subjects. Interestingly, HLA DR4/DR4 subjects were significantly older compared with all other subjects, suggesting that this haplotype, together with a high Th40 level, may represent someone in whom T1D will develop after age 30 years, which is reported for 42% of T1D cases.CONCLUSION:Considering the differences found in relation to prediabetic Th40 cell level, it may be possible to devise methods that more accurately predict who will proceed toward diabetes and, possibly, indicate prediabetic stage.

Autoimmunity treatments, fruitfully pioneered in mouse models, can be disappointing or result in immunosuppression and opportunistic infections in translational trials. Many possible reasons exist, but one major, overlooked reason may be the treatment timing in relation to circadian oscillations of the immune system. Mice and humans both have immunological circadian clocks and experience the same circulatory oscillations of immune cells with regards to their sleep/wake phases, but have opposite sleep/wake phases with regard to the daylight cycle. Therefore, researchers mainly study mice and potential autoimmunity treatments during the murine sleep/rest phase, which is when pro-inflammatory mediators and more adaptive immune cells are prevalent in the circulation. In translational trials, however, treatment administration happens primarily during a patient’s wake/activity phase, during the daytime, which is when more local and acute immune responses are active in the circulation. Therefore, we believe that the most opportune window for autoimmunity treatment may be missed in translational trials. Shifting the timing, and adjusting dosing to target only immune cells that are active at that time, may result in higher success with minimized immunosuppression or toxicities.

Prevention trials with statins reduce the relative risk of cardiovascular (CV) events by 10 to 40%. This leaves a ‘residual risk’ of 60–90% for which the CANTOS trial (IL1β inhibition) provided proof of concept that targeting inflammation reduces CV event rates; unfortunately, it did not change rates of type 1 diabetes (T1D) and infections in T1D patients were higher. A major molecular driver of auto-inflammation in both diseases is the CD40/CD154 inflammatory dyad with unique T cell subset, CD3+CD4+CD40+ (TH40 cell) known to be significantly expanded in T1D subjects. Normalization of the aberrant contact dependent interaction of this dyad has shown efficacy in animal models using antibody to CD154, reversing T1D and rate of atherosclerosis, but unfortunately has deleterious side effects when given in humans. We measured TH40 cells in T1D subjects with high calcium scores (a measure of atherosclerotic burden) and those with low calcium scores. Additional to the higher percentage of TH40 cells compared to controls, we have found a statistically significant elevation in in INFg production in the high calcium score cohort, indicating a more pathogenic state of these cells. We designed peptides capable of binding directly to the CD40 receptor, which have been shown to reverse T1D in animals. Peptide was administered to ApoE−/− mice, a model of atherosclerosis. Immunohistochemical staining demonstrated significant reduction in plaque as well as smooth muscle and collagen content. In-vitro analysis of TH40 mouse splenic cells showed modulation in IL2, INFγ and IL17, all potent cytokines of atherosclerosis. This new information relays promise for a more directed diagnostic and therapeutic target for T1D related atherosclerosis.

Abstract Multiple Sclerosis (MS) is a neuro-degenerative autoimmune disease, progressive in nature, that lacks biomarkers for diagnosis or progression. We described a population of CD4+ T cells that express the CD40 receptor, Th40 cells, which proved causal in murine models of autoimmunity. Hypothesis Peripheral blood Th40 numbers reflect MS progression and their control correlates with improvement. Peripheral blood and cerebral spinal fluid from MS patients and disease controls were examined for HLA haplotype and PBMC were characterized by flow cytometry. Random sampling of CIS, RRMS, SPMS and PPMS patients demonstrated diverse HLA haplotypes; DR2 / DQ6 were highly represented but surprisingly DR4, DR3 and DQ2 were prominent. In antigen recall, each HLA haplotype preferred unique myelin-based antigens when presenting to Th40 cells. Percentages of Th40 cells in peripheral blood of CIS and RRMS had a wide range while the range in PPMS and SPMS was tight yet significantly elevated. CSF from RRMS patients had Th40 cell percentages mirroring peripheral blood. RRMS subjects had elevated B cells that surprisingly were the major source of IL-17. Rituximab treated subjects demonstrated a significant but temporal reduction in Th40 cell numbers; FoxP3+ cells were elevated while Th40 inflammatory-cytokine production was reduced. Conclusions The wide range of Th40 cell numbers in CIS and RRMS but universally high numbers in SPMS suggest that Th40 cell number expansions in MS reflect progression. Th40 cells in CSF demonstrate access to the CNS and an important correlation between peripheral blood and CNS. Antigen recall to myelin demonstrates Th40 pathogenicity. Rituximab treatment ablates B cells that then affect pathogenic effector Th40 cells.

The incidence of Type 1 Diabetes (T1D) is on the rise. Quests to understand T1D etiology as well as how to predict onset are ongoing and, in many ways, those goals intertwine. CD4+CD40+ T cell (Th40) percentages are expanded in T1D subjects compared to controls. Antibodies to β-cell antigens are considered risk factors for T1D development but risk of seroconversion to antibody-positivity declines after age 10. While genetic components associate with T1D, not all T1D subjects have those components and not all subjects with those components develop T1D. Therefore, more robust methods for prediction of T1D are needed. Can high Th40 cell percentages be a biomarker in addition to other markers? We assessed Th40 cell percentages, along with other parameters, in blood collected from TrialNet preT1D subjects and stratified them according to their percentages. Th40-low versus Th40-high preT1D subjects demonstrate patterns of CD4 T cell levels that parallel those seen between control and T1D. Cytokine patterns are significantly different between the Th40 groups and a CD4+CD8+ population, described previously in Rheumatoid Arthritis, is more common in Th40-high subjects. Subjects experiencing impaired glucose tolerance have significantly higher Th40 cell percentages than controls. PreT1Ds expressing T1D associated HLA DR4/DR4 or DQ8/DQ8 are more likely found among Th40-high subjects. Interestingly, DR4/DR4 subjects were significantly older compared to all other preT1Ds, suggesting that this haplotype together with a high Th40 cell percentage may represent someone who will onset after age 30, which is reported for 42% of T1D cases. It will be important to determine if preT1D subjects with high Th40 cell percentages will develop overt T1D.

CD40/CD154-interaction is critical in the development of Experimental Autoimmune Encephalomyelitis (EAE; mouse model of Multiple Sclerosis). Culprit CD4+CD40+ T cells drive a more severe form of EAE than conventional CD4 T cells. Blocking CD40/CD154-interaction with CD154-antibody prevents or ameliorates disease but had thrombotic complications in clinical trials. We targeted CD40 using a CD154-sequence based peptide. Peptides in human therapeutics demonstrate good safety. A small peptide, KGYY6, ameliorates EAE when given as pretreatment or at first symptoms. KGYY6 binds Th40 and memory T cells, affecting expression of CD69 and IL-10 in the CD4 T cell compartment, ultimately hampering disease development.

Sustained inflammation is a crucial pathologic component in atherosclerosis and type 2 diabetes (T2D). A common molecular player driving auto-inflammation in both diseases is the CD40/CD154 inflammatory dyad. By normalizing the aberrant contact dependent interaction of the CD40/CD154 dyad, and resultant auto-inflammation, multiple studies have shown both prevention and therapeutic efficacy in both diseases. We designed a series of peptides derived from the CD154 protein sequence that are capable of binding directly to the CD40 receptor to interrupt the inflammatory signal pathways. Two peptides, KGYY6 and KGYY15, were highly effective, initially demonstrating efficacy in type one diabetes mouse models. KGYY6 was chosen for additional testing in the T2D model with atherosclerosis. ApoE-/- mice were utilized due to their ability to develop severe vascular disease and acquire the elements of T2D after 16 weeks of a high fat diet (HFD). KGYY6 was administered by IV tail injection at a dose of 1mg/kg and compared to controls given vehicle only. Aortic en-Face analysis with Sudan IV stain demonstrated significant reduction in plaque in KGYY6 treated mice. Decreases in plaque area and changes in both smooth muscle and collagen measurement were additionally noted by sequential 5um aortic cross sections from the aortic valve leaflets into the ascending aorta. In-vitro analysis of CD3+CD4+CD40+ splenic cells demonstrated a reduction in inflammatory cytokine expression in response to KGYY6 treatment, specifically IL2, IFNγ, and IL17, which are potent cytokines in atherosclerosis. Western blot analysis performed on adipose and muscle tissue demonstrated an increased expression of the glucose transport protein GLUT 4, with corresponding glucose tolerance testing demonstrating increased glucose tolerance and improved insulin sensitivity with lowered plasma insulin level. KGYY6 normalizes the aberrant CD40/CD154 interaction, reducing inflammatory cytokines and regulating glucose, all together resulting in abrogating atherosclerosis.

Autoimmunity treatments, fruitfully pioneered in mouse models, can be disappointing or result in immunosuppression and opportunistic infections in translational trials. Many possible reasons exist, but one major, overlooked reason may be the treatment timing in relation to circadian oscillations of the immune system. Mice and humans both have immunological circadian clocks and experience the same circulatory oscillations of immune cells with regards to their sleep/wake phases, but have opposite sleep/wake phases with regard to the daylight cycle. Therefore, researchers mainly study mice and potential autoimmunity treatments during the murine sleep/rest phase, which is when pro-inflammatory mediators and more adaptive immune cells are prevalent in the circulation. In translational trials, however, treatment administration happens primarily during a patient’s wake/activity phase, during the daytime, which is when more local and acute immune responses are active in the circulation. Therefore, we believe that the most opportune window for autoimmunity treatment may be missed in translational trials. Shifting the timing, and adjusting dosing to target only immune cells that are active at that time, may result in higher success with minimized immunosuppression or toxicities.