Objective
The major objective of this application is to demonstrate the efficacy of a novel combination protein-drug-conjugate (PDC) in enhancing or re-establishing regulatory T cell function in T1D patients. In this study, we will test different formulations of our PDC to select a lead candidate, on which IL-2Rx can, using Charles River, perform pharmacokinetic and toxicity studies. These data will allow us to pursue additional funding to further develop this therapy for restoration of endogenous immune regulation in patients with type 1 diabetes (T1D). This novel therapy was initially developed in the Fathman lab at Stanford where a druggable defect in regulatory T cell (Treg) function was first identified in autoimmune patients. The defect results from a loss in the expression of pSTAT5, a protein in Tregs that controls the transcription (expression) of various genes that give “function” to the Tregs. The defect is downstream of the IL-2 receptor (IL-2R), and is observed in the Tregs of T1D patients but also in Tregs from patients with other autoimmune and inflammatory diseases such as SLE, MS and asthma. We showed that we could activate Tregs and restore normal immune regulation to previously defective Tregs using a protein drug conjugate (PDC) formed by the attachment of a small molecule drug (a neddylation activating enzyme inhibitor; NAEi) onto an IL-2 fusion protein. This “drug” has two separate functions. The first is to bind to and activate the Treg IL-2R. Following activation, the complex of the IL-2R and the receptor bound PDC are internalized into the Treg by receptor mediated endocytosis (the Treg engulfs the entire IL-2R/PDC complex). Once inside the Treg, the small molecule drugs (warheads) attached to the IL-2 fusion protein are released by enzymatic cleavage by cytosolic enzymes. This can be considered a “Trojan Horse” approach to therapy with IL-2 opening the door for delivery of the NAEi payload. The small molecule drugs restore or enhance normal IL-2R signaling by replacing the function of the missing second messenger protein (GRAIL) that was defective in the T1D Tregs. These NAEi drugs are toxic in man when given systemically to treat cancer. But by attaching them to the IL-2 fusion protein, we can deliver them specifically to the Tregs and reduce the amount of the therapeutically effective drug required by over 1000-fold compared to the systemic dose used, thereby obviating the dose related toxicity. We believe that, once developed, this drug could lead to a paradigm shift in therapy of T1D, and allow physicians to block the inflammatory activities that drive tissue destruction in high risk normoglycemic first degree relatives of T1D patients. We have already demonstrated that the mouse version of our “drug” could prevent the development of hyperglycemia in the NOD mouse model of T1D. Thus, the human version of this drug should prevent progression to hyperglycemia in at-risk individuals by re-establishing immune regulation to block the autoimmune destruction of the islet beta cells. This novel drug could also impact how physicians treat established T1D patients who receive pancreas/islet grafts by generating tolerance to those grafts by enhancing the Treg function to block immune response to the transplant, thus allowing restoration of normal insulin production without the need of relatively toxic immuno-suppressive drugs that are currently used in transplant patients.
Background Rationale
Defects in immune regulation play a major role in the pathogenesis of autoimmune diseases including type one diabetes (T1D). Regulatory T cells (Tregs) are central to the normal maintenance of immune homeostasis. Since there are no studies demonstrating a defect in the number of Tregs in T1D disease, we asked if we could identify a detect in the function of T1D Tregs. By studying the downstream signaling from the IL-2 receptor (IL-2R) in low dose IL-2 activated Tregs of T1D patients compared to that of normal controls, we identified a novel druggable defect in the IL-2R driven pJAK1 activation of pSTAT5. pSTAT5 is a protein that controls the expression of the genes required for Treg function. Tregs from normal subjects exhibit a prolonged activation of the pSTAT5 pathway in response to low dose IL-2 due to GRAIL-mediated inhibition of IL-2R desensitization. GRAIL is a ubiquitin E3 ligase that allows prolonged Treg IL-2R signaling for up to 4 hours, enough time for pSTAT5 to activate the genes required for Treg function. GRAIL ubiquitinates the cullin backbone of the SOCS3/cullin5 ring ligase (CRL) in the exact site that need to be occupied by another small molecule called Nedd8, and thus, GRAIL acts as a competitive inhibitor of neddylation. By blocking neddylation, pJAK1 is not degraded and the genes required for Treg function are turned on. Tregs from T1D patients and NOD mice have a defect in GRAIL expression and thus cannot maintain IL-2R signaling, resulting in early loss of pSTAT5 expression and a loss of expression of the genes required for Treg function. This defect, mediated by loss of GRAIL (neddylation of the CRL, degradation of pJAK1, and premature IL-2R desensitization), can be repaired through the use of a small molecule drug called a neddylation activating enzyme inhibitor or NAEi. This drug can block neddylation of the CRL and maintain IL-2R signaling just as GRAIL does. Since GRAIL expression is diminished in NOD mouse Tregs, we were able to treat NOD mice with a combination of a neddylation activating enzyme inhibitor (NAEi) and low dose IL-2 to block pJAK1 degradation, restore pSTAT5 expression, and prevent disease progression. Unfortunately, the NAEi’s are quite toxic when given systemically. We developed a novel protein drug conjugate (PDC) consisting of a fusion protein of mouse IL-2 and thioredoxin, to which 3 molecules of NAEi are attached. The IL-2 allows targeting to and activation of the IL-2R on Tregs. Following ingestion (receptor mediated endocytosis) of the PDC-bound Treg IL-2R, the small molecule NAEi are cleaved from the PDC by intracellular enzymes to release the drugs in exactly the cell that needs to be treated. This targeted drug delivery has allowed us to decrease the amount of therapeutically effective NAEi required by over 1000-fold. In this proposal, we will generate 4 versions of a human PDC, test their effectiveness in restoring function in the Tregs of T1D patients in vitro, and, using the lead candidate, prevent disease in NOD mice and potentially used to develop tolerance to islet transplants in hyperglycemic NOD mice. PK/Tox studies will be performed on the lead candidate (funded by IL-2Rx), and the lead candidate drug will then be available for clinical development
Description of Project
A major cause of autoimmune diseases (including type one diabetes, T1D) is a defect in endogenous regulatory T cell function. Regulatory T cells (Tregs) are the “battlefield” controllers of the immune system. Tregs recognize “self” such that when they are not working properly, and following some unknown inductive event, the immune system attacks and destroys one’s own tissues. Autoimmune diseases are currently treated with toxic and relatively ineffective drugs to attempt to immunosuppress the autoimmune disease. The ability to restore defective endogenous immune regulation would represent a paradigm shift in the therapy of these diseases. The Fathman lab identified a druggable defect in Treg function in Tregs from patients with autoimmune diseases including T1D that can be corrected by combination treatment with two separate "drugs." Several years ago, the lab received JDRF funding (Combination drug therapy to rejuvenate endogenous Tregs; 2-SRA-2016-300-S-B) to study these two drugs in combination to block progression of disease from late-stage islet infiltration with lymphocytes (insulitis) but before overt hyperglycemia in 12-week-old female non-obese diabetic (NOD) mice, the current best animal model of T1D. Although the combination of the two drugs (a cytokine called IL-2 and a small molecule drug called a neddylation activating enzyme inhibitor or NAEi) were successful in blocking the progression to hyperglycemia in these mice, however the NAEi was toxic at therapeutic levels, similar to the toxicity seen in the ongoing human trials using this drug to treat cancer. During the past four years, in collaboration with a small biotech company (IL-2Rx), we have developed a non-toxic protein drug conjugate where the NAEi is bound to an IL-2 fusion protein by enzymatically cleavable linkers that can be only released inside a cell (a Trojan Horse approach to therapy). The IL-2 serves to both target Tregs that constitutively express the high affinity receptor for IL-2 (IL-2R) and activate the IL-2R on the Tregs. Following activation, the IL-2R bearing the attached protein drug conjugate is internalized into the Treg by activated-receptor-mediated endocytosis. Following internalization of the protein drug IL-2R complex, the small molecule drugs are released but only inside the targeted Treg allowing 1000-fold less of the NAEi to be used to achieve a significant clinical effect in this model, blocking progression to hyperglycemia for over 3 months following a single 5-day treatment regimen, and obviating the dose related toxicities seen in the combination therapy. In collaboration with IL-2Rx, we have recently constructed a human homalog of the mouse protein drug conjugate (mPDC) that was successful in treating the NOD mice. This human PDC (hPDC) uses a fusion protein of human IL-2 to which we attach the same small molecule drugs to repair the defect in Treg function. We have previously demonstrated the IL-2R signaling defect in human T1D Tregs and restored their regulatory function with the drug combination. Since human IL-2 activates the mouse IL-2R (but the mouse IL-2 does not activate the human IL-2R) we propose to test four new human protein drug conjugates (2 separate NAEi’s and 2 separate linkers) in NOD mice as well as in human Tregs from T1D and first degree relatives of T1D patients that have the defect in IL-2R signaling to demonstrate restoration of Treg activity which will allow us to choose which hPDC is most effective in restoring Treg function in mice and man as our lead candidate before testing this lead candidate in pharmokinetic and toxicity studies (PK/Tox funded by IL-2Rx). These pre-clinical studies can be completed within two years and will allow us to rapidly move into GMP production of the lead hPDC candidate and proof-of-concept studies in man.
Anticipated Outcome
Regulatory T cells (Tregs) of NOD mice and T1D patients have been shown to exhibit the same defect in regulatory T cell (Treg) IL-2 receptor signaling, loss of prolongation of pSTAT5 expression in response to low dose IL-2. Using the NOD mouse model of T1D, we have already demonstrated the feasibility of using an IL-2 fusion protein drug conjugate (mPDC) to prevent disease progression. In the studies proposed here, we will ask if the human protein drug conjugate (hPDC) homolog will effectively target Tregs and deliver the small molecule drugs (neddylation activating enzyme inhibitors, NAEi’s) specifically to human Tregs (that constitutively express the high affinity receptor for IL-2), and restore the function of defective Tregs in vitro. We will test 4 different candidate hPDC's generated using one of two linker strategies (di-peptide vs glucuronide) and two different NAEi’s (TK924 vs TAS4464) to conjugate to the hPDC, and select the most effective hPDC among the four candidates to develop as our lead candidate for clinical testing. Additionally, as the human IL-2 PDC will also target mouse Tregs, we will ask whether the lead candidate can block progression to hyperglycemia in the NOD mouse model by treating female NOD mice at 12-weeks of age, when mice have advanced insulitis but are still normoglycemic. Once we have identified the hPDC that is most effective in restoring Treg function in T1D patients in vitro, and in blocking disease progression in NOD mice, IL-2Rx will contract PK/TOX studies of the lead candidate, in preparation for proof-of-concept studies in man. A second potential use of the PDC is to enhance immune regulation in an attempt to create tolerance to islet grafts in hyperglycemic NOD mice. Islets from BALB/c mice that express a gene for firefly-luciferase will be transplanted into NOD mice, followed by treatment with the hPDC. Islet survival will be monitored in a non-invasive manner by the injection of luciferin into recipient NOD mice. Donor islets, containing the firefly-luciferase, will enzymatically generate luminescence in the presence of their substrate luciferin. The transplanted islets will bioluminesce (like the light from the firefly) , and using a very sensitive camera, the bioluminescence can be captured and quantified to generate a survival curve for the transplanted islets. We anticipate that islet survival will be prolonged in recipient NOD mice that are treated with the hPDC. These studies will be done in collaboration with Dr. Everett Meyer who routinely does such experiments.
Relevance to T1D
The combination drug therapy described in this proposal targets both pre-hyperglycemic at risk first- degree-relatives (FDR’s) of T1D patients (prevention) and established T1D patients (tolerance for islet transplants). Because T1D is an autoimmune disease that only manifests clinically as hyperglycemia following major destruction of the insulin producing pancreatic islet beta cells, treatment requires a two-pronged approach. One is to restore insulin secreting cells by transplantation to treat established T1D disease. That requires generating “tolerance” to the implanted organ or cells. Our novel therapy may help establish such transplantation tolerance in the absence of any drug-related toxicity by simply activating and enhancing regulatory T cell activity. The second opportunity is to prevent progression to hyperglycemia in pre-diabetic individuals at high risk. To do this, we must identify the subjects at risk of developing T1D. Currently, it is possible to identify “double antibody positive” first degree relatives (AA+ FDRs) of T1D patients who are at high risk of becoming hyperglycemic (as is being done by TrialNet). These subjects have an active ongoing autoimmune disease that can be “treated” by our protein drug conjugate to block progression to beta cell destruction. These are the two potential clinical targets of this proposal. The first is to establish transplantation tolerance to islet cell transplantation in established T1D subjects and, the second, to block progression to hyperglycemia in high-risk T1D FDRs. Current therapies for autoimmunity rely on relatively ineffective and, too often, toxic therapies to “treat” the autoimmune destructive inflammation. We are proposing an important paradigm shift in therapy to prevent progression to overt hyperglycemia by restoring endogenous immune regulation to stop the inflammatory disease in the islets. Since T1D (like other autoimmune diseases) reflects a loss of normal immunoregulation, restoration of the normal homeostatic regulatory function of the regulatory T cells would be a major advance in therapy of autoimmunity. Not only could this novel protein drug conjugate therapy potentially block progression to hyperglycemia in at-risk FDRs, it could have an enormous therapeutic potential to treat other autoimmune diseases, like systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis, to name a few. This would represent a major shift in the paradigm of therapy to treat autoimmune diseases, restoration of endogenous immunoregulation instead of trying to block the multitude of immune driven inflammatory events causing these diseases.