SBIR-STTR Award

Human stroke results in multi-organ systemic disease, rather than in solely a brain lesion. While patients may survive the initial brain insult, many succumb to CNS injury-induced immunodepression and fatal infection. Using a murine middle cerebral artery occlusion (MCAO) model of stroke, we found that cerebral ischemic injury leads to bi-phasic consequences for the immune system: early systemic activation of the peripheral immune system, including inflammatory myelin-reactive T cells, followed by a delayed phase of immunopathology associated with massive and progressive splenic apoptosis and loss or re-distribution of remaining immune cells. While this second phase may not directly influence infarct size, exhaustion of immunocompetent cells results in an inability to respond to antigenic challenges. Our overall hypothesis is that peripheral T lymphocytes are major contributors to brain damage after ischemia and to the systemic immunopathology that evolves in tandem. If so, therapy that can block early immune system activation and modulate immunocytes specific for brain antigens would be highly beneficial to stroke survivors. To address this hypothesis, we will utilize our newly developed "designer" recombinant T cell receptor ligand (RTL) platform technology to selectively modulate the inflammatory function of individual myelin-reactive T cell specificities. To test this hypothesis, we propose to 1) determine if RTL551 modulation of the inflammatory activity of brain antigen sensitized T cells results in reduced lymphocyte infiltration into brain and improved outcomes after MCAO in animals of both sexes. Milestone: RTL551 is protective in both sexes and inhibits translocation of GFP+ T cells into brain after MCAO; 2) determine if RTL551 improves peripheral immune dysfunction after MCAO in male and female mice. Milestone: RTL551 will improve splenocyte numbers and suppress cytokine-induced splenic apoptosis and immunosuppression after MCAO; and 3) develop a stroke treatment model in humanized DR2 transgenic mice and evaluate treatment effects and requirements for MHC and myelin specificity of RTL1000, an HLA-DR2/MOG-35-55 construct currently being tested in Phase I safety studies in patients with multiple sclerosis. Milestone: RTL1000 will treat MCAO and prevent immunosuppression in DR2 Tg mice. If effective in murine stroke, the RTL approach could be applied rapidly to human stroke patients. Our RTL construct for MS, RTL1000, is comprised of the HLA-DR2 moiety linked to the hMOG- 35-55 peptide, and currently is in FDA approved Phase I safety studies. The RTL1000 could be tested in DR2+ stroke patients relatively soon if the Phase I trial shows this construct to be safe.

Public Health Relevance:
Human stroke results in multi-organ systemic disease, rather than in solely a brain lesion. While patients may survive the initial brain insult, many succumb to stroke induced immunodepression caused by an initial over reaction of the immune system. We seek to develop a stroke therapy that would block the initial immune system over activation and thus be highly beneficial to stroke survivors.

Public Health Relevance Statement:
Human stroke results in multi-organ systemic disease, rather than in solely a brain lesion. While patients may survive the initial brain insult, many succumb to stroke induced immunodepression caused by an initial over reaction of the immune system. We seek to develop a stroke therapy that would block the initial immune system over activation and thus be highly beneficial to stroke survivors.

NIH Spending Category:
Aging; Autoimmune Disease; Brain Disorders; Multiple Sclerosis; Neurodegenerative; Neurosciences; Stroke

Project Terms:
APO2; ATGN; Acquired brain injury; Address; After Care; After-Treatment; Aftercare; Animals; Anti-Inflammatories; Anti-Inflammatory Agents; Anti-inflammatory; Antigens; Antiinflammatories; Antiinflammatory Agents; Apoplexy; Apoptosis; Apoptosis Pathway; Area; Assay; B blood cells; B-Cells; B-Lymphocytes; Behavioral; Bioassay; Biologic Assays; Biological Assay; Blood - brain barrier anatomy; Blood Neutrophil; Blood Poisoning; Blood Polymorphonuclear Neutrophil; Blood Segmented Neutrophil; Blood-Brain Barrier; Brain; Brain Injuries; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; CNS Injury; CSIF; CSIF-10; Cell Count; Cell Death; Cell Death, Programmed; Cell Number; Cells; Central Nervous System Injury; Cerebral Ischemia; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular accident; Cerebrum; Clinical Trials; Clinical Trials, Phase I; Clinical Trials, Phase II; Clinical Trials, Unspecified; Cohort Studies; Complex; Concurrent Studies; Contracting Opportunities; Contracts; Crossmatching, Tissue; Cytokine Synthesis Inhibitory Factor; Cytokine formation-inhibiting factor (mouse clone F115 protein moiety reduced); DR1; DR1 gene; DR4; Data; Dose; Drugs; EAE; Early-Stage Clinical Trials; Encephalomyelitis, Allergic; Encephalon; Encephalons; Experimental Allergic Encephalitis; Experimental Allergic Encephalomyelitis; Experimental Autoimmune Encephalitis; Experimental Autoimmune Encephalomyelitis; FDA approved; Family; Female; Frequencies (time pattern); Frequency; Funding; Gonadal Steroid Hormones; HLA-DR2; HLA-DR2 Antigen; Hemato-Encephalic Barrier; Heterophil Granulocyte; Histocompatibility Testing; Hortega cell; Hour; Human; Human, General; IL-10; IL-13; IL10; IL10A; IL13; Immune; Immune Diseases; Immune Disorders; Immune Dysfunction; Immune System Diseases; Immune System Disorder; Immune system; Immunization; Immunocompetent; Immunologic Diseases; Immunologic Stimulation; Immunological Diseases; Immunological Stimulation; Immunostimulation; Immunosuppression Effect; Immunosuppressions (Physiology); Immunosuppressive Effect; Individual; Infarction; Infection; Infiltration; Inflammatory; Injury; Injury of central nervous system; Intellectual Property; Interleukin 10 Precursor; Interleukin-10; Interleukin-13; Intervention; Intervention Strategies; Investigational New Drug Application; Ischemia; Lead; Legal patent; Lesion; Licensing; Ligands; Link; Lymphocyte; Lymphocytic; MGC9365; MHC Receptor; MS (Multiple Sclerosis); Major Histocompatibility Complex Receptor; Mammals, Mice; Man (Taxonomy); Man, Modern; Marrow Neutrophil; Mediating; Medication; Mice; Microglia; Middle Cerebral Artery Occlusion; Modeling; Multiple Sclerosis; Murine; Mus; Myelin; NC2-Beta; Natural immunosuppression; Nervous System, Brain; Neutrophilic Granulocyte; Neutrophilic Leukocyte; Organ; Outcome; Patents; Pathology; Patients; Pb element; Peptides; Peripheral; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Phase 1 Clinical Trials; Phase 2 Clinical Trials; Phase I Clinical Trials; Phase I Study; Phase II Clinical Trials; Play; Polymorph; Polymorphonuclear Cell; Polymorphonuclear Leukocytes; Polymorphonuclear Neutrophils; Process; Production; Programs (PT); Programs [Publication Type]; Property; Property Rights; Property, LOINC Axis 2; Reaction; Receptor Activation; Receptor Protein; Receptors, Antigen, T-Cell; Recombinants; Reporting; Research; Reticuloendothelial System, Spleen; SCID; SCID Mice; SUBGP; Safety; Sclerosis, Disseminated; Sensitization, Immunologic; Sensitization, Immunological; Septicemia; Series; Severe Combined Immunodeficient Mice; Sex Hormones; Sex Steroid Hormones; Site; Specificity; Spleen; Splenocyte; Stroke; Subgroup; Survivors; Systemic disease; T Cell Specificity; T-Cell Immunologic Specificity; T-Cell Receptor; T-Cells; T-Lymphocyte; TNFRSF10A; TNFRSF10A gene; TRAILR-1; TRAILR1; Technology; Testing; Thymus-Dependent Lymphocytes; Tissue Crossmatchings; Tissue Typing; Toxic effect; Toxicities; Transgenic Mice; Vascular Accident, Brain; Work; autoimmune encephalomyelitis; base; body system, allergic/immunologic; brain attack; brain damage; brain lesion (from injury); central nervous system injury; cerebral vascular accident; clinical investigation; cytokine; drug development; drug/agent; exhaustion; experiment; experimental research; experimental study; gitter cell; gonadal steroids; heavy metal Pb; heavy metal lead; histocompatibility typing; immune depression; immunodepression; immunogen; immunopathology; immunosuppression; improved; in vivo; infarct; inflammatory modulation; insular sclerosis; interventional strategy; lymph cell; macrophage; male; manufacturing process; mesoglia; microglial cell; microgliocyte; necrocytosis; neutrophil; novel; organ system, allergic/immunologic; perivascular glial cell; phase 1 study; phase 1 trial; phase 2 study; phase 2 trial; phase I trial; phase II trial; post stroke; poststroke; pre-clinical; preclinical; preclinical study; prevent; preventing; programs; protocol, phase I; protocol, phase II; public health relevance; receptor; research study; safety study; sensitizing antigen; septicaemia; severe combined immune deficiency; sex; sex steroid; stroke; stroke therapy; study, phase II; thymus derived lymphocyte; trafficking; treatment effect; treatment strategy

This application is a Phase II STTR to further the study of a novel therapeutic intervention for ischemic stroke. Phase I investigated the mechanism of protection against ischemic stroke provided by the novel ""designer"" therapy, recombinant T cell receptor ligand (RTL), a selective modulator of the inflammatory function of myelin- reactive T cell specificities. Our findings suggest that RTL protects against ischemia-induced brain injury only if the RTL contains a neuroantigen peptide and the matched Class II MHC moiety of the recipient. We demonstrate that RTL containing mouse MHC coupled to mouse myelin peptide is effective in reducing ischemic damage in mice (RTL551). Similarly, RTL1000 is a human MHC coupled to a human myelin peptide. RTL1000 is not effective in wild-type mice, but provides significant protection in 'humanized'HLA-DR2 mice that selectively express only this human MHC class II allele. As such, this treatment represents a new therapy targeting inflammatory immune cell populations specifically triggered by brain injury. This post-ischemic therapeutic approach is promising because it specifically suppresses brain targeted immune responses while leaving other portions of the immune system intact. The inflammatory response after ischemia in the brain has been studied extensively and modulation of this inflammatory response (immunotherapy) improves outcome in experimental models. However, these therapies have not translated successfully to patients in clinical trials. There is now support for the idea that stroke is a multi-organ systemic disease with interactions between the peripheral immune system and the injured and recovering brain. Many patients who survive the initial injury to the brain suffer CIDS, CNS injury-induced immunosuppression, resulting in fatal infection. Therefore, immunotherapy targeted towards minimizing brain damage following ischemia must minimize CNS inflammation while at the same time not exacerbating CIDS. This is a significant hurdle that our group is well poised to study. Our group has pioneered the study of cerebral ischemia-induced changes to peripheral immune organs using the middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke. This application will test three specific hypotheses 1) that RTL1000 has an extended therapeutic window and 2) that RTL1000 retains its protective potential in middle aged and old aged mice. Finally, 3) that RTL1000 is an effective therapy when combined with tissue plasminogen activator (tPA). Successful completion of the current proposal will determine the efficacy of RTL1000 in treatment of ischemic stroke, and its ability to be used in combination with tPA, ultimately providing a roadmap for designing pilot clinical trials. Upon completion of this Phase II STTR proposal, in partnership with Virogenomics, we will proceed to a Phase I safety trial in Stroke patients.

Public Health Relevance:
Stroke is one of the leading causes of death and the leading cause of disability in the United States. Unfortunately, there is currently only a single drug available to improve outcome following ischemic stroke, tissue plasminogen activator (tPA), with a limited therapeutic window of >4.5 hrs. The proposed studies will demonstrate the efficacy of RTL1000 in treatment of ischemic stroke, and its ability to be used in combination with tPA.