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"To our knowledge," the authors write, "our results provide the first indications that a short course of ATG given alone can restore self-tolerance...a facet that has been previously ascribed to anti-CD3." In light of its immunoregulatory potential, they support further studies of ATG, saying that the therapy "might be applicable not only to type 1 diabetes but also to other diseases associated with dysregulated immune responses."
The research, supported in part by grants from JDRF, was led by Mark Atkinson and is published in a recent issue of the journal Diabetes.
A closer look at ATG
Antibodies, which are proteins with the ability to bind specific cells and molecules of the immune system, represent an important therapeutic approach in type 1 diabetes. Monoclonal antibodies like anti-CD3, for example, have been shown to reverse or arrest the progression of diabetes in both mice and humans with the disease. (JDRF industry partners MacroGenics and Tolerx, in conjunction with Eli Lilly and GlaxoSmithKline, respectively, are currently developing and commercializing anti-CD3 antibodies, which have proven effective in clinical trials at slowing disease progression in the newly diagnosed.)
But while monoclonal antibodies may have taken center stage, researchers have also been using other antibody-based treatments to address type 1 diabetes-including anti-lymphocyte serum, of which ATG is one form. In studies conducted more than 30 years ago, anti-lymphocyte serum reversed type 1 diabetes in rats, and more recently, did so in diabetic mice. An FDA-approved form of anti-lymphocyte serum that is closely related to the ATG used in Dr. Atkinson's studies of mice, and widely known as Thymoglobulin, has long been known to deplete T cells in vivo and is used in a variety of therapeutic settings in humans to control a person's immune response. However, much of the previous scientific research on anti-lymphocyte serum did not account for the role of regulatory T cells, which have recently been identified as important cellular players whose immunosuppressive potential might be harnessed to treat autoimmune diseases.
Given the therapeutic potential of ATG, but a limited and incomplete understanding of its actions, particularly related to regulatory T cells, Dr. Atkinson and colleagues performed a number of in vivo and in vitro mouse experiments aimed at defining the physiologic, immunologic, and metabolic activities of this agent. Their key interests were to determine how ATG modulates the autoimmune response against beta cells and whether it delays or reverses type 1 diabetes at the various stages of disease.
What exactly is anti-lymphocyte serum?
A comparison helps: If monoclonal antibodies represent the specialist approach-one antibody, one target-then anti-lymphocyte serum represents the broader, generalist approach-many antibodies, many targets. In the case of ATG used in Dr. Atkinson's studies, the therapeutic is made up of a diverse repertoire of antibodies that are generated by injecting rabbits with immature T cells (thymocytes) extracted from the thymus glands of mice-the rabbits respond to the mouse antigens by forming anti-mouse thymocyte globulin (or ATG, with globulin being another name for antibodies). Blood serum samples from the rabbits thus contain a range of anti-lymphocyte antibodies that will bind to T cells and other immune cells of the exact specificity when injected into the mice in the experiments. The researchers conducted their tests in nonobese diabetic mice, which are characterized by the eventual spontaneous development of type 1 diabetes.
Key findings
Most striking, ATG was shown to delay the development of type 1 diabetes in a time-dependent manner. While the mice injected with ATG at 4 and 8 weeks of age did not show a significant delay in disease onset compared with control mice, mice injected at 12 weeks of age remained disease-free for significantly longer times. At 30 weeks of age, 89% of the mice treated with ATG at 12 weeks had blood glucose levels within the normal range, whereas only 22% of the control mice remained "normoglycemic." (Mice 12 weeks of age are considered to be in the late pre-diabetic phase.)
Treatment with ATG was also shown to reverse type 1 diabetes at disease onset-defined as the overt onset of hyperglycemia. Of the 7 mice injected with ATG at this time, 4 showed a significant disease reversal, their blood glucose levels dropping rapidly from an average of about 400 mg/dL to about 150 mg/dL one week later, with a sustained benefit up to the end of the six-week monitoring period. In contrast, none of the control mice, who were not given ATG, showed any sign of disease reversal.
Further investigations pointed to the regulatory T cells as mediating these benefits. When given to mice at 12 weeks of age, ATG reversed insulitis, an inflammation of the pancreatic islets caused by an infiltration of lymphocytes, which can destroy the insulin-producing beta cells. Not only were significantly lower levels of T cell infiltration observed in these mice compared with the control mice, but a less severe form of insulitis developed over time. This suggests, according the authors, that "above and beyond the initial [T cell] depletion afforded by ATG, the agent may induce protective mechanisms attenuating migration of cells to pancreatic islets." Supporting this further were the findings (in ATG mice) of improved response to glucose and a rapidly increased frequency of antigen-presenting cells in the spleen and pancreatic lymph nodes.
The researchers were also able to show that ATG therapy directly and dramatically increased the frequency and functional activity of the regulatory T cells-the latter via the suppression of T effector cells, which are the cells that target and destroy beta cells in type 1 diabetes.
The one caveat, however, is that because ATG delayed disease only at specific stages, there is a time-dependent window of opportunity for delaying or reversing type 1 diabetes with the use of this drug, based on its capacity to enhance the activity of regulatory T cells.
From bench to bedside
Dr. Atkinson and other researchers at the University of Florida, including Dr. Michael Haller, continue to study how ATG induces immunoregulation in mice. They are also moving this safe and promising type 1 treatment, which Dr. Atkinson said "seems to work both alone and in combination with other drugs," into clinical trials.
Their findings support a recent human trial of ATG that was independently initiated by Steve Gitelman, M.D., at the University of California, San Francisco, in affiliation with the Immune Tolerance Network, which is supported by JDRF. The trial aims to determine whether ATG treatment can halt the progression of newly diagnosed type 1 diabetes when given within six weeks of disease diagnosis. Dr. Gitelman noted that, "We are very hopeful that we can extend the findings of Dr. Atkinson and colleagues from mouse to man, and alter the natural course of type 1 diabetes by turning off the destructive autoimmune response with only a brief treatment with ATG. We need to determine if this approach will be even more successful than the initial clinical trials with the anti-CD3 monoclonal antibody." Further information about this trial can be found at www.clinicaltrials.gov by entering the search terms "Gitelman and START."
Describing research not yet published, Dr. Atkinson and Dr. Haller also recently observed that the combination of ATG and the cancer drug Granulocyte Colony-Stimulating Factor (G-CSF), when given to mice with recent-onset disease, will essentially reverse hyperglycemia in nearly all treated animals. "In almost every mouse receiving those two drugs," Dr. Atkinson said, "their diabetes reverses, and it's permanent."
Combination therapies do, however, face practical challenges in terms of their implementation, and as a first step in the process, Dr. Haller started a small trial of G-CSF in recent-onset type 1 patients (an effort also supported by JDRF; for more information, visit www.clinicaltrials.gov and enter the search terms "Haller and G-CSF"). In June, the two research groups (University of Florida and the University of California, San Francisco) will meet to discuss the plans for a human trial using the two drugs in combination.
