John Lyle Dzuris

Polyclonal Rabbit Antithymocyte Globulin Exhibits Consistent Immunosuppressive Capabilities Beyond Cell Depletion

Background. Polyclonal antithymocyte globulins (ATGs) are used clinically to prevent and treat acute allograft rejection and are believed to modulate the immune response primarily by depleting T cells. However, nondepleting mechanisms may also be important mediators of graft survival. In the present study, 14 lots of thymoglobulin (rabbit ATG) were analyzed and compared for nondepletive immunomodulatory activities in vitro. Methods. Coincubation of human peripheral blood mononuclear cells with thymoglobulin induces CD4+CD25highFoxp3+ regulatory T cells, which were evaluated for consistent ability to suppress T-cell activation in mixed lymphocyte reactions. The consistency of CD2, CD3, CD11a, and CD45 antigen specificities in thymoglobulin was determined using flow cytometry to measure inhibition of fluorescent monoclonal antibody binding to Jurkat T cells. A transwell chemotaxis assay was established and used to evaluate ATG-mediated inhibition of stromal cell-derived factor (SDF)-1α-driven Jurkat T-cell migration. Results. Physiologic levels of thymoglobulin produced nondepletive immunomodulatory activities, which were consistent from batch to batch. All lots of thymoglobulin induced functionally immunosuppressive regulatory T cells and inhibited monoclonal antibody binding to key T-cell surface antigens. In addition, these studies provide the first demonstration that thymoglobulin effectively inhibits CXCR4/SDF-1α-driven T-cell chemotaxis. Conclusions. This novel, systematic in vitro analysis of 14 different manufactured lots of thymoglobulin demonstrates the overall consistency of this product and provides further insights into nondepletive mechanisms by which thymoglobulin may generate durable immunoregulation and allograft survival.

Immunosuppressive activity of a novel peptide analog of alpha-melanocyte stimulating hormone (α-MSH) in experimental autoimmune uveitis

Autoimmune uveitis is an inflammatory disorder of the eye that can lead to pain and vision loss. Steroids and immunosuppressive drugs are currently the only therapeutics for uveitis and have serious ocular and systemic toxicities. Therefore, safer alternative therapeutics are desired. Alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide that suppresses effector T cell functions, induces regulatory T cells and has beneficial effects in certain autoimmune and transplant models. A novel d-amino acid peptide analog of native α-MSH (dRI-α-MSH) was produced that was protected from protease digestion and had increased selectivity for the melanocortin-1 receptor. Systemic delivery of the dRI-α-MSH analog dramatically suppressed disease progression and retained retinal architecture in the experimental autoimmune uveitis (EAU) model. Local delivery by periorbital injection was equally effective. Importantly, treatment with the novel dRI-α-MSH analog suppressed uveitis with a similar magnitude to the corticosteroid, dexamethasone. Data indicate that the novel dRI-α-MSH analogs show anti-inflammatory activities and have potential therapeutic use in uveitis and other autoimmune diseases.

A retro‐inverso α‐melanocyte stimulating hormone analog with MC1R‐binding selectivity

α‐melanocyte stimulating hormone (α‐MSH) is a tridecapeptide fragment of pro‐opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti‐inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro‐inverso (RI) sequence of α‐MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI‐α‐MSH exhibited a diminished binding affinity for MC1R compared to α‐MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non‐natural amino acids and substitution of the C‐terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical Ki as α‐MSH at MC1R and a lower EC50 in Mel‐624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L‐form α‐MSH, suggesting a significantly altered interaction with the MC1R. Copyright

Selected Mechanistic Studies and Future Directions for Thymoglobulin

A murine anti-thymocyte globulin, which was made in rabbits using the same manufacturing protocol used in production of Thymoglobulin (Genzyme Transplant, Cambridge, MA), was used to explore the biology and mechanism of action of Thymoglobulin. These preclinical studies suggest that determining optimal dosing and timing of Thymoglobulin can contribute to maximizing protection against allograft rejection. The murine anti-thymocyte globulin has a demonstrated ability to induce cells in vitro with a T-regulatory phenotype and was also shown to be effective in vivo in protecting mice against acute GVHD and in reversing overt diabetes in nonobese diabetic mice. The mechanistic rationale for Thymoglobulin as a therapeutic agent for the treatment of systemic lupus erythematosus, multiple myeloma, and myelodysplastic syndrome was also explored.