Batch-to-Batch Variation and Patient Heterogeneity in Thymoglobulin Binding and Specificity: One Size Does Not Fit...

Background: Thymoglobulin is used to prevent allograft rejection and is being explored at low doses as intervention immunotherapy in type 1 diabetes. Thymoglobulin consists of a diverse pool of rabbit antibodies directed against many different targets on human thymocytes that can also be expressed by other leukocytes. Since Thymoglobulin is generated by injecting rabbits with human thymocytes, this conceivably leads to differences between Thymoglobulin batches. Methods: We compared different batches for antibody composition and variation between individuals in binding to PBMC and T cell subsets, and induction of cytokines. Four different batches of Thymoglobulin were directly conjugated with Alexa-Fluor 647. Blood was collected from five healthy donors, and PBMCs were isolated and stained with Thymoglobulin followed or preceded by a panel of fluorescent antibodies to identify PBMC and T cell subsets. In addition, whole blood was incubated with unlabeled Thymoglobulin to measure cytokine induction. Results: Cluster analysis of flow cytometry data shows that Thymoglobulin bound to all PBMC subpopulations including regulatory T cells. However, Thymoglobulin binding was highly variable between donors and to a lesser extent between batches. Cytokines related to cytokine release syndrome were highly, but variably, increased by all Thymoglobulin batches, with strong differences between donors and moderate differences between batches. Discussion: The variation in Thymoglobulin binding and action between donors regarding PBMC recognition and cytokine response may underlie the different clinical responses to Thymoglobulin therapy and require personalized dose adjustment to maximize efficacy and minimize adverse side effects.