B cell repertoires in HLA-sensitized kidney transplant candidates undergoing desensitization therapy.

BACKGROUND: Kidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy. METHODS: We developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA. RESULTS: Dominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards naïve B cells. CONCLUSIONS: Relative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative abundance of memory and naïve B cell compartments. Candidates that responded to therapy experienced similar changes to those that did not respond. Further studies are required to understand differences between these two groups of highly sensitized kidney transplant candidates.

Thiolytically cleavable dithiobenzyl urethane-linked polymer-protein conjugates as macromolecular prodrugs: reversible PEGylation of proteins.

New thiolytically cleavable dithiobenzyl (DTB) urethane-linked conjugates of methoxypoly(ethylene glycol) (mPEG) and a model protein, lysozyme, were prepared and thoroughly characterized. In contrast to our earlier communication [Zalipsky, et al. (1999) Bioconjugate Chem. 10, 703], in the current study we used a more sterically hindered form of para-DTB urethane linkage containing a methyl group on the alpha-carbon to the disulfide moiety. The new reagent for covalent attachment of mPEG-DTB to amino groups of proteins was synthesized via a seven-step process. As a result of PEG conjugation, the lysozyme was shown to completely lose its bacterial cell wall-lysing activity. However, activity was almost fully restored upon cysteine-mediated cleavage of the PEG component. The conjugate decomposition process was monitored by RP-HPLC and by ion spray LC-MS, which showed the formation of the p-mercaptobenzyl urethane-lysozyme intermediate, and ultimately its conversion to the unmodified lysozyme as the sole protein component. Pharmacokinetic evaluation of (125)I-labeled cleavable and noncleavable PEG-lysozyme given intravenously in rats revealed similar clearance patterns; both cleared in a significantly slower manner compared to that of the native protein. However, subcutaneous administration of the same conjugates showed a significantly larger AUC of the cleavable conjugate, indicating that some cleavage of the DTB urethane may have occurred. Although the DTB-linked PEG-lysozyme exhibited almost the same plasma clearance as the noncleavable counterpart, hinting that methyl-DTB linkage might be stable in the bloodstream, SDS-PAGE examination of the conjugate incubated in plasma showed decomposition at least partially mediated by albumin. These results suggest the potential of PEG-DTB-proteins as macromolecular prodrugs capable of generating fully active native proteins under in vivo conditions.