Proteogenomic-based discovery of minor histocompatibility antigens with suitable features for immunotherapy of hematologic cancers.

Pre-clinical studies have shown that injection of allogeneic T cells primed against a single minor histocompatibility antigen (MiHA) could cure hematologic cancers (HC) without causing any toxicity to the host. However, translation of this approach in humans has been hampered by the paucity of molecularly defined human MiHAs. Using a novel proteogenomic approach, we have analyzed cells from 13 volunteers and discovered a vast repertoire of MiHAs presented by the most common HLA haplotype in European Americans: HLA-A*02:01;B*44:03. Notably, out of >6000 MiHAs, we have identified a set of 39 MiHAs that share optimal features for immunotherapy of HCs. These 'optimal MiHAs' are coded by common alleles of genes that are preferentially expressed in hematopoietic cells. Bioinformatic modeling based on MiHA allelic frequencies showed that the 39 optimal MiHAs would enable MiHA-targeted immunotherapy of practically all HLA-A*02:01;B*44:03 patients. Further extension of this strategy to a few additional HLA haplotypes would allow treatment of almost all patients.

Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems.

Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag. In contrast, the extraction yield of wild type cutinase was 2-3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.

Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix-assisted laser desorption ionization-time-of-flight MS.

SDS/gel capillary electrophoresis (SDS/gel CE) and matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells. The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE). The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes. A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off-line coupling of CZE and MALDI-TOF-MS for the analysis of biotechnological processes is demonstrated.