The Human Leukocyte Antigen-DPB1 Degree of Compatibility Is Determined by Its Expression Level and Mismatch Permissiveness: A German Multicenter Analysis

T-cell epitope matching according to the TCE3 algorithm classifies HLA-DPB1 mismatches in permissive and non-permissive. This classification has been shown to be predictive for mortality and acute GvHD (aGvHD) events in large international cohorts. We retrospectively genotyped HLA-DPB1 in 3523 patients transplanted in Germany between 2000 and 2014 and in their unrelated donors using an Illumina amplicon-NGS based assay. Aim of the study was to evaluate DP-compatibility beyond the established TCE3 algorithm by assessing the combined effect of several DP-mismatch parameters on post-transplant outcome. We implemented an extended DP-mismatch assessment model where TCE3, DP allotype expression with respect to rs9277534, mismatch vector and number of mismatches were conjointly taken into consideration. In this model, non-permissive HLA-DPB1 mismatches showed significantly increased aGvHD risk if they were accompanied by two HLA-DPB1 mismatches in GvH direction (HR: 1.46) or one mismatched highly expressed patient allotype (HR: 1.53). As previously reported, non-permissive HLA-DPB1 mismatches associated with a significantly higher risk of aGvHD and non-relapse mortality (HR 1.36 and 1.21, respectively), which in turn translated into worse GvHD and relapse free survival (HR 1.13). Effects on GvL and GvHD appeared strongest in GvH-directed non-permissive mismatches. Our study results support the consideration of additional HLA-DPB1 mismatch parameters along with the established TCE3 matching algorithm for refinement of future donor selection. In particular, our findings suggest that DP non-permissiveness associated with two HLA-DPB1 mismatches or at least on highly expressed mismatched patient allotype should be avoided

Enhanced ADCC activity of affinity maturated and Fc-engineered mini-antibodies directed against the AML stem cell antigen CD96.

CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential

Toxicity after chimeric antigen receptor T-cell therapy (Jun, 10.1007/s00108-021-01046-5, 2021)

Background. The transfusion of chimeric antigen receptor (CAR) T-cells has become established as a new treatment option in oncology; however, this is regularly associated with immune-mediated side effects, which can also run a severe course and necessitate a specific treatment and intensive medical treatment. Material and methods. A literature review was carried out on CAR T-cell therapy, toxicities and the management of side effects. Results. The cytokine release syndrome (CRS) and the immune effector cell-associated neurotoxicity syndrome (ICANS) regularly occur shortly after CAR T-cell treatment. The symptoms of CRS can range from mild flu-like symptoms to multiorgan failure. In addition to mild symptoms, such as disorientation and aphasia, ICANS can also lead to convulsive seizures and brain edema. The management of CRS and ICANS is based on the severity according to the grading of the American Society for Transplantation and Cellular Therapy (ASTCT). Tocilizumab and corticosteroids are recommended for CRS and corticosteroids are used for ICANS. In the further course persisting hypogammaglobulinemia and cytopenia are frequent even months after the initial treatment and promote infections even months after CAR T-cell therapy. Discussion. Potentially severe complications regularly occur after CAR T-cell therapy. An interdisciplinary cooperation between intensive care physicians, hematologists, neurologists and specialists in other disciplines is of decisive importance for the optimal care of patients after CAR T-cell therapy

Sequence alignment of the VH and VL domains of mutated CD96-scFv library.

<p>After the fifth round of screening, 30 clones were randomly selected and sequences were compared to same number of clones from the original library. The CDR regions are defined according the Kabat numbering scheme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042426#pone.0042426-Kabat1" target="_blank">[53]</a>. <b>A</b>) Light chains from original library and after the fifth round of screening. <b>B</b>) Heavy chains from original library and after the fifth round of screening.</p

Comparison of ADCC activity at saturating antibody concentrations.

<p>ADCC experiments with HSB-2 cells were performed using mononuclear cells as effector cells. (1, <b>▪</b>): CD96-S32F-scFv-IgG1-Fc-eng, (2, <b>▪</b>): CD96-wt-scFv-IgG1-Fc-eng, (3, <b>▪</b>): CD96-wt-scFv-IgG1-Fc, (4, <b>□</b>): TH-111. E/T ratio (80∶1). Antibody concentration 200 µg/ml. Data are presented as mean values +/− SEM of 5 independent experiments. (#) extend of lysis significantly different from lysis obtained with the murine TH-111 antibody. (*) extend of lysis significantly different between the two mini-antibodies (p<0.05).</p