Purpose Antibody-drug conjugates (ADCs), which are monoclonal antibodies (mAbs) conjugated with highly toxic payloads, achieve high tumor killing efficacy due to the specific delivery of payloads in ...accordance with mAbs' function. On the other hand, the conjugation of payloads often increases the hydrophobicity of mAbs, resulting in reduced stability and increased aggregation. It is considered that mAb aggregates have potential risk for activating Fcgamma receptors (FcgammaRs) on immune cells, and are internalized into cells via FcgammaRs. Based on the mechanism of action of ADCs, the internalization of ADCs into target-negative cells may cause the off-target toxicity. However, the impacts of aggregation on the safety of ADCs including off-target cytotoxicity have been unclear. In this study, we investigated the cytotoxicity of ADC aggregates in target-negative cells. Methods The ADC aggregates were generated by stirring stress or thermal stress. The off-target cytotoxicity of ADC aggregates was evaluated in several target-negative cell lines, and FcgammaR-activation properties of ADC aggregates were characterized using a reporter cell assay. Results Aggregation of ADCs enhanced the off-target cytotoxicity in several target-negative cell lines compared with non-stressed ADCs. Notably, ADC aggregates with FcgammaR-activation properties showed dramatically enhanced cytotoxicity in FcgammaR-expressing cells. The FcgammaR-mediated off-target cytotoxicity of ADC aggregates was reduced by using a FcgammaR-blocking antibody or Fc-engineering for silencing Fc-mediated effector functions. Conclusions These results indicated that FcgammaRs play an important role for internalization of ADC aggregates into non-target cells, and the aggregation of ADCs increases the potential risk for off-target toxicity.
The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney ...xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti‐pig antibodies significantly improved survival in a pig‐to–rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan–T cell depletion and an anti‐CD154–based maintenance regimen. Selective depletion of CD4+ T cells but not CD8+ T cells resulted in long‐term survival (median survival time >400 days vs 6 days). These studies suggested that CD4+ T cells may have a more prominent role in xenograft rejection compared with CD8+ T cells. Although animals that received selective depletion of CD8+ T cells showed signs of early cellular rejection (marked CD4+ infiltrates), animals receiving selective CD4+ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4+ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti‐pig antibody and CD4 depletion resulted in consistent, long‐term xenograft survival.
CD4 T cell depletion is critical to long‐term (beyond 1 year) survival of pig‐to‐nonhuman primate kidney xenografts.
In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely ...separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain β2-microglobulin. A series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.
Issue Information
The FEBS journal,
04/2019, Volume:
286, Issue:
7
Journal Article
Peer reviewed
Heatmap of differentially expressed genes between cetuximab‐resistant and cetuximab‐sensitive patients; image adapted from the Regular Paper by Kwang‐Hyun Cho and co‐authors (pp. 1305–1318).
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