The in vivo testing of biologics, such as monoclonal antibodies, is a regulatory requirement with the cynomolgus monkey being the most commonly used species. Whilst this monkey is used in toxicity studies for a range of drugs, it has poor predictivity for the potential of antibody-induced cytokine release in humans.
The technical challenge presented is to develop in vitro assays capable of predicting in vivo toxicity for a wide range of therapeutic mAb. Importantly, the assays must reflect the patient-to-patient variation in responses observed in mAb trials and reporter assays to date and relate this to the level of toxicity seen during treatment. Using peripheral blood cells (PBC) from healthy donors, Professor Martin Glennie and his team at University of Southampton will develop a panel of assays capable of monitoring most therapeutic mAb and where possible, validate these in on-going clinical trials. Finally, they will reproduce the optimised assays using NHP PBC to evaluate whether there is concordance between human in vitro models and comparable NHP assessments, thereby informing the need for in vivo preclinical testing.
Assay Evaluation: The team will present mAb to PBC from anonymous donors in the three settings: 1) soluble 2) soluble cross linked and 3) platebound.
Considerable evidence points to FcγR polymorphism playing a role in controlling the efficacy of mAb therapy and this is a likely candidate for toxicity. In addition to polymorphism, it is now well established that gene copy number variation (CNV) is a common source of inter‐individual variation. These will be further studied and characterised using the PBCs from this Challenge.
Full details about this CRACK IT Challenge can be found on the CRACK IT website.
The aim of this Challenge was to develop robust assays that predict human toxicity for monoclonal antibody (mAb) therapy and thereby reduce the need for pre-clinical animal testing. Current pre-clinical animal models are aslo poorly predictive for Cytokine Release Syndrome (CRS) in humans, as exemplified by the ‘TeGenero’ incident where primate studies gave no indication of human adverse events.
In vitro assays which reliably predict human toxicity would dramatically reduce testing in animals- potentially to just to those required for minimal toxicity testing. These in vitro tests might also be particularly useful for the development of bio-similars where it is likely that efficacy and toxicity will be the same as the original therapy, but due to the underlining concerns that biosimilars are made in different clones and thus may behave differently, similar performance of the original therapy and the biosimilar in in vitro testing would be strong evidence that further animal tests would not be needed. In vitro testing would also provide insight into the mechanisms of action of a mAb and the option to further engineer to refine function via modification of the anitobody.
Several leukocyte based assays were developed, and following discussion with the Sponsor, the team at University of Southampton validated one of these assays with the intention to use it to evaluate toxicity for a wide range of mAbs. The Cytokine Release Assay (CRA) assay was highly reproducible and correctly predicted that T cell targeting mAbs like TGN1412 have the potential to cause more severe toxicity and in comparison to non-T cell targeting mAbs like Avastin.
Furthermore, cynomolgus macaque peripheral blood mononuclear cells (PBMCs) reacted in a similar manner to human PBMCs in response to TGN142 stimulation in the CRA, providing the potential to reduce the need for cynomolgus macaques PBMCs to predict mAb induced toxicity in humans.
Further collaborative work will continue between, HLS and the University of Southampton if HLS chooses to commercialise the CRA developed by this project. There is a possibility that the testing of the potential of novel mAbs (in development), to cause CRS using the CRA developed by this project may be offered as a service to potential clients in the future by either HLS or the University of Southampton.
Hussain K et al. (2015). Upregulation of FcγRIIb on monocytes is necessary to promote the superagonist activity of TGN1412. Blood 125(1):102-10. doi: 10.1182/blood-2014-08-593061. Epub 2014 Nov 13.
Hargreaves CE et al. (2015). Evaluation of High-Throughput Genomic Assays for the Fc Gamma Receptor Locus. PLoS One 10(11):e0142379. doi: 10.1371/journal.pone.0142379. eCollection 2015.