Robin Thorpe

After TGN1412: Recent developments in cytokine release assays

The failure of regulatory science to keep pace with and support the development of new biological medicines was very publically highlighted in March 2006 when the first-in-man Phase I clinical trial of the immunomodulatory CD28-specific monoclonal antibody (mAb) TGN1412 ended in disaster when all six volunteers suffered a life-threatening adverse reaction termed a ‘Cytokine Storm’. The poor predictive value of standard pre-clinical safety tests and animal models applied to TGN1412 demonstrated the need for a new generation of immunotoxicity assays and animal models that are both sensitive and predictive of clinical outcome in man. The non-predictive result obtained from pre-clinical safety testing in cynomolgus macaques has now been attributed to a lack of CD28 expression on CD4+ effector memory T-cells that therefore cannot be stimulated by TGN1412. In contrast, high levels of CD28 are expressed on human CD4+ effector memory T-cells, the source of most TGN1412-stimulated pro-inflammatory cytokines. Standard in vitro safety tests with human cells were also non-predictive as they did not replicate in vivo presentation of TGN1412. It was subsequently shown that, if an immobilized therapeutic mAb-based assay or endothelial cell co-culture assay was used to evaluate TGN1412, then these would have predicted a pro-inflammatory response in man. New in vitro assays based on these approaches are now being applied to emerging therapeutics to hopefully prevent a repeat of the TGN1412 incident. It has emerged that the mechanism of pro-inflammatory cytokine release stimulated by TGN1412 is different to that of other therapeutic mAbs, such that standard pro-inflammatory markers such as TNFα and IL-8 are not discriminatory. Rather, IL-2 release and lymphoproliferation are optimal readouts of a TGN1412-like pro-inflammatory response.

Severity of the TGN1412 trial disaster cytokine storm correlated with IL‐2 release

Aim To determine if cytokine release with a solid phase assay is predictive of adverse responses for a range of therapeutic mAbs. Methods Cytokine ELISAs and a multi-array system were used to compare responses generated by different therapeutic mAbs using a solid phase assay. Flow cytometry was employed to determine the cellular source of those cytokines. Results Only TGN1412 and muromonab-CD3 stimulated CD4+ T-cell mediated cytokine release characterized by significant (all P < 0.0001) IFNγ, TNFα, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and IL-22 release, comparable with T-cell mitogen. Significantly greater (P < 0.0001) IL-2 release with TGN1412 (2894–6051 pg ml−1) compared with muromonab-CD3 (62–262 pg ml−1) differentiated otherwise comparable cytokine responses. Likewise, TGN1412 stimulated significantly more (P = 0.0001) IL-2 producing CD4+ T-cells than muromonab-CD3 and induced Th1, Th2, Th17 and Th22 subsets that co-release this cytokine. Significant TNFα release was observed with bevacizumab (P = 0.0001), trastuzumab (P = 0.0031) and alemtuzumab (P = 0.0177), but no significant IL-2 release. TGN1412 and muromonab-CD3 caused pro-inflammatory cytokine release despite significantly (both P < 0.0001) increasing numbers of T-cells with a regulatory phenotype. Conclusions The severity of the adverse response to TGN1412 compared with muromonab-CD3 and other therapeutic mAbs correlates with the level of IL-2 release.

Development of a Lyophilization Formulation that Preserves the Biological Activity of the Platelet‐inducing Cytokine Interleukin‐11 at Low Concentrations

Recombinant human interleukin‐11 (rhIL‐11) is a licensed biological therapeutic product in at least one country and is used to combat thrombocytopenia during chemotherapeutic regimens, as well as undergoing clinical trials for a range of other disorders. Following attempts to lyophilize IL‐11 at low concentrations, it was clear that a significant loss of recoverable biological activity occurred.

Establishment of the first international standard for PEGylated granulocyte colony stimulating factor (PEG-G-CSF): Report of an international collaborative study

We assessed the feasibility of developing a suitable international reference standard for determination of in vitro biological activity of human sequence recombinant PEG-G-CSF products with a 20 kD linear PEG linked to the N-terminal methionyl residue of G-CSF (INN Filgrastim), produced using a conjugation process and coupling chemistry similar to that employed for the lead PEGfilgrastim product. Based on initial data which showed that the current WHO 2nd international standard, IS for G-CSF (09/136) or alternatively, a PEG-G-CSF standard with a unitage traceable to the G-CSF IS may potentially serve as the IS for PEG-G-CSF products, two candidate preparations of PEG-G-CSF were formulated and lyophilized at NIBSC. These preparations were tested by 23 laboratories using in vitro bioassays in a multi-centre collaborative study. Results indicated that on the basis of parallelism, the current WHO 2nd IS for G-CSF or any of the PEG-G-CSF samples could be used as the international standard for PEG-G-CSF preparations. However, because of the variability in potency estimates seen when PEG-G-CSF preparations were compared with the current WHO 2nd IS for G-CSF, a candidate PEG-G-CSF was suitable as the WHO IS. The preparation 12/188 was judged suitable to serve as the WHO IS based on in vitro biological activity data. Therefore, the preparation coded 12/188 was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2013 as the WHO 1st IS for human PEGylated G-CSF with an assigned in vitro bioactivity of 10,000 IU per ampoule.

The 2nd International Standard for human granulocyte colony stimulating factor.

Five candidate preparations of human sequence recombinant granulocyte-colony stimulating factor (G-CSF) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement International Standard (IS). The preparations were tested by 13 laboratories using in vitro bioassays. The candidate preparation 09/136 was judged suitable to serve as a replacement international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/136 was established by the WHO Expert Committee on Biological Standardization (ECBS) as the 2nd IS for human G-CSF with an assigned value for G-CSF activity of 95,000 IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies entirely on the estimates calculated relative to the WHO 1st IS for continuity of the IU.

The 1st International standard for transforming growth factor-β3 (TGF-β3)

One candidate preparation of human sequence recombinant transforming growth factor-β3 (TGF-β3) was formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for its suitability to serve as an international standard. The preparation was tested by 8 laboratories using in vitro bioassays and immunoassays. The candidate preparation 09/234 was judged suitable to serve as an international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/234 was established by the WHO Expert Committee on Biological Standardisation (ECBS) as the WHO 1st IS for human TGF-β3 with an assigned value for TGF-β3 activity of 19,000 IU/ampoule.

The 2nd International standard for Interleukin-2 (IL-2) Report of a collaborative study

Two candidate preparations of human sequence recombinant Interleukin-2 (IL-2) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement international standard. The preparations were tested by eight laboratories using in vitro bioassays and immunoassays. The candidate preparation 86/500 was judged suitable to serve as a replacement international standard based on the data obtained for activity and stability. On the basis of the results reported here, the preparation coded 86/500 was established by the WHO Expert Committee on Biological Standardisation (ECBS) in 2012 as the WHO 2nd IS for human IL-2 with an assigned value for IL-2 activity of 210IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies exclusively on the estimates calculated relative to the WHO 1st IS for continuity of the IU.