Inefficient transcription is a production bottleneck for artificial therapeutic BiTE® proteins.

Antibodies are potent biopharmaceuticals used to treat severe diseases, including cancers. During the past decade, more complex modalities have been developed including bispecific T-cell engager (BiTE®) molecules, e.g. by Amgen. However, non-natural and complex molecule formats often prove to be difficult-to-express (DTE), which is the case for BiTE® molecules. Due to the growing importance of multispecific modalities such as half-life extended (HLE) BiTE® and HLE dual-targeting bispecific T-cell engager (dBiTE) molecules, this artificial class of therapeutic proteins was investigated for molecular bottlenecks in stable production cell lines, by analyzing all relevant steps of recombinant protein production. As a result, drastically reduced intracellular BiTE® molecule-encoding mRNA levels were identified as a potential production bottleneck. Using in vitro transcription (IVT), the transcription rate of the BiTE® molecule-encoding mRNA was identified as the root cause for reduced amounts of intracellular mRNA. In an attempt to improve the transcription rate of a BiTE® molecule, it could be demonstrated that the artificial and special structure of the BiTE® molecule was not the rate limiting step for reduced IVT rate. However, modulation of the primary DNA sequence led to significant improvement of IVT rate. The analyses presented provide insight into the HLE BiTE® / HLE d(BiTE®) class of DTE proteins and perhaps into other classes of DTE proteins, and therefore may lead to identification of further production bottlenecks and optimization strategies to overcome manufacturability challenges associated with various complex therapeutics.

The interleukin-2 antagonizing antibody MT204 delays allogeneic skin graft rejection in non-human primates and is well tolerated.

MT204 is a humanized IgG1 antibody specific for interleukin-2 (IL-2) of human and rhesus monkey origin. It potently antagonizes IL-2 signaling in both CD25(+) and CD25(-) cells by a unique mode of action. MT204 can not only prevent soluble IL-2 from binding to the intermediate affinity IL-2 receptors but can also antagonize IL-2 that is already bound to the CD25 subunit of high affinity IL-2 receptors on the cell surface. As initial steps toward development of a therapeutic antibody, pharmacokinetics (PK) and tolerability of MT204, as well as efficacy were investigated in rhesus monkeys. MT204 was infused by single escalating (2, 10 and 50mg/kg) or repeat dose administrations (50mg/kg, 1 ×/week for 3 weeks). Over the course of the experiment, the animals were regularly observed for clinical adverse reaction and bled for laboratory investigations (PK, hematology, chemical chemistry and leukocyte subset analysis). For the efficacy study, six rhesus monkeys were grafted with MHC-mismatched rhesus skin and infused with MT204 (50mg/kg), on the day of transplantation and again on days 5 and 12 post grafting. Efficacy was determined by assessment of graft necrosis at different time-points. No obvious adverse effects were observed clinically after single infusion, or after three repeated infusions of 50mg/kg and no MT204-related toxic effects were revealed by hematology or clinical chemistry. In the efficacy study, MT204-treated animals showed a significant delay in graft rejection versus control animals (p=0.025 by Log-rank test). The characteristics of MT204, displaying linear pharmacokinetics, half-life in the range expected for a human IgG, benign safety profile and signs of efficacy, warrant further development of this antibody for therapy.