C117
Evidence that hexa histidine tags on therapeutic proteins are not immunogenic in patients
Surinder Sharma1, Natalie Griffin1, Steve Cleverley2, Hassan Shahbakhti1, Heide Kogelberg1, Berend Tolner1, Richard Begent1, Kerry Chester1
1University College London Cancer Institute, London, UK, 2TouchDown Bioinnovation, Bemmel, Netherlands
Background
Engineered hexahistidine tags (his-tags) provide a rapid and effective means for purification of recombinant proteins using immobilised metal affinity chromatography (IMAC). We have applied IMAC to obtain highly pure recombinant therapeutics for use in Phase I/II cancer trials. Immunogenicity evaluation of therapeutic proteins is an important component of their clinical development as it can impact on safety and efficacy. We aimed to assess immunogenicity of the his-tag in man. To correlate immune response with clinical events, it is essential to develop reliable and validated methods. We have previously developed such assays to study and dissect the human immune response to the enzyme and antibody component of MFECP, a multi-component recombinant fusion his-tagged therapeutic used in antibody directed enzyme prodrug therapy (ADEPT).
Here, we describe assays to investigate the immunogenicity of the his-tag component of MFECP.
Method
Two assays (1) A direct ELISA format and (2) A novel quantitative real-time assay employing diffractive optics technology using the Axela dotLab System were developed and validated. The assays were used to measure anti-his antibodies in sera from patients receiving single or repeated doses of MFECP during treatment with ADEPT.
Results
Both assays were sensitive and reproducible. All samples tested were negative for anti-his tag antibodies although the same samples were positive for anti-CPG2 and anti-MFE antibodies in validated assays.
Conclusion
The his-tag did not appear to be immunogenic although the patients made a robust immune response to other components of the protein therapeutic.
This study suggests that his-tagged proteins may be safe and non-immunogenic in humans.
The assay formats are applicable for immunogenicity assessment of any his-tagged protein.
Acknowledgements
This work is funded by Cancer Research UK, ECMC and the UCL Cancer Institute Research trust
