07/18/2024
By Kwok Fan Chow

The Kennedy College of Science, Department of Chemistry, invites you to attend a Ph.D. Dissertation defense by Andrew Zihao Feng entitled “Development of Methodologies in the Identification and Characterization of the Heterogeneites Present in a Complex IL-2-MHC-1-VPE-Fc Fusion Protein.”

Degree: Doctoral
Location: Perry Hall, Room 415
Date: Wednesday, July 31, 2024
Time: 3 p.m.

Committee:

  • Chair Dr. Jin Xu, Department of Chemistry, University of Massachusetts Lowell
  • Carl Lawton, Department of Chemistry, University of Massachusetts Lowell
  • Matthew Gage, Department of Chemistry, University of Massachusetts Lowell
  • Dongming Xie, Department of Chemistry, University of Massachusetts Lowell

Abstract:
Modern immunotherapeutics which have reached the market typically rely on stimulation of the immune system in a highly generalized manner. Antibody therapeutics bind to specific epitopes to either block function at the site or recruit an effector response. Other immunostimulatory molecules act in an undirected manner, which can lead to cytotoxicity. The complexity of a therapeutic required to stimulate a highly specific immune response to a single antigen increases with each target and signal. C421 attempts to combine the antigen epitope presentation abilities of the MHC 1, the CD8+ T-cell stimulatory effects of IL-2, and the improvements to in vivo half-life that the IgG1 Fc region offers into a single fusion protein that is also capable of separately conjugating a variable peptide epitope (VPE) into the MHC 1 epitope binding groove following expression of the fusion protein. This highly complex construct eschews the previously non-specific strategies offered by past immunotherapies and instead attempts to generate a highly specific response to a target chosen by specialists at point-of-care.

Characterization of C421 inherits the innate complexities of each of its components, with additional sources of heterogeneities added by the chemistry of the various linkers required to construct such a complex fusion protein. The level of heterogeneous species available to C421 also makes it very resistant to most forms of high resolution instrumentation, including mass spectrometry. Without the ability to determine C421’s amino acid sequence and multitude of post-translation modifications and degradations, there is no way to ensure its safety and efficacy following manufacture. We developed a series of methods through which we were able to identify and separate the key sources of heterogeneity present in C421 from its core IL-2-MHC-1-Fc scaffold and independently characterize the individual heterogeneities. It was discovered that C421 possesses a great deal of heterogeneous glycosylation, both N and O-linked, and stability assays revealed that C421 is resistant to enzymatic digestion in is hinge region and experiences significant degradations when high temperatures are used during preparations for analytical procedures. Well-characterized N-linked oligosaccharides were profiled with 2-AB labels, while novel O-linked glycan structures were identified with the MS compatible procainamide label. Peptide mapping investigating cysteine-maleimide conjugation of the VPE showed the importance of the addition of copper ions during conjugation reactions to form coordinating bonds with pre-formed disulfide bonds in the protein, leading to more on-target VPE conjugation.

Development of the methods for characterization of C421 also provides deep insights into the strategies required to characterize any similarly complex fusion proteins manufactured in the future. The increasing demand for highly specific, even patient specific, protein therapeutics inevitably creates proteins of increasing complexity that will become increasing challenging to characterize. Improving the methods involved in identifying and characterizing these heterogeneities must keep pace with the rapidly developing fusion protein industry.

All interested students and faculty members are invited to attend.