Page 45 - Book of Abstracts 2020

th
8 Biannual Conference on Chemistry - CHEM 08

Conjugation and Modification Technology of A Therapeutic

Protein: An Approach to Improve Drug Detoxification and
Antibody Directed Enzyme Prodrug Therapy for Efficient
Cancer Treatment by Glucarpidase

Fatma B. Rashidi
Chemistry Department, Faculty of Science, Cairo University,Giza, Egypt

ABSTRACT

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is a Food and Drug
Administration-approved enzyme used in targeted cancer strategies by Antibody Directed
Enzyme Prodrug Therapy (ADEPT) and methotrexate detoxification resulting from
excessive levels of its use. Although this medicinal importance, CPG2 application has some
limitations due to its potential immunogenicity and a relatively short half-life in patient’s
serum caused by the need for the repeated cycles of the ADEPT. This in turn can diminish
its bioavailability by neutralizing antibodies produced by patients. The modification and
conjugation technology of CPG2 can be employed to overcome these drawbacks by elongate
the enzyme residency time in patient’s blood. PEGylation and fusion with human serum
albumin (HSA) are two approaches commonly used increase the half-lives of the
therapeutic proteins in vivo. These technology has been addressed to increase protein
stability and bioavailability. ‘biobetter’ CPG2 variants, mono-PEGylated CPG2, and HSA
fused CPG2 by genetic fusion with albumin, were generated. Biochemical and bioactivity
analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability
using human blood serum and immunoassays, demonstrated that the functional activities
of the designed modified CPG2 conjugates were maintained. The immunotoxicity studies
indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation,
suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free
glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared
with the negative control. In the latter case, this might be attributed to trace impurities of
the used type of expression system associated with the highly purified (99.99%)
recombinant HSA- CPG2. Both PEGylated glucarpidase and HSA-glucarpidase exhibit
more stability in human serum and were more resistant to key human proteases relative to
native glucarpidase. To our knowledge, this study is the first to report stable and less
immunogenic CPG2 variants produced by PEGylation and fusion with HSA. The results
suggest that the employed modifications may have better efficacy in CPG2 drug
detoxification and ADEPT, thereby improving this cancer treatment strategy.

BOOK OF ABSTRACTS CHEM 08 (2020) Page 44