Abstract LB002: <i>Escherichia coli</i> as a platform for the production of full-length IgG antibodies

Abstract Introduction: Genetically modified strains of Escherichia coli bacteria are widely used as a platform for recombinant protein production. A expressive portion of high value recombinant proteins are represented by IgG antibodies, critically important in the pharmaceutical industry. However, expressing full-length IgG antibodies in bacteria presents significant challenges, primarily related to proper disulfide bond formation. To overcome this limitation, the SHuffle strain was engineered to support disulfide bond formation on cytoplasm. This study explores the potential of the SHuffle strain to produce functional full-length IgG antibodies in E. coli (cyclonals) against three targets of interest for biomedical applications: immune checkpoints PD-1 and TIM-3 and pro-inflammatory cytokine TNFalpha. Objectives: Investigate the capacity of the SHuffle strain to produce full-length IgG antibodies and characterize the generated IgG antibodies by structure and biological activity analysis. Methods: Cyclonals were purified from the soluble fraction of lysates derived from SHuffle T7 cells transformed with bicistronic cyclonal plasmids containing heavy and light chain expression sequences. Briefly, cells were incubated in LB media supplemented with IPTG for 24 h and harvested for purification with protein A agarose resin (Mabselect SuRe). Functional analysis was performed by indirect ELISA. In summary, antigens were adsorbed at ELISA plates and 5-fold serial dilutions of cyclonals were incubated. Absorbance was measured at 450 nm after anti-human IgG antibody-HRP conjugate. Interaction with FcRn receptors was measured by time-resolved fluorescence energy transfer (TR-FRET) following manufacturer instructions for Human FcRn binding Kit. Calculation of EC50 values was performed by the AATBio algorithm. Results and Conclusion: Cyclonals obtained from the E. coli SHuffle strain were analyzed by SDS-PAGE in denaturation conditions revealed bands corresponding to both the heavy (∼50 kDa) and light (∼25 kDa) chains of the IgG antibodies. Analysis in native conditions exhibited the presence of high mass bands, suggesting presence of fully assembled IgG molecules. Additionally, immunoblot analysis confirmed the identity of the produced antibodies.The binding of the antibodies to their target antigen was evaluated using an indirect ELISA. The EC50 value calculated from the absorbance curve suggests high-affinity binding, with values in the subnanomolar range (anti-TIM-3 0.15 nM; anti-TNFalpha 0.07 nM; anti-PD-1 0.11 nM). Analysis of cyclonals interaction with the neonatal Fc receptor (FcRn), critical for extended half-life exhibited a binding profile similar to that of glycosylated IgG, suggesting that the SHuffle strain is able to produce highly functional aglycosylated cyclonals, with pharmacokinetics properties necessary for therapeutic efficacy. Support: FAPESPResearch Internship Abroad, process number 2024/01723-0Doctorate, process number 2023/01816-6 Citation Format: Gabriel Correia Lima, Jinjoo Jung, Matthew de Lisa, Daniela Luz. Escherichia coli as a platform for the production of full-length IgG antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB002.