Penpulimab, an Fc-Engineered IgG1 Anti-PD-1 Antibody, With Improved Efficacy and Low Incidence of Immune-Related Adverse Events

Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Tailong Qu, Na Chen, Shun Ma, Xinrong He, Dennis Xia, Max Wang, Michelle Xia, Baiyong Li, Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Tailong Qu, Na Chen, Shun Ma, Xinrong He, Dennis Xia, Max Wang, Michelle Xia, Baiyong Li

Abstract

Background: IgG4 anbibodies are deficient in stability and may contribute to tumor-associated escape from immune surveillance. We developed an IgG1 backbone anti-programmed cell death protein-1 (PD-1) antibody, penpulimab, which is designed to remove crystallizable fragment (Fc) gamma receptor (FcγR) binding that mediates antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and proinflammatory cytokine release.

Methods: Aggregation of different anti-PD-1 antibodies was tested by size exclusion chromatography, and melting temperature midpoint (Tm) and aggregation temperature onset (Tagg) were also determined. The affinity constants of penpulimab for PD-1 and human FcγRs were measured by surface plasmon resonance and biolayer interferometry. ADCC and ADCP were determined in cellular assays and antibody-dependent cytokine release (ADCR) from human macrophages was detected by ELISA. Binding kinetics of penpulimab to human PD-1 was determined by Biacore, and epitope/paratope mapping of PD-1/penpulimab was investigated using x-ray crystallography. Additionally, patients from six ongoing trials were included for analysis of immune-related adverse events (irAEs).

Results: Penpulimab demonstrated better stability and a lower level of host-cell protein residue compared with IgG4 backbone anti-PD-1 antibodies. As expected, penpulimab exhibited no apparent binding to FcγRIa, FcγRIIa_H131, FcγRIIIa_V158 and FcγRIIIa_F158, elicited no apparent ADCC and ADCP activities, and induced no remarkable IL-6 and IL-8 release by activated macrophages in vitro. Penpulimab was shown in the co-crystal study to bind to human PD-1 N-glycosylation site at N58 and had a slower off-rate from PD-1 versus nivolumab or pembrolizumab. Four hundred sixty-five patients were analyzed for irAEs. Fifteen (3.2%) patients had grade 3 or above irAEs. No death from irAEs was reported.

Conclusions: IgG1 backbone anti-PD1 antibody penpulimab has a good stability and reduced host cell protein residue, as well as potent binding to the antigen. Fc engineering has eliminated Fc-mediated effector functions of penpulimab including ADCC, ADCP and reduced ADCR, which may contribute to its more favorable safety profile.

Clinical trial registration: www.ClinicalTrials.gov, identifier: AK105-101: NCT03352531, AK105-201: NCT03722147, AK105-301: NCT03866980, AK105-202:NCT03866967, AK105-203: NCT04172571, AK105-204: NCT04172506.

Keywords: Fc engineering; IgG1 anti-PD-1 antibody; binding kinetics; immune-related adverse events; penpulimab.

Conflict of interest statement

All authors are employees of Akeso Biopharma Co., Ltd., who participated in the discovery, production and commercialization of penpulimab.

Copyright © 2022 Huang, Pang, Zhong, Qu, Chen, Ma, He, Xia, Wang, Xia and Li.

Figures

Figure 1
Figure 1
(A) Aggregates tested by size exclusion chromatography (SEC), (B) melting (Tm) and (C) aggregation temperature (Tagg) characterization using dynamic light scattering (DLS) and static light scattering (SLS), respectively, and (D) Host-cell protein (HCP) residue assay in penpulimab and other IgG4 backbone anti-PD1 antibodies.
Figure 2
Figure 2
(A) Antibody-dependent cell-mediated cytotoxicity (ADCC) activities of penpulimab and nivolumab were determined by measuring lactase dehydrogenase (LDH) release from 293T-PD1 cells. (B) Complement-dependent cytotoxicity (CDC) activities of penpulimab, penpulimab (hG1WT), a version of penpulimab with wildtype IgG1 backbone, and nivolumab were determined by measuring LDH release from CHO-K1-PD1-CTLA4 cells. (C) Antibody-dependent cellular phagocytosis (ADCP) activities of penpulimab (hG1WT), penpulimab, and nivolumab were studied by examining phagocytosis of CHO-K1-PD1 cells by murine bone marrow derived macrophages (HPMMs). Effects of Fc engineering of penpulimab on the release of inflammatory cytokines. (D) IL-6 and (E) IL-8 by HPMMs in the presence of IFN-γ. Data are expressed as mean or mean ± SEM and analyzed using one-way ANOVA. *P<0.05 and ***P<0.001 vs. isotype control; ###P<0.001 vs. negative control.
Figure 3
Figure 3
(A) PD-L1 target occupancy following intravenous administration of 1.0, 3.0 or 10.0 mg/kg penpulimab once every two weeks (Q2W) in the dose escalation study or 200 mg penpulimab Q2W in the expansion study, with 28 days per cycle. Post-infusion blood samples were collected at day 1, 2, 8, and 15 of cycle 1, and day 1 of cycle 2, 3, 5, 7, 9, 11, and 13, respectively, as indicated on the x-axis. C: cycle; D: day. Penpulimab potentiates T cell activation via PD1/PDL1 blockade. Raji-PD-L1 cells overexpressing PD-L1 were co-cultured with peripheral blood mononuclear cells (PBMCs) from a normal subject, and the production of IL-2 (B) and IFN-γ (C) was examined by ELISA. Data are shown as mean ± SEM for n = 2, and analyzed using one-way ANOVA. *P<0.05, **P<0.01 and ***P<0.001 vs. isotype control.

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