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

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.

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

(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

(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.