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. 2021 Dec;12(2):12383-12393.
doi: 10.1080/21655979.2021.2003666.

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery

Lipei Wu  1   2 , Weiwei Wang  3 , Jiale Tian  2 , Chunrun Qi  3 , Zhengxin Cai  4 , Wenhui Yan  4 , Shihai Xuan  1 , Anquan Shang  2
Affiliations

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery

Lipei Wu et al. Bioengineered. 2021 Dec.

Abstract

The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that _targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases.Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

Keywords: LNP; PD-1; PD-L1; bispecific antibody; cancer immunotherapy; mRNA.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Design and optimization of XA-1 mRNA for further in vivo evaluation. (a) Schematic of the structure of mRNA-encoded the heavy and light chains of the XA-1. (b) SDS-PAGE detection of the XA-1 translated from mRNA in supernatants from Expi293F cells. The expression levels of XA-1 in (c) supernatants of AML-12 cell and (d) C57BL/6 J mice with different SPs. The expression levels of XA-1 in (e) AML-12 and (f) C57BL/6 J with different molar ratios. *p < 0.05, **p < 0.01, ***p < 0.001. Results were showed as SEM, n = 6
Figure 2.
Figure 2.
In vivo expression of XA-1 mRNA-LNPs in rodent animals. (a) Serum concentration of XA-1 in the C57BL/6 mice received single injection of XA-1 mRNA-LNPs at three three doses. (b) Pharmacokinetic comparison of XA-1 protein (10 mg/kg) and XA-1 mRNA-LNPs (2 mg/kg) in C57BL/6 mice. All the data were showed as Mean ± SEM, n = 6
Figure 3.
Figure 3.
The serum concentrations of XA-1 in NOD/SCID mice received the three repeated injections of XA-1 mRNA-LNPs. All the data were showed as Mean ± SEM, n = 6
Figure 4.
Figure 4.
In vitro functional activity of mRNA translated XA-1. (a) Binding of XA-1 from two sources to CHO-K1 cells expressing cell surface PD-1 or PD-L1 by flow cytometry. (b) NFAT reporter assay. (c) Association and dissociation of Pembrolizumab to hPD-1 and hPD-L1 by SPR. (d) IFN-γ levels. (e) Tumor cell line killing tests. All the data were showed as Mean ± SEM, n = 6 for Figure (a, b, d, e)
Figure 5.
Figure 5.
In vivo antitumor efficacy test of LNP-Pembrolizumab mRNA in hPD-1 and hPD-L1 knock-in mouse model. (a–b) Tumor growth inhibition (TGI), (c) body weight changes and (d–e) changes in ratios of tumor infiltrating CD4+ T cells, CD8+ T cells, and Treg cells of tumor bearing mice treated with XA-1 mRNA-LNPs at the doses of 0.2, 0.6, and 2.0 mg/kg using XA-1 proteins and empty LNPs as control. Red and blue arrows indicated the injection day of XA-1 mRNA-LNPs or XA-1 proteins, respectively. All the data were showed as Mean ± SEM, n = 10
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The author(s) reported there is no funding associated with the work featured in this article.
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