Engineering of \u03b1-PD-1 antibody-expressing long-lived plasma cells by CRISPR/Cas9-mediated targeted gene integration

Baohong Luo,Guanwen Wang,Jun Liu,Minqi Luo,Els Verhoeyen,Yikang Zhan,Huimin Dong,Junsong Zhang,Hui Zhang,Yiwen Zhang,Yingtong Lin

doi:10.1038/s41419-020-03187-1

Abstract

Long-lived plasma cells (LLPCs) are robust specialized antibody-secreting cells that mainly stay in the bone marrow and can persist a lifetime. As they can be generated by inducing the differentiation of B-lymphocytes, we investigated the possibility that human LLPCs might be engineered to express α-PD-1 monoclonal antibody to substitute recombinant α-PD-1 antitumor immunotherapy. To this end, we inserted an α-PD-1 cassette into the GAPDH locus through Cas9/sgRNA-guided specific integration in B-lymphocytes, which was mediated by an integrase-defective lentiviral vector. The edited B cells were capable of differentiating into LLPCs both in vitro and in vivo. Transcriptional profiling analysis confirmed that these cells were typical LLPCs. Importantly, these cells secreted de novo antibodies persistently, which were able to inhibit human melanoma growth via an antibody-mediated checkpoint blockade in xenograft-tumor mice. Our work suggests that the engineered LLPCs may be utilized as a vehicle to constantly produce special antibodies for long-term cellular immunotherapy to eradicate tumors and cellular reservoirs for various pathogens including human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV).

Highlights

B-lymphocytes are a special class of immune cells that provide specific immune surveillance mainly by producing various antibodies[1,2]
clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated targeted transgene insertion by integrase-defective lentiviral vector (IDLV) delivery In order to efficiently mediate the targeted integration of the α-Programmed death 1 (PD-1) transgene into the locus of the housekeeping gene GAPDH and ensure persistent gene expression, we generated a donor plasmid to carry a promoterless P2A-α-PD-1 sequence flanked by two GAPDH homology arms (HAs), named homologous recombination (HR) donor
To conveniently evaluate the knock-in efficiency, we fused a T2A-CD90 reporter gene downstream of the α-PD-1 gene to allow the co-expression of CD90 on the cell surface, which was similar to α-PD-1 being under the control of the GAPDH promoter, and functioned as a convenient marker for evaluating the insertion efficiency (Fig. 1a)

Summary

Introduction

B-lymphocytes are a special class of immune cells that provide specific immune surveillance mainly by producing various antibodies[1,2]. Blymphocytes differentiate into short-lived PBs in the germinal centers of lymph nodes and the spleen, and subsequently travel to the bone marrow, where they receive survival signals from special niches and LLPCs may persist a lifetime and maintain a continuous supply of serum antibodies[4,5]. The bacteria-originated clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system, which allows for a highly efficient modification at specific genetic loci in primary human cells[11,12], has been used as a tool to engineer B-lymphocytes[13,14,15,16,17,18,19,20].

Methods

Results

Conclusion