Nanobody-based chimeric antigen receptor T cells designed by CRISPR/Cas9 technology for solid tumor immunotherapy

Fengzhen Mo,Aiqun Liu,Shihua Yin,Hua Yao,Xiaobing Jiang,Xiaoqiong Hou,Xinyue Zhao,Cueva Jumbo Juan Carlos,Zihang Yu,Bruce D Hammock,Siliang Duan,Zhuoran Tang,Shenxia Xie,Xiaoling Lu,Ziqiang Ding,Wu Wang,Wei Shi,Xiaomei Yang

doi:10.1038/s41392-021-00462-1

Abstract

Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.

Highlights

It is known that malignant tumors are able to invade and metastasize due to their ability to escape the immune system surveillance
The recombinant plasmid was transformed into competent TG1 cells by electroploration. The titer of this Nb library against CD105 was calculated by counting the number of colonies in gradient dilution plates (Fig. 1c), which showed that the library should have a probability to obtain Nbs with high specificity and sequence diversity
SDSpolyacrylamide gel electrophoresis (SDS-PAGE) analysis displayed a band with approximately 15 kDa in molecular weight (Fig. 1e)

Summary

Introduction

It is known that malignant tumors are able to invade and metastasize due to their ability to escape the immune system surveillance. CD105, termed endoglin, is highly expressed on cancer cells, and on peri- and intratumoral endothelial cells, which line tumor blood vessels.[1,2] On the other hand, CD105 is expressed in varying degrees in the vasculature of normal tissues and on normal blood vessels, except for the umbilical cord of newborns.[3] A phase II study of TRC105 in patients with hepatocellular carcinoma (HCC) or advanced/metastatic urothelial carcinoma were carried out by Tracon Pharma and several oncologists. TRC105 was well tolerated, it did not improve progression-free survival in patients, and suggest that TRC105 therapy will not be effective as a single agent using the present schedule of tumor treatment. Common adverse effects included infusion reactions, headache, epistaxis, and oral hemorrhage during TRC105 treatment of solid tumors.[4,5] At present, multiple clinical trials to evaluate the potential of TRC105 in combination with vascular endothelial growth factor or programmed cell death protein 1 (PD-1) checkpoint inhibitors are being carried out and some have obtained positive effects.[6,7,8] Compelling evidence support the notion that CD105 plays a key role in angiogenesis, and that CD105 is an optimal molecular target.[9,10] CD105 can be used to design innovative biological immunotherapy to treat human malignant tumors

Objectives

Methods

Results

Conclusion