Development of plant-produced bispecific monoclonal antibody targeting PD-L1 and CTLA-4 for cancer immunotherapy

Abstract

Checkpoint blockade immunotherapy has transformed the course of cancer treatments. The use of monoclonal antibodies (mAbs) specific against immune checkpoints has displayed satisfactory clinical benefits. However,its effectiveness is limited to just a small percentage of cancer patients. In this work, a recombinant DVD-AT2C bispecific antibody (bsAb) was developed for dual inhibition of PD-1/PD-L1 and CTLA-4 axes and produced in plants. Plant system was utilized as an alternative protein production platform. Transient expression of DVD-AT2C targeting PD-L1 and CTLA-4 in Nicotiana benthamiana was determined highest after 4 days of infiltration. The size, assembly, and protein properties of the purified bsAb were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced DVD AT2C are as expected, and it was mostly present as monomer. Expression of bsAb in wildtype plants produced high mannose-type and some complex N-glycans. The plant-produced DVD-AT2C showed in vitro binding to PD-L1 and CTLA-4 proteins. The antitumor activity of plant-produced bsAb was tested in vivo colon tumor and compared to plant-produced Atezolizumab and 2C8. Plant-produced DVD-AT2C significantly inhibited tumor growth by reducing tumor volume and weight. The antitumor effects, however, were not superior to monospecific parent 2C8. Body weight changes indicated that plant-produced bsAb was safe and tolerable. Overall, this proof-of-concept study demonstrated the viability of plants to produce functional plant-produced bsAb for cancer immunotherapy.