Efficient development and expression of scFv recombinant proteins against PD-L1 surface domain and potency in cancer therapy

Abstract

PD-L1 is a 40 kDa trans-membrane protein of B7 family and an important T cell regulator. Binding of PD-L1 and PD-1 inhibits proliferation and activation of T cell results cell exhaustion. This phenomenon can be reversed by blocking PD-L1/PD-1 interactions with single chain variables fragment (scFv) fusion proteins and by direct inhibition of tumor cells with drug conjugates. The human phagedisplayed scFv library was utilized to generate scFv against the PD-L1 antigen by affinity bio-panning. The positive clones were selected by continuous transfection of bacterial cells and sequence analysis. The binding affinity and specificity of the scFv and antibody fragments were determined by using surface plasmon resonance biosensor, western blot analysis, and immunofluorescence assay. After three rounds of panning selection, about 30% of clones have a binding affinity with targeted PD-L1 antigen. Eight positive clones with accurate sequences were isolated and analyzed for binding affinity with PD-L1 antigen. Three of those with accurate sequences and binding affinity were selected for the recombinant formation and soluble expression by Escherichia coli host machinery. The highly positive recombinant clones with the exact orientation of FR and CDR domains were developed and can be used as a drug carrier tools in ADC formation or direct inhibition of immune checkpoint in cancer immunotherapy. The conjugate achieved its initial potency and need efficient improvement to enhance direct tumor suppression and bio-therapeutics strategies enrichment.

Development of a human phage display-derived anti-PD-1 scFv antibody: an attractive tool for immune checkpoint therapy

“…Due to in vitro selection of scFvs through phage display technique, specific scFvs can be isolated against poorly immunogenic antigens, such as antigens showing high homology to host animals’ proteins or those that are toxic to their hosts [ 26 , 32 ] . The smaller size of scFvs compared to mAbs (~ 25 vs. 150 kDa) leads to increased penetration ability of scFvs to tumor cells [ 29 , 36 ]. The absence of an Fc domain in scFvs can decrease the chance of non-specific binding and the resulting undesirable immunogenic responses [ 37 ] .…”

“…Immune checkpoint inhibition therapies, especially those employing monoclonal antibodies, have shown significant and durable responses in numerous malignancies [ 29 , 39 ]. Several anti-PD-1 and anti- PD-L1 antibodies have been developed for immunotherapy of melanoma, lung, bladder, skin, and uterus cancers.…”

“…Immunotherapy has emerged as an innovative treatment for several decades, aiming to activate the immune system to recognize and attack cancer cells. Cancer immunotherapy strategies include immune checkpoint inhibitors, cancer vaccines, monoclonal antibodies, adoptive cell therapy (ACT), oncolytic virus therapy, and cytokines, all of which aim to increase the efficacy of treatment [29][30][31].…”

Engineered scPDL1-DM1 drug conjugate with improved in vitro analysis to target PD-L1 positive cancer cells and intracellular trafficking studies in cancer therapy

“…Cells were cultured in a humidified incubator with 5% CO 2 at 37°C. The high-affinity scFv-PD-L1 single-chain proteins were previously engineered by our group using phage display technology and positive clones were selected by bio-panning strategies and successfully expressed in bacterial host cell machinery (Kalim et al, 2019). Anti-PD-L1 IgG antibody and anti-6xHis Tag rabbit antibody (Cat No AB 10002) were from Life Science Production & Services, China.…”

Virulent Drexlervirial Bacteriophage MSK, Morphological and Genome Resemblance With Rtp Bacteriophage Inhibits the Multidrug-Resistant Bacteria

“…They are obligate intracellular parasites, present everywhere wherever bacteria live ( Suttle, 2005 ). The roles of phages could not be ignored as they are used as vehicles in gene transfer ( Gómez-Gómez et al, 2019 ), an alternative to antibiotics ( Alvi et al, 2020 ), and antibody engineering using phage display technology ( Kalim et al, 2019 ). Regarding benefits, bacteriophages also cause severe problems in industrial production and fermentation units.…”