Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains

Abstract

Systemic injection of therapeutic antibodies may cause serious adverse effects due to on-target toxicity to the antigens expressed in normal tissues. To improve the targeting selectivity to the region of disease sites, we developed protease-activated pro-antibodies by masking the binding sites of antibodies with inhibitory domains that can be removed by proteases that are highly expressed at the disease sites. The latency-associated peptide (LAP), C2b or CBa of complement factor 2/B were linked, through a substrate peptide of matrix metalloproteinase-2 (MMP-2), to an anti-epidermal growth factor receptor (EGFR) antibody and an anti-tumor necrosis factor-α (TNF-α) antibody. Results showed that all the inhibitory domains could be removed by MMP-2 to restore the binding activities of the antibodies. LAP substantially reduced (53.8%) the binding activity of the anti-EGFR antibody on EGFR-expressing cells, whereas C2b and CBa were ineffective (21% and 9.3% reduction, respectively). Similarly, LAP also blocked 53.9% of the binding activity of the anti-TNF-α antibody. Finally, molecular dynamic simulation showed that the masking efficiency of LAP, C2b and CBa was 33.7%, 10.3% and −5.4%, respectively, over the binding sites of the antibodies. This strategy may aid in designing new protease-activated pro-antibodies that attain high therapeutic potency yet reduced systemic on-target toxicity.

Blockade of ITGA2 Induces Apoptosis and Inhibits Cell Migration in Gastric Cancer

“…Direct application of anti-ITGA2 antibodies to treat gastric cancer may induce considerable cytotoxicity to these cells. Several strategies may be used to reduce the risks of this complication and improve the safety of the antibodies, including designing pro-antibodies by masking the binding sites of antibodies which may cleaved by proteases that highly expressed at tumor sites [ 85 – 87 ]. This strategy may direct the therapeutic activity of the anti-ITGA2 antibodies to tumor lesions, but not the normal tissues.…”

Protease‐activated prodrugs: strategies, challenges, and future directions

“…In another study, Chen et al . developed anti‐EGFR pro‐Ab masked with MMP‐2 cleavage peptide (GPLG/VR). This pro‐Ab was shown to be rapidly activated by MMP‐2, displayed high serum stability in in vitro models, and had high binding affinity to EGFR target in cell culture models.…”

Biotherapeutics: Challenges and Opportunities for Predictive Toxicology of Monoclonal Antibodies

“…Importantly, the antigens targeted by therapeutic mAbs are not always expressed exclusively at the disease sites [ 11 ] and systemic injection of therapeutic mAbs may cause considerable adverse effects which can decrease treatment efficacies. Chen and colleagues [ 14 ] highlighted this with examples such as epidermal growth factor receptor (EGFR) which is over-expressed in some tumors and has an important role in tumor progression. Importantly, EGFR is also expressed in some epithelial cells and systemic administration of anti-EGFR mAbs will induce adverse effects such as skin rash in a majority of patients with metastatic colorectal cancer.…”

“…Importantly, TNF-α is also an important cytokine in the defense against microbial infections. Therefore, as Chen and colleagues [ 14 ] pointed out, systemic targeting of TNF-α with anti-TNF-α mAbs such as Remicade and Humira is known to create a higher risk of serious infections, and long-term treatment with Remicade may increase lymphoma incidence [ 14 ]. Several of the immune-mediated adverse effects, which include hypersensitivity reactions, abrogation of the intended pharmacologic effect, and altered pharmacokinetic profiles, which impact therapeutic exposure of the mAb, are due to anti-drug antibodies (ADAs) that generally result in the formation of the drug/ADA immune complex (IC).…”