Experession and characterization of single-chain diabody binding to hERG1 and Integrin beta 1

Espressione e caratterizzazione di un single-chain diabody con affinita singola hERG1 e Integrina beta 1. Antibodies (Abs), whether monoclonal (mAbs) or bispecific (bsAb), are one of the most effective treatments for many forms of cancer. However, there are several drawbacks like poor response in some patients and resistance during treatment with immunotherapy are restricting the use of full-length mAbs for clinical purposes, so the use of engineered antibody fragments can overcome these drawbacks and efficiently act on cancerous cells. There were many studies performed on the use of biologics in comparison with other drugs such as small molecule inhibitors. I believe biologics are extremely expensive and only a small percentage of the drugs are being used in oncology. However, I think they are extremely effective in many cases, especially when applied in a “personalized” treatment. Engineered antibody fragments like single-chain diabodies (scDbs) were found to be a good option to treat solid cancers. There were many attempts made to create a highly specific single-chain diabody by replacing one amino acid phenylalanine with cystine at 92 position in the sequence of the hERG1-scFv antibody. In some solid cancers, hERG1 which is a potassium channel, and β1 integrins are highly expressed in close proximity on the surface of the tumor cells. A bispecific scDb can offer tumor-specificity by binding to the hERG1/β1 complex very efficiently and previous studies showed that the scDb binds to the hERG1/β1 complex in cancer cells and tissues but not to the hERG1 channel or integrin in non-pathological tissues such as the heart. In vitro, the scDb-hERG1-β1 inhibits the formation of the hERG1/β1complex, suppresses Akt phosphorylation and HIF-1 expression, and reduces cell survival, proliferation, and migration. These effects are only shown in cancer cells (colon, pancreatic, or breast), not in normal cells. When scDb was given intravenously the scDb-hERG1-β1 showed an excellent pharmacokinetic profile, with a half-life of 13.5 hours and no general, cardiac, or renal toxicity. The scDb-hERG1-β1 accumulates in subcutaneous xenografted tumors derived from the colon or pancreatic human cancer cells and inhibits tumor development and vascularization. Overall, the scDb-hERG1-β1 antibody is a novel single-chain bispecific antibody with therapeutic implications in solid tumors that overexpress the hERG1-β1 integrin signaling complex. The main aim of my work was to express this scDb-hERG1-β1 in large quantities to study its interaction with the hERG1 potassium channel and the β1 subunit of the integrin receptor, and the macromolecular complex of the two present in cancer cells, using structural biology, biochemical, and biophysical techniques. I started my work by expressing scDb in small-scale experiments (5ml) and scaled up to larger volumes (1L & 6L). Unfortunately, while performing these experiments, I could manage to produce very little of its intact form and observed degradation. To overcome this problem, I tried many batches of scDb production to optimize the condition, but the outcome was the instability of protein in the tested expression and purification conditions.