Comprehensive engineering of a therapeutic neutralizing antibody targeting SARS-CoV-2 spike protein to neutralize escape variants.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

High-performance liquid chromatographic assay for the determination of nilotinib in human plasma.

A precise and convenient high-performance liquid chromatography (HPLC) method has been established to assay nilotinib in human plasma. Chromatographic separation of nilotinib was performed on a LiChrosphere(®)100 RP-18(e) column (250 mm×4.0 mm, 5 µm) using a mixture of acetonitrile and 0.01 M phosphate buffer (pH 3.0) (42 : 58, v/v) under isocratic conditions at a flow rate of 1.0 ml/min with ultraviolet (UV) detection at 266 nm. The calibration curve showed linearity at concentrations between 250 ng/ml and 5000 ng/ml (r(2)>0.999). The mean±S.D. absolute recovery of nilotinib from plasma was 99.2±3.3%. The coefficients of variation of both intra- and inter-day precision were below 9.1%. These results indicate that this new HPLC-based quantification may be useful for therapeutic drug monitoring of nilotinib to help manage treatment in patients with chronic myeloid leukemia in clinical practice.

Association of SLCO1B3 polymorphism with intracellular accumulation of imatinib in leukocytes in patients with chronic myeloid leukemia.

Intracellular concentration of imatinib in leukemic cells is thought to affect the clinical efficacy of this drug in patients with chronic myeloid leukemia (CML); however, there is no report that directly indicates the relationship between intracellular concentration and clinical outcome and/or, plasma concentration. In addition, the impacts of genetic variations of drug transporters, which mediate leukocyte concentration of imatinib, are unknown. In the present study, we investigated the correlation between intracellular imatinib concentrations in leukocytes, plasma imatinib levels, and genotypes of drug transporters, including ATP binding cassette B1 (ABCB), ABCG2, solute carrier 22A1 (SLC22A1), solute carrier organic anion transporter family members 1B1 (SLCO1B1) and SLCO1B3. The imatinib levels in leukocytes were determined using HPLC in 15 patients with chronic phase CML. No significant correlation between intracellular and plasma concentrations of imatinib was observed. The intracellular concentration was comparable in both patients with or without complete cytogenetic response. The intracellular imatinib concentration was significantly higher in patients with SLCO1B3 334TT than in those with 334TG/GG (p=0.0188). Plasma concentrations were similar in both SLCO1B3 genotypes (p=0.860), thereby resulting in the intracellular to plasma concentration ratio being higher in patients with SLCO1B3 334TT than those with 334 TG/GG (p=0.0502). These results suggested that the SLCO1B3 334T>G polymorphism could have a significant impact on the intracellular concentration of imatinib in leukocytes as a promising biomarker for personalized treatment of CML patients.

Contribution of BCR-ABL-independent activation of ERK1/2 to acquired imatinib resistance in K562 chronic myeloid leukemia cells.

BCR-ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR-ABL is inhibited by imatinib; however, several mechanisms of imatinib resistance have been proposed that account for loss of imatinib efficacy in patients with CML. Previously, we showed that overexpression of the efflux drug transporter P-glycoprotein partially contributed to imatinib resistance in imatinib-resistant K562 CML cells having no BCR-ABL mutations. To explain an additional mechanism of drug resistance, we established a subclone (K562/R) of the cells and examined the BCR-ABL signaling pathway in these and wild-type K562 (K562/W) cells. We found the K562/R cells were 15 times more resistant to imatinib than their wild-type counterparts. In both cell lines, BCR-ABL and its downstream signaling molecules, such as ERK1/2, ERK5, STAT5, and AKT, were phosphorylated in the absence of imatinib. In both cell lines, imatinib effectively reduced the phosphorylation of all the above, except ERK1/2, whose phosphorylation was, interestingly, only inhibited in the wild-type cells. We then observed that phospho-ERK1/2 levels decreased in the presence of siRNA targeting BCR-ABL, again, only in the K562/W cells. However, using an ERK1/2 inhibitor, U0126, we found that we could reduce phospho-ERK1/2 levels in K562/R cells and restore their sensitivity to imatinib. Taken together, we conclude that the BCR-ABL-independent activation of ERK1/2 contributes to imatinib resistance in K562/R cells, and that ERK1/2 could be a target for the treatment of CML patients whose imatinib resistance is due to this mechanism.

Relationship between an effective dose of imatinib, body surface area, and trough drug levels in patients with chronic myeloid leukemia.

The standard dose of imatinib for the treatment of chronic-phase chronic myeloid leukemia (CML) is 400 mg/day. Some patients receive reduced doses of imatinib because of serious adverse effects. Recently, the effective plasma threshold for trough imatinib levels was demonstrated to be 1,002 ng/mL. In this study, we evaluated the association of an imatinib dose with trough plasma concentrations and clinical outcomes in 31 patients with chronic-phase CML who were treated at Kumamoto University Hospital. Twenty-seven patients were optimally treated with various doses of imatinib. The mean (+/-SD) trough plasma concentrations of imatinib were 1.40 +/- 0.57 microg/mL in 13 patients receiving 400 mg/day and 1.15 +/- 0.44 microg/mL in 9 patients receiving 300 mg/day as an effective dose. Mean trough levels of the two groups were not significantly different and exceeded the effective plasma threshold. Body surface area (BSA) was significantly smaller in patients receiving the reduced dose compared with those receiving the standard dose (p = 0.001). The effective imatinib dose was associated with age and gender as well as BSA. A reduced dose of 300 mg/day of imatinib may be sufficient for the treatment of CML patients with smaller body size, particularly when intolerability arises.