Discovery of a highly potent and selective Bruton’s tyrosine kinase inhibitor avoiding impairment of ADCC effects for B-cell non-Hodgkin lymphoma

The datasets used and/or analyzed to support the findings of this study are available in this paper or the Supplementary Information. Any other raw data that support the findings of this study are available from the corresponding author upon reasonable request. Pal Singh, S., Dammeijer, F. & Hendriks, R. W. Role of Bruton’s tyrosine kinase in B cells and malignancies. Mol. Cancer. https://doi.org/10.1186/s12943-018-0779-z (2018). Mathur, R. Burton’s Tyrosine Kinase Inhibition by Ibrutinib: current status. J. Leuk. https://doi.org/10.4172/2329-6917.1000e113 (2015). Kohrt, H. E. et al. Ibrutinib antagonizes rituximab-dependent NK cell-mediated cytotoxicity. Blood 123, 1957–1960 (2014). CAS Article Google Scholar Byrd, J. C. et al. Acalabrutinib (ACP-196) in relapsed chronic lymphocytic leukemia. New Engl. J. Med. 374, 323–332 (2016). CAS Article Google Scholar Guo, Y. et al. Discovery of zanubrutinib (BGB-3111), a novel, potent, and selective covalent inhibitor of Bruton’s tyrosine kinase. J. Med. Chem. 62, 7923–7940 (2019). CAS Article Google Scholar Download references This work was supported by the National Natural Science Foundation of China (Grant Nos. 81773777, 81872748, and 81803366), the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (Grant No. 2018ZX09711002), the Natural Science Foundation of Anhui Province (Grant No. 1808085MH268), the China Postdoctoral Science Foundation (Grants Nos. 2018T110634 and 2018M630720), the Frontier Science Key Research Program of CAS (Grant No. QYZDB-SSW-SLH037), the Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology (Grant No. 2019HSC-CIP011), the CASHIPS Director’s Fund (Grant No. BJPY2019A03), and the Key Program of 13th five-year plan of CASHIPS (Grant No. KP-2017-26). We are also grateful for the support of Hefei leading talent for F.Z. These authors contributed equally: Juan Liu, Qianmao Liang, Aoli Wang, Fengming Zou. Anhui Province Key Laboratory of Medical Physics and Technology, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 230031, Hefei, Anhui, P. R. China Juan Liu, Qianmao Liang, Aoli Wang, Fengming Zou, Ziping Qi, Kailin Yu, Qingwang Liu, Cheng Chen, Jing Liu & Qingsong Liu University of Science and Technology of China, 230036, Hefei, Anhui, P. R. China Juan Liu, Qianmao Liang, Cheng Chen, Jing Liu & Qingsong Liu Hefei Cancer Hospital, Chinese Academy of Sciences, 230031, Hefei, Anhui, P. R. China Aoli Wang, Fengming Zou, Ziping Qi, Qingwang Liu, Cheng Chen, Jing Liu & Qingsong Liu Precision Medicine Research Laboratory of Anhui Province, 230088, Hefei, Anhui, P. R. China Qingwang Liu & Qingsong Liu Precision Targeted Therapy Discovery Center, Institute of Technology Innovation, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 230088, Hefei, Anhui, P. R. China Qingwang Liu & Qingsong Liu Institutes of Physical Science and Information Technology, Anhui University, 230601, Hefei, Anhui, P. R. China Qingsong Liu You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar Correspondence to Jing Liu or Qingsong Liu. The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions Liu, J., Liang, Q., Wang, A. et al. Discovery of a highly potent and selective Bruton’s tyrosine kinase inhibitor avoiding impairment of ADCC effects for B-cell non-Hodgkin lymphoma. Sig Transduct Target Ther 5, 200 (2020). https://doi.org/10.1038/s41392-020-00309-1 Download citation Received: 22 April 2020 Revised: 28 July 2020 Accepted: 12 August 2020 Published: 14 September 2020 DOI: https://doi.org/10.1038/s41392-020-00309-1

The results showed that it was highly selective (S Score (1) = 0.00) at the concentration of 1 μM and only BTK kinase was revealed as the strong binding target ( Fig. 1e and Supplementary  Table S2).
Given the fact that Ibrutinib could impair anti-CD20 antibodies exerted antibody drug-dependent NK-cell-mediated cytotoxicity (ADCC) due to the selectivity problem, we then examined compd. 85 in the human NK cells co-cultured with the Mino cells or SK-OV-3 cells in the presence of Rituximab or Herceptin. Ibrutinib strongly inhibited Rituximab and Herceptin-induced IFN-γ secretion in the NK cells in a dose-dependent manner between 0.1 and 3 μM, meanwhile compd. 85 showed no apparent inhibition up to 3 μM, which recapitulated its weak ITK inhibitory activity ( Fig. 1f and Supplementary Fig. S4). Furthermore, Ibrutinib significantly impaired the antibody-dependent NK-cell-mediated cytotoxicity (ADCC) against Mino and SK-OV-3 cells in the in vitro lactate dehydrogenase (LDH) release experiment ( Fig. 1g and Supplementary Fig. S5). In comparison, compd. 85 did not affect the killing efficacy of NK cells which further confirmed that it would not abrogate the ADCC effect.
We next evaluated the anti-proliferative effects of compd. 85 against a panel of B-cell lymphoma cell lines. Overall, it was potent to all these cell lines (GI 50 s: <2 μM) while Ibrutinib exhibited a relatively random trend ( Fig. 1h and Supplementary Table S3). In addition, it displayed similar potency to the second generation BTK kinase inhibitor Acalabrutinib in TMD8 (DLBCL) and REC-1 (MCL) cells. In TMD8, REC-1, and DOHH2 cells, compd. 85 potently blocked the BTK Y223 autophosphorylation (<10 nM) and inhibited the phosphorylation of downstream mediators such as PLCγ2, ERK, AKT (Fig. 1i), and p-NF-κB p65 ( Supplementary Fig. S6). In addition, dose-dependent apoptotic induction and cell cycle arrest were observed in these cell lines ( Supplementary  Fig. S7a, b).
The in vivo pharmacokinetic study showed that compd. 85 bore acceptable bioavailability (F = 29%) and suitable half-life (T 1/2 = 2.9 h), and good drug exposure (AUC 0−t = 2145 ng/mL) for oral administration at 10 mg/kg in rats (Table S4). The dose escalation study showed that compd. 85 was well tolerated up to 800 mg/kg/ day dosage for continuous 14 days with no apparent toxicity observed ( Supplementary Fig. S8a, b). In addition, compd. 85 exhibited dose-dependent anti-tumor efficacy in the TMD8 cell (DLBCL)-inoculated xenograft mouse model and the tumor growth inhibition (TGI) of 96% was achieved at 100 mg/kg/day dosage, which was better than Ibrutinib (TGI = 90%) at the same dosage (Fig. 1j). Again, no weight loss or any other obvious signs of toxicity were observed ( Supplementary Fig. S9a). In the TMD8 tumor tissues, the BTK-mediated signaling was dose-dependently inhibited by compd. 85, which was consistent with its in vivo antitumor phenotype and confirmed its on-target effect ( Supplementary Fig. S9b). In order to further evaluate the in vivo efficacy of compd. 85, we then examined it in the REC-1 cell (MCL)inoculated xenograft mouse model, 100 mg/kg/day dosage of compd. 85 slowed down the tumor progression and showed a TGI of 65% without obvious signs of toxicity, which was slightly better than Ibrutinib (TGI = 59%) and Acalabrutinib (TGI = 58%) at the same dosage ( Supplementary Fig. S10a, b). In total, 150 mg/kg/day dosage of compd. 85 could achieve TGI of 79%. Evaluation of drug The disseminated NOD/SCID mice were intravenously inoculated with REC-1 cells and received daily oral administration of CHMFL-BTK-85 dosing at 50 and 150 mg/kg. The total study length was 84 days, and each treatment group contained 4-5 animals. Date are shown as mean ± SEM, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, and ****P < 0.0001 Letter enrichment in the tumor showed that at the same dosage (100 mg/kg) compd. 85 could reach a much higher concentration (2.37 μM) relative to Ibrutinib (1.23 μM) and Acalabrutinib (1.30 μM). This better in tumor PK profile may partially explain the better in vivo efficacy of compd. 85. In the REC-1 cell-mediated orthogonal mouse model of bone marrow engraftment, compd. 85 dose-dependently extended the median survival time of mice to 42 days at 50 mg/kg/day dosage and meanwhile exhibited better efficacy than Ibrutinib (median survival time was 39 days at 100 mg/kg/day dosage). At 150 mg/kg/day dosage, compd. 85 could even extend the median survival time of mice to 63 days ( Fig. 1k and Supplementary Fig. S10c).
In short, we have discovered a novel highly selective covalent BTK kinase inhibitor CHMFL-BTK-85, which did not affect the NKcell-mediated ADCC effects and showed good in vitro and in vivo anti-tumor efficacies. These data support further investigation of CHMFL-BTK-85 as a potential clinical drug candidate, especially in combination with anti-CD20 antibodies, for B-cell non-Hodgkin lymphoma.

DATA AVAILABILITY
The datasets used and/or analyzed to support the findings of this study are available in this paper or the Supplementary Information. Any other raw data that support the findings of this study are available from the corresponding author upon reasonable request.