从BTK原位突变（C481S、A428D等）到体内筛选：一个应对BTK靶向治疗耐药性的综合平台

Bruton's tyrosine kinase (BTK) is a core regulatory molecule in the B-cell receptor (BCR) signaling pathway. It is constitutively expressed in myeloid and lymphoid cells and plays a decisive role in the proliferation, survival, differentiation, activation, and apoptosis of B cells. Abnormal activation of BTK is closely associated with B-cell malignancies such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In hematologic tumors, BTK inhibitors block key steps in the BCR signaling pathway, thereby inhibiting the proliferation and survival of tumor cells. Ibrutinib is the first effective covalent Bruton's tyrosine kinase inhibitor (BTKi), approved by the U.S. Food and Drug Administration (FDA) in 2013, ushering in an era of chemotherapy-free treatment for B-cell malignancies. Second-generation covalent BTK inhibitors, including acalabrutinib, zanubrutinib, and orelabrutinib, have also been approved for marketing in China. The advent of BTK inhibitors has had profound significance for the treatment of B-cell malignancies, improving the treatment modalities and prognosis for patients with B-cell lymphoma. However, traditional BTK inhibitors exhibit poor kinase selectivity and a high incidence of off-target adverse reactions. Moreover, in recent years, although BTK inhibitors (such as ibrutinib and acalabrutinib) have significantly improved patient survival, the issue of drug resistance has become increasingly prominent-approximately 30% of patients develop resistance due to BTK gene mutations (e.g., C481S, T474I, L528W, etc.), limiting therapeutic efficacy. Through breakthroughs in in-situ mutation technology, kyinno has precisely introduced specific BTK mutations directly into the genome of TMD8 cells, preserving the natural expression regulatory mechanisms. This provides a more clinically relevant "touchstone" for analyzing BTK resistance mechanisms and developing targeted therapies. Kyinno has developed over 20 BTK in-situ mutant cell lines (including single mutations such as C481S, T474I, A428D, L528W, and compound mutations). These novel cell models, based on in-situ mutation technology, enable systematic evaluation of the dynamic impact of different mutations on drug binding and guide the structural optimization of next-generation inhibitors. Additionally, we have developed in vitro and in vivo screening platforms based on these mutant cell lines, enabling rapid validation of lead compounds from the molecular to the organism level, thereby accelerating BTK inhibitor development.