Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line.

Chaetominine is a cytotoxic alkaloid that has been demonstrated to promote apoptotic cell death in human leukemia K562 cells. In the present study, chaetominine inhibited K562 (IC 34 nM) and SW1116 (IC 46 nM) cell growth. However, it remains unclear whether the inhibition of cell growth is associated with the cell cycle. To assess this potential relationship, the effect of chaetominine on the cell cycle of K562 and SW1116 cells was examined. Chaetominine treatment caused cell apoptosis and G-phase arrest in SW1116 cells. Conversely, K562 cells underwent S-phase arrest according to flow cytometric analysis. The present study also aimed to elucidate the molecular mechanisms underpinning cell cycle regulation following the incubation of the associated cells with chaetominine. Western blot and reverse transcription-quantitative polymerase chain reaction analyses suggested that chaetominine treatment facilitated the expression of p53, p21, checkpoint kinase 2 (Chk2) and phosphorylated ataxia telangiectasia mutated (p-ATM) and caused a reduction in the mRNA levels of cyclin E and cyclin-dependent kinases (CDKs) 2 and 4. These results suggest that chaetominine may be involved in the regulation of p53/p21 and ATM and Rad3-related (ATM)/Chk2 signaling in SW1116 cells. Previous studies have demonstrated that these signaling pathways are responsible for G-phase arrest. Results of the present study demonstrated that the expression of p-ATR and Chk1 were increased in K562 cells. Additionally, cdc25A levels were decreased, while protein and gene expression levels of cyclin A and CDK2 were repressed. These results elucidated the role of chaetominine in in the regulation of ATR/cdc25A/Chk1 expression in K562 cells. These proteins are thus important determinants in the initiation of S-phase arrest. These data support the hypothesis that chaetominine is a potential anti-cancer therapeutic agent that targets the cell cycle.

Cluster of differentiation 47 (CD47) is a widely expressed self‐protection transmembrane protein that functions as a critical negative regulator to induce macrophage‐mediated phagocytosis. Overexpression of CD47 enables cancer cells to escape immune surveillance and destruction by phagocytes both in solid tumours and leukaemia. The usefulness of anti‐CD47 antibody has been demonstrated in multiple immunotherapies associated with macrophages. However, antigen sinks and toxicity induced by inadvertent binding to normal cells restrict its clinical applications. Here, a novel anti‐human CD47 antibody, 4D10, was generated, and its variable regions were grafted onto a human IgG4 scaffold. Compared with the anti‐CD47 antibody Hu5F9, the resulting chimeric antibody (c4D10) has consistently demonstrated good tolerance in in vitro and in vivo toxicity studies. Additionally, c4D10 showed effective therapeutic potential through inducing the eradication of human cancer cells. Thus, c4D10 is a promising candidate therapeutic antibody with higher efficacy and reduced side effects compared to earlier antibodies, and its use may reduce the dose‐limiting toxicity of CD47 antagonists for immunotherapy.

The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c and SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no detectable binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. The final lead is a humanized antibody which behaves well in stability test. Lab scale processes are successfully developed and confirmed. The safety and efficacy will be further validated. Citation Format: Teddy Yang, Jing Gao, Jingyun Yao, Dongxu Wang, Ken Dai. Discovery of Anti-NKG2A antibody from immunized phage display approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5653.

NK cells express activating receptors such as CD16a (FcγRIIIA), NKG2D, SLAM family members and the natural cytotoxicity receptors (NCRs). Full activation of NK cells require co-engagement of different activating receptors. NKp46, a member of NCRs, is a 46 kDa glycoprotein and belongs to immunoglobulin (Ig) superfamily, NKp46 is frequently expressed on tumor infiltrating lymphocytes. Activation of NKp46 with antibody triggers not only NK cell cytotoxicity but also cytokine release. Taken together, NKp46 is a promising target for anti-tumor therapy. Here we described the discovery of fully human NKp46 antibody by phage display approach with common light chain and naïve human B cell library. Bispecific antibody has been researched for many years and very limited number of bispecific antibodies are currently in the market. Heavy and light chain miss pairing is the bottleneck of bispecific antibody discovery and development. To overcome the hurdle, we generated a fully human common light chain phage display library with two commonly used light chain sequences combined with heavy chain fragment from human naïve scFv library. Two light chain sequences were selected based on the approved antibodies sequences and the expression levels. The phage library was then used for panning against recombinant human NKp46 extracellular domain protein and CHOK1 cells overexpressing human NKp46. After conversion to full IgG format, 4 candidates bind specifically to NKp46 and cross react with both human and cyno NKp46. Affinities of the candidates were comparable to bench mark antibodies as determined by FACS. To summarize, we have generated fully human common light chain phage display library that can be used for bispecific antibody discovery. The anti-NKp46 antibodies discovered can be further developed for NK cell engager and NK cell redirecting bispecific antibody. Citation Format: Teddy Yang, Seng Zhu, Jing Gao, Jingyun Yao, Xumin Gong. Discovery of NKp46 antibody for NK cell engager using common light chain phage display approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 524.