Addition of BTK inhibitor orelabrutinib to rituximab improved anti-tumor effects in B cell lymphoma

Abstract

Bruton tyrosine kinase (BTK) inhibitor ibrutinib has been validated as an effective drug to treat B cell malignancies. Combined therapies comprising ibrutinib and anti-CD20 antibodies like rituximab were designed as a backbone in many clinical trials. However, the off-target inhibition of ibrutinib on interleukin-2 (IL-2)-inducible T cell kinase (ITK) may reduce rituximab's antibody-dependent cellular cytotoxicity (ADCC) efficacy. Orelabrutinib (Orel), a novel BTK inhibitor, was designed with high selectivity to BTK. In our study, we demonstrated in preclinical models that orelabrutinib in combination with rituximab could preserve NK-cell-mediated ADCC induced by rituximab and enhanced the apoptosis of tumor cells in vitro. The addition of orelabrutinib to rituximab had produced promising combined anti-tumor effects in B cell lymphomas in vivo. Collectively, combination therapy of orelabrutinib with rituximab would benefit patients with B cell lymphoma, especially those with relapsed or refractory disease.

Keywords: B cell lymphoma; BTK inhibitor; anti-CD20 antibody; combined effects.

Conflict of interest statement

The authors declare no competing interests.

© 2021 The Authors.

Figures

Graphical abstract

Figure 1

Orelabrutinib inhibited B cell lymphoma cell proliferation in vitro (A) B cell lymphoma cell lines (TMD8, HBL-1, SU-DHL-6, WSU-NHL, Mino, Z138, and Raji) were treated with indicated concentrations of orelabrutinib (Orel) or ibrutinib (IBR) for 72 h. Cell viability was measured by Cell Titer-Glo luminescent cell viability assay. Data are shown as mean ± SD of three biological replicates. (B) Western blot analysis for phosphorylation levels of Bruton’s tyrosine kinase (BTK) and its related signaling pathway proteins in Z138 and HBL-1 cells after IBR or Orel treatment for 2 h. (C) The relative phosphorylation levels of signaling proteins from three biologically repeated experiments were quantified by measuring the relative intensity of phosphorylated bands to the β-actin bands. ∗p 

Figure 2

Orelabrutinib lacked off-target inhibition effect on cellular IL-2-inducible T cell kinase (ITK) compared with ibrutinib (A and B) p-ITK and p-IκBα were assessed of proteins obtained from YT and NK-92 cells (two kinds of NK/T lymphoma cell lines expressing ITK) treated with ibrutinib or orelabrutinib for 2 h. PBMCs obtained from healthy adults were also used to verify the different effects of orelabrutinib and ibrutinib on ITK inhibition. After being pretreated with or without CD3/CD28-activating agent, PBMCs were treated with different concentrations of BTK inhibitors, orelabrutinib or ibrutinib. Proteins were extracted from pre-treated cells, and proteins of p-ITK and its associated downstream pathway were analyzed by western blot. These western blots were quantified from three biologically repeated tests. ∗p 

Figure 3

Orelabrutinib preserved rituximab-mediated cytotoxicity (A) NK cells pretreated with 1 μmol/L BTK inhibitor, ibrutinib, or Orel were co-cultured with B lymphoma cells at different E:T ratio in the presence of rituximab (RTX) at a concentration of 10 μg/mL or not. (B) Pretreated NK cells with 1 μmol/L ibrutinib or Orel were co-cultured with B lymphoma cells at 5:1 in the presence of 10 μg/mL rituximab or PBS. (C) Rituximab (10 μg/mL) or PBS was combined with the increasing concentration of Orel to evaluate tumor cell lysis induced by NK-cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Cell supernatant was collected after 4-h co-culture and then was measured for lactate dehydrogenase (LDH) with Non-Radioactive Cytotoxicity Assay. Each cell line was biologically tested for at least 3 times. ∗p 

Figure 4

Orelabrutinib enhanced apoptosis of tumor cells induced by rituximab (A and B) B cell lymphoma cells (TMD8 and Z138) were treated with increasing concentration of ibrutinib or Orel for 48 h; after then, cells were stained with PI and Annexin V-FITC. Statistical analysis was performed using one-way ANOVA. Values from three independent experiments are presented as percentages of vehicle in mean ± SD. ∗p + cells were gated to analyze tumor apoptosis by flow cytometer. The column graph represents the percent of cell apoptosis for each group. Statistical analysis was performed using one-way ANOVA. Values of three biologically independent tests are presented as percentages of vehicle in mean ± SD. ∗p < 0.05 compared with vehicle group; ∗∗p < 0.01 compared with control group. #p < 0.05 compared with rituximab group; ##p < 0.01 compared with rituximab group.

Figure 5

Orelabrutinib combined with rituximab effectively inhibited tumor growth in animal models (A) CB.17/SCID mice were inoculated subcutaneously with 1 × 107 TMD8 cells in 0.1 mL PBS with Matrigel (1:1 ratio); n = 5 per group. (B) 1 mm3 patient tumor tissue was inoculated subcutaneously into the right flank of per mouse. n = 6 per group. Orelabrutinib (10 mg/kg, bid) and rituximab (200 μg/dose, weekly) were administered to the tumor-bearing mice when the tumor volume achieved 150 mm3. Body weight and tumor volume of mice were measured every other day during treatment. (C) Splenocytes of mice from different group were co-cultured with MCL cell line Z138 for 4 h. NK cell cytotoxic activity was then measured using the Nonradioactive Cytotoxicity Assay Kit. Each column represents the mean value of the triplicate experiments. (D) Hematoxylin and eosin (H&E) staining of tumor tissues from TMD8 cell-line-derived xenograft (CDX) tumor model and patient-derived xenograft (PDX) model. Scale bar: 70 μm. ∗p < 0.05 compared with control group, ∗∗p < 0.01 compared with control group, and ∗∗∗p < 0.001 compared with control group; #p < 0.05 compared with rituximab group and ###p < 0.001 compared with rituximab group.

Figure 6

Combined effects of orelabrutinib and rituximab in vivo (A and B) Apoptosis of tumor tissues from TMD8 tumor model was assessed by the terminal deoxynucleotidyl transferase nick-end-labeling (TUNEL) assay, and the nuclei were counterstained with DAPI. Representative images show apoptotic (fragmented) DNA (green staining) and the corresponding cell nuclei (blue) staining. Scale bar: 50 μm. Results are expressed as mean ± SD (n = 3 biologically independent samples). (C and D) Ki67, granzyme B, and NKp44 were assessed by immunohistochemistry of TMD8 tumor tissue. Scale bar: 60 μm. The data show the density of positivity cells for each section. Student’s t test was performed for statistical analysis. Values present as percentages of control group in mean ± SD (n = 3 biologically independent samples), ∗∗p