
==== Front
Neoplasia
Neoplasia
Neoplasia (New York, N.Y.)
1522-8002
1476-5586
Neoplasia Press

S1476-5586(24)00095-2
10.1016/j.neo.2024.101053
101053
Original Research
A highly selective PI3Kδ inhibitor BGB-10188 shows superior preclinical anti-tumor activities and decreased on-target side effects on colon
Yang Xiao
Bai Huichen
Yuan Xi
Yang Xiaolong
Liu Ye
Guo Mingming
Hu Nan
Jiang Beibei
Lian Zeqin
Ma Zhilong
Wang Jingyuan
Sun Xuebing
Zhang Taichang
Su Dan
Wu Yue
Li Jing
Wang Fan
Wang Zhiwei
Wang Lai
Liu Xuesong
Song Xiaomin xiaomin.song@beigene.com
⁎
BeiGene Global Research, Beijing 102206, PR China
⁎ Corresponding author. xiaomin.song@beigene.com
10 9 2024
11 2024
10 9 2024
57 10105318 6 2024
5 9 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• BGB-10188 is a novel PI3Kδ inhibitor with high selectivity and potency and showed long-lasting and strong target inhibition activities in vivo.

• BGB-10188 showed significant anti-tumor effects in malignant B cell xenograft models as a monotherapy or in combination of zanubrutinib.

• BGB-10188 showed significant Treg inhibition in blood but not in colon, along with less drug accumulation in colon compared with idelalisib.

PI3Kδ is a key signal transduction molecule in normal and malignant B cells, as well as in T-regulatory cells, making it a promising target for treatment of hematologic malignancies through both direct killing and anti-tumor immunity regulation. BGB-10188 is a highly selective inhibitor of PI3Kδ, showing more than 3000 folds selectivity over other PI3K isoforms and no significant inhibition across tested kinases. BGB-10188 potently inhibited PI3Kδ with IC50s ranging from 1.7-16 nM through various in vitro assays and showed a long-lasting and strong target inhibition in mouse B cells in vivo. BGB-10188 showed significant antitumor effects in human B cell lymphoma xenograft models as single agent or in combination with the BTK inhibitor zanubrutinib. BGB-10188 showed significant Treg inhibition in blood but not in colon, along with less drug accumulation in colon compared with idelalisib, which is an approved PI3Kdelta inhibitor with high incidence of gastrointestinal side effects in clinic. In summary, BGB-10188 is a novel PI3Kδ inhibitor with high selectivity, potency and improved safety profile shown in preclinical studies, which is showing the potential as a best-in-class PI3Kδ inhibitor.

Keywords

BGB-10188
PI3Kδ
B cell malignancy
Treg inhibition
Colon distribution
==== Body
pmcIntroduction

PI3Kδ is one of the four isoforms (PI3Kα, β, δ and γ) of PI3K class I family [1]. It is restrictedly expressed in leukocytes and plays important roles in both the adaptive immune system including B and T cells [2], and innate immune system in regulating mast cells [3] and myeloid cells such as neutrophils and macrophages [4,5]. PI3Kδ regulate multiple functions of normal B cells, including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses, and maintenance of B cell homeostasis [6]. In B cell malignancies, constitutive activated PI3Kδ can promote malignant B-cell proliferation, growth, survival, adhesion, and homing, making it an attractive drug target [7,8]. Given the pivotal function of PI3Kδ in B cell growth and development, multiple PI3Kδ inhibitors have been tested in clinic. However, clinical data also indicated that serious gastrointestinal symptoms and infections are common among patients receiving PI3Kδ inhibitor therapy [[9], [10], [11], [12]], which were warned by FDA. The four once launched PI3Kδ inhibitors, including idelalisib (δ/γ), duvelisib (δ/γ), umbralisib (δ/casein kinase-1ε (CK1ε)) and copanlisib (pan PI3K) for the treatment of relapsed/refractory indolent Non-Hodgkin lymphoma (NHL) malignancies after at least two prior therapies, have voluntarily withdrawn their applications in relapsed follicular lymphoma (FL) and small lymphocytic lymphoma (SLL). These toxicities are likely due to on target immune mediated effects by PI3Kδ inhibition. Many hypotheses were raised and verified in PI3Kδ kinase dead knock-in mice, mainly focus on the decrease or function impairment of T regulatory cells (Tregs) [13] and amplified inflammatory regulation by macrophages [14,15].

BGB-10188 is a highly selective PI3Kδ inhibitor that is being investigated as a monotherapy, and in combination with BTK inhibitor zanubrutinib or PD-1 antibody tislelizumab in patients with B cell malignancies or solid tumors in a Phase I/II clinical trial (NCT04282018). BGB-10188 showed no significant inhibition on 376 protein kinases and 17 lipid kinases and exhibited more than three-thousand folds selectivity over PI3Kα, PI3Kβ, and PI3Kγ. In this work, we disclosed the preclinical profile of BGB-10188 including biochemical and cellular potency, in vivo PK/PD correlation, in vivo efficacy in malignant B cell xenograft models as a monotherapy or in combination of zanubrutinib. To evaluate the potential colitis occurrence risk, we compared the blood, and colonic Treg inhibition, and tissue distribution of BGB-10188 with idelalisib in rodents. The preclinical results indicated that BGB-10188 is a highly potent and effective PI3Kδ inhibitor with potential improved safety profile warranting the clinical development of the compound both as monotherapy and as a combination partner with BTK inhibitors.

Methods

See the Supplementary Appendix for a detailed description of the methods.

Kinase activity inhibition on PI3Kδ, PI3Kα, PI3Kβ and PI3Kγ. PI3K p110δ/p85α (Promega, Cat#: V1771), p110α/p85α (Promega, Cat#: V1721), p110β/p85α (Promega, Cat#: V1751) and p110γ (ThermoFisher Scientific, Cat#: PV4786) kinase activity inhibition by BGB-10188 (BeiGene (Beijing) Co. Ltd) and idelalisib (Shanghai Scochem Tecknology) were tested in ADP-GloTM Kinase Assay.

Cellular activity against PI3Kδ, PI3Kα, PI3Kβ and PI3Kγ. pAKT inhibition of BGB-10188 in Raji, Farage, mouse B cells, SK-OV-3, PC-3 and Raw264.7 cells were tested in serum-free or serum-containing conditions through a TR-FRET-based method [16] using a phospho-AKT (Ser473) cellular assay kit (Cisbio, 64AKSPEH rev03) or FACS detection. Before detection, Raji cells, mouse B cells and Raw264.7 were stimulated by anti-human IgM, anti-mouse IgD and human C5a respectively.

Anti-proliferation activity assays. The growth-inhibitory activity of BGB-10188 on a panel of human B-cell lymphoma cell lines and combination effect of BGB-10188 and zanubrutinib in MINO and TMD8 cells were determined using the CellTiter-Glo luminescent cell viability assay (Promega, Catalog# G7573) following a 6-day exposure to the compounds.

Animal purchase and husbandry. BALB/c mice, non-obese diabetic (NOD)-severe combined immune-deficient (SCID) mice (NOD SCID) and SCID Beige mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. NCG mice were purchased from Jiangsu GemPharmatech Co., Ltd. Immunodeficient mice for efficacy studies used in this work were pre-treated with cyclophosphamide and disulfiram for myeloablation. All animals were maintained under specific pathogen free (SPF) “full barrier” condition with free access to food and water. Operations regarding to general husbandry, quarantine, housing and sanitation, food and water, environmental conditions were complied with BeiGene's Institutional animal care and use committee (IACUC).

In vivo pharmacodynamics assay. Female BALB/c mice treated with BGB-10188 were euthanized at 0.5, 3, 7 and 24 h for plasma and whole blood collection. The plasma was collected for BGB-10188 concentration measurement and the whole blood was stimulated by mouse IgD Monoclonal Antibody (10.4.22) (eBioscience, Catalog# 16-5924-85) for p-AKT detection using Guava easyCyte HT flow cytometer.

In vivoactivity on Treg. Tregs were collected in tumors from A20 syngeneic model, and in blood and colon tissues from BALB/c mice not bearing tumor. Mice were treated with BGB-10188 and idelalisib for 22 days for Treg detection in A20 tumor and for 6 days for blood and colon Treg evaluation in BALB/c mice not bearing tumor. Tumors were processed to single-cell suspension by collagenase digestion solution, and the lamina propria lymphocytes from colon tissues were obtained by modified methods described from Han's group [17]. Cells were finally analyzed by FACS using BD FACSCelesta™ Flow Cytometer.

Tissue distribution of PI3Kδ inhibitors in rodents. The animals treated with the PI3Kδ inhibitors were euthanized at 0.5, 2, 4 and 8 h in mice or 0.5, 2, 7 and 24 h in rats after administration. Tissues from mice (plasma, tumor, colon, spleen and draining lymph nodes) and SD rats (plasma, small intestine, colon, spleen and submandibular lymph nodes) were carefully collected and washed with PBS to remove blood. Drug concentration in tissue homogenate was detected by LC-MS/MS.

Statistics and reproducibility. As indicated in the figure legends, all data used for statistical analysis were performed in more than three biological replicates unless stated otherwise. P value was calculated using one-way ANOVA to analyze the log transformed value between groups, including tumor volume, proliferating rate of cells and Treg percentage, etc. Log rank test was used to compare the survival curves between groups. p < 0.05 was statistically significant. Loewe model [18] is used to analyze the synergistic effect of two drugs, the interaction between two drugs is likely to be additive or synergistic when the synergy score is from -10 to 10 or larger than 10.

Results

BGB-10188 is a highly potent and selective PI3Kδ inhibitor

The biochemical potency of BGB-10188 and idelalisib on PI3K kinases inhibition were tested in ADP GloTM based assays. BGB-10188 is a highly selective inhibitor of PI3Kδ with IC50 of 1.7 nM, which is 4900, 5100 and 3800-fold selective over PI3Kα, PI3Kβ, and PI3Kγ. Idelalisib demonstrated similar PI3Kδ inhibition with IC50 of 2.3 nM, but showed only 430, 360 and 52-fold selectivity over PI3Kα, PI3Kβ, and PI3Kγ (Supplementary Table S1). BGB-10188 was also profiled against additional 13 human lipid kinases and 376 protein kinases at 1 μM by Reaction Biology Corporation and displayed ≤ 31 % inhibition against all these kinases (data not shown).

To evaluate the cellular activity on PI3Kδ inhibition, the inhibitory effect of BGB-10188 on AKT phosphorylation (pAKT) was investigated in a panel of cells. In Raji cells stimulated with anti-IgM antibody which crosslinked with the cell surface B cell receptor (BCR) to activate downstream pAKT through PI3K, BGB-10188 potently inhibited the AKT phosphorylation with an IC50 of 16 nM (Supplementary Table S2). BGB-10188 was also tested in Farage cells with constitutive AKT phosphorylation spiking in human whole blood, as well in mouse whole blood in which the primary mouse B cells were activated by anti-IgD antibody, IC50s of 9.2 nM and 68.5 nM (Supplementary Table S2) in Farage cells and mouse B cells were found, respectively, indicating the potent cellular activities of BGB-10188 in both human and mouse whole blood.

To determine the selectivity of BGB-10188 at the cellular level, the effect of BGB-10188 on pAKT was investigated in SK-OV-3 (PI3Kα), PC-3 (PI3Kβ) and C5a-stimulated Raw264.7 (PI3Kγ) cells. BGB-10188 did not show obvious inhibition in PI3Kα and PI3Kγ mediated AKT phosphorylation in SK-OV-3 and Raw264.7 cells, with IC50s greater than 30 µM and 10 µM, respectively, and only showed weak inhibitory effect in PC-3 cells, with an IC50 of 3478 nM (Supplementary Table S3). These results demonstrate that BGB-10188 is a potent and selective PI3Kδ inhibitor over the other three Class I PI3K isoforms.

BGB-10188 showed long-lasting and strong target inhibition activities in vivo

The pharmacokinetics (PK) and pharmacodynamics (PD) relationship of BGB-10188 was evaluated in a mouse B cell-based in vivo assay. The results showed that oral administration of BGB-10188 inhibited the phosphorylation of AKT in mouse peripheral blood B cells in a dose-dependent manner (Fig. 1A), with a maximum inhibition of 78 %. The IC50 of BGB-10188 obtained from the PK/PD correlation analysis in this study was ∼84.8 nM, similar to that detected in the whole blood B cells with in vitro BGB-10188 treatment (Supplementary Table S2). The plasma concentrations of BGB-10188 covered the IC50 for more than 7 h at the dosage of 10 mg/kg and above (Fig. 1B), indicating a sustained target inhibition activities in mice peripheral blood of BGB-10188.Fig. 1 BGB-10188 showed potent BCR downstream PI3Kδ/AKT signaling inhibition in normal mouse B cells. BALB/c mice were treated with 0, 0.3, 3, 10 and 30 mg/kg of BGB-10188 once and euthanized at different time points after dosing as indicated. Whole blood was collected for both pAKT detection and BGB-10188 concentration measurement. (A) pAKT Inhibition of BGB-10188 in mouse peripheral blood B cells. Whole blood was stimulated by anti-mouse IgD antibody for 7 min at 37 °C for activating B-cell receptors, thus inducing the phosphorylation of AKT. pAKT level in mouse peripheral blood B cells (IgD+B220+ cells) was detected by flow cytometry. (B) The plasma concentration of BGB-10188 measured by LC-MS/MS.

Fig. 1

BGB-10188 showed anti-tumor activity in B-cell lymphoma cell lines and Farage xenograft models

PI3Kδ expression is restricted to immune cells and plays a critical role in regulating B-cell signaling and function. Constitutive activation of the PI3Kδ/AKT pathway was found in aberrant activated B cells, and therefore PI3Kδ is a promising therapeutic target for B-cell malignancies [2,19,20]. In order to assess its therapeutic potential in B-cell lymphomas, the effect of BGB-10188 on cell proliferation was investigated in a total of 21 B-cell lymphoma cell lines, BGB-10188 potently inhibited the proliferation of 5/13 diffuse large B-cell lymphoma (DLBCL) lines, and 2/6 mantle cell lymphoma (MCL) lines, with IC50s ranging from 3.5 to 535 nM. The most sensitive cell lines included SU-DHL-4, Pfeiffer, and TMD8, with IC50 less than 10 nM. Moderate sensitivity to BGB-10188 was observed in 3/13 DLBCL and 1/6 MCL cell lines, with antiproliferative IC50 at micromolar level (Fig. 2A). These findings suggest that BGB-10188 inhibits the proliferation of a subset of DLBCL and MCL tumor types.Fig. 2 Inhibitory effects of BGB-10188 on B-cell malignancy. (A) IC50 of BGB-10188 on B-cell lymphoma cell proliferation. A panel of human B-cell lymphoma cell lines were seeded into 96-well plates at a density of 3 × 103 per well in the full RPMI-1640 medium. Cells were then treated with a titration of BGB-10188 for 6 days and the cell viability was detected by CellTiter-Glo. (B) Efficacy in human Farage subcutaneous xenograft model. Farage tumor cells (3 × 106/mouse) were implanted subcutaneously in female NCG mice. On Day 4 after tumor inoculation, animals were treated twice daily (BID) with vehicle (0.5 % MC) and BGB-10188 (10, 30 and 100 mg/kg) for 22 days (n = 10). (C) Efficacy in human Farage subcutaneous xenograft model with co-injection of human PBMC (hPBMC). Farage tumor cells (3 × 106/mouse) and hPBMC (6 × 105/mouse) were mixed and implanted subcutaneously in female NCG mice. On Day 4 after tumor inoculation, animals were treated BID with vehicle (0.5 % MC) and BGB-10188 (10, 30 and 100 mg/kg) for 21 days (n = 10). (D) Efficacy in human Farage disseminated xenograft model. Farage tumor cells (3 × 106/mouse) were injected via tail vein into female SCID Beige mice. On Day 1 after inoculation, mice were treated twice daily with BGB-10188 (10, 30 and 60 mg/kg; all dosing were stopped on Day 63). The survival time was defined as the time from the day of tumor cell inoculation to the day of animal death or euthanization according to Beigene's IACUC. Kaplan-Meier curves were generated using survival time in days for each mouse (n = 15).Tumor volume was compared on logarithmic scale between each treatment group and the vehicle group using one-way ANOVA with Dunnett's multiple comparisons test, *p < 0.05, ***p < 0.001, ****p < 0.000. The survival curve between treatment group and the vehicle group was analyzed using Log rank test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

The DLBCL cell line Farage, sensitive to BGB-10188 in vitro, was used in xenograft models to test the efficacy of the compound in vivo. BGB-10188 showed significant anti-tumor activity at the doses of 30 and 100 mg/kg with the tumor growth inhibition (TGI) of 44 % and 48 % respectively in Farage subcutaneous xenograft model (Fig. 2B). No significant changes of animal body weight or serum ALT and AST was identified in all treatment groups throughout the study (Supplementary Fig. 1A, B). Intermittent dosing with 30 mg/kg BGB-10188 twice daily (BID) for 3, 4 and 5 days each week also induced significant tumor growth inhibition in the same model, but slightly weaker than continuous daily treatment (Supplementary Fig. S2). In the humanized model with Farage and human PBMC (hPBMC) co-transplantation subcutaneously, BGB-10188 showed better anti-tumor activity than in the Farage-alone xenograft model, with TGI of 56 %, 71 % and 89 % at 10, 30 and 60 mg/kg, indicating that BGB-10188 has dual effect on both tumor and immune cells and could achieve stronger anti-tumor activity with the existence of immune system (Fig. 2C).

B cell malignancies are usually located in lymphoid organs and the potential difference in tumor microenvironment existed between subcutaneous and orthotopic sites. In addition, the exposure of BGB-10188 in orthotopic tumor site could be different and result in different anti-tumor effects. We thus established the disseminated Farage xenograft model by injecting Farage cells into tail veil. In this model, body weight loss was found from day 21 post the cell injection along with the disease progress (Supplementary Fig. S1D, E), treatment with BGB-10188 significantly prolonged animal survival with the median survival of 31, 35 and 45 days compared to the vehicle group of 28 days and increase in life span of 10.7 %, 25.0 % and 60.7 % at 10, 30 and 60 mg/kg, respectively (Fig. 2D). It is noticeable that three mice (1 with 30 mg/kg and 2 with 60 mg/kg) were still alive with recovered body weight from disease progress when we terminated this study, further pathological detection by HE staining demonstrated that organs in all the three mice were tumor-free (Supplementary Fig. S1F).

BGB-10188 showed both in vitro and in vivo combinational anti-tumor activity with BTK inhibitor in DLBCL and MCL

BTK is another kinase positioned within the BCR signaling cascade playing a crucial role in B cell development [21,22]. The additive anti-tumor effects in B cell malignancies by combination of BTK and PI3Kδ inhibitors had been validated pre-clinically [23,24] and tested in on-going clinical trials. The combinational anti-tumor activity of BGB-10188 and BTK inhibitor zanubrutinib was evaluated in MCL and DLBCL cell lines and xenograft models in this work.

In a six-day cell viability assay, the IC50s in TMD8 cells were 133 nM and 0.7 nM for BGB-10188 and zanubrutinib, respectively, and the IC50 for zanubrutinib can be achieved as low as 0.07 nM when treated the cells with the combination of 300 nM BGB-10188. Loewe model [18] is used to analyze the combination effect of two drugs on cell viability, and BGB-10188 ranging from 10 to 3000 nM combining with zanubrutinib (0.1-1 nM) showed the synergistic effect (Fig. 3A-B). The synergized anti-proliferation effect was also found in MINO cells (Fig. 3C-D). We then checked the downstream signaling of PI3K and BTK pathways after treatment with BGB-10188 and zanubrutinib in these two cell lines, besides the phosphorylation of AKT and BTK, the combination treatment of BGB-10188 and zanubrutinib showed an enhanced downregulation in pIKBα/IKBα, pERK or cMyc than each single agent treatment at 4 or 24 h by Western Blot analysis (Fig. 3E-F). The c-Myc, MAPK and NF-κB pathways were reported to be related with PI3Kδ inhibitor resistance [23,25], these results demonstrated a strong rational for the combination use of the two drugs.Fig. 3 BGB-10188 and zanubrutinib synergized in DLBCL TMD8 cell line and MCL MINO cell line. (A, C) Inhibition of TMD8 (A) and MINO (C) cell proliferation by co-treatment with BGB-10188 and zanubrutinib at a series of corresponding concentrations in a 6-day viability assay. (B, D) The synergy score analyzing the synergistic effect of BGB-10188 and Zanubrutinib in TMD8 (B) and MINO (D) cells. The synergy score is calculated by Loewe model and the interaction between two drugs is likely to be additive or synergistic when synergy score is from -10 to 10 or larger than 10. (E, F) Western Blot analysis of PI3Kδ and BTK downstream signaling. TMD8 (E) and MINO (F) cells were seeded into 6-well plates at a density of 1 × 106 per well in full RPMI-1640 medium. Cells were co-treated with 100 nM BGB-10188 and 10 nM zanubrutinib for 4 or 24 h respectively, dimethyl sulfoxide alone was added to the negative control cells.

Fig. 3

We then tested the combination anti-tumor effects of BGB-10188 and zanubrutinib in subcutaneous xenograft models using TMD8 (ABC DLBCL), Farage (GCB DLBCL), MINO (MCL) and JeKo-1 (MCL) cell lines. The single agent BGB-10188 at 10 and 30 mg/kg, and zanubrutinib at 7.5 mg/kg were orally treated twice daily in Farage, MINO and JeKo-1 models. In TMD8 model, besides the two doses of BGB-10188 in the other models, an additional higher dose of 100 mg/kg was treated and zanubrutinib was treated with 2.5 mg/kg. In the TMD8 model, single treatment of BGB-10188 at 10, 30 and 100 mg/kg showed antitumor effects with TGI of 26 % to 43 % and zanubrutinib at 2.5 mg/kg induced TGI of 68 % on Day 14. Improved antitumor activities were found in the combined treatment groups with the TGI of 77 %, 95 % and 101 % for 10, 30 and 100 mg/kg of BGB-10188, respectively, and further improved antitumor activities and tumor shrinkage were observed with a prolonged treatment (Fig. 4A). The enhanced anti-tumor effects were also found for BGB-10188 at 10 and 30 mg/kg in combination with zanubrutinib comparing with each single agent in the other three xenograft models (Fig. 4B-D).Fig. 4 BGB-10188 showed combinational anti-tumor activity with BTK inhibitor in DLBCL and MCL xenograft models. All mice were pre-treated with cyclophosphamide (prepared in saline, 100 mg/kg i.p.) and disulfiram (prepared in 0.8% Tween-80 in saline, 125 mg/kg p.o., one hour after each dose of cyclophosphamide) once daily for two days. (A) Farage cells (3 × 106/ mouse) were inoculated subcutaneously in NCG mice. The mice in Farage xenograft model were divided into 6 groups according to the body weight on day 4 post cyclophosphamide and disulfiram administration. The mice were treated BID with BGB-10188 (10 and 30 mg/kg), zanubrutinib (7.5 mg/kg), and the combinations of the two compounds as indicated. (B) TMD-8 cells (1 × 107/mouse) were implanted subcutaneously in NOD/SCID mice. The mice were randomized into 8 groups when the average tumor volume achieved 250 mm3, and treated BID with BGB-10188 (10, 30 and 100 mg/kg), zanubrutinib (2.5 mg/kg), and the combinations of the two compounds as indicated. (C) Mino (1 × 107/mouse) and (D) JeKo-1 cells (1 × 107/mouse) were implanted subcutaneously in NOD/SCID mice. The mice were randomly allocated according to inoculation order and treated BID from the next day post grouping with BGB-10188 (10 and 30 mg/kg), zanubrutinib (7.5 mg/kg), and the combinations of the two compounds as indicated. n = 10 for each group in all four studies. Tumor volume was compared on logarithmic scale between each treatment groups using one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig 4

BGB-10188 induced Treg inhibition in blood but not in colon

PI3Kδ plays essential roles on the homeostasis and function of Foxp3+ Treg [26,27], we then validated that Treg isolated from the p110δD910A/D910A (PI3Kδ kinase dead) transgenic mice showed significant decreased proliferation than that from the wild type littermates, which was not found for non-Treg CD4+ and CD8+ T cells (Fig. 5A–C). It was reported that the loss of PI3Kδ activity especially by specific deletion in Tregs can restrict the growth of transplanted tumors in mice [13]. The inhibitory effect of BGB-10188 on tumoral Treg was next validated in the A20 mouse B cell lymphoma model, BGB-10188 at 60 mg/kg decreased the tumoral Treg, showing an indirect anti-tumor effect by regulating the anti-tumor immunity (Fig. 5D-E).Fig. 5 BGB-10188 showed inhibition in both Treg percentage and Treg function in mouse blood but not in colon tissues where idelalisib does. (A–E) PI3Kδ deficiency or inhibition by BGB-10188 led to decreased Treg proliferation and tumor growth inhibition. (A–C) Effects of PI3Kδ deficiency on Treg and T cell proliferation. Splenocytes from P110δWT/WT, P110δD910A/WT, P110δD910A/D910A mice (n = 12, 6 female plus 6 male) were stained with CFSE (Cat:65-0850-84, Life Technologies) and activated by soluble 10 µg/ml anti-CD3 (145-2C11, Biolegend), 1 µg/ml anti-CD28 (16-0281-86, eBioscience™) and 500 IU/mL IL-2 in the u-bottom 96-well plates at 37°C for 3 days. Cell percentage with low CFSE fluorescence of Treg (A, CD4+FoxP3+ population), non-Treg CD4+ cells (B, CD4+FoxP3-) and CD8+ cells (C) detected by FACS were indicated as the proliferation rate. (D-E) Efficacy (D) and tumor Treg inhibition (E) of BGB-10188 in A20 model. A20 tumor cells (2.5 × 106/mouse) were implanted subcutaneously in female BALB/c mice. On the next day after tumor inoculation, animals were randomly assigned according to the body weight and treated BID with vehicle (0.5 % MC) and 30 mg/kg or 60 mg/kg BGB-10188 for 22 days, n = 12. At the end of the study, mice were sacrificed to collect tumor and isolate the tumor infiltrated lymphocytes for Treg detection by flow cytometry. Log transformed tumor volume on the last day or Treg cell (Foxp3+) percentages between groups were analyzed by one-way ANOVA, *P < 0.05 was considered statistically significant. (F–K) BALB/c mice were orally treated with 30 or 60 mg/kg BGB-10188, and 60 or 100 mg/kg idelalisib. The mice were sacrificed after 6 days’ drug administration to collect blood (F–H) and colon (I–K) for immune cell profiling by flow cytometry.

Fig 5

Considering the systemic immune regulatory effects of Treg in the whole body, inhibiting Tregs can also break the protective effect in normal tissues against inflammatory reactions. PI3Kδ-deficient mice developed colitis partially due to the reduced Treg functions in colon [27]. Consistently, PI3Kδ inhibitors exhibited high occurrence of GI related AEs, which is one of the hazard factors to limit their clinical application. Idelalisib was once approved by FDA and reported to induce sever colitis in patients [9,10,12]. We thus compared BGB-10188 and idelalisib in Treg inhibition in blood and colon in BALB/c mice. BGB-10188 at 30 and 60 mg/kg, the doses achieving anti-tumor effects in varieties of efficacy models, induced decreased Treg percentage in blood but not in colon (Fig. 5F and I). CTLA4 which was well demonstrated to be involved in crucial function of Tregs [28] was also reduced only in blood Tregs, indicating a significant Treg inhibition by BGB-10188 in both Treg number and Treg suppressive function in blood whereas not in colon (Fig. 5G and J). ST2+ Tregs, identified as tissue resident Tregs which are critical against tissue inflammation and reported to be inhibited by PI3Kδ inhibitor in mice colon before [29], were significantly inhibited by BGB-10188 in blood as well but not affected in the colon. On the contrary, the Treg inhibition regarding the number, function and ST2 subtype by idelalisib showed no tissue specificity in blood and colon (Fig. 5H and K). These results revealed that BGB-10188 impaired Treg functions peripherally without affecting Tregs in colon tissues in mice, which is different from idelalisib.

BGB-10188 has a lower relative colon distribution than idelalisib

To better understand the reason for Treg inhibition with different tissue specificity between BGB-10188 and idelalisib, we checked the tissue distribution of these compounds in BALB/c mice. The drug exposures within 24 h in colon compared to that in spleen, draining lymph nodes and tumor (subcutaneous A20) were 1.7-, 0.3- and 5.8-fold, respectively for BGB-10188 and 47.5-, 6.6- and 47.5-fold, respectively for idelalisib. These results indicated an significantly higher drug concentration in colon than tumor and lymphoid organs for idelalisib, However, BGB-10188 has comparable drug concentrations in colon, tumor and lymphoid organs (the target organs for lymphomas treatment), leading a potential better therapeutic window, which is also consistent with its weaker Treg inhibition in colon (Fig. 6A-B).Fig. 6 BGB-10188 showed lower relative colon exposure than idelalisib in mice and rats. (A, B) The tissue PK study in A20 mouse lymphoma model. A20 tumor cells (2.5 × 106 /mouse) were implanted subcutaneously in female BALB/c mice. 30 mg/kg BGB-10188 (A) and 60 mg/kg idelalisib (B) were orally treated the mice once when the tumor volume arrived 500–700 mm3, and the tissues were collected at 0.5, 2, 4, and 8 h post dosing. (C–F) The tissue PK study in rat. 20 mg/kg BGB-10188 and idelalisib were treated once by oral administration (C, D) or by intravenous administration (E, F) in rat, and the tissues were collected at 0.5, 2, 7 and 24 h post dosing. Plasma, tumor, spleen, draining lymph nodes (mice) or submandibular lymph nodes (rat), colon and small intestine were collected for drug concentration detection by LC-MS/MS.

Fig 6

Considering the relatively low exposure of idelalisib in mice by oral administration, we further evaluate the tissue distribution in rats by both oral and intravenous injection and similar results were found (Fig. 6C-F). That is, BGB-10188 showed the comparable drug concentrations in colon and lymphoid organs, however higher relative colon concentration of idelalisib was found. Tissue distribution of other PI3Kδ inhibitors including umbralisib (low colitis incidence), copanlisib (low colitis incidence) and parsaclisib (high colitis incidence) by oral and/ or intravenous injection were also tested in rats. The drug concentration in colon compared to that in spleen or lymph nodes was lower for copanlisib, was comparable for umbralisib, whereas was significantly higher for parsaclisib (Supplementary Fig. S4) correlating with its high colitis incidences in clinic. Whether BGB-10188 will have less GI related adverse effects than idelalisib and other PI3Kδ inhibitors still need further validation in clinic.

Discussion

PI3Kδ is frequently hyperactive in B-cell cancers thus becomes an alternative treatment target for B cell malignancies. Favorable progression-free survival (PFS) and objective response rate (ORR) were obtained by PI3Kδ inhibition in clinic in relapse and refractory FL, SLL, marginal zone lymphoma (MZL) or chronic lymphocytic leukemia (CLL) indications. However, maximal or sub-maximal tolerated doses (MTD) had been the general choices for approved PI3Kδ inhibitors to achieve the best possible efficacy, meanwhile, the risk of shortened survival span with severe and fatal toxicities including pneumonitis and colitis was also found [10,30]. Therefore, FDA raised the post-marketing requirement for dose optimization or long term safety profiling for these drugs, and four once launched PI3Kδ inhibitors (idelalisib, copanlisib, umbrelisib and duvelisib) had voluntarily withdrawn their applications in relapsed FL and SLL indications in responding to the FDA requirement without conducting further clinical trials. Loss of unmet medical needs and multiple treatment options like CAR-T therapy in these indications may be important reasons for the company's decision [31].

Next generation PI3Kδ inhibitors like YY-20394 [32], parsarclisib [11,33] and ME-401 [34] demonstrated better efficacy regarding to increased ORR, PFS and DoR at RP2D (generally not reaching the MTD [11,32,35]), and manageable safety profiles with dramatically decreased rates for ≥ G3 infection. These drugs usually have thousands-fold selectivity to PI3Kγ which is constitutively expressed in the myeloid lineage (eg neutrophils, macrophages and mast cells) besides T cells and plays a particularly important role in response to both bacterial and viral pathogens [36,37]. The low selectivity to PI3Kγ of the launched PI3Kδ inhibitors (10-50 folds) is one potential reason contributing to the treatment related fatal and severe infections. BGB-10188 is a highly potent and selective PI3Kδ inhibitor with over 3000 folds selectivity over PI3Kγ, warranting BGB-10188 as a promising therapeutic agent with less off-target side effects.

Colitis is well documented as a PI3Kδ on-target toxicity in both preclinical and clinical studies. p110δD910A/D910A mice developed microbiota-dependent mild colitis with colonic IL-10 reduction and IL12/IL23 increment especially at an elder age [2,14]. p110δD910A/D910A Tregs lost the protection against naïve CD4+ T cell-induced colitis in vivo [27]. Idelalisib was the first approved PI3Kδ inhibitor with nonnegligible severe colitis incidence in ∼10 % patients and often necessitating permanent drug discontinuation [9,12]. Duvelisib is structurally similar to idelalisib [38], also demonstrated similar clinical efficacy and colitis incidence. Umbralisib induced serious colitis in only 2 % patients possibly due to its CK1ε inhibition preserving the function of the regulatory T cells [39,40]. Several next-generation PI3Kδ inhibitors with different chemical structures from idelalisib [38], like AMG319, ME-401 or parsarclisib at clinical stage, still show high colitis incidence with daily continuous dosing schedule. However, mitigation of the severity and occurring rate of colitis were extensively found for these drugs by intermittent treatment in clinic [11,29,41]. Similarly, copanlisib rarely induced colitis which was considered to correlate with its intravenous dosing way which PK is similar with the intermittent oral treatment [42]. The achievement of intermittent treatment indicated colon distribution of drugs may affect their on-site side effects a lot. In our work, by detecting the colon distribution of drug in rodents, BGB-10188 showed comparable exposure in colon, spleen and lymph nodes, which was also found for Umbralisib and copanlisib with low colitis incidence in clinic. However higher relative colon exposure was found for both idelalisib and parsaclisib, they also demonstrated high colitis incidence in clinic. The results suggested a correlation between colon distribution in rodents and colitis incidence in clinic. BGB-10188 with low colon distribution in rodents and no colon Treg inhibition in mice may show superior clinical safety profile with potential less colitis induction.

Although some unfavorable outcomes were found in FL/SLL patients due to the toxicity of launched PI3Kδ inhibitors, however, there is still high unmet medical need in aggressive R/R NHL types like MCL and DLBCL. Emerging CAR T therapy has been approved by FDA as second- or third- line therapy across NHLs, however limitedly benefited MCL and DLBCL populations [31,43] due to the intrinsic limitation of CAR T therapy as well as their aggressive and heterogenous phenotypes. BTK inhibition is a favorable option for 2nd-line MCL treatment [31] and showed preclinical benefits in DLBCL when combined with PI3Kδ inhibitors [23,24]. Therefore, for patients with relapsed or refractory MCL or DLBCL who are not eligible to receive CAR T therapy, PI3Kδ inhibitor become a promising combinational partner with BTK inhibitor treatment. In our work, BGB-10188 alone showed dose-dependent anti-tumor activity in subcutaneous and orthotopic DLBCL Farage xenografts. Combinational anti-tumor effects by targeting both BTK and PI3Kδ inhibitors were also found in DLBCL and MCL cell lines and xenograft models. A phase I/II (NCT04282018) clinical trial for evaluating the combination efficacy of BGB-10188 with Zanubrutinib (BTK inhibitor) across NHLs is in progress.

In addition to the application in B cell lymphoma, PI3Kδ inhibitors also showed their potential as an effective combinational partner in solid tumors regarding their essential function in Tregs. Besides, PI3Kδ inhibition is also tested in autoimmune diseases[[44], [45], [46]] due to its regulatory effects on extensive immune cells. These wide applications revealed a broad market prospect for PI3Kδ inhibitors. Whereas a safer PI3Kδ inhibitor is still the prerequisite.

Overall, BGB-10188 is a highly selective PI3Kδ inhibitor with superior pre-clinical anti-tumor activities and potential improved safety profiles, which is promising as a combinational treatment partner in aggressive NHLs, solid tumors and autoimmune diseases.

Ethics approval and consent to participate

All animal studies were approved by BeiGene's Institutional animal care and use committee (IACUC). Operations regarding to general husbandry, quarantine, housing and sanitation, food and water, environmental conditions and animal experiment methods were complied with relevant standard operation procedures and BeiGene's IACUC animal welfare regulations. All animal studies reported in this work were in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Consent for publication

Not applicable.

Data availability

The data supporting the findings of this study are available upon request to the author for correspondence.

CRediT authorship contribution statement

Xiao Yang: Data curation, Formal analysis, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Huichen Bai: Data curation, Project administration. Xi Yuan: Conceptualization, Writing – review & editing. Xiaolong Yang: Data curation. Ye Liu: Conceptualization, Writing – review & editing. Mingming Guo: Data curation, Writing – review & editing. Nan Hu: Conceptualization, Writing – review & editing. Beibei Jiang: Conceptualization, Writing – review & editing. Zeqin Lian: Data curation, Project administration. Zhilong Ma: Data curation. Jingyuan Wang: Data curation. Xuebing Sun: Data curation. Taichang Zhang: Data curation. Dan Su: Data curation. Yue Wu: Data curation, Writing – review & editing. Jing Li: Writing – review & editing. Fan Wang: Writing – review & editing. Zhiwei Wang: Writing – review & editing. Lai Wang: Conceptualization. Xuesong Liu: Conceptualization. Xiaomin Song: Conceptualization, Data curation, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Funding

There is no finding for this work.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neo.2024.101053.
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