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Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, China
Correspondence: Yu Hu, PhD, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan 430022, China.
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Correspondence: Heng Mei, PhD, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan 430022, China.
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaHubei Clinical Medical Center of Cell Therapy for Neoplastic Disease, Wuhan, ChinaHubei Key Laboratory of Biological Targeted Therapy, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Combination therapy is being actively explored to improve the efficacy and safety of anti-CD19 chimeric antigen receptor T-cell (CART19) therapy, among which Bruton tyrosine kinase inhibitors (BTKIs) are highly expected. BTKIs may modulate T-cell function and remodel the tumor micro-environment (TME), but the exact mechanisms involved and the steps required to transform different BTKIs into clinical applications need further investigation.
Methods
We examined the impacts of BTKIs on T-cell and CART19 phenotype and functionality in vitro and further explored the mechanisms. We evaluated the efficacy and safety of CART19 concurrent with BTKIs in vitro and in vivo. Moreover, we investigated the effects of BTKIs on TME in a syngeneic lymphoma model.
Results
Here we identified that the three BTKIs, ibrutinib, zanubrutinib and orelabrutinib, attenuated CART19 exhaustion mediated by tonic signaling, T-cell receptor (TCR) activation and antigen stimulation. Mechanistically, BTKIs markedly suppressed CD3-ζ phosphorylation of both chimeric antigen receptor and TCR and downregulated the expression of genes associated with T-cell activation signaling pathways. Moreover, BTKIs decreased interleukin 6 and tumor necrosis factor alpha release in vitro and in vivo. In a syngeneic lymphoma model, BTKIs reprogrammed macrophages to the M1 subtype and polarized T helper (Th) cells toward the Th1 subtype.
Conclusions
Our data revealed that BTKIs preserved T-cell and CART19 functionality under persistent antigen exposure and further demonstrated that BTKI administration was a potential strategy for mitigating cytokine release syndrome after CART19 treatment. Our study lays the experimental foundation for the rational application of BTKIs combined with CART19 in clinical practice.
Anti-CD19 chimeric antigen receptor T-cell (CART19) therapy has demonstrated dramatic efficacy in patients with B-cell lymphoma. However, short duration of remission and toxicities remain obstacles to clinical translation. Tonic signaling, sustained antigen stimulation and a suppressive tumor micro-environment (TME) can induce CART19 exhaustion and dysfunction [
]. CART19- and myeloid cell-derived cytokines such as interleukin 6 (IL-6), interferon gamma and tumor necrosis factor alpha (TNF-α) are mainly responsible for cytokine release syndrome (CRS) [
]. An optimal strategy is urgently needed to improve CART19 therapy.
Combination therapy provides a potential solution. Immune checkpoints such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain 3 (TIM-3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) are upregulated on CART19 [
Chimeric Antigen Receptor T Cell Targeting to CD19 and CD22 Combined with Anti-PD-1 Antibody Induce High Response in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma.
]. The immunomodulatory agent lenalidomide has been proven to enhance the efficacy of chimeric antigen receptor (CAR) T cells via multiple mechanisms [
]. Bruton tyrosine kinase inhibitors (BTKIs) are widely used in lymphoma, and ibrutinib (IB) has shown the potential to improve the clinical efficacy of CART19 therapy in patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma [
Pharmacological Profile and Clinical Outcomes of KTE-X19 By Prior Bruton Tyrosine Kinase Inhibitor (BTKi) Exposure or Mantle Cell Lymphoma (MCL) Morphology in Patients With Relapsed/Refractory (R/R) MCL in the ZUMA-2 Trial.
]. However, the underlying mechanisms involved and the steps required to optimally administer BTKIs in patients receiving CART19 therapy remain unclear.
IB, the first-in-class BTKI, blocks Bruton tyrosine kinase (BTK) as well as interleukin 2 (IL-2)-inducible T-cell kinase (ITK) as off-target site. Zanubrutinib (ZB) and orelabrutinib (OB) are novel BTKIs with higher selectivity for B-cell malignancies that have received approval from the US Food and Drug Administration [
] demonstrated that long-term IB pre-treatment preserves functionality of T cells from CLL patients. Moreover, CART19 with concurrent IB therapy mediates more durable remission and lower CRS in CLL [
]. Considering that BTK is expressed in monocytes and macrophages, the three BTKIs have the potential to regulate immunosuppressive profiles. Previous studies of CAR T-cell therapy using immunodeficient mice were unable to fully address this question. Additionally, all three BTKIs are irreversible non-receptor tyrosine kinase inhibitors that will theoretically impair T-cell activation and affect differentiation and exhaustion [
]. Therefore, we hypothesized that CART19 therapy would benefit from BTKI combination through BTK-dependent and BTK-independent mechanisms. Herein we systematically investigated the effects of BTKIs on T cells, CART19 and the TME to verify the hypothesis and to clarify potential mechanisms.
Methods
Cell lines
The human cell lines Nalm-6, Raji, THP-1 and HEK 293T and the mouse cell line A20 were purchased from American Type Culture Collection (Manassas, VA, USA). CD19-K562 cells were kindly provided by Wuhan Si'an Medical Technology Co, Ltd (Wuhan, China). Cell identities were verified by short tandem repeat DNA analysis. Nalm-6 and Raji cells were transduced with lentiviral vector encoding green fluorescent protein–luciferase and then purified by fluorescence-activated cell sorting. Cell lines were cultured in Roswell Park Memorial Institute 1640 (Thermo Fisher Scientific, Waltham, MA USA) or Dulbecco's Modified Eagle's Medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Sigma-Aldrich, St Louis, MO, USA) at 37°C in 5% ambient carbon dioxide. All cells were tested regularly for mycoplasma, which was determined to be negative.
Reagents and antibodies
ZB and OB were kindly provided by BeiGene (Beijing, China) and Beijing InnoCare Pharma Tech Co, Ltd (Beijing, China), respectively. IB was purchased from MedChemExpress (Monmouth Junction, NJ, USA). IB, ZB and OB were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and stored at –80°C at a suitable stock concentration for ex vivo experiments, and IB 1 μM, ZB 2 μM and OB 200 nM were used. All antibodies used are listed in supplementary Table 1.
Manufacture of CART19
The humanized CAR19 construct is provided in supplementary Figure 1. The pGEM-T, PSPAX2 and PMD2G plasmids were transfected into HEK 293T cells. The supernatants were collected at 48 h and 72 h and stored at –80°C. Peripheral blood monocular cells were separated from healthy donors by density gradient centrifugation, and T cells were isolated using a CD3 separation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Anti-CD3 and anti-CD28 monoclonal antibodies (BioLegend, San Diego, CA, USA) were pre-coated overnight at 4°C for T-cell activation. Primary CD3+ T cells were cultured in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 200 U/mL human IL-2 (PeproTech, Cranbury, NJ, USA). Virus supernatant was added to infect T cells 48 h after CD3/CD28 stimulation. The transduction efficiency was measured on day 4 after virus infection. To test the effects of BTKIs on CART19 generation, DMSO, IB, ZB or OB was administered concurrently with CD3/CD28 stimulation.
Cell culture, proliferation and apoptosis assay
T cells with CD3/CD28 stimulation and CART19 were cultured in the presence of DMSO/IB/ZB/OB for 4 days (short-term administration) and 14 days (long-term administration). Absolute cell counts during ex vivo expansion of T cells and CART19 were obtained with propidium iodide (PI) and counting beads (BioLegend). Population doubling was calculated using the equation Nt = N0 × 2n, where n was the population doubling, N0 was the initial number of cells and Nt was the total number of cells. Cells were stained with fluorescein isothiocyanate-labeled annexin V/PI (BioLegend) according to the manufacturer's protocol, and the percentage of cells at different apoptotic stages was measured by flow cytometry (FCM).
Multi-parametric FCM and western blot
Extracellular and intracellular proteins were labeled with antibodies conjugated with fluorochrome as previously described [
]. FCM was performed on the ID7000 spectral cell analyzer (Sony Biotechnology, Tokyo, Japan), and data were analyzed with FlowJo 10.4 software (FlowJo, Ashland, OR, USA). For western blots, cells were lysed in RIPA buffer with protease inhibitors. The total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by standard immunoblotting with anti-glyceraldehyde 3-phosphate dehydrogenase antibody (1:5000 dilution; AntGene, Wuhan, China), anti-ITK antibody (1:1000 dilution; Proteintech, Rosemont, IL, USA), anti-p-ITK antibody (1:250 dilution; BioLegend), anti-CD3-ζ antibody (1:1000 dilution; Proteintech) and anti-p-CD3-ζ antibody (1:1000 dilution; Abcam, Cambridge, UK).
In vitro cytotoxicity assay
To evaluate the cytotoxicity of CART19, three different co-culture experiments were performed: (i) simultaneous co-culture of CART19, target cells and BTKIs; (ii) pre-treatment of target cells with BTKIs for 4 days prior to addition of CART19; and (iii) pre-treatment of CART19 with BTKIs for 4 days before co-culture with target cells. Target cells were pre-stained with 2 μM carboxyfluorescein succinimidyl ester (BD Biosciences, San Jose, CA, USA). CART19 and target cells were co-cultured in a flat 96-well plate at different effector-to-target ratios for 24 h, and PI was then added to analyze cytotoxicity. Targets alone were incubated simultaneously as controls to determine spontaneous cell death.
Serial stimulation assay
Irradiated (100 Gy) CD19-K562 cells were added twice to CART19 at a 1:4 ratio in the presence of DMSO/IB/ZB/OB for 4 days. CART19 were then harvested to assess messenger RNA (mRNA) expression, CAR expression, cytotoxicity and phosphorylation of ITK and CD3-ζ. The cytolytic ability of stimulated CART19 was assessed using a lactate dehydrogenase cytotoxicity test kit (Beyotime, Nanjing, China) according to the manufacturer's protocol.
SYBR Green real-time quantitative polymerase chain reaction
Total RNA of T cells and CART19 was extracted with TRIzol reagent (Sigma-Aldrich). Complementary DNA was synthesized by reverse transcription from 100 ng RNA using a First Strand cDNA Synthesis Kit (Nanjing Vazyme Biotech Co, Ltd, Nanjing, China). A SYBR Green master mix kit (Nanjing Vazyme Biotech Co, Ltd) was then used to carry out real-time quantitative polymerase chain reaction (RT-qPCR). RT-qPCR was performed on a 7500 Fast System (Thermo Fisher Scientific). The list of primers is shown in supplementary Table 2. Relative mRNA expression was calculated by 2−ΔΔCt normalized to control group.
RNA sequencing and transcriptome analysis
CART19 from three healthy donors were treated with DMSO/IB/ZB/OB for 14 days and then collected for RNA sequencing. RNA extraction, library construction and next-generation sequencing were performed by HaploX Biotechnology (Shenzhen, China). Raw sequencing reads were pre-processed by fastp v0.12.64 to remove sequencing adapters and low-quality reads [
]. Gene-level differential expression analyses were performed in R 4.0.3 (www.r-project.org). Differentially expressed genes were identified by imposing a log2 fold change cutoff of 0.05 and P < 0.05. Gene set enrichment analysis was performed using the clusterProfiler R package, and Kyoto Encyclopedia of Genes and Genomes sets were used for gene set enrichment analysis. Additionally, gene set variation analysis was performed using the gene set variation analysis R package to analyze the different signaling pathways.
In vitro TME
THP-1 cells (5 × 105/mL) were first treated with 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich). A total of 48 h later, macrophages were generated, as confirmed by microscope and FCM. CART19 (1 × 106/mL) and Nalm-6 cells (1 × 106/mL) were then added to THP-1-derived macrophages in the presence of DMSO/IB/ZB/OB for 4 days. Cell culture medium was changed every 2 days. After 96 h of co-culture, we measured cytokines from culture supernatants using cytometric bead array (BD Biosciences).
Xenograft and syngeneic tumor model
All mice were housed in a pathogen-free animal facility according to institutional guidelines. All animal studies were approved by the institutional animal care and use committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) (institutional animal care and use committee no. 2962).
Approximately 4- to 6-week-old female NOD-Prkdcscid IL2rgem1/Smoc (M-NSG) mice (three per group) were purchased from Shanghai Model Organisms Center, Inc (Shanghai, China). On day 0, mice were subcutaneously injected with 5 × 106 Raji–luciferase cells diluted in 100 μL of phosphate-buffered saline (PBS) and then underwent bioluminescence imaging with MI SE 7.2.1 (Bruker, Billerica, MA, USA). All animals were anesthetized with isoflurane. Upon confirmation of tumor engraftment on day 7, mice were randomized into a CART19 monotherapy group or BTKI combination group. In both groups, all mice were intravenously infused with 5 × 106 CART19 on day 7. Simultaneously, 25 mg/kg IB, 10 mg/kg ZB and 10 mg/kg OB were administered orally once daily for 28 days in the BTKI combination group accordingly. The proportion of CART19 in the epicanthal vein on day 7, day 14 and day 21 after infusion was analyzed by FCM. Body weight was measured every 3 days or 7 days to monitor CRS. Mice were killed on day 35, and tumor masses were obtained by excision to perform FCM or immunohistochemistry analysis.
Approximately 4- to 6-week-old female CB17.Cg-PrkdcscidLystbg-J/Crl (SCID-beige) mice were purchased from Viton Lihua Experimental Animal Technology Co, Ltd (Beijing, China). On day 0, mice were subcutaneously injected with 5 × 106 Raji–luciferase cells diluted in 100 μL of PBS. Upon confirmation of tumor engraftment on day 7, mice were randomized into a CART19 monotherapy group or BTKI combination group. Plasma of the epicanthal vein was harvested and cytokines were measured.
Approximately 4- to 6-week-old male BALB/c mice (five per group) were obtained from Viton Lihua Experimental Animal Technology Co, Ltd. BALB/c mice were injected subcutaneously in the right flank with 5 × 106 A20 cells diluted in 100 μL of PBS. After 7 days of lymphoma growth, gavage was carried out with three BTKIs once daily for 28 days. Vehicle treatment was performed using 0.5% carboxymethylcellulose sodium. Mice in healthy controls were fed with no additional treatment. Mice were killed on day 35, and tumor samples were collected in formalin or ground into cell suspensions for FCM analysis. Splenocytes and bone marrow specimens were also collected for FCM analysis.
Tumor tissues harvested from M-NSG and BALB/c mice were fixed in paraformaldehyde for 48 h and then embedded in paraffin. Immunohistochemistry was performed on 5-μm paraffin sections. The list of corresponding antibodies is shown in supplementary Table 1.
Statistical analysis
Continuous variables were presented as mean ± standard deviation. Results were plotted with Prism 9 (GraphPad Software, San Diego, CA, USA). Subgroup comparisons were performed using paired or unpaired Student's t-test or two-way analysis of variance. All tests were two-sided. P < 0.05 was considered statistically significant.
Results
BTKIs prevent T-cell exhaustion and activation-induced cell death
Considering that IB is a BTK/ITK inhibitor and BTKIs are non-receptor tyrosine kinase inhibitors, we hypothesized that BTKIs would prevent excessive T-cell receptor (TCR) activation and reverse T-cell dysfunction. In TCR activation assays (Figure 1A), we found that population doubling of T cells was inhibited in the presence of short-term administration of BTKIs (Figure 1B,C). RT-qPCR and FCM analysis showed that BTKIs downregulated the expression of activation markers (CD25 and CD69) and inhibitory receptors (PD-1, TIM-3 and CTLA-4) on day 4 (Figure 1D,E). BTKIs downregulated the expression of cell death receptor CD95 (Figure 1F) and apoptosis marker annexin V (Figure 1G,H), indicating that BTKIs were able to reduce T-cell apoptosis after CD3/CD28 stimulation for 4 days. Furthermore, a lower proportion of apoptosis was observed in T cells after CD3/CD28 stimulation for 2 days, but IB still blocked T-cell apoptosis (Figure 1H). Mechanistically, we showed that the three BTKIs significantly or slightly decreased phosphorylation of ITK and CD3-ζ in T cells after CD3/CD28 stimulation for 4 days (Figure 1I,J; also see supplementary Figure 2). However, population doubling, expression of activation markers and inhibitory receptors and apoptosis of T cells did not show obvious changes in the presence of long-term administration of BTKIs (see supplementary Figure 3). A possible explanation for this is that CD3/CD28 stimulation was removed on day 4 and BTKIs were unable to inhibit TCR activation in the following 10 days. Therefore, we determined that BTKIs were able to ameliorate TCR activation-mediated exhaustion and apoptosis of T cells.
Fig. 1BTKIs prevent T-cell exhaustion and activation-induced cell death. (A) Experimental design. T cells were activated with CD3/CD28 stimulation for 4 days and cultured in the presence of DMSO/IB/ZB/OB for 14 days in vitro. (B) Comparison of the proliferative capacity of T cells over time during ex vivo expansion in the presence of DMSO/IB/ZB/OB for 4 days (n = 3). (C) Representative FCM profile of T-cell proliferation. Numbers on the histograms correspond to the percentage of CFSEhigh cells or the MFI of CFSElow cells. T cells labeled with 2 μM CFSE were stimulated with CD3/CD28 antibody for 4 days in the presence of DMSO/IB/ZB/OB. (D) FCM analysis and (E) mRNA level of activation markers (CD25 and CD69) and inhibitory receptors (PD-1, TIM-3 and CTLA-4) in T cells treated with DMSO/IB/ZB/OB for 4 days (n = 3 or 4). (F) MFI of death receptor CD95 on T cells in the presence of DMSO/IB/ZB/OB for 4 days (n = 4). (G,H) FCM analysis of expression of apoptosis marker annexin V on T cells in the presence of DMSO/IB/ZB/OB for 4 days and 14 days (n = 3). (I) Western blot analysis of ITK and p-ITK (Y180) levels in T cells treated with DMSO/IB/ZB/OB for 4 days. (J) Western blot analysis of CD3-ζ and p-CD3-ζ (Y142) levels in T cells treated with DMSO/IB/ZB/OB for 4 days. Control = T cells with DMSO treatment. Values are shown as mean ± SD. Statistical differences were analyzed with two-tailed paired or unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: P ≥ 0.05 . BTKIs, Bruton’s tyrosine kinase inhibitors; DMSO, dimethyl sulfoxide; IB, ibrutinib; ZB, zanubrutinib; OB, orelabrutinib; ITK, interleukin-2-inducible T-cell kinase; FCM, flow cytometry; CFSE, carboxyfluorescein succinimidyl ester; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MFI, mean fluorescence intensity; SD, standard deviation.
BTKIs attenuate CART19 exhaustion mediated by tonic signaling
CD19 expression was not altered on target cells in co-culture with BTKIs for 4 days (see supplementary Figure 4A), and CART19 did not show higher cytotoxicity against the pre-treated target cells (see supplementary Figure 4B). However, a higher concentration of BTKIs promoted apoptosis of Raji cells but not CD19-K562 or Nalm-6 cells (see supplementary Figure 4C). In 24-h co-culture, concurrent BTKIs did not affect the cytotoxicity of CART19 against CD19-K562, Nalm-6 or Raji cells (see supplementary Figure 5). In co-culture of CART19 and three BTKIs (Figure 2A), long-term administration of BTKIs increased population doubling of CART19 (Figure 2B). Short-term administration of BTKIs downregulated the expression of activation markers CD25 and CD69, inhibitory receptors TIM-3 and CTLA-4 and death receptor CD95 on CART19 (Figure 2C–E; also see supplementary Figure 6A). Long-term administration of BTKIs downregulated the expression of CD25 but not CD69 on CART19 (Figure 2F) and decreased the expression of three inhibitory markers (PD-1, TIM-3 and CTLA-4) on CART19 (Figure 2G), whereas the mRNA expression of CTLA-4 was not altered with statistical significance (Figure 2H). Long-term administration of BTKIs influenced CART19 viability (Figure 2I). Moreover, CART19 pre-treated with BTKIs for 4 days showed higher cytotoxicity against target cells, and IB displayed statistically significant enhancement (Figure 2J–L). Fan et al. [
] reported that in vitro administration of IB increased CAR transduction efficiency in patient-derived T cells and upregulated CD62L expression on patient-derived CAR T cells on day 14. Here we illustrated that IB administration did not increase transduction efficiency of CAR19 in donor-derived T cells (Figure 2M) but did upregulate CD62L expression and the proportion of naive cells (Figure 2N,O; also see supplementary Figure 6B). RNA sequencing highlighted that BTKIs downregulated T-cell activation-associated genes and signaling pathways, such as JAK-STAT (Figure 2P; also see supplementary Figure 7). In support of FCM and RT-qPCR results, transcriptome analysis showed significant decreases in HAVCR2 (encoding TIM-3) expression in CART19 with IB administration for 14 days (see supplementary Figure 8). Additionally, western blot analysis demonstrated that BTKIs significantly decreased phosphorylation of ITK and CD3-ζ in CART19 (Figure 2Q,R; also see supplementary Figure 9). Tonic signaling was defined as antigen-independent, CAR-induced, sustained activation of CAR T cells [
]. In summary, short- and long-term administration of BTKIs alleviated CART19 exhaustion and differentiation and enhanced cytotoxicity of CART19, mainly through inhibition of tonic signaling.
Fig. 2BTKIs attenuate CART19 exhaustion mediated by tonic signaling. (A) Experimental design. CART19 were cultured in vitro and treated with DMSO/IB/ZB/OB for 4 days and 14 days. (B) Comparison of the proliferative capacity of CART19 over time in the presence of DMSO/IB/ZB/OB (n = 3). (C–H) FCM analysis and mRNA expression level of activation markers (CD25 and CD69) and inhibitory receptors (PD-1, TIM-3 and CTLA-4) in CART19 treated with DMSO/IB/ZB/OB for (C–E) 4 days and (F–H) 14 days (n = 3–6). (I) Viability of CART19 treated with DMSO and BTK inhibitors during ex vivo culture (n = 3). (J–L) Cytotoxicity of CART19 pre-treated with DMSO/IB/ZB/OB for 4 days against (J) CD19-K562 cells, (K) Nalm-6 cells and (L) Raji cells (n = 5). (M) Transduction efficiency of CAR19 in T cells after 4 days of infection (n = 5). (N,O) FCM analysis of differentiation status of CART19 on day 14 following CD3+ T-cell isolation (n = 7). (P) Representative GSEA plot demonstrating downregulation of the JAK-STAT signaling pathway with IB versus DMSO treatment (CART19 from three donors treated with IB or DMSO for 14 days). (Q) Western blot analysis of ITK and p-ITK (Y180) in CART19 treated with DMSO/IB/ZB/OB for 14 days. (R) Western blot analysis of endogenous (approximately 19 kDa) and exogenous (approximately 50 kDa) CD3-ζ and p-CD3-ζ (Y142) in CART19 treated with DMSO/IB/ZB/OB for 14 days. Control = CART19 with DMSO treatment. Values are shown as mean ± SD or mean. Statistical differences were analyzed with two-tailed paired or unpaired t-test and two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: P ≥ 0.05. BTKIs, Bruton’s tyrosine kinase inhibitors; DMSO, dimethyl sulfoxide; IB, ibrutinib; ZB, zanubrutinib; OB, orelabrutinib; ITK, interleukin-2-inducible T-cell kinase; FCM, flow cytometry; ANOVA, analysis of variance; E:T, effector-to-target; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSEA, gene set enrichment analysis; NES, normalized enrichment score; SD, standard deviation; Tcm, central memory CART19; Te, effector CART19; Tem, effector memory CART19; Tn, naive CART19.
]. Here we explored the effects of BTKIs on CART19 under repeated antigen stimulation (Figure 3A). RT-qPCR analysis demonstrated that BTKIs downregulated mRNA expression of inhibitory receptors (PD-1, TIM-3 and CTLA-4) (Figure 3B). FCM analysis showed that BTKIs mediated a higher proportion of CART19 during serial antigen stimulation (Figure 3C). Additionally, lactate dehydrogenase assay demonstrated that BTKIs enhanced the cytotoxicity of CART19 after two rounds of stimulation (Figure 3D–F). Moreover, western blot analysis demonstrated that BTKIs significantly reduced phosphorylation of ITK and CD3-ζ in CART19 during serial antigen stimulation (Figure 3G,H; also see supplementary Figure 10). Chronic antigen stimulation contributes to CAR T-cell exhaustion, dysfunction and death [
]. Here we demonstrated that BTKIs were able to decelerate CART19 exhaustion and increase the efficacy of CART19 after repeated antigen stimulation.
Fig. 3BTKIs protect CART19 from repeated antigen stimulation. (A) Experimental design. Irradiated CD19-K562 cells were added twice to CART19 with administration of DMSO/IB/ZB/OB for 96 h. (B) mRNA expression level of inhibitory receptors (PD-1, TIM-3 and CTLA-4) in CART19 with two rounds of irradiated CD19-K562 cells (n = 6). (C) FCM analysis of CAR-positive cells in all viable cells at 96 h (n = 3). (D–F) Following repeated antigen stimulation in the presence of DMSO/IB/ZB/OB, cytotoxicity of CART19 against (D) CD19-K562 cells, (E) Nalm-6 cells and (F) Raji cells was measured by LDH assay in triplicate (n = 3). (G) Western blot analysis of ITK and p-ITK (Y180) in stimulated CART19 in the presence of DMSO/IB/ZB/OB for 4 days. (H) Western blot analysis of endogenous (approximately 19 kDa) and exogenous (approximately 50 kDa) CD3-ζ and p-CD3-ζ (Y142) in stimulated CART19 in the presence of DMSO/IB/ZB/OB for 4 days. Control = experiment performed in the presence of DMSO. Values are shown as mean ± SD. Statistical differences were analyzed with two-tailed paired or unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: P ≥ 0.05. BTKIs, Bruton’s tyrosine kinase inhibitors; DMSO, dimethyl sulfoxide; IB, ibrutinib; ZB, zanubrutinib; OB, orelabrutinib; ITK, interleukin-2-inducible T-cell kinase; FCM, flow cytometry; E:T, effector-to-target; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LDH, lactate dehydrogenase; SD, standard deviation.
BTKIs improve CART19 persistence and safety profiles
We next tested the possible effects of the three BTKIs on CART19 using the subcutaneous Raji xenogeneic tumor model (Figure 4A). BTKIs administration increased the proportion of circulating CART19 on day 14 and day 21 following infusion, but not on day 7, highlighting that BTKIs prolonged the in vivo persistence of CART19 (Figure 4B). CART19 with concurrent IB therapy showed improved anti-tumor efficacy compared with CART19 monotherapy in vivo (Figure 4C,D). Mice treated with IB had less weight loss following CART19 infusion, suggesting dampened toxicity (Figure 4E). FCM and immunohistochemistry analysis of tumor tissues harvested on day 35 also revealed that BTKI administration mediated increased persistence of CART19 in vivo (Figure 4F,G). Additionally, oral gavage with BTKIs for 28 days significantly downregulated the expression of TIM-3 on tumor-infiltrating CART19 but not the expression of PD-1 or CTLA-4 (Figure 4H; also see supplementary Figure 11). To investigate the possibilities underlying the decreased weight loss of lymphoma-bearing M-NSG mice treated with CART19 and BTKIs, we established a subcutaneous Raji xenogeneic tumor model with SCID-beige mice. Cytokine analysis revealed that BTKIs decreased concentrations of cytokines IL-6 and TNF-α (Figure 4I; also see supplementary Figure 12A), supporting better safety profiles. Furthermore, we established an ex vivo TME model as described earlier. The addition of BTKIs decreased the release of IL-6 and TNF-α, which are mainly responsible for CRS (Figure 4J; also see supplementary Figure 12B). Collectively, our data demonstrated that BTKI administration led to lower CRS severity in CART19 therapy.
Fig. 4BTKIs improve CART19 persistence and safety profiles. (A) A total of 5 × 106 luciferase-expressing Raji cells were injected subcutaneously into M-NSG mice (three mice per group). On day 7, a total of 5 × 106 CART19 diluted in 100 μL PBS were injected i.v., and BTKIs were administered orally once daily for 28 days. (B) The proportion of circulating CART19 was monitored weekly via the epicanthal vein. (C,D) Tumor burden was monitored using BLI. Fold change was calculated using log2(D27/D6), where D27/D6 represents the ratio of fluorescence intensity on day 27 and day 6. (E) Body weight was monitored following CART19 and BTKI administration. (F) FCM analysis of CD45+ human-derived cells in tumor samples and bone marrow on day 35. (G) Immunohistochemistry staining for CD3 expression in tumor samples collected on day 35 (scale bar = 100 μm). Histology images were acquired with an Olympus BX43F microscope at ×10 and ×40. (H) FCM analysis of TIM-3 expression on tumor-infiltrating CART19 on day 35. Cells from five randomly selected images were counted and subjected to statistical analysis. (I) Levels of cytokines IL-6 and TNF-α in a SCID-beige xenograft model 7 days after CART19 infusion (n = 3). (J) Pairwise comparisons of IL-6 level in the presence of BTKIs versus DMSO. Control = co-culture treated with DMSO. Values are shown as mean ± SD. Statistical differences were analyzed with two-tailed unpaired t-test or two-way ANOVA. *P < 0.05, **P < 0.01, NS: P ≥ 0.05. BTKIs, Bruton’s tyrosine kinase inhibitors; IB, ibrutinib; ZB, zanubrutinib; OB, orelabrutinib; FCM, flow cytometry; ANOVA, analysis of variance; APC, allophycocyanin; BLI, bioluminescence imaging; CMC-Na, carboxymethylcellulose sodium; i.v., intravenously; SD, standard deviation.
Finally, we systematically evaluated whether and how BTKIs would modulate the TME in our established syngeneic mouse model (Figure 5A). All three BTKIs reduced tumor-infiltrating macrophages, especially type 2 macrophages (M2) (Figure 5B,C). In spleen and tumor samples, administration of all three BTKIs polarized T helper (Th) cells to the Th1 subtype (Figure 5D,E). However, BTKIs did not show effects on tumor-infiltrating natural killer cells, natural killer T cells or other T-cell populations (Figure 5F). Immunohistochemistry results demonstrated that BTKIs suppressed and reprogrammed tumor-associated macrophages (Figure 5G,H), which was consistent with FCM analysis.
Fig. 5BTKIs reprogram macrophages and Th cells. (A) Experimental design. A total of 5 × 106 A20 cells were injected subcutaneously into BALB/c mice (five mice per group). Engraftment was confirmed after 7 days, and oral gavage was carried out once daily for 28 days. (B,C) FCM analysis of macrophages in all CD45+ cells and percentage of M2 subtype in total tumor-infiltrating macrophages on day 35. (D) Ratio of Th1 to Th2 in tumor and spleen specimens on day 35. (E) Representative FCM profile of Th cells in spleen specimens. (F) Heat map showing FCM analysis of immune landscape in tumor tissue, splenocytes and bone marrow specimens on day 35 (R software pheatmap package). (G,H) Immunohistochemistry staining for F4/80 and CD163 expression in tumor samples collected on day 35 (scale bar = 100 μm). Histology images were acquired with an Olympus BX43F microscope at ×10 and ×40. Cells from five randomly selected images were counted and subjected to statistical analysis. Vehicle = mice treated with 0.5% CMC-Na. Values are shown as mean ± SD. Statistical differences were analyzed with two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, NS: P ≥ 0.05 BTKIs, Bruton tyrosine kinase inhibitors; CMC-Na, carboxymethylcellulose sodium; FCM, flow cytometry; IB, ibrutinib; OB, orelabrutinib; SD, standard deviation; Th, T helper; ZB, Zanubrutinib.
]. IB has improved the efficacy of CART19 therapy in patients with CLL and mantle cell lymphoma, possibly because of its effects on T cells and the TME. However, the exact mechanism of IB and the potential effects of other BTKIs on CART19 therapy need further investigation. In this study, we systematically compared the potential effects of three different BTKIs on CART19 in vitro and in vivo and explored how they would modulate the immune landscape in B-cell lymphoma.
Our in vitro studies indicated that both short- and long-term administration of BTKIs downregulated the expression of activation markers and immune checkpoints on T cells and CART19 and improved cell viability and cytotoxicity. Mechanistically, BTKIs reduced ITK and CD3-ζ phosphorylation of TCR and CAR during ex vivo culture and inhibited the T-cell activation-associated JAK-STAT signaling pathway [
]. CART19 attained more enriched naive phenotypes and higher CD62L expression when BTKIs were administered concurrently with CD3/CD28 stimulation during manufacturing. IB also increased CD62L expression in CLL patients and infectious disease models [
]. Compared with IB, ZB and OB showed limited impacts on CART19 in vitro and an immunodeficient mouse model, possibly because of their higher selectivity and lower off-target effects. Taken together, BTKIs protected T cells and CART19 from CD3/CD28 activation and tonic signaling.
The off-BTK activity of IB on ITK has been regarded as a mechanism of enhancement of CAR T-cell therapy [
]. In our study, IB showed stronger effects with regard to inhibiting excessive T-cell and CART19 activation, differentiation and exhaustion, which is consistent with the downregulation of ITK and CD3-ζ phosphorylation. Additionally, our evidence showed that expression of CD95 on T cells and CART19 was mostly downregulated with IB administration—a finding also reported in animal models with ITK deficiency [
]. Hence, we speculate that different ITK activities lead to discrepancies in the phenotype and functionality of T cells and CART19.
We established an ex vivo repeated stimulation model to imitate persistent antigen exposure to CART19 and observed that BTKIs significantly enhanced CART19 effector function, which is in line with previous research [
]. BTKIs administration alleviated CART19 exhaustion, which might partially explain the improved efficacy. Higher levels of CART19 in the epicanthus vein were observed in xenograft lymphoma mice after oral gavage of BTKIs for 14 days and 21 days, but not for 7 days. Hence, we supposed that BTKIs prolonged in vivo persistence of CART19 but did not boost CART19 expansion. Moreover, we found that the three BTKIs were inclined to downregulate expression of TIM-3 compared with PD-1 and CTLA-4 in vitro and in vivo. Previous studies have demonstrated that T-cell activation is accompanied by upregulation of Blimp-1, T-bet and nuclear factor of activated T cell (NFAT), which are closely related to the expression of PD-1, TIM-3 and CTLA-4, respectively [
A Notch/STAT3-driven Blimp-1/c-Maf-dependent molecular switch induces IL-10 expression in human CD4(+) T cells and is defective in Crohn´s disease patients.
]. Therefore, we speculated that TIM-3 was more sensitive to T-cell activation and BTKIs were thus apt to decrease the expression of TIM-3. Nonetheless, the exact mechanism remains unknown and more studies are needed. TIM-3 expression on T cells was increased after CART19 infusion [
], highlighting the translational significance of impairing the TIM-3 inhibitory signals. Collectively, under chronic antigen stimulation, BTKIs attenuated CART19 exhaustion, prolonged CART19 persistence and further preserved cell functionality.
CART19 with concurrent IB therapy has been reported to mediate a deeper response with milder adverse effects [
], providing the rationale for exploring the impact of the other two novel BTKIs. In a subcutaneous Raji xenogeneic tumor model, M-NSG mice treated with BTKIs had less weight loss following CART19 infusion. Moreover, cytokine analysis revealed that BTKIs decreased concentrations of cytokines IL-6 and TNF-α in SCID-beige mice. In our established ex vivo TME model, the three BTKIs impeded the release of IL-6 and IB significantly decreased levels of TNF-α. Activated CAR T cells and myeloid cells with expression of BTK are mainly responsible for cytokine release [
]. Furthermore, BTKIs reduced tumor-infiltrating macrophages, reprogrammed macrophages into the M1 phenotype and polarized T cells toward the Th1 subtype in A20-bearing BALB/c mice. BTKIs might modulate the TME to endow CART19 with better safety and efficacy profiles. Nevertheless, the specific mechanism underlying BTKIs modulation of the crosstalk among T cells, CART19 and the TME in B-cell lymphoma warrants further investigation.
Based on the aforementioned findings, we made the reasonable assumption that long-term oral BTKIs before and after CART19 infusion would improve patient outcomes in further clinical applications by mitigating T-cell and CART19 exhaustion. Patients previously given two BTKIs attain a more favorable prognosis compared with those given prior monotherapy [
Pharmacological Profile and Clinical Outcomes of KTE-X19 By Prior Bruton Tyrosine Kinase Inhibitor (BTKi) Exposure or Mantle Cell Lymphoma (MCL) Morphology in Patients With Relapsed/Refractory (R/R) MCL in the ZUMA-2 Trial.
]. Thus, it seems a superior strategy to administer different BTKIs interchangeably, and more studies are warranted to explore the combination strategy.
Some limitations exist in our study. First, sequencing RNA was a mixture, and thus the transcriptional analysis was not specific to T cells or CART19. Second, the synergism of CART19 with concurrent BTKIs was verified in our established subcutaneous lymphoma model but not in our systemic lymphoma model by tail vein injection. Third, the potential benefits of one or more BTKIs combined with CART19 were not confirmed in patients. An ongoing clinical trial has been launched by our group (NCT050220392).
Conclusions
This is the first pre-clinical study in which we systematically deciphered the effects of three different BTKIs on T cells, CART19 and the TME in B-cell lymphoma. Our data revealed that BTKIs ameliorated T-cell and CART19 exhaustion mediated by CD3/CD28 activation, sustained tonic signaling and persistent antigen-specific stimulation. ZB and OB exerted a slight impact on CART19 and T cells, possibly because of their decreased ITK selectivity. All three BTKIs impeded cytokine release of macrophages, reprogrammed macrophages to the M1 subtype and polarized T cells to the Th1 subtype. Our study provides evidence to support that BTKIs improve CART19 therapy by preserving CART19 functionality and reprogramming TME and further raise the combination strategy of one or more BTKIs with CART19 in future clinical practice.
Funding
This work was supported by grants from the National Key R&D Program of China (2019YFC1316200 and 2022YFC2502700), National Natural Science Foundation of China (82070124) and Natural Science Foundation of Hubei Province (2020CFA065).
Declaration of Competing Interest
The authors have no commercial, proprietary or financial interest in the products or companies described in this article.
Author Contributions
Conception and design of the study: HM and YH. Acquisition of data: WL, CL, JW, LT, XW, YZ, ZW, ZH, JX, YK, JD and WX. Analysis and interpretation of data: WL, CL and JW. Drafting or revising the manuscript: HM, WL and CL. All authors have approved the final article.
Acknowledgments
The authors thank the Huazhong University of Science and Technology Analytical and Testing Center, medical subcenter, for its technical support. The authors also thank BeiGene (Beijing, China) and Beijing InnoCare Pharma Tech Co, Ltd (Beijing, China) for their provision of ZB and OB.
Data availability
All data generated or analyzed during this study are available from the corresponding authors on reasonable request.
Chimeric Antigen Receptor T Cell Targeting to CD19 and CD22 Combined with Anti-PD-1 Antibody Induce High Response in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma.
Pharmacological Profile and Clinical Outcomes of KTE-X19 By Prior Bruton Tyrosine Kinase Inhibitor (BTKi) Exposure or Mantle Cell Lymphoma (MCL) Morphology in Patients With Relapsed/Refractory (R/R) MCL in the ZUMA-2 Trial.
A Notch/STAT3-driven Blimp-1/c-Maf-dependent molecular switch induces IL-10 expression in human CD4(+) T cells and is defective in Crohn´s disease patients.
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