Here we present a protocol for the generation and functional verification of hypoxia-sensitive chimeric antigen receptor (CAR)-T cells. This protocol presents the lentivirus-based generation of hypoxia-sensitive CAR-T cells and their characterization, including the validation of hypoxia-dependent CAR expression and selective cytotoxicity.
Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.
Chimeric antigen receptor T cell (CAR-T) therapy has represented a significant breakthrough in cancer treatment. Since the Food and Drug Administration (FDA) approved the first CAR-T therapy for treating advanced/resistant lymphoma and acute lymphoblastic leukemia in 20171,2,3, 10 CAR-T therapies targeting CD19 or B-cell maturation antigen (BCMA) have received approval globally4. However, despite extensive research, replicating the remarkable efficacy of CAR-T therapy in treating hematological malignancies remains challenging for its application to solid tumors5,6,7,8.
The immunosuppressive tumor microenvironment (TME) is a primary contributor to the poor efficacy of CAR-T in the solid tumor setting. TME impedes the activity and survival of CAR-T cells due to insufficient nutrients, hypoxia, an acidic pH, and the accumulation of metabolic waste9,10,11,12. Further hostility comes from infiltrating immunosuppressive cells such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAM), which, alongside tumor cells, secrete immunosuppressive cytokines that cause additional inhibition of CAR-T cells once they enter the tumor13,14.
Apart from the unsatisfactory therapeutic efficiency, safety issues are another Achilles' heel of CAR-T cells when dealing with solid tumors15,16. The safety concern arises from the fact that none of the tumor-specific antigens (TSA) identified so far are strictly restricted to tumor cells. In other words, the tumor-associated antigens (TAA) chosen as the target of CAR, although showing higher expression in tumor cells, are often also expressed by normal tissues17. On-target, off-tumor effects could therefore occur from the unexpected activation of CAR-T cells upon CAR efficiently recognizing normal tissues, leading to cytokine release syndrome (CRS), CAR-T-related encephalopathy syndrome (CRES)18, and other adverse outcomes19.
Many strategies have been explored to avoid such effects, including decreasing the affinity of CAR to allow CAR-T cells to distinguish tumor cells from normal cells based on the expression levels of the targeted TAA; equipping CAR-T cells with an off switch, such as a suicide gene or elimination marker to promote their elimination upon unexpected activation; partitioning the CD3ζ and co-stimulatory signals into two CAR moieties, whose simultaneous engagement is consequently required for effective activation of CAR-T cells; utilizing a synthetic Notch (synNotch)-based circuit that restricts the activity of CAR-T cells to targeted cells co-expressing two different TAAs; and engineering CAR-T cells to attain TME sensitivity by implementing a mechanism to tune CAR expression to changing environmental cues20,21,22,23,24,25,26.
A key consideration in the TME sensitivity option outlined above is the low oxygen level in the TME due to the rapid proliferation of tumor cells. The accommodation of tumor cells to hypoxia hinges on the activation of hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcriptional factor consisting of an inducible subunit, HIF-1α, and a constitutively expressed subunit, HIF-1β27. Under normoxic conditions, the HIF-1α protein undergoes ubiquitination and rapid proteasomal degradation, dependent on its oxygen-dependent degradation domain (ODD)28. When the cellular supply of oxygen becomes limited, HIF-1 is stabilized and activates the transcription of its downstream target genes by binding to hypoxia-response elements (HREs)29. Given the nature of ODD and HRE as oxygen-sensitive elements, they have been explored to realize the conditional expression of CARs within the hypoxic TME30. Here, we present a protocol focusing on methods for phenotypic and functional characterization of hypoxia-sensitive CAR-T cells, preceded by a brief description of the CAR design and the preparation procedures of these cells. This protocol intends to provide a useful guideline for exploiting hypoxia-responsive CAR to generate CAR-T cells with restrained off-tumor toxicity.
In this study, HER2-BBz-ODD, a hypoxia-sensitive CAR targeting HER2 (Gene ID: 2064) was compared with its regular counterpart, HER2-BBz. The schematics of the two CARs are illustrated in Figure 1A, which shows that HER2-BBz-ODD is derived from HER2-BBz by adding the ODD sequence to the C-terminal of CD3ξ. The construction of lentiviral vectors expressing the two CARs and the generation of the corresponding lentivirus by 293T cell transfection has been previously described31.
1. Generation of hypoxia-sensitive CAR-T cells by lentiviral infection
2. Assessment of oxygen-dependent CAR expression in CAR-T cells using flow cytometry
3. Analysis of oxygen-dependency of CAR expression in CAR-modified Jurkat T cells by western blot
4. In vitro assessment of the oxygen dependency of cytotoxicity mediated by hypoxia-sensitive CAR-T cells
5. Detection of IL-2 and IFN-γ secretion by hypoxia-sensitive CAR-T cells
Fusing the ODD domain of HIF-1α to the CAR moiety represents a primary strategy for generating a hypoxia-sensitive CAR. The hypoxia-sensitive HER2-targeting CAR analyzed in this study, named HER2-BBz-ODD, was constructed using this strategy by integrating the ODD sequence into its conventional HER2-BBz (Figure 1A). In this study, we used lentiviral transduction to express HER2-BBz-ODD CAR or HER2-BBz CAR and subsequently examined their oxygen sensitivity in two cell types: human PBMCs and Jurkat T cells.
The first examination is the expression of CAR under hypoxic conditions versus normoxic conditions, which was conducted in both CAR-transduced PBMC-derived T cells by flow cytometry and CAR-transduced Jurkat T cells by western blotting. In the setting of CAR-transduced PBMC-derived T cells, we observed that HER2-BBz-ODD CAR had significantly higher expression under 1% O2 than under 21% O2 in terms of both the percentage of CAR-positive cells and the median fluorescence intensity (MFI) (Figure 1B). The immunoblotting analysis of CAR-transduced Jurkat T cells also confirmed the hypoxia-dependent induction of HER2-BBz-ODD.
It is worth noting that, in this context, the hypoxic condition can be conveniently mimicked by adding CoCl2, a chemical inducer of hypoxia, to the culture medium. As illustrated in Figure 1C, our immunoblotting results demonstrated that exposure to 50 or 200 µM CoCl2 recapitulated the effect of exposure to 1% O2, markedly inducing the expression of the HER2-BBz-ODD CAR but not that of the HER2-BBz CAR. The functional characterization of hypoxia-sensitive CAR was conducted with PBMC-derived CAR-T cells. In the study, a firefly luciferase-carrying SKOV-3 cell line was utilized as the target cell line. This setup allowed us to measure target cell-associated luciferase activity as a proxy for assessing the cytotoxicity mediated by co-cultured CAR-T cells.
As shown in Figure 1D, the measurements indicated that the HER2-BBz CAR-T cells effectively kill target cells, regardless of whether the atmosphere was normoxic or hypoxic. In contrast, the HER2-BBz-ODD CAR-T cells displayed significantly weaker cytotoxicity under normoxic conditions for all three E:T ratios examined. However, their cytotoxicity was significantly enhanced when exposed to hypoxic conditions. The supernatant levels of IL-2 and IFN-γ were also measured by ELISA after co-culturing CAR-T cells with target cells for 24 h. For both cytokines, higher secretion under 1% O2 compared to 21% O2 was observed for HER2-BBz-ODD CAR-T cells, which is consistent with the cytotoxicity data. In contrast, HER2-BBz CAR-T cells showed lower secretion of the two cytokines under 1% O2 compared to 21% O2, indicating an adverse impact of hypoxia on cellular activity (Figure 1E). Taken together, these results convincingly validated the hypoxia-sensitive nature of HER2-BBz-ODD CAR.
Figure 1: Construction and characterization of hypoxia-sensitive CAR-T cells. (A) Schematic representation of the designs of a hypoxia-sensitive CAR, HER2-BBz-ODD, and its conventional counterpart, HER2-BBz. Both CARs consist of an N-terminal CD8α signal peptide, a FLAG tag, a human HER2-targeting scFv, a CD8 hinge and transmembrane domain, and an intracellular portion comprised of a costimulatory domain from 4-1BB, a CD3ξ signaling domain, an IRES, and an EGFP. HER2-BBz-ODD differs from HER2-BBz in the fusion of an ODD domain to the C-terminal of the CD3ξ signaling domain, allowing for its hypoxic-dependent expression by promoting its ubiquitin-dependent degradation under normoxic conditions. (B,C) Assessments of oxygen-dependent expression of HER2-BBz-ODD CAR. (B) One assessment was performed with human PBMC-derived CAR-T cells after culturing under 1% or 21% O2 for 24, 48, or 72 h using flow cytometry. (C) The other assessment was with CAR-transduced Jurkat T cells, where cell lysates were harvested 24 h after culturing under 21% O2, 50 or 200 µM CoCl2, or 1% O2 and analyzed for CAR expression using western blotting, with HER2-BBz CAR-transduced cells included as a control. (D,E) In vitro cytotoxicity and cytokine secretion of CAR-T cells under different oxygen conditions. (D) CAR-T cells were co-cultured with firefly luciferase-expressing SKOV3 cells at indicated E:T ratios under 1% or 21% O2. After 24 h of co-culturing, target cell killing efficiency was determined by measuring the change in cell-associated firefly luciferase activity relative to that with non-transduced T cells. (E) The supernatants were collected for the detection of secreted IL-2 and IFN-γ. The results are displayed as the mean ± SEM (n = 3 healthy donors) (****p < 0.0001). Abbreviations: scFv = single-chain fragment variable; CAR = chimeric antigen receptor. Panels C and D are adapted from Liao et al.31. Please click here to view a larger version of this figure.
Safety concerns are significant issues that must be addressed for any CAR-T cell therapy to advance to clinical use. Utilizing the unique properties of tumor cells or the TME has become a primary research direction focusing on the development of CAR-T cells that target tumor tissues selectively. Designing a hypoxia-sensitive CAR-T is an attractive strategy in this direction, with several approaches being explored, including the one presented in this study-fusing the CAR moiety with the naturally occurring hypoxia-sensing ODD protein domain. An alternative approach involves substituting a constitutive promoter commonly used to drive CAR expression with a hypoxia response element (HRE), which has shown promise in previous studies. It is believed that combining HRE and ODD elements (wild-type or engineering versions that offer tighter control of expression) represents an optimal design for a hypoxia-inducible CAR.
This protocol outlines experimental procedures for generating and validating hypoxia-sensitive CAR-T cells. The implementation of this protocol involves several key considerations. A primary consideration is the creation of hypoxic conditions. Between the two approaches, the addition of CoCl2 to the culture medium has been prevalently used in previous hypoxia-related studies, largely thanks to its convenience32,33. However, it is impossible to measure the degree of hypoxia mimicked by this approach. In contrast, using a mobile CO2/O2/N2 incubation chamber is advantageous in that the O2 levels can be precisely set and is thus suitable for a fine analysis of the hypoxia-sensitivity of CAR-T cells. In this respect, the hypoxia level within tumors varies among different types of solid tumors and different periods of tumor progression34, while just 1% O2 is exemplified in the protocol. It is an optimal practice for researchers to adjust the oxygen level according to the actual demand. If the CoCl2 method is the only available approach, we recommend including a range of CoCl2 concentrations in the assay to simulate various oxygen levels.
Choosing an appropriate method for examining hypoxia-dependent CAR expression is another key consideration. While immunoblotting analysis of CAR-transduced Jurkat T cells is a convenient option during CAR construct optimization, analyzing the effect of oxygen levels on surface CAR expression in CAR-transduced human PBMCs by flow cytometry serves as the ultimate validation. It is optimal to examine the dynamics of CAR expression in response to transitioning from hypoxic to normoxic conditions, as we did previously with an improved version of hypoxia-sensitive CAR, namely HiTA-CAR35. This would further demonstrate hypoxia-restricted CAR expression.
For functional verification of hypoxia-sensitive CAR-T cells, the cytotoxicity assay outlined in the protocol involves using firefly luciferase-carrying target cells. This reporter-based assay can be replaced by other killing assessment methods, such as the CCK8 method and the real-time cellular analysis (RTCA) method, where non-modified tumor cells can be used. RTCA analysis is also advantageous for measuring the real-time killing kinetics of CAR-T cells. To measure the specific cytotoxicity caused by CAR-T cells, non-transduced PBMCs should be included as a control. High transduction efficiency of PBMCs is desirable to avoid concerns that differences in detected cytotoxicity between experimental and control groups arise from unspecific killing mediated by varying amounts of effector cells added.
There are several limitations in this protocol. Hypoxic conditions can affect the viability of both target cells and T cells36,37, which introduces the concern that cell death unrelated to CAR-T cell-mediated cytotoxicity may confound the interpretation of assay results. Ensuring that both CAR-T cells and target cells have excellent viability immediately before the assay is suggested to avoid or minimize such concerns. It should also be noted that in vitro validation does not guarantee successful in vivo translation. In vivo assessments are always needed to confirm whether the hypoxia-sensitive CAR-T candidate could avoid targeting normal tissues that express the targeted antigen
The authors have nothing to disclose.
This work was supported by grants from the National Key Research and Development Program of China (2016YFC1303402), the National Megaproject on Key Infectious Diseases (2017ZX10202102, 2017ZX10304402-002-007), and the General Program of Shanghai Municipal Health Commission (201740194).
1.5 mL Centrifuge tube | QSP | 509-GRD-Q | Supernatants and cells cellection Protocol Step 2,3,4 |
10% ExpressCast PAGE | NCM biotech | P2012 | Immunoblotting Protocol Step 3 |
10x PBS | NCM biotech | 20220812 | Cell culture Protocol Step 4 |
10 mL pipette | Yueyibio | YB-25H | Pipetting Protocol Step 1 |
10xTRIS-Glycine-SDS electrophoresis buffer | Epizyme | 3673020 | Immunoblotting Protocol Step 3 |
15 mL Centrifuge tube | Thermo Scientific | 339650 | Supernatants and cells cellection Protocol Step 1 |
25 cm2 EasYFlask | Thermo Scientific | 156367 | Cell culture Protocol Step 3,4 |
4x Protein SDS PAGE Loading Buffer | Takara | 9173 | Immunoblotting Protocol Step 3 |
6-well flat-bottom tissue culture plates | Thermo Scientific | 140675 | T Cells culture Protocol Step 1 |
96-well black flat-bottom tissue culture plates | Greiner | 655090 | Cytotoxicity assay Protocol Step 4 |
96-well ELISA plates | Corning | 3590 | ELISA Protocol Step 5 |
96-well plate shaker | QILINBEIER | MH-2 | Shake Protocol Step 4 |
96-well U-bottom tissue culture plates | Thermo Scientific | 268200 | Supernatants cellection Protocol Step 4,5 |
anti-FLAG antibody | Sigma | F1804-50UG | Immunoblotting Protocol Step 3 |
Carbinol | Sinopharm | 10010061 | Immunoblotting Protocol Step 3 |
Carbon dioxide incubator | Thermo Scientific | 360 | Cell culture Protocol Step 1,2,3,4 |
Cell counting plate | Hausser scientific | 1492 | Cell counting Protocol Step 1,3,4 |
CELLection Pan Mouse IgG Kit | Thermo Scientific | 11531D | Mouse IgG magnetic beads Protocol Step 1 |
Centrifuge | Thermo Scientific | 75002432 | Cell culture Protocol Step 1,3,4 |
Chemiluminescence gel imaging system | BIO-RAD | 12003154 | Immunoblotting Protocol Step 3 |
Cobalt chloride solution (0.5 M) | bioleaper | BR4000203 | Hypoxic condition Protocol Step 2,3,4 |
DMEM | Corning | 10-103-CV | Cell culture Protocol Step 4 |
Electronic balance | Sartorius | PRACTUM612-1CN | weigh Protocol Step 5 |
FBS | BI | 04-001-1ACS | Cell culture Protocol Step 3,4 |
GAPDH Mouse mAb | ABclonal | AC002 | Immunoblotting Protocol Step 3 |
Gel electrophoresis apparatus | BIO-RAD | 1645070 | Immunoblotting Protocol Step 3 |
GloMax Microplate Readers | Promega | GM3000 | luciferase activity measurement Protocol Step 4 |
Goat anti-Mouse IgG (H+L) | Yeasen | P1126151 | Immunoblotting Protocol Step 3 |
High speed microfreezing centrifuge | eppendorf | 5810 R | Cell culture Protocol Step 1 |
Human IFN-γ ELISA Set | BD | 555142 | ELISA Protocol Step 5 Items: Recombinant Human IFN-γ Lyophilized Standard, Detection Antibody Biotin Anti-Human IFN-γ , Capture Antibody Purified Anti-Human IFN-γ, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) |
Human IL-2 ELISA Set | BD | 555190 | ELISA Protocol Step 5 Items: Recombinant Human IL-2 Lyophilized Standard, Detection Antibody Biotin Anti-Human IL-2 , Capture Antibody Purified Anti-Human IL-2, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) |
IL-15 | R&D systems | P40933 | T Cells culture Protocol Step 1 |
IL-21 | Novoprotein | GMP-CC45 | T Cells culture Protocol Step 1 |
IL-7 | R&D systems | P13232 | T Cells culture Protocol Step 1 |
Inverted microscope | Olympus | CKX41 | Cell culture Protocol Step 1,3,4 |
Jurkat | ATCC | TIB-152 | CAR-Jurkat construction Protocol Step 3 |
LSRFortessa | BD | LSRFortessa | Flow cytometry Protocol Step 2 |
Luciferase Assay System | Promega | E1501 | luciferase reporter assay Protocol Step 4 Items: Passive lysis buffer, firefly luciferase substrate |
Microplate reader | BioTek | HTX | ELISA Protocol Step 5 |
mobile CO2/O2/N2 Incubator Chamber | China Innovation Instrument Co., Ltd. | Smartor118 | Hypoxic condition Protocol Step 2, 3, 4 |
Mouse Anti-Hexa Histidine tag | Sigma | SAB2702218 | Immunoblotting Protocol Step 3 |
NcmBlot Rapid Transfer Buffer | NCM biotech | WB4600 | Immunoblotting |
NcmECL Ultra | NCM biotech | P10300 | Immunoblotting Protocol Step 3 Items: NcmECL Ultra Luminol/Enhancer Reagent (A) ,NcmECL Ultra Stabilized Peroxide Reagent (B) |
NovoNectin -coated 48-well flat plates | Novoprotein | GMP-CH38 | CAR-T cells construction Protocol Step 1 |
OPD (o-phenylenediamine dihydrochloride) tablet set | Sigma | P9187 | Substrate Reagent Protocol Step 5 Items: OPD tablet (silver foil),urea hydrogen peroxide tablet (gold foil) |
PE-conjugated anti-DYKDDDDK | Biolegend | 637310 | Flow cytometry Protocol Step 2 |
Protamine sulfate | Sigma | P3369-1OG | Lentivirus infection Protocol Step 1 |
Protein Marker 10 Kda-250 KDa | Epizyme | WJ102 | Immunoblotting Protocol Step 3 |
Purifed NA/LE Mouse Anti-Human CD3 | BD | 566685 | T Cells culture Protocol Step 1 |
Purified NA/LE Mouse Anti-Human CD28 | BD | 555725 | T Cells culture Protocol Step 1 |
PVDF membrane | Millipore | 168627 | Immunoblotting Protocol Step 3 |
RPMI 1640 | Corning | 10-040-CVRC | Cell culture Protocol Step 3 |
Skim milk powder | Yeasen | S9129060 | Immunoblotting Protocol Step 3 |
SKOV3-Luc | ATCC | HTB-77 | Cytotoxicity assay Protocol Step 4 |
Trypsin-EDTA | NCM biotech | C125C1 | Cell culture Protocol Step 4 |
Tween 20 | Sinopharm | 30189328 | Immunoblotting Protocol Step 3 |
Water bath | keelrein | NB014467 | Heating Protocol Step 1 |
X-VIVO 15 | LONZA | 04-418Q | Serum-free lymphocyte culture medium Protocol Step 1 |