Method Article

Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells

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DOI:

10.3791/66697

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June 14th, 2024

 ,  ,  ,  ,  , 
1Institutes of Biomedical Sciences, Fudan University, 2Clinical Center of Biotherapy at Zhongshan Hospital, Fudan University, 3Department of Oncology and Bio-therapeutic Center, Shenzhen Third People's Hospital, Southern University of Science and Technology, 4Shanghai Public Health Clinical Center, Fudan University

* These authors contributed equally

In This Article

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

  1. Thaw cryopreserved human peripheral blood mononuclear cells (PBMCs) rapidly at 37 °C in a water bath. Transfer the thawed PBMCs into a 15 mL tube containing 9 mL of serum-free lymphocyte culture medium supplemented with 400 IU of IL-2, 5 ng/mL IL-7, and 10 ng/mL IL-15 (human T cell growth medium, referred to as TGM thereafter).
  2. After taking an aliquot for cell counting, centrifuge the tube at 300 × g for 5 min. Resuspend the pelleted PBMCs with TGM at a density of 4 × 106 cells/mL and transfer the suspension into a 6-well plate.
  3. Prepare anti-hCD3/hCD28-coated immunobeads.
    1. Transfer 100 µL of mouse IgG magnetic beads into a 1.5 mL microcentrifuge tube and wash them 2 times using a magnetic stand with PBS.
    2. Resuspend the beads in 100 µL of PBS and add 0.2 µg of mouse anti-Human CD3 antibody and 2 µg of mouse anti-Human CD28 antibody. Gently mix the mixture using a pipette and rock overnight at 4 °C.
    3. Wash the beads 2 times using a magnetic stand with PBS and resuspend them in 100 µL of PBS.
  4. Add anti-hCD3/hCD28-coated immunobeads to the plate in step 1.2 at a bead-to-cell ratio of 1:1. Place the plate in a humidified incubator with 5% CO2 at 37 °C.
  5. After 48 h of incubation, transfer the cell suspension into a 15 mL centrifuge tube after gently mixing the cells with a pipette. Place the tube on a magnetic stand for 3 min, then carefully transfer the supernatant into a new 15 mL tube to remove immunobeads from the PBMCs.
  6. Take an aliquot for cell counting, then seed the PBMCs at 5 × 105 cells/well in 300 µL of TGM into a 48-well flat plate. Add 200 µL of lentiviral stock into the corresponding wells and add protamine sulfate to a final concentration of 10 µg/mL.
  7. Centrifuge the plate at 1,000 × g at 32 °C for 1.5 h and carefully remove and discard 300 µL of supernatant from each well using a pipette. Then, add 1 mL of fresh TGM using a pipette and place the plate in a humidified incubator with 5% CO2 at 37 °C.
  8. Add fresh TGM to adjust the cell density to 0.5-2 × 106 cells/mL every 2-3 days. Start by transferring cells first to a 12-well plate and then to a 6-well plate. Continue to culture the cells until the total number reaches 6 × 106 in a 4 mL volume.
    NOTE: In parallel, conduct the CAR transduction of Jurkat T cells following the same procedures described above, except that TGM is replaced with RPMI1640 medium containing 10% fetal bovine serum (FBS).

2. Assessment of oxygen-dependent CAR expression in CAR-T cells using flow cytometry

  1. Plate the CAR-transduced T cells from step 1.8 into two 12-well plates at a density of 2.5 × 106 cells/well in 2 mL of TGM. Place one plate directly in a humidified incubator with 5% CO2 at 37 °C (normoxic condition, as the standard condition for culturing cells has 21% O2) and place the other plate in a mobile CO2/O2/N2 incubator chamber with the O2 level preset at 1% (hypoxic condition) and then keep the chamber in a humidified, 5% CO2/94% N2 incubator at 37 °C.
  2. Every 24 h, collect 5 × 105 cells from the plates under hypoxic or normoxic conditions into 1.5 mL microcentrifuge tubes. Centrifuge the tubes at 500 × g for 5 min, remove the supernatant, and gently resuspend the cells in 1 mL of PBS by pipetting. Repeat the PBS wash one more time.
  3. Resuspend the pelleted cells in 50 µL of FACS buffer (PBS supplemented with 2% FBS) in each tube. Add 50 µL of a 1:100 dilution of PE-conjugated anti-Flag antibody (0.2 µg/mL) and mix thoroughly by pipetting. Incubate in the dark at room temperature for 20 min.
  4. At the end of incubation, add 1 mL of FACS buffer to each tube. Mix thoroughly by pipetting, then centrifuge at 500 × g for 5 min.
  5. Carefully remove and discard the supernatant using a pipette and repeat step 2.4 one more time.
  6. Carefully remove and discard the supernatant using a pipette. Resuspend the cells in 200 µL of FACS buffer, then transfer the resulting cell suspension into 5 mL flow tubes.
  7. Perform flow cytometry on the cell suspension in step 2.6 to determine the surface CAR expression. Include non-transfected T cells as a negative control.
    1. Use the FSC/SSC and FSC-A/FSC-H gates to screen live single cells. Collect 1 × 104 live single events for every sample. Gate the cells positive for EGFP (a constitutive marker carried in the lentiviral vector as an indicator of successfully transduced T cells) and then, the cells positive for phycoerythrin (PE) (CAR-expressing cells) to measure PE positivity and median fluorescence intensity (MFI).

3. Analysis of oxygen-dependency of CAR expression in CAR-modified Jurkat T cells by western blot

  1. Plate the CAR-transduced Jurkat T cells from section 1 in two 48-well plates at a density of 5 × 105 cells per well (500 µL culture volume) in RPMI1640 medium with 10% FBS.
  2. For one plate, add CoCl2 to the experimental wells to a final concentration of 0 µM, 50 µM, or 200 µM. Then, place the plate in a humidified, 5% CO2 incubator at 37 °C (normoxic condition). For the other plate, do not add CoCl2 and place it in a mobile CO2/O2/N2 incubator chamber with the O2 level preset at 1% (hypoxic condition) before transferring it to the same incubator.
  3. After 24 h of incubation, transfer the cell suspensions into 1.5 mL microcentrifuge tubes. Centrifuge the tubes at 500 × g for 5 min. Completely remove and discard the supernatants first using a 1-mL pipette, then a 100 µL pipette, and resuspend the cell pellets in 50 µL of 1x SDS-PAGE sample buffer.
  4. Heat the samples in a boiling water bath for 10 min. Immediately place the tube on ice for 30 s, then centrifuge at 16,000 × g for 30 s.
  5. Load 30 µL of the cleared samples into each slot of a 10-well, 10% SDS PAGE gel with a thickness of 1.5 mm. Run the gel at 80 V for 30 min, then increase the voltage to 100 V and run for 1.5 h.
  6. At the end of electrophoresis, transfer proteins from the gel to a PVDF membrane using the standard wet transfer method, with the current and duration set at 400 mA and 1 h, respectively.
  7. Block the membrane in blocking buffer (5% milk (w/v) in PBST (PBS+0.05% Tween-20)) for 1 h at room temperature. Then, cut out the piece between 30 kD and 40 kD for detection of the GAPDH loading control and the piece between 50 kD and 70k D for detection of the CAR molecules.
  8. Incubate the 30-40 kD and 50-70 kD pieces with a mouse anti-GAPDH antibody (1:2,000 dilution) and a mouse anti-Flag antibody (1:2,000 dilution), respectively, in 3 mL of blocking buffer either at room temperature for 2 h or at 4 °C overnight.
  9. Wash the membranes with PBST at room temperature on a platform rocker for 3 x 5 min.
  10. Incubate the membranes with HRP-conjugated goat anti-mouse antibody (1:5,000 dilution) in 3 mL of blocking buffer at room temperature for 1 h; then wash the membrane with PBST for 5 x 10 min.
  11. Develop the membranes by using incubating them with an HRP substrate, then visualize the detected protein bands using a luminescent image analyzer.

4. In vitro assessment of the oxygen dependency of cytotoxicity mediated by hypoxia-sensitive CAR-T cells

  1. On Day 0, seed 1 × 104 target cells (SKOV3-Luc cells) per experimental well in 200 µL of DMEM containing 10% FBS in two black flat-bottom 96-well tissue culture plates.
  2. On Day 1, Carefully remove 100 µL of the supernatant from the top of each well. Add CAR-T cells or non-transduced T cells at effector-to-target ratios of 1:1, 2:1, and 4:1 in 100 µL of DMEM medium containing 10% FBS.
  3. Place one plate in a 21% O2 atmosphere and the other in 1% O2 atmosphere using a mobile CO2/O2/N2 incubator chamber, as described in step 2.1.
    NOTE: If a mobile CO2/O2/N2 incubator chamber is not available, adding CoCl2 to the culture medium can be used to mimic a hypoxic condition.
  4. On Day 2, after 24 h of co-culturing, carefully transfer all the supernatant (approximately 150-200 µL) into a new U-bottom 96-well plate using a pipette. Store at -20 °C for later cytokine detection, following the procedures outlined in section 5.
  5. Add 60 µL of 1x passive lysis buffer to each experimental well of the black flat-bottom 96-well plates from step 4.4. Then, place the plates on a shaker and shake for 30 min to ensure efficient cell lysis.
  6. Add 60 µL of firefly luciferase substrate to each experimental well, and measure the luciferase activity immediately using a microplate reader.
  7. Calculate normalized cytotoxicity (%) using equation (1):
    Normalized cytotoxicity (%) = 100 - CAR-T cell therapy efficiency, treatment ratio equation, gene expression analysis, experimental results. ×100    (1)

5. Detection of IL-2 and IFN-γ secretion by hypoxia-sensitive CAR-T cells

  1. On Day 0, prepare a 1:250 dilution of IL-2 or IFN-γ capture antibody in Coating Buffer. Add 100 µL of the diluted antibody to each well of a 96-well ELISA plate and Incubate the plate at 4 °C overnight.
    NOTE: Coating Buffer is made by dissolving 7.13 g of NaHCO3 and 1.59 g of Na2CO3 in 1 L of distilled water and adjusting the pH to 9.5.
  2. On day 1, remove the unadsorbed capture antibody by vigorously flipping the plate upside down, then wash the wells 3x with 200 µL of Wash Buffer (PBS containing 0.05% Tween 20).
  3. Add 200 µL of Assay Diluent (PBS containing 10% FBS) to each well and incubate at room temperature for 1 h.
  4. Discard the solution by vigorously flipping the plate upside down; then, wash the wells 3x with 200 µL of Wash Buffer.
  5. Thaw the frozen supernatant samples/plate from step 4.4 at room temperature. Once completely thawed, dilute the samples and standards with Assay Diluent. Add 100 µL of diluted samples or standards to each well of the coated ELISA plate and incubate at room temperature for 2 h.
    NOTE: For IL-2 detection, a 10-fold dilution is recommended, while for IFN-γ detection, a 50-fold dilution is preferred.
  6. Remove the samples and standards by vigorously flipping the plate upside down, then wash the wells 5x with 200 µL of Wash Buffer per well.
  7. Prepare a working detection solution by diluting the IL-2 detection antibody or IFN-γ detection antibody/Streptavidin-HRP (SAv-HRP) at a 1:250 ratio in assay diluent. Add 100 µL of the working detection solution to each well and incubate the plate at room temperature for 1 h with gentle shaking.
  8. Discard the solution and wash the wells 7x with 200 µL of Wash Buffer.
  9. Add 100 µL of Substrate Reagent to each well and incubate the plate at room temperature for 30 min in the dark.
  10. Add 50 µL of Stop Solution (1 M H2SO4) to each well. Immediately read the absorbance at 450 nm using a microplate reader.

Results

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.

HER2-BBz signaling construct comparison; flow cytometry, Western blot, cytotoxicity, cytokine analysis.
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.

Discussion

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

Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

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).

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1.5 mL Centrifuge tubeQSP509-GRD-QSupernatants and cells cellection
Protocol Step 2,3,4
10% ExpressCast PAGENCM biotechP2012Immunoblotting
Protocol Step 3
10x PBSNCM biotech20220812Cell culture
Protocol Step 4
10 mL pipetteYueyibioYB-25HPipetting
Protocol Step 1
10xTRIS-Glycine-SDS electrophoresis bufferEpizyme3673020Immunoblotting
Protocol Step 3
15 mL Centrifuge tubeThermo Scientific339650Supernatants and cells cellection
Protocol Step 1
25 cm2 EasYFlaskThermo Scientific156367Cell culture
Protocol Step 3,4
4x Protein SDS PAGE Loading BufferTakara9173Immunoblotting
Protocol Step 3
6-well flat-bottom tissue culture platesThermo Scientific140675T Cells culture
Protocol Step 1
96-well black flat-bottom tissue culture platesGreiner655090Cytotoxicity assay
Protocol Step 4
96-well ELISA platesCorning3590ELISA
Protocol Step 5
96-well plate shakerQILINBEIERMH-2Shake
Protocol Step 4
96-well U-bottom tissue culture platesThermo Scientific268200Supernatants cellection
Protocol Step 4,5
anti-FLAG antibodySigmaF1804-50UGImmunoblotting
Protocol Step 3
CarbinolSinopharm10010061Immunoblotting
Protocol Step 3
Carbon dioxide incubatorThermo Scientific360Cell culture
Protocol Step 1,2,3,4
Cell counting plateHausser scientific1492Cell counting
Protocol Step 1,3,4
CELLection Pan Mouse IgG KitThermo Scientific11531DMouse IgG magnetic beads
Protocol Step 1
CentrifugeThermo Scientific75002432Cell culture
Protocol Step 1,3,4
Chemiluminescence gel imaging systemBIO-RAD12003154Immunoblotting
Protocol Step 3
Cobalt chloride solution (0.5 M)bioleaperBR4000203Hypoxic condition
Protocol Step 2,3,4
DMEMCorning10-103-CVCell culture
Protocol Step 4
Electronic balanceSartoriusPRACTUM612-1CNweigh
Protocol Step 5
FBSBI04-001-1ACSCell culture
Protocol Step 3,4
GAPDH Mouse mAbABclonalAC002Immunoblotting
Protocol Step 3
Gel electrophoresis apparatusBIO-RAD1645070Immunoblotting
Protocol Step 3
GloMax Microplate ReadersPromegaGM3000luciferase activity measurement
Protocol Step 4
Goat anti-Mouse IgG (H+L)YeasenP1126151Immunoblotting
Protocol Step 3
High speed microfreezing centrifugeeppendorf5810 RCell culture
Protocol Step 1
Human IFN-γ ELISA SetBD555142ELISA
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 SetBD555190ELISA
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-15R&D systemsP40933T Cells culture
Protocol Step 1
IL-21NovoproteinGMP-CC45T Cells culture
Protocol Step 1
IL-7R&D systemsP13232T Cells culture
Protocol Step 1
Inverted microscopeOlympusCKX41Cell culture
Protocol Step 1,3,4
JurkatATCCTIB-152CAR-Jurkat construction
Protocol Step 3
LSRFortessaBDLSRFortessaFlow cytometry
Protocol Step 2
Luciferase Assay SystemPromegaE1501luciferase reporter assay
Protocol Step 4
Items: Passive lysis buffer, firefly luciferase substrate
Microplate readerBioTekHTXELISA
Protocol Step 5
mobile CO2/O2/N2 Incubator ChamberChina Innovation Instrument Co., Ltd.Smartor118Hypoxic condition
Protocol Step 2, 3, 4
Mouse Anti-Hexa Histidine tagSigmaSAB2702218Immunoblotting
Protocol Step 3
NcmBlot Rapid Transfer BufferNCM biotechWB4600Immunoblotting
NcmECL UltraNCM biotechP10300Immunoblotting
Protocol Step 3
Items: NcmECL Ultra Luminol/Enhancer Reagent (A) ,NcmECL Ultra Stabilized Peroxide Reagent (B) 
NovoNectin -coated 48-well flat platesNovoproteinGMP-CH38CAR-T cells construction
Protocol Step 1
OPD (o-phenylenediamine dihydrochloride) tablet setSigmaP9187Substrate Reagent
Protocol Step 5
Items: OPD tablet (silver foil),urea hydrogen peroxide tablet (gold foil)
PE-conjugated anti-DYKDDDDKBiolegend637310Flow cytometry
Protocol Step 2
Protamine sulfateSigmaP3369-1OGLentivirus infection
Protocol Step 1
Protein Marker 10 Kda-250 KDaEpizymeWJ102Immunoblotting
Protocol Step 3
 Purifed NA/LE Mouse Anti-Human CD3BD566685T Cells culture
Protocol Step 1
Purified NA/LE Mouse Anti-Human CD28BD555725T Cells culture
Protocol Step 1
PVDF membraneMillipore168627Immunoblotting
Protocol Step 3
RPMI 1640Corning10-040-CVRCCell culture
Protocol Step 3
Skim milk powderYeasenS9129060Immunoblotting
Protocol Step 3
SKOV3-LucATCCHTB-77Cytotoxicity assay
Protocol Step 4
Trypsin-EDTANCM biotechC125C1Cell culture
Protocol Step 4
Tween 20Sinopharm30189328Immunoblotting
Protocol Step 3
Water bathkeelreinNB014467Heating
Protocol Step 1
X-VIVO 15 LONZA04-418QSerum-free lymphocyte culture medium
Protocol Step 1

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CAR T CellsHypoxia-Sensitive CARTumor MicroenvironmentSolid TumorsCancer ImmunotherapyOxygen-Dependent DegradationCAR ExpressionCytotoxicity AssayHypoxia ModelTumor Selectivity
Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells
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