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

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 2017^1,2,3, 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 tumors^5,6,7,8.

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 waste^9,10,11,12. 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 tumor^13,14.

Apart from the unsatisfactory therapeutic efficiency, safety issues are another Achilles' heel of CAR-T cells when dealing with solid tumors^15,16. 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 cues^{20,21,22,23,24,25,26}.

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 - ×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 a...

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...

Materials

Name	Company	Catalog Number	Comments
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