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).

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 CD3&#958;. 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
<ol>
	<li>Thaw cryopreserved human peripheral blood mononuclear cells (PBMCs) rapidly at 37 &#176;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&#160;ng/mL IL-7, and 10 ng/mL IL-15 (human T cell growth medium, referred to as TGM thereafter).</li>
	<li>After taking an aliquot for cell counting, centrifuge the tube at 300 &#215; g for 5 min. Resuspend the pelleted PBMCs with TGM at a density of 4 &#215; 106 cells/mL and transfer the suspension into a 6-well plate.</li>
	<li>Prepare anti-hCD3/hCD28-coated immunobeads.
	<ol>
		<li>Transfer 100 &#181;L of mouse IgG magnetic beads into a 1.5 mL microcentrifuge tube and wash them 2 times&#160;using a magnetic stand with PBS.</li>
		<li>Resuspend the beads in 100 &#181;L of PBS and add 0.2 &#181;g of mouse anti-Human CD3 antibody and 2 &#181;g of mouse anti-Human CD28 antibody. Gently mix the mixture using a pipette and rock overnight at 4 &#176;C.</li>
		<li>Wash the beads 2 times&#160;using a magnetic stand with PBS and resuspend them in 100 &#181;L of PBS.</li>
	</ol>
	</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Take an aliquot for cell counting, then seed the PBMCs at 5 &#215; 105 cells/well in 300 &#181;L of TGM into a 48-well flat plate. Add 200 &#181;L of lentiviral stock into the corresponding wells and add protamine sulfate to a final concentration of 10 &#181;g/mL.</li>
	<li>Centrifuge the plate at 1,000 &#215; g at 32 &#176;C for 1.5 h and carefully remove and discard 300 &#181;L 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 &#176;C.</li>
	<li>Add fresh TGM to adjust the cell density to 0.5-2 &#215; 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 &#215; 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).</li>
</ol>
2. Assessment of oxygen-dependent CAR expression in CAR-T cells using flow cytometry
<ol>
	<li>Plate the CAR-transduced T cells from step 1.8 into two 12-well plates at a density of 2.5 &#215; 106 cells/well in 2 mL of TGM. Place one plate directly in a humidified incubator with 5% CO2 at 37 &#176;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 &#176;C.</li>
	<li>Every 24 h, collect 5 &#215; 105 cells from the plates under hypoxic or normoxic conditions into 1.5 mL microcentrifuge tubes. Centrifuge the tubes at 500 &#215; 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.</li>
	<li>Resuspend the pelleted cells in 50 &#181;L of FACS buffer (PBS supplemented with 2% FBS) in each tube. Add 50 &#181;L of a 1:100 dilution of PE-conjugated anti-Flag antibody (0.2 &#181;g/mL) and mix thoroughly by pipetting. Incubate in the dark at room temperature for 20 min.</li>
	<li>At the end of incubation, add 1 mL of FACS buffer to each tube. Mix thoroughly by pipetting, then centrifuge at 500 &#215; g for 5 min.</li>
	<li>Carefully remove and discard the supernatant using a pipette and repeat step 2.4 one more time.</li>
	<li>Carefully remove and discard the supernatant using a pipette. Resuspend the cells in 200 &#181;L of FACS buffer, then transfer the resulting cell suspension into 5 mL flow tubes.</li>
	<li>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.
	<ol>
		<li>Use the FSC/SSC and FSC-A/FSC-H gates to screen live single cells. Collect 1 &#215; 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).</li>
	</ol>
	</li>
</ol>
3. Analysis of oxygen-dependency of CAR expression in CAR-modified Jurkat T cells by western blot
<ol>
	<li>Plate the CAR-transduced Jurkat T cells from section 1 in two 48-well plates at a density of 5 &#215; 105 cells per well (500 &#181;L culture volume) in RPMI1640 medium with 10% FBS.</li>
	<li>For one plate, add CoCl2 to the experimental wells to a final concentration of 0 &#181;M, 50 &#181;M, or 200 &#181;M. Then, place the plate in a humidified, 5% CO2 incubator at 37 &#176;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.</li>
	<li>After 24 h of incubation, transfer the cell suspensions into 1.5 mL microcentrifuge tubes. Centrifuge the tubes at 500 &#215; g for 5 min. Completely remove and discard the supernatants first using a 1-mL pipette, then a 100 &#181;L pipette, and resuspend the cell pellets in 50 &#181;L of 1x SDS-PAGE sample buffer.</li>
	<li>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 &#215; g for 30 s.</li>
	<li>Load 30 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C overnight.</li>
	<li>Wash the membranes with PBST at room temperature on a platform rocker for 3 x 5 min.</li>
	<li>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.</li>
	<li>Develop the membranes by using incubating them with an HRP substrate, then visualize the detected protein bands using a luminescent image analyzer.</li>
</ol>
4. In vitro assessment of the oxygen dependency of cytotoxicity mediated by hypoxia-sensitive CAR-T cells
<ol>
	<li>On Day 0, seed 1 &#215; 104 target cells (SKOV3-Luc cells) per experimental well in 200 &#181;L of DMEM containing 10% FBS in two black flat-bottom 96-well tissue culture plates.</li>
	<li>On Day 1, Carefully remove 100 &#181;L 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 &#181;L of DMEM medium containing 10% FBS.</li>
	<li>Place one plate in a 21% O2&#160;atmosphere and the other in 1% O2&#160;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.</li>
	<li>On Day 2, after 24 h of co-culturing, carefully transfer all the supernatant (approximately 150-200 &#181;L) into a new U-bottom 96-well plate using a pipette. Store at -20 &#176;C for later cytokine detection, following the procedures outlined in section 5.</li>
	<li>Add 60 &#181;L 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.</li>
	<li>Add 60 &#181;L of firefly luciferase substrate to each experimental well, and measure the luciferase activity immediately using a microplate reader.</li>
	<li>Calculate normalized cytotoxicity (%) using equation (1): 
	Normalized cytotoxicity (%) = 100 - <img alt="Equation 1" src="/files/ftp_upload/66697/66697eq01.jpg" style="margin: auto;" /> &#215;100&#160; &#160; (1)</li>
</ol>
5. Detection of IL-2 and IFN-&#947; secretion by hypoxia-sensitive CAR-T cells
<ol>
	<li>On Day 0, prepare a 1:250 dilution of IL-2 or IFN-&#947; capture antibody in Coating Buffer. Add 100 &#181;L of the diluted antibody to each well of a 96-well ELISA plate and Incubate the plate at 4 &#176;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.</li>
	<li>On day 1, remove the unadsorbed capture antibody by vigorously flipping the plate upside down, then wash the wells 3x with 200 &#181;L of Wash Buffer (PBS containing 0.05% Tween 20).</li>
	<li>Add 200 &#181;L of Assay Diluent (PBS containing 10% FBS) to each well and incubate at room temperature for 1 h.</li>
	<li>Discard the solution by vigorously flipping the plate upside down; then, wash the wells 3x with 200 &#181;L of Wash Buffer.</li>
	<li>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 &#181;L 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-&#947; detection, a 50-fold dilution is preferred.</li>
	<li>Remove the samples and standards by vigorously flipping the plate upside down, then wash the wells 5x with 200 &#181;L of Wash Buffer per well.</li>
	<li>Prepare a working detection solution by diluting the IL-2 detection antibody or IFN-&#947; detection antibody/Streptavidin-HRP (SAv-HRP) at a 1:250 ratio in assay diluent. Add 100 &#181;L of the working detection solution to each well and incubate the plate at room temperature for 1 h with gentle shaking.</li>
	<li>Discard the solution and wash the wells 7x with 200 &#181;L of Wash Buffer.</li>
	<li>Add 100 &#181;L of Substrate Reagent to each well and incubate the plate at room temperature for 30 min in the dark.</li>
	<li>Add 50 &#181;L of Stop Solution (1 M H2SO4) to each well. Immediately read the absorbance at 450 nm using a microplate reader.</li>
</ol>

Functional Validation of Hypoxia-Sensitive CAR-T Cells for Selective Tumors

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+noninvasive+preclinical+tumor+hypoxia+imaging+methods:+A+review.">In vivo noninvasive preclinical tumor hypoxia imaging methods: A review.</a> Int J Radiat Biol. 97 (5), 593-631 (2021).</li><li>He, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+car-t+cells+by+hypoxia-inducible+transcription+amplification+(hita)+system+significantly+enhances+systemic+safety+and+retains+antitumor+efficacy.">Conditioned car-t cells by hypoxia-inducible transcription amplification (hita) system significantly enhances systemic safety and retains antitumor efficacy.</a> J Immunother Cancer. 9 (10), e002755 (2021).</li><li>Barsoum, I. B., Smallwood, C. A., Siemens, D. R., Graham, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+of+hypoxia-mediated+escape+from+adaptive+immunity+in+cancer+cells.">A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells.</a> Cancer Res. 74 (3), 665-674 (2014).</li><li>Kroemer, G., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+metabolism:+Cancer's+achilles'+heel.">Tumor cell metabolism: Cancer's achilles' heel.</a> Cancer Cell. 13 (6), 472-482 (2008).</li></ol>

The authors have no conflicts of interest to declare.

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...

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&#39; 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 CD3&#950; 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-1&#945;, and a constitutively expressed subunit, HIF-1&#946;27. Under normoxic conditions, the HIF-1&#945; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>QSP</td>
			<td>509-GRD-Q</td>
			<td>Supernatants and cells cellection 
			Protocol Step 2,3,4</td>
		</tr>
		<tr>
			<td>10% ExpressCast PAGE</td>
			<td>NCM biotech</td>
			<td>P2012</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>NCM biotech</td>
			<td>20220812</td>
			<td>Cell culture 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>10 mL pipette</td>
			<td>Yueyibio</td>
			<td>YB-25H</td>
			<td>Pipetting 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>10xTRIS-Glycine-SDS electrophoresis buffer</td>
			<td>Epizyme</td>
			<td>3673020</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Thermo Scientific</td>
			<td>339650</td>
			<td>Supernatants and cells cellection 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>25 cm2 EasYFlask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td>Cell culture 
			Protocol Step 3,4</td>
		</tr>
		<tr>
			<td>4x Protein SDS PAGE Loading Buffer</td>
			<td>Takara</td>
			<td>9173</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>6-well flat-bottom tissue culture plates</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td>T Cells culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>96-well black flat-bottom tissue culture plates</td>
			<td>Greiner</td>
			<td>655090</td>
			<td>Cytotoxicity assay 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>96-well ELISA plates</td>
			<td>Corning</td>
			<td>3590</td>
			<td>ELISA 
			Protocol Step 5</td>
		</tr>
		<tr>
			<td>96-well plate shaker</td>
			<td>QILINBEIER</td>
			<td>MH-2</td>
			<td>Shake 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>96-well U-bottom tissue culture plates</td>
			<td>Thermo Scientific</td>
			<td>268200</td>
			<td>Supernatants cellection 
			Protocol Step 4,5</td>
		</tr>
		<tr>
			<td>anti-FLAG antibody</td>
			<td>Sigma</td>
			<td>F1804-50UG</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>Carbinol</td>
			<td>Sinopharm</td>
			<td>10010061</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>Carbon dioxide incubator</td>
			<td>Thermo Scientific</td>
			<td>360</td>
			<td>Cell culture 
			Protocol Step 1,2,3,4</td>
		</tr>
		<tr>
			<td>Cell counting plate</td>
			<td>Hausser scientific</td>
			<td>1492</td>
			<td>Cell counting 
			Protocol Step 1,3,4</td>
		</tr>
		<tr>
			<td>CELLection Pan Mouse IgG Kit</td>
			<td>Thermo Scientific</td>
			<td>11531D</td>
			<td>Mouse IgG magnetic beads 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002432</td>
			<td>Cell culture 
			Protocol Step 1,3,4</td>
		</tr>
		<tr>
			<td>Chemiluminescence gel imaging system</td>
			<td>BIO-RAD</td>
			<td>12003154</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>Cobalt chloride solution (0.5 M)</td>
			<td>bioleaper</td>
			<td>BR4000203</td>
			<td>Hypoxic condition 
			Protocol Step 2,3,4</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-103-CV</td>
			<td>Cell culture 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Sartorius</td>
			<td>PRACTUM612-1CN</td>
			<td>weigh 
			Protocol Step 5</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>BI</td>
			<td>04-001-1ACS</td>
			<td>Cell culture 
			Protocol Step 3,4</td>
		</tr>
		<tr>
			<td>GAPDH Mouse mAb</td>
			<td>ABclonal</td>
			<td>AC002</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus</td>
			<td>BIO-RAD</td>
			<td>1645070</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>GloMax Microplate Readers</td>
			<td>Promega</td>
			<td>GM3000</td>
			<td>luciferase activity measurement 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L)</td>
			<td>Yeasen</td>
			<td>P1126151</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>High speed microfreezing centrifuge</td>
			<td>eppendorf</td>
			<td>5810&#160;R</td>
			<td>Cell culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>Human IFN-&#947; ELISA Set</td>
			<td>BD</td>
			<td>555142</td>
			<td>ELISA 
			Protocol Step 5 
			Items: Recombinant Human IFN-&#947; Lyophilized Standard, Detection Antibody Biotin Anti-Human IFN-&#947; , Capture Antibody Purified Anti-Human IFN-&#947;, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP)</td>
		</tr>
		<tr>
			<td>Human IL-2 ELISA Set</td>
			<td>BD</td>
			<td>555190</td>
			<td>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)</td>
		</tr>
		<tr>
			<td>IL-15</td>
			<td>R&#38;D systems</td>
			<td>P40933</td>
			<td>T Cells culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>IL-21</td>
			<td>Novoprotein</td>
			<td>GMP-CC45</td>
			<td>T Cells culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>IL-7</td>
			<td>R&#38;D systems</td>
			<td>P13232</td>
			<td>T Cells culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Cell culture 
			Protocol Step 1,3,4</td>
		</tr>
		<tr>
			<td>Jurkat</td>
			<td>ATCC</td>
			<td>TIB-152</td>
			<td>CAR-Jurkat construction 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>LSRFortessa</td>
			<td>BD</td>
			<td>LSRFortessa</td>
			<td>Flow cytometry 
			Protocol Step 2</td>
		</tr>
		<tr>
			<td>Luciferase Assay System</td>
			<td>Promega</td>
			<td>E1501</td>
			<td>luciferase reporter assay 
			Protocol Step 4 
			Items: Passive lysis buffer, firefly luciferase substrate</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>HTX</td>
			<td>ELISA 
			Protocol Step 5</td>
		</tr>
		<tr>
			<td>mobile CO2/O2/N2 Incubator Chamber</td>
			<td>China Innovation Instrument Co., Ltd.</td>
			<td>Smartor118</td>
			<td>Hypoxic condition 
			Protocol Step 2, 3, 4</td>
		</tr>
		<tr>
			<td>Mouse Anti-Hexa Histidine tag</td>
			<td>Sigma</td>
			<td>SAB2702218</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>NcmBlot Rapid Transfer Buffer</td>
			<td>NCM biotech</td>
			<td>WB4600</td>
			<td>Immunoblotting</td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>NCM biotech</td>
			<td>P10300</td>
			<td>Immunoblotting 
			Protocol Step 3 
			Items: NcmECL Ultra Luminol/Enhancer Reagent (A) ,NcmECL Ultra Stabilized Peroxide Reagent (B)&#160;</td>
		</tr>
		<tr>
			<td>NovoNectin -coated 48-well flat plates</td>
			<td>Novoprotein</td>
			<td>GMP-CH38</td>
			<td>CAR-T cells construction 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>OPD (o-phenylenediamine dihydrochloride) tablet set</td>
			<td>Sigma</td>
			<td>P9187</td>
			<td>Substrate Reagent 
			Protocol Step 5 
			Items: OPD tablet (silver foil),urea hydrogen peroxide tablet (gold foil)</td>
		</tr>
		<tr>
			<td>PE-conjugated anti-DYKDDDDK</td>
			<td>Biolegend</td>
			<td>637310</td>
			<td>Flow cytometry 
			Protocol Step 2</td>
		</tr>
		<tr>
			<td>Protamine sulfate</td>
			<td>Sigma</td>
			<td>P3369-1OG</td>
			<td>Lentivirus infection 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>Protein Marker 10 Kda-250 KDa</td>
			<td>Epizyme</td>
			<td>WJ102</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>&#160;Purifed NA/LE Mouse Anti-Human CD3</td>
			<td>BD</td>
			<td>566685</td>
			<td>T Cells culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>Purified NA/LE Mouse Anti-Human CD28</td>
			<td>BD</td>
			<td>555725</td>
			<td>T Cells culture 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Millipore</td>
			<td>168627</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>10-040-CVRC</td>
			<td>Cell culture 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Yeasen</td>
			<td>S9129060</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>SKOV3-Luc</td>
			<td>ATCC</td>
			<td>HTB-77</td>
			<td>Cytotoxicity assay 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>NCM biotech</td>
			<td>C125C1</td>
			<td>Cell culture 
			Protocol Step 4</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sinopharm</td>
			<td>30189328</td>
			<td>Immunoblotting 
			Protocol Step 3</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>keelrein</td>
			<td>NB014467</td>
			<td>Heating 
			Protocol Step 1</td>
		</tr>
		<tr>
			<td>X-VIVO 15&#160;</td>
			<td>LONZA</td>
			<td>04-418Q</td>
			<td>Serum-free lymphocyte culture medium 
			Protocol Step 1</td>
		</tr>
	</tbody>
</table>

generation and functional verification of hypoxia-sensitive chimeric antigen receptor-t cells

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.

Author Spotlight: Advancements in Hypoxia-Sensitive CAR-T Therapy for Enhanced Cancer Immunotherapy

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

Our research primarily focuses on cancer immunotherapy, particularly in CAR T-cell-based immunotherapy. We have been exploring novel strategies during last 10 years to enable CAR T-cells to specifically target a tumor with minimizing the side effect or even no side effect at all. Novel strategy have been persistently explored to improve the safety and efficacy of the CAR T-cell-based immunotherapy, in particularly by targeting tumor microenvironment, such as hypoxia-conditioned CAR, as exemplified in this protocol.Interestingly, recent study demonstrated that hypoxia-condition CAR T not only greatly improved the safety profile, but also reduced the CAR T exhaustion and enhanced the efficacy in solid tumors. Hypoxia is a characteristic feature of tumor microenvironment. Our research aims to help researchers in the field of cancer immunotherapy understand the concept of hypoxia-sensitive CAR T-cells, their construction and how to establish hypoxia models for evaluation.Our laboratory will continue exploring new strategies for constructing CAR T-cells to enhance their tumor-curing efficacy, safety profile and cost effectiveness.

Fusing the ODD domain of HIF-1&#945; 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...

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 ...

generation-functional-verification-hypoxia-sensitive-chimeric-antigen

Research

JoVE Journal

Cancer Research

Development of Hypoxia-Responsive CAR-T Cells via Lentiviral Transduction

To begin, place a vial of cryopreserved human peripheral blood mononuclear cells in a water bath at 37 degrees Celsius. Once thawed, transfer the PBMC suspension into a 15 milliliter tube containing nine milliliters of TGM. Centrifuge the tube at 300 G for five minutes after pipetting out an aliquot of the cell suspension for counting.Resuspend the pelleted PBMC in three milliliters of TGM. Then, transfer the required volume of cell suspension into a six well plate. The day before the cell resuscitation, transfer 100 microliters of mouse immunoglobulin G magnetic beads into a 1.5 milliliter micro centrifuge tube.Add one milliliter PBS to the tubes and place them on a magnetic stand to wash the beads. Resuspend the beads in 100 microliters of PBS. Then add the antibodies to the suspension.Use a pipette to gently mix the suspension well. Place the suspension on a rocker at four degrees Celsius overnight. After washing the beads in PBS as done earlier, resuspend them in 100 microliters of PBS.Now add NHCD3 hCD28 coated immuno beads to the plate and incubate. After 48 hours of incubation, mix and transfer the cell suspension into a 15 milliliter centrifuge tube. Place the tube on a magnetic stand for three minutes.Then transfer the supernatant into a new 15 milliliter tube. Seed five times 10 to the power of five PBMC in 300 microliters of TGM into the wells of a 48 well flat plate. Pipette 200 microliters of lentiviral stock into the corresponding wells.Then add protamine sulfate to a final concentration of 10 micrograms per milliliter before centrifuging. Pipette out 300 microliters of the supernatant from each well, then add one milliliter of fresh TGM into each well. Place the plate in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide.Add fresh 0.5 to two milliliters TGM to the plate every two to three days. Then transfer the cells into a 12 well plate, followed by a six well plate until the total cell density reaches 6 million in a four milliliter volume.

Flow Cytometric Assessment of Oxygen-Dependent CAR Expression in CAR-T Cells

To begin, plate the cultured CAR-transduced T cells into two 12-well plates containing two milliliters of TGM. Place one plate directly in a humidified incubator under 5%carbon dioxide at 37 degrees Celsius. Place the other plate in a mobile carbon dioxide, oxygen nitrogen incubator chamber with the oxygen level preset at 1%Collect 500, 000 cells from the plates every 24 hours under both conditions into 1.5 milliliter micro centrifuge tubes.Centrifuge the tubes at 500 G for five minutes. After removing the supernatant, gently pipette one milliliter of PBS into the cell pellet. Resuspend the pelleted cells in 50 microliters of FACS buffer.Then add 50 microliters of a one to 100 dilution of phycoerythrin conjugated anti-flag antibody. Pipette the suspension to mix thoroughly. After incubation, add one milliliter of FACS buffer to each tube.Centrifuge the mixed suspension at 500 G for five minutes. Now, pipette out the supernatants from each tube. Then resuspend the cells in 200 microliters of FACS buffer.Transfer the resulting cell suspension into five milliliter flow cytometry tubes. Perform flow cytometry on the cell suspension to determine the surface chimeric antigen receptor expression. Set up new tubes as blank and CAR.Click FSCA, FSCH, SSCA, SSCH, PE and FITC channel. Then create four scatter plots to sequentially identify single cells, living cells, GFP positive and PE positive cells. Set parameters for collecting 10, 000 single living cells.Click acquire data. After cell collection is stable, click record data. To determine the location of gates according to negative control, gate the cells positive for EGFP and phycoerythrin to measure phycoerythrin positivity and median fluorescence intensity.The hypoxia sensitive per two targeting CAR showed significantly high expressions of CAR under hypoxic conditions relative to normoxic conditions.

In Vitro Assessment of Cytotoxicity Mediated by Hypoxia-Sensitive CAR-T Cells

To begin, seed 1000 target cells into each well of two flat bottom 96 well tissue culture plates containing 200 microliters of FBSDMEM. The next day carefully pipette out 100 microliters of the supernatant from the top of each well. Add chimeric antigen receptor T cells or nontransduced T cells in 100 microliters of FBSDMEM at different ratios.Place one plate in a 21%oxygen atmosphere and the other in a mobile carbon dioxide oxygen nitrogen incubator chamber under 1%oxygen atmosphere. After 24 hours of co-culturing, use a pipette to carefully transfer all the supernatant into a new U-bottom 96 well plate. Store the supernatant at minus 20 degrees Celsius for cytokine detection.Now add 60 microliters of passive lysis buffer into each experimental well of the black flat-bottom 96 well plates. Place the plates on a shaker for 30 minutes to ensure efficient cell lysis. Add 60 microliters of firefly luciferase substrate into each experimental well.Use a microplate reader to measure the luciferase activity immediately. Finally, calculate the normalized cytotoxicity percentage with the given equation. The hypoxia sensitive HER2 targeting CAR was able to effectively kill target cells regardless of whether the atmosphere was hypoxic or normoxic.In contrast, the HER2-BBz-ODD CAR T cells displayed significantly weaker cytotoxicity under normoxic conditions for all conditions.

538 Views.  Fudan University.  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. 

Functional Validation of Hypoxia-Sensitive CAR-T Cells for Selective Tumors

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