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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<li>D'alonzo, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+noninvasive+preclinical+tumor+hypoxia+imaging+methods%3A+A+review%5BTitle%5D)+AND+%22Int+J+Radiat+Biol%22%5BJournal%5D)">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/pubmed?term=((Conditioned+car-t+cells+by+hypoxia-inducible+transcription+amplification+%28hita%29+system+significantly+enhances+systemic+safety+and+retains+antitumor+efficacy%5BTitle%5D)+AND+%22J+Immunother+Cancer%22%5BJournal%5D)">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/pubmed?term=((A+mechanism+of+hypoxia-mediated+escape+from+adaptive+immunity+in+cancer+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">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/pubmed?term=((Tumor+cell+metabolism%3A+Cancer%27s+achilles%27+heel%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">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><tbody><tr><td>1.5 mL Centrifuge tube</td><td>QSP</td><td>509-GRD-Q</td><td>Supernatants and cells cellection&lt;br/&gt; Protocol Step 2,3,4</td></tr><tr><td>10% ExpressCast PAGE</td><td>NCM biotech</td><td>P2012</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>10x PBS</td><td>NCM biotech</td><td>20220812</td><td>Cell culture&lt;br/&gt; Protocol Step 4</td></tr><tr><td>10 mL pipette</td><td>Yueyibio</td><td>YB-25H</td><td>Pipetting&lt;br/&gt; Protocol Step 1</td></tr><tr><td>10xTRIS-Glycine-SDS electrophoresis buffer</td><td>Epizyme</td><td>3673020</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>15 mL Centrifuge tube</td><td>Thermo Scientific</td><td>339650</td><td>Supernatants and cells cellection&lt;br/&gt; Protocol Step 1</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; EasYFlask</td><td>Thermo Scientific</td><td>156367</td><td>Cell culture&lt;br/&gt; Protocol Step 3,4</td></tr><tr><td>4x Protein SDS PAGE Loading Buffer</td><td>Takara</td><td>9173</td><td>Immunoblotting&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; Protocol Step 4</td></tr><tr><td>96-well ELISA plates</td><td>Corning</td><td>3590</td><td>ELISA&lt;br/&gt; Protocol Step 5</td></tr><tr><td>96-well plate shaker</td><td>QILINBEIER</td><td>MH-2</td><td>Shake&lt;br/&gt; 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&lt;br/&gt; Protocol Step 4,5</td></tr><tr><td>anti-FLAG antibody</td><td>Sigma</td><td>F1804-50UG</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>Carbinol</td><td>Sinopharm</td><td>10010061</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>Carbon dioxide incubator</td><td>Thermo Scientific</td><td>360</td><td>Cell culture&lt;br/&gt; Protocol Step 1,2,3,4</td></tr><tr><td>Cell counting plate</td><td>Hausser scientific</td><td>1492</td><td>Cell counting&lt;br/&gt; 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&lt;br/&gt; Protocol Step 1</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>75002432</td><td>Cell culture&lt;br/&gt; Protocol Step 1,3,4</td></tr><tr><td>Chemiluminescence gel imaging system</td><td>BIO-RAD</td><td>12003154</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>Cobalt chloride solution (0.5 M)</td><td>bioleaper</td><td>BR4000203</td><td>Hypoxic condition&lt;br/&gt; Protocol Step 2,3,4</td></tr><tr><td>DMEM</td><td>Corning</td><td>10-103-CV</td><td>Cell culture&lt;br/&gt; Protocol Step 4</td></tr><tr><td>Electronic balance</td><td>Sartorius</td><td>PRACTUM612-1CN</td><td>weigh&lt;br/&gt; Protocol Step 5</td></tr><tr><td>FBS</td><td>BI</td><td>04-001-1ACS</td><td>Cell culture&lt;br/&gt; Protocol Step 3,4</td></tr><tr><td>GAPDH Mouse mAb</td><td>ABclonal</td><td>AC002</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>Gel electrophoresis apparatus</td><td>BIO-RAD</td><td>1645070</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>GloMax Microplate Readers</td><td>Promega</td><td>GM3000</td><td>luciferase activity measurement&lt;br/&gt; Protocol Step 4</td></tr><tr><td>Goat anti-Mouse IgG (H+L)</td><td>Yeasen</td><td>P1126151</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>High speed microfreezing centrifuge</td><td>eppendorf</td><td>5810&amp;nbsp;R</td><td>Cell culture&lt;br/&gt; Protocol Step 1</td></tr><tr><td>Human IFN-&amp;gamma; ELISA Set</td><td>BD</td><td>555142</td><td>ELISA&lt;br/&gt; Protocol Step 5&lt;br/&gt; Items: Recombinant Human IFN-&amp;gamma; Lyophilized Standard, Detection Antibody Biotin Anti-Human IFN-&amp;gamma; , Capture Antibody Purified Anti-Human IFN-&amp;gamma;, 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&lt;br/&gt; Protocol Step 5&lt;br/&gt; 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&amp;amp;D systems</td><td>P40933</td><td>T Cells culture&lt;br/&gt; Protocol Step 1</td></tr><tr><td>IL-21</td><td>Novoprotein</td><td>GMP-CC45</td><td>T Cells culture&lt;br/&gt; Protocol Step 1</td></tr><tr><td>IL-7</td><td>R&amp;amp;D systems</td><td>P13232</td><td>T Cells culture&lt;br/&gt; Protocol Step 1</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>CKX41</td><td>Cell culture&lt;br/&gt; Protocol Step 1,3,4</td></tr><tr><td>Jurkat</td><td>ATCC</td><td>TIB-152</td><td>CAR-Jurkat construction&lt;br/&gt; Protocol Step 3</td></tr><tr><td>LSRFortessa</td><td>BD</td><td>LSRFortessa</td><td>Flow cytometry&lt;br/&gt; Protocol Step 2</td></tr><tr><td>Luciferase Assay System</td><td>Promega</td><td>E1501</td><td>luciferase reporter assay&lt;br/&gt; Protocol Step 4&lt;br/&gt; Items: Passive lysis buffer, firefly luciferase substrate</td></tr><tr><td>Microplate reader</td><td>BioTek</td><td>HTX</td><td>ELISA&lt;br/&gt; Protocol Step 5</td></tr><tr><td>mobile CO&lt;sub&gt;2&lt;/sub&gt;/O&lt;sub&gt;2&lt;/sub&gt;/N&lt;sub&gt;2&lt;/sub&gt; Incubator Chamber</td><td>China Innovation Instrument Co., Ltd.</td><td>Smartor118</td><td>Hypoxic condition&lt;br/&gt; Protocol Step 2, 3, 4</td></tr><tr><td>Mouse Anti-Hexa Histidine tag</td><td>Sigma</td><td>SAB2702218</td><td>Immunoblotting&lt;br/&gt; 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&lt;br/&gt; Protocol Step 3&lt;br/&gt; Items: NcmECL Ultra Luminol/Enhancer Reagent (A) ,NcmECL Ultra Stabilized Peroxide Reagent (B)&amp;nbsp;</td></tr><tr><td>NovoNectin -coated 48-well flat plates</td><td>Novoprotein</td><td>GMP-CH38</td><td>CAR-T cells construction&lt;br/&gt; Protocol Step 1</td></tr><tr><td>OPD (o-phenylenediamine dihydrochloride) tablet set</td><td>Sigma</td><td>P9187</td><td>Substrate Reagent&lt;br/&gt; Protocol Step 5&lt;br/&gt; 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&lt;br/&gt; Protocol Step 2</td></tr><tr><td>Protamine sulfate</td><td>Sigma</td><td>P3369-1OG</td><td>Lentivirus infection&lt;br/&gt; Protocol Step 1</td></tr><tr><td>Protein Marker 10 Kda-250 KDa</td><td>Epizyme</td><td>WJ102</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>&amp;nbsp;Purifed NA/LE Mouse Anti-Human CD3</td><td>BD</td><td>566685</td><td>T Cells culture&lt;br/&gt; 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&lt;br/&gt; Protocol Step 1</td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>168627</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>10-040-CVRC</td><td>Cell culture&lt;br/&gt; Protocol Step 3</td></tr><tr><td>Skim milk powder</td><td>Yeasen</td><td>S9129060</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>SKOV3-Luc</td><td>ATCC</td><td>HTB-77</td><td>Cytotoxicity assay&lt;br/&gt; Protocol Step 4</td></tr><tr><td>Trypsin-EDTA</td><td>NCM biotech</td><td>C125C1</td><td>Cell culture&lt;br/&gt; Protocol Step 4</td></tr><tr><td>Tween 20</td><td>Sinopharm</td><td>30189328</td><td>Immunoblotting&lt;br/&gt; Protocol Step 3</td></tr><tr><td>Water bath</td><td>keelrein</td><td>NB014467</td><td>Heating&lt;br/&gt; Protocol Step 1</td></tr><tr><td>X-VIVO 15&amp;nbsp;</td><td>LONZA</td><td>04-418Q</td><td>Serum-free lymphocyte culture medium&lt;br/&gt; 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.

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...

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

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.

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 ...

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

Research

JoVE Journal

Cancer Research

876 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. 

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

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

Bioengineering

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

Immunology and Infection

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Author Spotlight: Advancements in CAR-T Cell Manufacturing and Gene Therapy Production 

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

Medicine

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Source: Ghassemi, S., et al. <a href="https://app.jove.com/v/59949">Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy.</a> J. Vis. Exp. (2019)
This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. T Cell Activation, Transduction, ...

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Immunology

Immunotherapy

Cellular Immunotherapy

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

On the day after the splenectomy, prepare PBS containing 25 micrograms per milliliter recombinant human Fibronectin fragment or RHFF and coat non-tissue culture-treated 24-well plates by adding 0.5 milliliters of the solution to each well. The following day, remove the solution from the wells, and add 1 milliliter per well of 2% BSA in PBS.
Incubate at room temperature for 30 minutes, then, remove the BSA by firmly upending the plate before adding 2 milliliters of PBS to wash. After collecting the viral supernatant from the HEK293T cells, add fresh TCM to a final volume in milliliters equal to the number of RHFF-coated wells plus an additional 3 milliliters.
Remove the cultured splenocytes from the incubator, and resuspend by gently pipetting up and down two to three times in each well. Transfer the cell suspensions to a 50-milliliter conical tube. Next, resuspend the splenocytes in viral supernatant to 1 x 106 cells per milliliter.
Then, add 1 milliliter per well of the splenocytes suspension to the wells of the RHFF-coated plates. Now, spin the plates for 90 minutes at 32 degrees Celsius and 770 gs with an acceleration of 4 and a break setting of zero. During the spin, prepare 50 international units per milliliter of recombinant human IL-2 in TCM.
After the spin, add 1 milliliter of the medium to each well of the plates and incubate overnight. If T cells reach greater than 80% confluence, split the cells by gently pipetting up and down in each well, two to three times, then transfer 1 milliliter from each well into new wells of a fresh 24-well tissue culture-treated plate.
Add 1 milliliter of fresh TCM, with 50 international units per milliliter of IL-2 to each well. If the cells do not reach greater than 80% confluence, change half the medium by slowly pipetting off 1 milliliter of medium from the top of each well and replacing it with 1 milliliter of fresh TCM, containing 50 international units per milliliter of recombinant human IL-2.
Resuspend CAR T cells by gently pipetting up and down three times in each well, and transfer to a 250-milliliter centrifuge tube. Spin the cells at 300 gs for 10 minutes. Next completely aspirate the supernatant without disturbing the pellet. Use PBS to wash the cells, then count them before washing a second time. A typical CAR T cell yield is approximately 1 x 106 cells per well.