flow cytometric assessment of oxygen-dependent car expression in car-t cells

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

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.

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.

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.

flow-cytometric-assessment-oxygen-dependent-car-expression-car-t

Research

JoVE Journal

Cancer Research

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.

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 ...

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.

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.