Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells
Summary
Here we present a protocol for the generation and functional verification of hypoxia-sensitive chimeric antigen receptor (CAR)-T cells. This protocol presents the lentivirus-based generation of hypoxia-sensitive CAR-T cells and their characterization, including the validation of hypoxia-dependent CAR expression and selective cytotoxicity.
Abstract
Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.
Introduction
Chimeric antigen receptor T cell (CAR-T) therapy has represented a significant breakthrough in cancer treatment. Since the Food and Drug Administration (FDA) approved the first CAR-T therapy for treating advanced/resistant lymphoma and acute lymphoblastic leukemia in 20171,2,3, 10 CAR-T therapies targeting CD19 or B-cell maturation antigen (BCMA) have received approval globally4. However, despite extensive research, replicating the remarkable efficacy of CAR-T therapy in treating hematological malignancies remains challenging for its application to solid tumors5,6,7,8.
The immunosuppressive tumor microenvironment (TME) is a primary contributor to the poor efficacy of CAR-T in the solid tumor setting. TME impedes the activity and survival of CAR-T cells due to insufficient nutrients, hypoxia, an acidic pH, and the accumulation of metabolic waste9,10,11,12. Further hostility comes from infiltrating immunosuppressive cells such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAM), which, alongside tumor cells, secrete immunosuppressive cytokines that cause additional inhibition of CAR-T cells once they enter the tumor13,14.
Apart from the unsatisfactory therapeutic efficiency, safety issues are another Achilles' heel of CAR-T cells when dealing with solid tumors15,16. The safety concern arises from the fact that none of the tumor-specific antigens (TSA) identified so far are strictly restricted to tumor cells. In other words, the tumor-associated antigens (TAA) chosen as the target of CAR, although showing higher expression in tumor cells, are often also expressed by normal tissues17. On-target, off-tumor effects could therefore occur from the unexpected activation of CAR-T cells upon CAR efficiently recognizing normal tissues, leading to cytokine release syndrome (CRS), CAR-T-related encephalopathy syndrome (CRES)18, and other adverse outcomes19.
Many strategies have been explored to avoid such effects, including decreasing the affinity of CAR to allow CAR-T cells to distinguish tumor cells from normal cells based on the expression levels of the targeted TAA; equipping CAR-T cells with an off switch, such as a suicide gene or elimination marker to promote their elimination upon unexpected activation; partitioning the CD3ζ and co-stimulatory signals into two CAR moieties, whose simultaneous engagement is consequently required for effective activation of CAR-T cells; utilizing a synthetic Notch (synNotch)-based circuit that restricts the activity of CAR-T cells to targeted cells co-expressing two different TAAs; and engineering CAR-T cells to attain TME sensitivity by implementing a mechanism to tune CAR expression to changing environmental cues20,21,22,23,24,25,26.
A key consideration in the TME sensitivity option outlined above is the low oxygen level in the TME due to the rapid proliferation of tumor cells. The accommodation of tumor cells to hypoxia hinges on the activation of hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcriptional factor consisting of an inducible subunit, HIF-1α, and a constitutively expressed subunit, HIF-1β27. Under normoxic conditions, the HIF-1α protein undergoes ubiquitination and rapid proteasomal degradation, dependent on its oxygen-dependent degradation domain (ODD)28. When the cellular supply of oxygen becomes limited, HIF-1 is stabilized and activates the transcription of its downstream target genes by binding to hypoxia-response elements (HREs)29. Given the nature of ODD and HRE as oxygen-sensitive elements, they have been explored to realize the conditional expression of CARs within the hypoxic TME30. Here, we present a protocol focusing on methods for phenotypic and functional characterization of hypoxia-sensitive CAR-T cells, preceded by a brief description of the CAR design and the preparation procedures of these cells. This protocol intends to provide a useful guideline for exploiting hypoxia-responsive CAR to generate CAR-T cells with restrained off-tumor toxicity.
Protocol
Representative Results
Discussion
Safety concerns are significant issues that must be addressed for any CAR-T cell therapy to advance to clinical use. Utilizing the unique properties of tumor cells or the TME has become a primary research direction focusing on the development of CAR-T cells that target tumor tissues selectively. Designing a hypoxia-sensitive CAR-T is an attractive strategy in this direction, with several approaches being explored, including the one presented in this study-fusing the CAR moiety with the naturally occurring hypoxia-sensing…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the National Key Research and Development Program of China (2016YFC1303402), the National Megaproject on Key Infectious Diseases (2017ZX10202102, 2017ZX10304402-002-007), and the General Program of Shanghai Municipal Health Commission (201740194).
Materials
| 1.5 mL Centrifuge tube | QSP | 509-GRD-Q | Supernatants and cells cellection Protocol Step 2,3,4 |
| 10% ExpressCast PAGE | NCM biotech | P2012 | Immunoblotting Protocol Step 3 |
| 10x PBS | NCM biotech | 20220812 | Cell culture Protocol Step 4 |
| 10 mL pipette | Yueyibio | YB-25H | Pipetting Protocol Step 1 |
| 10xTRIS-Glycine-SDS electrophoresis buffer | Epizyme | 3673020 | Immunoblotting Protocol Step 3 |
| 15 mL Centrifuge tube | Thermo Scientific | 339650 | Supernatants and cells cellection Protocol Step 1 |
| 25 cm2 EasYFlask | Thermo Scientific | 156367 | Cell culture Protocol Step 3,4 |
| 4x Protein SDS PAGE Loading Buffer | Takara | 9173 | Immunoblotting Protocol Step 3 |
| 6-well flat-bottom tissue culture plates | Thermo Scientific | 140675 | T Cells culture Protocol Step 1 |
| 96-well black flat-bottom tissue culture plates | Greiner | 655090 | Cytotoxicity assay Protocol Step 4 |
| 96-well ELISA plates | Corning | 3590 | ELISA Protocol Step 5 |
| 96-well plate shaker | QILINBEIER | MH-2 | Shake Protocol Step 4 |
| 96-well U-bottom tissue culture plates | Thermo Scientific | 268200 | Supernatants cellection Protocol Step 4,5 |
| anti-FLAG antibody | Sigma | F1804-50UG | Immunoblotting Protocol Step 3 |
| Carbinol | Sinopharm | 10010061 | Immunoblotting Protocol Step 3 |
| Carbon dioxide incubator | Thermo Scientific | 360 | Cell culture Protocol Step 1,2,3,4 |
| Cell counting plate | Hausser scientific | 1492 | Cell counting Protocol Step 1,3,4 |
| CELLection Pan Mouse IgG Kit | Thermo Scientific | 11531D | Mouse IgG magnetic beads Protocol Step 1 |
| Centrifuge | Thermo Scientific | 75002432 | Cell culture Protocol Step 1,3,4 |
| Chemiluminescence gel imaging system | BIO-RAD | 12003154 | Immunoblotting Protocol Step 3 |
| Cobalt chloride solution (0.5 M) | bioleaper | BR4000203 | Hypoxic condition Protocol Step 2,3,4 |
| DMEM | Corning | 10-103-CV | Cell culture Protocol Step 4 |
| Electronic balance | Sartorius | PRACTUM612-1CN | weigh Protocol Step 5 |
| FBS | BI | 04-001-1ACS | Cell culture Protocol Step 3,4 |
| GAPDH Mouse mAb | ABclonal | AC002 | Immunoblotting Protocol Step 3 |
| Gel electrophoresis apparatus | BIO-RAD | 1645070 | Immunoblotting Protocol Step 3 |
| GloMax Microplate Readers | Promega | GM3000 | luciferase activity measurement Protocol Step 4 |
| Goat anti-Mouse IgG (H+L) | Yeasen | P1126151 | Immunoblotting Protocol Step 3 |
| High speed microfreezing centrifuge | eppendorf | 5810 R | Cell culture Protocol Step 1 |
| Human IFN-γ ELISA Set | BD | 555142 | ELISA Protocol Step 5 Items: Recombinant Human IFN-γ Lyophilized Standard, Detection Antibody Biotin Anti-Human IFN-γ , Capture Antibody Purified Anti-Human IFN-γ, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) |
| Human IL-2 ELISA Set | BD | 555190 | ELISA Protocol Step 5 Items: Recombinant Human IL-2 Lyophilized Standard, Detection Antibody Biotin Anti-Human IL-2 , Capture Antibody Purified Anti-Human IL-2, Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) |
| IL-15 | R&D systems | P40933 | T Cells culture Protocol Step 1 |
| IL-21 | Novoprotein | GMP-CC45 | T Cells culture Protocol Step 1 |
| IL-7 | R&D systems | P13232 | T Cells culture Protocol Step 1 |
| Inverted microscope | Olympus | CKX41 | Cell culture Protocol Step 1,3,4 |
| Jurkat | ATCC | TIB-152 | CAR-Jurkat construction Protocol Step 3 |
| LSRFortessa | BD | LSRFortessa | Flow cytometry Protocol Step 2 |
| Luciferase Assay System | Promega | E1501 | luciferase reporter assay Protocol Step 4 Items: Passive lysis buffer, firefly luciferase substrate |
| Microplate reader | BioTek | HTX | ELISA Protocol Step 5 |
| mobile CO2/O2/N2 Incubator Chamber | China Innovation Instrument Co., Ltd. | Smartor118 | Hypoxic condition Protocol Step 2, 3, 4 |
| Mouse Anti-Hexa Histidine tag | Sigma | SAB2702218 | Immunoblotting Protocol Step 3 |
| NcmBlot Rapid Transfer Buffer | NCM biotech | WB4600 | Immunoblotting |
| NcmECL Ultra | NCM biotech | P10300 | Immunoblotting Protocol Step 3 Items: NcmECL Ultra Luminol/Enhancer Reagent (A) ,NcmECL Ultra Stabilized Peroxide Reagent (B) |
| NovoNectin -coated 48-well flat plates | Novoprotein | GMP-CH38 | CAR-T cells construction Protocol Step 1 |
| OPD (o-phenylenediamine dihydrochloride) tablet set | Sigma | P9187 | Substrate Reagent Protocol Step 5 Items: OPD tablet (silver foil),urea hydrogen peroxide tablet (gold foil) |
| PE-conjugated anti-DYKDDDDK | Biolegend | 637310 | Flow cytometry Protocol Step 2 |
| Protamine sulfate | Sigma | P3369-1OG | Lentivirus infection Protocol Step 1 |
| Protein Marker 10 Kda-250 KDa | Epizyme | WJ102 | Immunoblotting Protocol Step 3 |
| Purifed NA/LE Mouse Anti-Human CD3 | BD | 566685 | T Cells culture Protocol Step 1 |
| Purified NA/LE Mouse Anti-Human CD28 | BD | 555725 | T Cells culture Protocol Step 1 |
| PVDF membrane | Millipore | 168627 | Immunoblotting Protocol Step 3 |
| RPMI 1640 | Corning | 10-040-CVRC | Cell culture Protocol Step 3 |
| Skim milk powder | Yeasen | S9129060 | Immunoblotting Protocol Step 3 |
| SKOV3-Luc | ATCC | HTB-77 | Cytotoxicity assay Protocol Step 4 |
| Trypsin-EDTA | NCM biotech | C125C1 | Cell culture Protocol Step 4 |
| Tween 20 | Sinopharm | 30189328 | Immunoblotting Protocol Step 3 |
| Water bath | keelrein | NB014467 | Heating Protocol Step 1 |
| X-VIVO 15 | LONZA | 04-418Q | Serum-free lymphocyte culture medium Protocol Step 1 |
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