CAR T-cell Therapy: How Does It Work?

An introduction to genetic modulation of immune cells by CAR T-cell therapy

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What Is CAR T-cell Therapy?

Chimeric antigen receptor (CAR) T-cell therapy is a gene immunotherapy technique in which the patient’s T cells are genetically engineered to express a CAR that targets a particular antigen of interest in order to treat diseases1,2

  • CARs are synthetic fusion proteins that can be designed to recognize a specific antigen or antigens expressed on the surface of targeted cells2–4
    • The specificity of CAR-mediated T-cell recognition is defined by the antibody domain and is independent of the major histocompatibility complex (MHC) presentation
  • When expressed in T cells, CARs mimic T-cell receptor (TCR) activation and redirect T-cell responses toward a specified target of interest5

CAR T-CELL THERAPY

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Regulatory Determinants of CAR T-cell Function

CARs are transmembrane receptor proteins comprising the following functional components6:

extracellular domain

spacer

transmembrane domain

intracellular domain

Each of these domains plays a role in regulating the function of the CAR6

CAR STRUCTURE

The extracellular domain contains the antigen-recognition domain, which consists of a single-chain fragment variant (scFv6,7)

  • The scFv is composed of the variable light and heavy chain regions derived from an antigen-specific antibody, which are separated by a flexible linker6,7
  • The scFv domain has the following functions8:
    • recognizes the specific target
    • provides tunable affinity
    • determines immunogenicity of the CAR

The spacer (or hinge) binds the transmembrane domain to the extracellular domain; it has the following functions6,8:

  • transmits the receptor-binding signal
  • provides flexibility and length to the CAR
  • helps positioning the CAR for efficient targeting of the antigen

This domain is a hydrophobic alpha helix that spans the cell membrane and is fundamental for6,9:

  • surface expression patterns
  • stability of the receptor

The intracellular domain usually contains one or more discrete intracellular signaling and co-stimulatory domains6,8.

Following antigen binding, the intracellular domain6,8:

  • clusters and undergoes conformational changes, enabling downstream signaling proteins to be recruited and phosphorylated
  • helps in T-cell survival and persistence

Figure adapted from Tokarew N, et al. 20196.
scFv, single-chain fragment variant.

Evolution of CARs

The progress in the development of CARs over the past 3 decades can be roughly grouped into five CAR generations6

This grouping is based on the structure and composition of the intracellular domain

CAR EVOLUTION

The first generation of CARs contain a cluster of differentiation 3 zeta chain (CD3ζ) intracellular signaling domain6

  • CD3ζ is composed of three immunoreceptor tyrosine-based activation motifs that are important for signal transduction
  • CD3ζ is the core component of most CARs across all generations of CARs

The next generation of CARs include additional co-stimulatory domains

  • One co-stimulatory domain (e.g. CD28 or CD137) is present in the second-generation CARs
  • A second (e.g. CD134 or CD137) co-stimulatory domain is present in the third-generation CARs

The role of the co-stimulatory domain is to improve the6,7:

  • secretion of cytokines
  • proliferation and persistence of CAR T cells
  • overall activity of CAR T cells against diseased cells compared with the first-generation CARs

The fourth-generation CARs are based on the second-generation CARs but are paired with a constitutively or inducibly expressed cytokine (e.g. interleukin [IL]-12)2,6

  • Expression of the desired cytokine promotes killing of diseased cells through several synergistic mechanisms (e.g. exocytosis or cell death receptor/ligand systems)

Cells transduced with these fourth-generation CARs are referred to as T cells redirected for universal cytokine-mediated killing (TRUCKs)6

The fifth-generation or next-generation CARs (currently in the exploratory phase of development) are based on the second-generation CARs but comprise a truncated cytoplasmic IL-2 receptor beta chain, with a binding site for the signal transducer and activator of transcription 3 or 5 transcription factor6

  • The fifth generation of CARs simultaneously trigger TCR, co-stimulatory, and cytokine signaling6

Additional variants have been generated to potentially further enhance the specificity and control of the transfused T cells. Some examples include3,6:

  • dual CARs (expressing two full-length CARs in each T cell)
  • pooled CARs (combining two T-cell products, each expressing one CAR)
  • single-chain bispecific CAR (expressing a single CAR molecule that can recognize two different antigens)
  • split CARs (involves the separation of the co-stimulatory domains from the signaling domain from two different CARs)

Figure adapted from Tokarew N, et al. 20196.
CD3ζ, cluster of differentiation 3 zeta chain; IL-12, interleukin-12; IL-2Rβ, interleukin-2 receptor beta chain; STAT3/5, signal transducer and activator of transcription 3 or 5.

Advantages of CAR T-cell Therapy

Advantages of CAR T-cell therapy include2,10–13:

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High antigen affinity

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High clinical efficacy (for the treatment of some hematologic malignancies)

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MHC-independent antigen detection of soluble or cell surface antigens
Allows for more flexibility for precise and individualized treatment of different malignancies. Can be used to recognize antigens in cancer cells where the expression of the MHC has been downregulated or lost

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Ability to recognize different types of targets on tumors, such as proteins, carbohydrates, and glycolipids, that arise during tumorigenesis

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Modular design enables precise control of neoantigen response

Limitations of CAR T-cell Therapy

Widespread application of CAR T-cell therapy remains limited due to diverse obstacles that constrain its efficacy and challenge its safety2,5,7,10,13,14

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Potentially life-threatening toxicity
Tumor lysis syndrome, cytokine release syndrome, neurotoxicity , and on-target off-tumor toxicity have been reported

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Antigen loss
This can lead to escape of diseased cells, which limits the treatment's long-term success

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Toxic signaling, exhaustion, and activation-induced cell death
These limit CAR T-cell functionality, proliferation, and persistence

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Limited efficacy in solid tumors
The hostile microenvironment, limited trafficking, and antigen heterogeneity in tumors can limit the efficacy of CAR T-cell therapy

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Limited neoantigen recognition
CAR T cells can only recognize antigens that are naturally expressed on the cell surface

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Tumor escape
Recurrence of the disease can occur

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Limited commercialization
Due to the complex manufacturing and associated cost

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Based on an unnaturally expressed protein
This may increase the risk of an immune reaction

Approaches for Regulation and Control of CAR T Cells

A number of regulatory mechanisms are being investigated to control CAR T cells more effectively following infusion and overcome some of the limitations of CAR T-cell therapy to treat solid tumors6,15

T-cell control mechanisms

  • Passive control methods provide opportunities to limit CAR T-cell toxicity but do not offer downstream control over engrafted cells following transfusion15
  • Inducible control methods allow for regulation of CAR T-cell activity or elimination of CAR T cells via co-administration of drugs15
  • Autonomous control methods aim to generate autonomous CARs with a higher target specificity, capable of better distinguishing diseased cells from healthy cells15

Passive control15

Examples

  • Transient transfection (CAR-encoding mRNA)
  • Affinity tuning by lowering the binding domain’s affinity toward the targeted antigen

Inducible control15

Examples

  • Co-expression of suicide genes (e.g. caspase 9, herpes simplex virus tyrosine kinase, human thymidylate kinase) or elimination markers
  • Systemic immunosuppressive agents
  • Adapter-dependent CAR T cells
  • Drug-dependent CAR T cells (e.g. inducers, Tet-On)
  • Protease inhibitors

Autonomous control15

Examples

  • Dual CAR design (two antigens are required for activation)
  • Inhibitory CAR (restricts its activity to diseased cells lacking the healthy antigen)
  • Incorporation of a hypoxia-inducible factor (CAR T cells are destroyed in a normoxic environment)

Strategies to address limitations in targeting solid tumors

New methods are being investigated to enhance the ability of CAR T cells to target solid tumors by6:

  • Improving recognition, infiltration, and persistence within tumors
  • Enhancing resistance to the suppressive tumor microenvironment

Examples to address limitations in targeting solid tumors6:

  • Improve T-cell trafficking to solid tumor (e.g. transgene chemokine receptors)
  • Improve T-cell recognition and targeting of tumor cells (e.g. dual CAR T cells, split CARs)
  • Improve proliferation and survival (e.g. co-stimulatory domains, chemokine receptors, next-generation CARs)
  • Counteracting the immunosuppressive microenvironment

Clinical Applications of CAR T-cell Therapy

CAR T-cell therapy is considered a breakthrough in the treatment of hematologic tumors2

  • This success culminated with the approvals of tisagenlecleucel and axicabtagene ciloleucel in the US and EU and brexucabtagene autoleucel in the US, all CAR T-cell therapies targeting CD19-expressing B cells16–20
    • Tisagenlecleucel and axicabtagene ciloleucel are indicated for the treatment of relapsed or refractory (R/R) large B-cell lymphoma
    • Tisagenlecleucel is also indicated for the treatment of R/R B-cell precursor acute lymphoblastic leukemia
    • Brexucabtagene autoleucel is indicated for the treatment of R/R mantle cell lymphoma

For full details of these indications, go to Section 1: Genetic Modulation of Immune Cells: Overview

  • While CD19-targeting CARs remain the benchmark for CAR treatments, CAR research has expanded beyond CD19 and includes other hematologic malignancies and solid tumors2,21
    • Use of CAR T-cell therapy to treat solid tumors has been limited due to toxicities and modest therapeutic benefit2,3
    • The following are some examples of solid tumors being explored as potential targets for CAR T-cell therapy (mostly in Phase 1/2)2,4
      • neuroblastoma
      • glioblastoma
      • sarcoma
      • mesothelioma
      • pancreatic cancer
      • gastric cancer
      • prostate cancer
      • liver cancer
      • lung cancer

References

  1. Seif M, et al. Front Immunol 2019;10:2711.
  2. Zhao L, Cao YJ. Front Immunol 2019;10:2250.
  3. Chang ZL, Chen YY. Trends Mol Med 2017;23(5):430–450.
  4. Titov A, et al. Cancers (Basel) 2020;12(1):125.
  5. Srivastava S, Riddell SR. J Immunol 2018;200(2):459–468.
  6. Tokarew N, et al. Br J Cancer 2019;120(1):26–37.
  7. Stoiber S, et al. Cells 2019;8(5):472.
  8. Dwivedi A, et al. Front Immunol 2018;9:3180.
  9. Zhang C, et al. Biomark Res 2017;5:22.
  10. Anguela XM, High KA. Annu Rev Med 2019;70:273–288.
  11. Li D, et al. Signal Transduct Target Ther 2019;4:35.
  12. Ren YB, et al. Curr Pharm Des 2018;24(1):78–83.
  13. Wang Z, Cao YJ. Front Immunol 2020;11:176.
  14. Nam S, et al. Driving the next wave of innovation in CAR T-cell therapies. December 13, 2019. Available at: https://www.mckinsey.com/industries/pharmaceuticals-and-medical-products/our-insights/driving-the-next-wave-of-innovation-in-car-t-cell-therapies. Accessed December 11, 2020.
  15. Brandt LJB, et al. Front Immunol 2020;11:326.
  16. Kymriah® [package insert]. 2018. Available at: https://www.novartis.us/sites/www.novartis.us/files/kymriah.pdf. Accessed December 11, 2020.
  17. Yescarta® [package insert]. 2020. Available at: https://www.fda.gov/media/108377/download. Accessed December 11, 2020.
  18. Kymriah® [product information]. 2020. Available at: https://www.ema.europa.eu/en/documents/product-information/kymriah-epar-product-information_en.pdf. Accessed December 11, 2020.
  19. Yescarta® [product information]. 2020. Available at: https://www.ema.europa.eu/en/documents/product-information/kymriah-epar-product-information_en.pdf. Accessed December 11, 2020.
  20. Tecartus [package insert]. 2020. Available at: https://www.fda.gov/media/140409/download. Accessed December 11, 2020.
  21. Leick MB, Maus MV. Am J Hematol 2019;94(S1):S34–S41.