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CAR T cell therapy


Editorial Board Members: Patricia Tsang, M.D., M.B.A., Mrigender Singh Virk, M.D.
Melissa R. George, D.O.

Last author update: 9 July 2024
Last staff update: 9 July 2024

Copyright: 2020-2024, PathologyOutlines.com, Inc.

PubMed Search: CAR T cell therapy

Melissa R. George, D.O.
Page views in 2024 to date: 682
Table of Contents
Definition / general | Essential features | Terminology | History | Procedure to create CAR T product | Pathophysiology | Types of CAR T cells | Diagrams / tables | Challenges | Adverse effects | Clinical features | Laboratory | Case reports | Treatment | Board review style question #1 | Board review style answer #1 | Board review style question #2 | Board review style answer #2
Cite this page: George MR. CAR T cell therapy. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/transfusionmedcartcelltherapy.html. Accessed December 17th, 2024.
Definition / general
  • Chimeric antigen receptor T (CAR T) cells are a form of cellular immunotherapy involving the genetic engineering of T cells to produce surface receptors targeted at specific cell surface receptors
  • CAR T cell therapy is primarily used to treat hematologic malignancies, with acute lymphoblastic leukemia (ALL) being the first disease targeted
Essential features
  • CAR T cell therapy was originally developed for B cell acute lymphoblastic leukemia
  • It is also now Food and Drug Administration (FDA) approved for B cell non-Hodgkin lymphoma (NHL), follicular lymphoma, mantle cell lymphoma and multiple myeloma; its application to solid organ tumors is being studied
  • Process starts with collecting autologous T cells via leukocytapheresis, shipping the cells to be engineered / manufactured, expanding the cell population to achieve appropriate dose, cryopreserving CAR T cells and shipping to the treating facility
  • Patient is given lymphocyte depleting chemotherapy prior to receiving the treatment
  • Key steps of CAR T cell therapy are trafficking, recognition, control, microenvironment, proliferation / persistence
  • Safety measures are built in to allow ablation of CAR T cells if severe toxicity develops
  • Severe toxicity can include cytokine release syndrome and a CAR T encephalopathy
Terminology
  • CART-19: CAR T directed against CD19 antigen on B cells
  • BCMA: B cell maturation antigen found on plasma cells in multiple myeloma

FDA approved CAR T cell therapies (NIH: CAR T Cells - Engineering Patients' Immune Cells to Treat Their Cancers [Accessed 9 April 2024])
Generic name Brand name Target antigen Targeted disease Patient population
Tisagenlecleucel Kymriah® (Novartis, Basel, Switzerland) CD19 B cell acute lymphoblastic leukemia (ALL) Children and young adults with refractory or relapsed B cell ALL
B cell non-Hodgkin lymphoma (NHL) Adults with relapsed or refractory B cell NHL
Axicabtagene ciloleucel Yescarta® (Kite Pharma / Gilead, Los Angeles, CA) CD19 B cell NHL Adults with relapsed or refractory B cell NHL
Follicular lymphoma Adults with relapsed or refractory follicular lymphoma
Brexucabtagene autoleucel Tecartus CD19 Mantle cell lymphoma (MCL) Adults with relapsed or refractory MCL
B cell ALL Adults with relapsed or refractory B cell ALL
Lisocabtagene maraleucel Breyanzi CD19 B cell NHL Adults with relapsed or refractory B cell NHL
Idecabtagene vicleucel Abecma® (Bristol‐MyersSquibb) BCMA Multiple myeloma Adults with relapsed or refractory multiple myeloma
Ciltacabtagene autoleucel Carvykti® (Janssen Pharmaceutical Companies of Johnson & Johnson) BCMA Multiple myeloma Adults with relapsed or refractory multiple myeloma
History
  • 1989: discovery that it was possible to redirect T cell signaling to an antigen of choice, independent of major histocompatibility complex (MHC) restrictions
  • 2006: first results published of human clinical trials using chimeric antigen receptor T cell technology (J Clin Oncol 2006;24:e20, Clin Cancer Res 2006;12:6106)
  • 2010: first use of CAR T cell therapy was in a 5 year old girl with relapsed B cell acute lymphoblastic leukemia (ALL); received CAR T cell therapy directed against CD19 antigen on B lymphoblasts (CART-19)
    • Developed complications, treated with tocilizumab, an anti-IL6 monoclonal antibody
    • Patient survived and became global ambassador for CAR T cell therapy
  • 2014: CTL019 was granted breakthrough therapy designation by the United States Food and Drug Administration (Immunol Rev 2015;263:68)
  • 2021: ide-cel was FDA approved as a CAR T cell construct with murine BCMA targeting plasma cells in multiple myeloma (CA Cancer J Clin 2023;73:275)
  • 2022: cilta-cel became second FDA approved anti-BCMA CAR T cell therapy (CA Cancer J Clin 2023;73:275)
Procedure to create CAR T product
  • Collect autologous T cells via leukocytapheresis
  • Ship T cells to manufacturing site
    • CAR structure is composed of an extracellular antigen recognition domain fused to intracellular TCR signaling domains (CD3z) and costimulatory domains such as CD28
    • Manufacture CAR T cells by CD3 / CD28 bead stimulation and lentiviral transduction (Tisagenlecleucel)
    • Manufacture CAR T cells by CD3 antibody / IL2 stimulation and retroviral transduction (Axicabtagene)
  • Expand cell population over 6 - 10 days
  • Achieve patient dose of 2 - 4 x 106 CAR expressing T cells/kg patient body weight
  • Cryopreserve CAR T cells
  • Ship to treating facility
  • Patient is given lymphodepleting chemotherapy (usually fludarabine and cyclophosphamide)
    • Deplete endogenous T cells that might reject the CAR T cells
    • Increase likelihood of expanding CAR T population in recipient
    • Enhance antigen presentation capabilities
Pathophysiology
  • Targets (Front Immunol 2021;12:744823)
    • CD19
    • CD20, CD22 and BCMA
    • Developing targets include CD70, CD7, CD5
    • NKG2DL, GD2 and mesothelin for solid tumors potentially
  • Structure
    • Receptor: combines facets of normal T cell activation into single protein
    • Links extracellular antigen recognition domain to an intracellular signaling domain to activate the T cell when an antigen is bound
    • 4 parts
      • Antigen recognition domain
        • Exposed to the outside of the cell in the ectodomain portion of the receptor
        • Allows the CAR T cell to attack any cell that expresses the matching molecule
        • Derived from variable regions of a monoclonal antibody linked together as a single chain variable fragment (scFv)
        • scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobulins connected with a short linker peptide
        • VL and VH regions are selected to bind the target antigen like CD19
      • Extracellular hinge region
        • Structural domain between the antigen recognition region and the cell's outer membrane
        • Optimizes flexibility of the scFv receptor head to promote antigen binding between CAR T and target antigen
      • Transmembrane domain
        • Anchors the CAR to the plasma membrane, links the extracellular hinge and antigen recognition domains with the intracellular signaling region
        • Stabilizes the entire structure
        • CD28 transmembrane domain is often used and is very stable
        • Should not use CD3 zeta transmembrane domain because it can cause incorporation of the artificial TCR into the native T cell receptor
      • Intracellular T cell signaling domain
        • Receives and perpetuates signal after antigen binds external recognition portion
        • Cytoplasmic domain of CD3 zeta is often used to mimic the normal T cell activation dependent on phosphorylation of immunoreceptor tyrosine based activation motifs
        • Includes 1 or more chimeric domains from costimulatory proteins
  • Function of CAR T cells
    • Trafficking
      • Engineered T cell must be able to get to site of tumor cells
      • Target tumor cells to be killed
      • Possibility of introducing chemokine receptors into CAR T cells to improve trafficking to tumors that produce cognate chemokines
    • Recognition
      • Recognize target tumor cells
      • Discriminate and ignore bystander tissues
    • Control
      • T cells are relatively autonomous once they are infused
      • New work on regulatory processes to modulate the survival of T cells, timing, strength and location of their activity
    • Microenvironment
      • Resist immunosuppression
      • Prime / mobilize endogenous immunity
    • Proliferation / persistence
  • Safety measures: allow ablation of CAR T cells if severe toxicity develops
    • Inclusion of suicide gene, iCaspase9
    • Surface tag such as epidermal growth factor receptor
Types of CAR T cells
  • First generation: contained only TCR complex CD3ξ chain domain with additional costimulatory domains
  • Second generation: incorporated costimulatory domains like CD28 or CD137 to boost survival, proliferation and antitumor activity
  • Third generation: combined CD3ξ chain domain with additional costimulatory domains (Cell 2016;164:780)
  • Fourth generation: T cells redirected for universal cytokine killings (TRUCKS)
    • Improve tumor microenvironment
    • May have potential for solid tumors
  • Newer generation: additional gene editing, such as CRISPR-Cas9
  • Armored CARs: CAR T cells modified to express cytokines, ligands or scFv that help turn suppressive tumor environment into proinflammatory to better fight tumor
  • Tandem CARs: CAR molecule engineered to recognize multiple antigens via 2 binders on a single molecule
  • Designer CARs: modified by CRISPR-Cas9 to edit CAR T genomes for advantageous features, such as being less susceptible to immunosuppressive effects
  • Smart CARs: logic gated, regulated
    • Have new receptors that function independently of CAR / TCR pathways but interfere with CAR activity in controlled way
    • Use of modular receptors called synthetic Notch (synNotch) receptors
  • Split, universal and programmable (SUPRA) CARs
    • Universal receptor found on T cells and tumor targeting scFv adaptor molecule
    • 2 component receptor system with universal receptor (zipCAR) expressed on T cells and tumor targeting scFv adaptor (zipFv)
      • Fusion of intracellular signaling domains and leucine zipper as extracellular domain
        • scFv of zipFv binds tumor antigen
        • Leucine zipper binds and activates zipCAR on T cells
    • Regulate activity to limit over activation, decrease cytokine secretion and improve tumor targeting (Maitta: Immunologic Concepts in Transfusion Medicine, 1st Edition, 2019, Cell 2018;173:1426)
  • Universal CAR T (UCAR T) (Front Immunol 2021;12:744823)
    • Potential advantages
      • Could be taken from healthy allogeneic donors
      • Could have batched rather than customized manufacturing
      • More immediate availability
      • Lower cost
      • Possible application in T cell malignancies
    • Potential disadvantages
      • Need for additional gene editing to avoid graft versus host disease and rejection
      • Lower amplification and shorter persistence in vivo
Diagrams / tables

Images hosted on other servers:
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Chimeric antigen receptor modified

Engineering immune cells

Engineering immune cells

Multiple gene or non-gene editing

Multiple gene or nongene editing

Challenges
  • Tumor specific antigens
    • CD19 is a great target, since a loss of normal B cells can be compensated for by replacement antibody therapy (IVIG)
    • How specific must they be?
      • Tumor cells that do not express the target antigen may evade therapy
      • Tumor cells with splice variants, lacking a specific epitope may also escape targeting (Blood 2018;131:2621)
    • Can we pinpoint antigens expressed by tumors versus normal cells reliably?
    • Do all tumor cells in a given tumor express the same antigens?
    • Can a tumor be effectively treated even if only some of the cells are susceptible to targeting?
    • How significant is bystander toxicity? (Blood 2018;131:2621)
  • Rare chance of accidentally introducing CAR gene into a tumor cell during manufacturing
    • Proliferation of tumor cells
    • Tumor cells escape detection by CAR T cells
  • Response in non-Hodgkin lymphoma (NHL) is worse than in ALL
    • CD19 loss variants
    • Microenvironment factors that limit proliferation and the effect of CAR T cells
    • Remission rates 70 - 80%
  • Solid tumors
    • Challenges identifying suitable cell surface molecules for the CAR T cells to target
    • Attempts to engineer T cells with T cell receptors capable of recognizing tumor specific antigens from intracellular proteins
    • Modify tumor microenvironment to be more hospitable to CAR T cells
Adverse effects
  • Toxicity
    • On target effects: reversible when target cells are eliminated or CAR T cell engraftment is terminated
  • B cell aplasia
    • More severe than that caused by anti-CD20 monoclonal antibody rituximab
    • Rapidly reversed after ablation of CAR T cells
    • May require immunoglobulin therapy
  • Cytokine release syndrome
    • Initial flu-like presentation
    • Fevers, hypotension, hypoxia, neurologic changes
    • Can progress to capillary leak
    • T cell activation and high levels of cytokines, IL6 and interferon γ
  • Neurotoxicity: CAR T related encephalopathy syndrome (Blood 2014;123:2625, Maitta: Immunologic Concepts in Transfusion Medicine, 1st Edition, 2019)
Clinical features
  • Patient selection considerations may differ from autologous stem cell transplant (ASCT) and take into account previous therapies, upper age limit, severity of comorbidities and resistance to chemotherapy
  • Some clinical trials may include transplant ineligible patients
  • Other clinical trials are studying CAR T cell therapy as second line treatment and for both transplant eligible and ineligible patients
  • Other factors that may be considered in therapy include
    • Performance status
    • Organ function
    • T cell count
  • B acute lymphoblastic leukemia (ALL); target antigen is CD19
    • CART-19: CAR T directed against CD19 antigen on B lymphoblasts
    • Tisagenlecleucel, CTL019, also known as Kymriah® (Novartis, Basel, Switzerland)
    • Axicabtagene ciloleucel, also known as axi-cel, Yescarta® (Kite Pharma / Gilead, Los Angeles, CA)
  • Diffuse large B cell lymphoma; target antigen is CD19, no specific age limit
    • Tisagenlecleucel, CTL019, also known as Kymriah® (Novartis, Basel, Switzerland)
    • Axicabtagene ciloleucel, also known as axi-cel, Yescarta® (Kite Pharma / Gilead, Los Angeles, CA)
  • Other diagnoses under investigation for possible CAR T cell therapies include
    • Hodgkin lymphoma (HL); target antigen is CD30
    • Anaplastic large cell lymphoma; target antigen is CD30
    • Myeloma; target antigens are SLAMF7, B cell maturation antigen (BCMA)
    • Acute myeloid leukemia (AML); target antigens are CD123, CD33, Lewis Y and FOLR2
    • Other potential costimulatory domains are PD1 / CD28 and CD200R / CD28
    • T cell malignancies are a challenge since candidate target antigens are found on normal T cells
    • CARs posttransplant: maximize graft versus tumor effect while minimizing graft versus host disease (GVHD)
    • CARs in donor leukocyte infusions (DLI): may help improve survival in relapses of hematologic malignancy
    • CARS in virus specific T cells
    • Chimeric autoantibody receptor T cells (CAARs): autoimmune disease such as pemphigus vulgaris (Immunol Rev 2015;263:68, Clin Cancer Res 2016;22:1875)
Laboratory
  • Polymerase chain reaction (PCR) molecular assays are available for all CARs produced; however, such testing might not accurately reflect if the CAR is actually expressed on the cell surface and is generally performed retrospectively
  • Flow cytometry may be a useful modality to monitor real time expansion and response
    • CD19 CARs are known to expand rapidly, clear target tumor cells, then contract
    • CARs have single chain variable fragments (scFv) for specificity against a target antigen and have little else on the cell surface
    • There are 3 potential ways to detect the scFv using flow cytometry
      • Develop an anti-idiotype antibody to the scFv
      • Use Fc conjugated soluble antigen, such as Fc-CD22, that interacts with CD22 CAR T cells followed by a secondary detection antibody (anti-Fc)
      • Use biotinylated protein L followed by a streptavidin conjugated fluorophore
    • There are benefits and deficits to each method and the process is labor intensive
  • Well constructed assay provides meaningful information for individual patient care and a better understanding of CARs as a whole
  • Additionally, there is need for standardized methods to profile memory phenotype of CAR T cells to evaluate quality and promote manufacturing improvements
  • Use of a standardized memory T cell panel can help evaluate how T cell phenotype impact the efficacy and longevity of response in patients receiving CAR T cell therapies
  • One attempt at this includes a dried memory T cell panel containing a prevalidated mixture of 7 antibodies for the identification of naïve, stem cell memory, central memory and effector memory CD4+ and CD8+ T cell subsets (BD Biosciences)
  • One study used this prevalidated mix and additional drop in antibodies can complement the panel and enable more in depth evaluation of the T cell phenotype and monitor changes in expression of PD-1, TIM3, LAG3, HLA-DR, CD45RO and CXCR3 on T cells transduced to express a novel anti-CD37 CAR (Transfus Med Hemother 2019;46:15, Blood 2019;134:5626)
Case reports
  • 18 month old girl and 52 year old woman treated with CAR T cell therapy for B ALL with mixed lineage leukemia gene (MLL) mutations developed AML clonally related to their B ALL, suggesting CD19 negative immune escape (Blood 2016;127:2406)
  • 20 year old man relapsing 9 months after CD19 targeted CAR T cell (CTL019) therapy (Nat Med 2018;24:1499)
  • 38 year old woman with CD19 directed CAR T cell therapy combined with BTK inhibitor and PD-1 antibody against secondary central nervous system lymphoma (Front Immunol 2022;13:983934)
  • Brief review of control mechanisms for future CAR T cell products (Front Immunol 2020;11:326)
  • Advancements in safety for new CAR T cell therapy models (Mol Cancer 2019;18:125)
  • Applications of CAR T cell therapy for community oncology physicians (Oncologist 2016;21:608)
Treatment
  • Cytokine release syndrome: treated with anti-IL6 monoclonal antibody, tocilizumab
Board review style question #1
A 10 year old boy diagnosed with acute lymphoblastic leukemia with several relapses is participating in a clinical trial for CD19 CAR T cell therapy. He develops rapid onset of flu-like symptoms (including fever) and progresses with mental status change and clinical concern for cytokine release syndrome. What would be an appropriate treatment for suspected cytokine release syndrome?

  1. Anti-CD20 (rituximab)
  2. Anti-CD38 (daratumumab)
  3. Anti-IL6 (tocilizumab)
  4. Azathioprine
  5. Interferon γ
Board review style answer #1
C. Anti-IL6 (tocilizumab). Cytokine release syndrome is likely due to high levels of IL6 and interferon γ. Anti-IL6 (tocilizumab) has mainly been used in inflammatory conditions such as juvenile rheumatoid arthritis and has been effective in decreasing the IL6 levels involved in cytokine release syndrome. Answer A is incorrect because anti-CD20 (rituximab) is used in the treatment of B cell lymphomas. Answer B is incorrect because anti-CD38 (daratumumab) is used in the treatment of multiple myeloma. Answer D is incorrect because azathioprine can be used to treat immune disorders such as Crohn's disease, renal transplant rejection and rheumatoid arthritis. Answer E is incorrect because interferon γ is used to treat various autoimmune diseases.

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Reference: CAR T cell therapy
Board review style question #2
Which of the following functions of CAR T cells refers to the movement of the engineered T cells to the site of tumor cells targeted for destruction?

  1. Control
  2. Microenvironment
  3. Proliferation / persistence
  4. Recognition
  5. Trafficking
Board review style answer #2
E. Trafficking is the first function of a CAR T cell. The engineered cell must be able to travel to the site of the target tumor cells. Answer A is incorrect because control refers to the relative autonomy of T cells once they are infused. Answer B is incorrect because microenvironment refers to background immunity that impacts the effect of CAR T cells. Answer C is incorrect because proliferation / persistence refers to the ability of the engineered cells to circulate. Answer D is incorrect because recognition is the ability of the cells to recognize their target.

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Reference: CAR T cell therapy
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