1. CAR-T Cells: Light at the Horizon?
Wolfram C. M . Dempke, MD, PhD, MBA
Professor of Haematology & Oncology
SaWo Oncology Ltd
Cambridge, September 2017

2. CAR-T Cells: Introduction
CAR stands for: “Chimeric Antigen Receptor”
A CAR is a recombinant receptor construct composed of an antibody-derived extracellular single-chain variable fragment (scFv) which is linked to the intracellular T cell signalling domains of the TCR.
This results in re-directing of the T cell specificity to the tumour in an HLA-independent manner.
T cells are the most frequently used CAR “drivers” (CAR-T cells), but other cell types can also be used (e.g., NK cells).

3. CAR-T Cells: Introduction
CARs are encoded with viral RNA vectors, which guide the RNA to reverse-transcribe into DNA and permanently integrate into the genome of the patient cells.
A “tool” is therefore needed for the delivery of the foreign gene into human T cells.
Currently, there are two ways to accomplish gene incorporation with vectors: viral systems versus non-viral systems.
CAR-T cell therapy is designed to re-direct a patient’s (“autologous”) or donor’s (“allogeneic”) T cells to specifically target and destroy tumour cells (or other target cells).

4. CAR: General Structure

5. CAR: Extracellular Domain
The extracellular domain of CAR (scFv format) is the antigen-recognition domain of CARs.
Monoclonal antibodies used for the scFv design are well-characterised in pre- clinical models.
However, one drawback could be the risk of developing an immune response against the murine and linker sequences within scFv since most clinically tested CARs encompass non-humanised scFv sequences.
The connection between the antigen binding domain and the transmembrane domain relies on a spacer.
The simplest form of a spacer is the hinge region of IgG1 and is commonly used.

6. CAR: Transmembrane Domain
The transmembrane domain consists of a hydrophobic alpha helix that spans the membrane.
The stability of CAR is related to this domain.
At present, the CD28 transmembrane domain is the most stable receptor, although the transmembrane sequences of CD3ζ, CD8, FcRIγ, or (less frequently) OX40 are also used.

7. CAR: Intracellular Domain
The intracellular domain is the functional end of the CAR and the most common component is CD3ζ with three ITAMs (immunoreceptor tyrosine-based activation motifs) included.
After antigen recognition, the receptors cluster and the signal is transmitted to the T cell. This requires co-stimulatory signalling (e.g., CD28, 4-1BB etc.)
Activated LcK (lymphocyte-specific kinase; activated by CD45) phosphorylates ITAMs in CD3ζ, thereby activating ZAP-70, which then in turn activates LAT (linker of activated T cells).

8. CAR: Four Generations
Since the initial development of CARs in 1989, CAR-T cells can be divided into four generations according to the structure of the intracellular domain.

9. CAR-T Cells: First Generation
First generation CARs contained only the CD3ζ chain.
This led to a cytotoxic reaction against tumour cells, however, it did not provide enhanced proliferation of activated CAR-T cells.
Since these CAR-T cells could not produce enough interleukin-2 (IL-2) in order to kill tumour cells effectively, it was necessary to administer exogenous IL-2.
Although first generation CAR-T cells were studied to treat various tumours the observed outcome was poor.

10. CAR-T Cells: Second Generation
Second generation CARs (with the CD28-CD3ζ or the CD137-CD3ζ sequence) still remain the most frequent format used in clinical practice.
Second generation CARs add intracellular signalling domains to the cytoplasmatic tail of CAR-T cells to provide co-stimulatory signals (e.g., OX40, CD37, CD28).
The CD28-CD3ζ-based CARs provide an “explosive“ expansion of CAR-T cells in vivo.
However, this can lead to CAR-T cell exhaustion and terminal differentiation (→ lack of anti-tumour effects).

11. CAR-T Cells: Third Generation
Third generation CARs were made by combining multiple signalling domains such as CD3ζ-CD28-OX40 or CD3ζ- CD28-CD137 to augment potency with stronger and broader cytokine production and killing ability.
These CAR-T cells were used to treat malignant lymphomas and other solid tumours, however, outcomes were not improved relative to the second generation.
Since the number of patients was small, further studies are needed to explore the role of these CAR-T cells.

12. CAR-T Cells: Fourth Generation
Fourth generation CARs were generated by adding IL-12 (interleukin-12) to the structure of second generation constructs (also known as “T cell re-directed for universal cytokine-mediated killing”: TRUCKs).
TRUCKs can augment T cell activation and activate immune cells.
They are also able to “shape” the tumour microenvironment and may play a role for treatment of viral infections, metabolic disorders and auto-immune diseases.

13. CAR-T Cell Therapy

14. CAR-T Cell Therapy: Step 1
Apheresis collection of mononuclear cells (MNCs) is the initial step for obtaining the large number of T cells necessary to initiate CAR-T cell culture.
Several FDA-cleared systems are available to perform MNC apheresis (e.g., COBE Spectra, ASTEC etc.).
Parameters for collection of MNCs for donor lymphocyte infusions (DLIs) may be used as a starting point for CAR-T cell collection from donors or patients.

15. CAR-T Cell Therapy: Step 1
Density gradients (commonly used for apheresis) alone cannot distinguish monocytes and lymphocytes.
Since monocytes inhibit T cell activation and expansion, it is mandatory to isolate lymphocytes form monocytes prior to culture in the apheresis product.
Two methods are currently used in clinical practice: elutriation and antibody-beat conjugates.
Elutriation is based on cell size and is widely used in CAR-T trials.
Antibody-beat conjugate selection has some disadvantages: it is costly and beads needs to be removed (“masking of epitopes”)

16. Apheresis for CAR-T cell therapy
23-year old healthy male during apheresis (stem cell donor for an AML patients).

17. CAR-T Cell Therapy: Step 2
Following the separation of T cell subsets at the level of CD4/CD8 composition culture, is then needed to activate the T cells.
This can be achieved by several methods:
- beats coated with anti-CD3/anti-CD28 antibodies
- growth factors (e.g., IL-2)
- artificial dendritic cells
- purified APCs form the patients

18. CAR-T Cell Therapy: Step 2
T cells are then transfected with viral vectors encoding for CAR using different methodologies.
The vector is subsequently washed out of the culture and the ex vivo expansion process starts.
Lentiviral gene delivery vectors have become the current gold standard for modifying T cells.
However, genomic integration may represent a potential for malignant transformation of transduced cells.

19. CAR-T Cell Therapy: Step 3
Ex vivo expansion of generated CAR-T cells is necessary to obtain the required number of cells.
Currently, three bioreactor culture systems are used (WAVE Bioreactor; G-Rex; CliniMACS Prodigy).
Amongst them, the CliniMACS Prodigy system is a single device that can effectively enrich, activate, transduce and expand the cells.

20. CliniMACS Prodicy®

21. CAR-T Cell Therapy: Step 4
Lymphodepletion (i.e. Treg cells) does improve CAR-T cell persistence and engraftment.
Increasing the intensity of lymphodepletion will lead to a much greater depletion of Treg cells and better engraftment of T cells infused.
Current treatment of choice is cyclophosphamide plus fludarabine (optimal lymphodepletion method is unknown).
Experimental approach: selective depletion of Treg cells along with cytokine administration.

22. CAR-T Cell Therapy: Step 5
After the CAR-T cells produced and propagated in the various bioreactors reach the number required for clinical use, they are collected and transfused into the patient.
Persistence duration is reported to be 1-6 months with peak levels seen prior to day 10.
Infused cell doses: 106 – 107 CAR-T cells/kg
Efforts to develop CAR-T cells that can either be “turned off” with activation of a “suicide gene” (e.g., iCASP9, CD20) or “turned on” through conditional dimerisation are underway.

23. CAR-T Cell Therapy: Results
CAR-T cell therapies have generated a great deal of enthusiasm in cancer treatment.
Impressive results of the CAR-T cell therapies of r/r-ALL, CLL, DLCB, follicular lymphoma, and multiple myeloma (ORRs of 50% to 100% including high CR rates) have stimulated attempts to adapt this technology to the treatment of solid tumours (e.g, EGFR, HER-2/neu, mesothelin, EpCAM etc.).
The major adverse effects observed after CAR-T cell therapies include severe cytokine release syndrome (CRS) and neurotoxicity.

25. Executive Summary
The development of genetically re-directed T cells has generated significant excitement over the last several years.
ORRs were found to be 50% to 100% in several tumours.
Advances in CAR design (e.g., more than one CAR per T cell; multiple co-stimulatory domains, new targets, etc.) will provide a toolbox for treatment of a huge bundle of different diseases.
Implementation of CAR-T cell therapies as a mainstay treatment for not only haematologic, but also solid malignancies will be a significant business opportunity for many companies.

26. Selected References
Fesnak AF, Lin CY, Siegel DL, Maus MV. CAR-T cell therapies from the transfusion medicine perspective. Transfus Med Rev 3: (2016).
Kulemzin SV, Kuznetsova VV, Mamonkin M, Taranin AV, Gorchakov AA. Engineering chimeric antigen receptors. Acta Naturae 32: 6-14 (2017).
Maus MV, June CH. Making better chimeric antigen receptors for adoptive T-cell therapy. Clin Cancer Res 22: (2016).
Park JH, Geyer MB, Brentjens RJ. CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcome to date. Blood 127: (2016).
Zhang C, Liu J, Zhong JF, Zhang X. Engineering CAR-T cells. Biomarker Res 5: (2017).