CAR-T-cells

CAR-T is the acronym for "chimeric antigen receptor T cells". These are tumor-specific T lymphocytes with an artificial (chimeric) antigen receptor (ACAR) consisting of a single-chain variable antibody fragment (chimeric Antigen Receptor T Cells). Artificial (chimeric) antigen receptors are fusion proteins composed of a single-chain fragment variable of a specific monoclonal antibody and one or more intracellular T cell receptor signaling domains. This genetic modification of T cells can be accomplished either via virus-based gene transfer methods or non-viral methods such as DNA-based transposons, CRISPR/Cas9 technology, or direct transfer of in vitro transcribed mRNA by electroporation (Miliotou AN et al. 2018).

Most CARs consist of an antigen-binding domain, an extracellular spacer/joint region, a transmembrane domain, and an intracellular signaling domain that leads to T-cell activation after antigen binding (Curran KJ et al. 2012).

Lymphocytes modified in this way recognize the tumor antigen directly, without the involvement of the major histocompatibility complex (Mohanty R et al. 2019). There is now the possibility of expressing receptors against more than one antigen (bi-, tri-, quad-specific CARs). Current technologies are working to make such CART-T cells even more efficient through built-in and combinable logical "gates" ("AND", "OR", "NOT"). A CAR-T treatment thus combines the characteristics of three innovative forms of therapy - those of immune, cell and gene therapy.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a novel therapeutic T cell engineering approach in which patient blood-derived T cells are modified in vitro to express artificial receptors targeting a specific tumor antigen. This therapy has been used successfully in recent years, with remission rates of up to 80% in hematologic cancers, particularly acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas, such as large B-cell lymphoma (Mohanty R et al. 2019).

First, leukocytes from the tumor patient are extracted by leukapheresis and genetically engineered ex vivo to express chimeric antigen receptors. These antigen receptors bind to specific tumor cell antigens after infusion of the CAR-T cells into the patient, initiating cancer cell death or apoptosis.

CAR-T cell therapy is thus an alternative immunotherapy with curative potential, in which T cells are removed from the patient, genetically modified in the laboratory and reinfused into the patient as a so-called personalized drug. Two products of this new type of drug have already reached market maturity in advanced blood cancers.

The domains of CAR combine the specificity of an antibody with the cytotoxic and memory functions of T cells (Haji-Fatahaliha M et al. 2016).

Production

Leukapheresis: White blood cells from the tumor patient are obtained using a special procedure. They are frozen and shipped to the manufacturer of the CART T cells.

Gene transfer: An inactive virus is introduced into the T cells. Its genetic material has been expanded with a special gene. The DNA of the T cells takes up the genetic material of the viruses. With the help of the prepared gene, they produce a protein (chimeric antigen receptor) against CD19, which they present on their surface (CAR-T cell). These cells are able to recognize the CD19 protein of the tumor. According to the lock-and-key principle, the antibody of the CART cell binds to the tumor protein and destroys the tumor cell.

Preparatory chemotherapy: Before the actual therapy, chemotherapy is used to destroy as many of the patient's T cells as possible. This gives the CAR-T cells a better starting base.

Infusion of CAR-T cells: The genetically modified CAR-T cells are given back to the patient via an infusion. The CAR-T cells attach to the tumor cells cancer cells and destroy them. They are living cells that continue to proliferate in the body, forming the long-term protective shield against blood cancer.

In the course of development, several CAR generations were created. All protein constructs carry an extracellular, variable antibody single chain fragment (scFv) and a hinge peptide (hinge). The transmembrane domain (tm) anchors the CAR in the T cell and links to the intracellular signaling domains. The intracellular costimulatory domains derive from CD28 and/or 4-1BB. They modulate T cell activation and survival as well as cytokine release. The CD3-zeta domain is derived from the intracellular signaling part of the T cell receptor, which mediates signal transduction during T cell activation.

G1: First generation CARs (G1) were generated using only the CD3ζ domain. They were unable to activate resting T cells and drive sustained T cell responses or sustained cytokine release due to their limited signaling ability .

G2: Coupling with additional costimulatory signaling domains(CD28 or 4-1BB) resulted in sufficient T cell activation in the 2nd generation . Such CAR-T could be effectively expanded ex vivo . CARs with CD19 as scFV form the basis for the currently approved CAR-T cell preparations Kymriah and Yescarta.

G3: Third generation CAR-T cells combine the signaling potential of two costimulatory domains (CD28/4-1BB tandem).

G4: The antitumor activity of fourth-generation CAR, also called TRUCKs (T-cells redirected for universal cytokine-mediated killing), is enhanced by further genetic modification. Fourth-generation CARs are additionally transduced with genes that enable the expression of cytokines. These include additional transgenes for cytokine secretion (e.g. IL-12) or additional costimulatory ligands.

CAR-T cell therapy has shown amazing therapeutic success in patients with advanced leukemia. It has been used successfully in recent years, with remission rates of up to 80% in hematologic cancers, particularly acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas, such as large B-cell lymphoma (Mohanty R et al. 2019).

Two compounds have received accelerated approval in Europe (Kymriah (Novartis) and Yescarta (Gilead/ Kite) for the treatment of hematologic neoplasms. Despite severe side effects, optimism prevails that this form of therapy can be improved.

The extent to which CART T-cell therapies are effective in metastatic solid tumors, such as metastatic prostate cancer, remains to be seen (Yu YD et al. 2021)

Recent findings show that CAR-T cells are also effective in severe systemic lupus erythematosus.

1. Curran KJ et al (2012) Chimeric antigen receptors for T cell immunotherapy: current understanding and future directions. J Gene Med 14:405-415.
2. Haji-Fatahaliha M et al (2016) CAR-modified T-cell therapy for cancer: an updated review. Artif Cells Nanomed Biotechnol 44:1339-1349.
3. Miliotou AN et al (2018) CAR T-cell therapy: a new era in cancer immunotherapy. Curr Pharm Biotechnol 19: 5-18.
4. Mohanty R et al (2019) CAR T cell therapy: A new era for cancer treatment (Review). Oncol Rep 42(6):2183-2195.
5. Yu YD et al (2021) Chimeric Antigen Receptor-Engineered T Cell Therapy for the Management of Patients with Metastatic Prostate Cancer: A Comprehensive Review. Int J Mol Sci 22: 640.
6. Mougiakakos D et al (2021) CD19-targeted CAR T cells in refractory systemic lupus erythematosus. N Engl J Med. 5;385: 567-569.