Chimeric antigen receptor

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral vectors. In this way, a large number of cancer-specific T cells can be generated for use as adoptive cell therapy. . Phase I clinical studies of this approach show efficacy.

Anatomy of Chimeric Antigen Receptors

The most common form of these molecules are fusions of single-chain Variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g. neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19.

cartoon showing different components of an artificial TCR

The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows to the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signalling endodomain which protrudes into the cell and transmits the desired signal.

The fact that these molecules actually work is at first glance surprising. At second glance one remembers that type I proteins are in fact two protein domains linked by a transmembrane alpha helix in between. The cell membrane lipid bilayer, through which the transmembrane domain passes, acts to isolate the inside portion (endodomain) from the external portion (ectodomain). It is not so surprising hence that attaching an ectodomain from one protein to an endodomain of another protein results in a molecule that combines the recognition of the former to the signal of the latter.

To more completely appreciate the engineering work that goes into these molecules, the different components will be discussed separately.

Ectodomain - signal peptide

A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence usually works fine. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g. in a scFv with orientation light-chain - linker - heavy chain, the native signal of the light-chain is used

Ectodomain - antigen recognition region

The antigen recognition domain is usually an scFv. There are however many alternatives. An antigen recognition domain from native TCR alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen recognition region.

Ectodomain - spacer

A spacer region links the antigen binding domain to the transmembrane domain. It should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgG1. Alternatives include the CH₂CH₃ region of immunoglobulin and portions of CD3. For most scFv based constructs, the IgG1 hinge suffices. However the best spacer often has to be determined empirically.

Transmembrane domain

This is a hydrophobic alpha helix that spans the membrane. Generally, the transmembrane domain from the most membrane proximal component of the endodomain is used. Interestingly, using the CD3-zeta transmembrane domain may result in incorporation of the artificial TCR into the native TCR a factor that is dependent on the presence of the native CD3-zeta transmembrane charged aspartic acid residue . Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly expressed, stable receptor.

Endodomain

This is the business-end of the receptor. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together.

Evolution of CAR T-cell design

“First-generation” CARs typically had the intracellular domain from the CD3 ξ- chain, which is the primary transmitter of signals from endogenous TCRs. “Second-generation” CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent,“third-generation” CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further augment potency.

Methods of Gene Delivery to T Cells

T cells are highly proliferative, and thus, sustained and stable modification of these cells requires vectors that provide for integration of the transgene(s) into the cellular DNA as well as persistent expression of the transgene(s) at moderately high levels.

1)Gamma retroviruses

Gamma retroviruses integrate into the genome and produce reliable gene expression in T cells. These vectors, however, have limitations with respect to the size of the gene(s) that can be transferred (limited cargo capacity). In addition, because these vectors can only transduce dividing cells, T cells must be activated ex vivo, which can be detrimental for their in vivo persistence once returned to the patient.

2)Lentiviral vectors

Lentiviral vectors can transduce nondividing or minimally proliferating T cells, reducing the requirement for ex vivo activation of the T cells, which could benefit their in vivo persistence by reducing activation induced cell death and cell exhaustion that comes with repeated stimulation. Lentiviral vectors also offer the advantages of accommodating larger genes or gene cassettes with reduced susceptibility to gene silencing as well as the reduced, but not absent, potential for insertional mutagenesis.

3)Nonviral vectors

Unlike viral vectors, which infect target cells and can produce high transduction rates, nonviral vectors cannot enter target cells on their own and require electroporation or chemical (liposomal)-based transfection methods for cellular entry. Transposon-based gene delivery systems, such as Sleeping Beauty and PiggyBac, are integrating nonviral gene delivery systems that can overcome low random integration rates and have been evaluated for their ability to modify T cells.

History of Chimeric Antigen Receptors

http://www.nejm.org/doi/full/10.1056/NEJMe1106965

Clinical Studies testing Chimeric Antigen Receptors

There were presentations of individual clinical trials (Renier Brentjens, Memorial Sloan-Kettering Cancer Center; Gianpietro Dotti, Baylor College of Medicine; Steven Rosenberg, NCI; Carl June, University of Pennsylvania; Laurence Cooper, M.D. Anderson Cancer Center; and Michael Jensen, City of Hope National Medical Center), and it was evident that there were disparate components in each investigator’s approach, in terms of the B-lineage malignancies being treated, the design of the anti-CD19 CAR molecule, the CAR gene delivery method, the preparative regimens, and the type of T-cell type targeted for transduction.

Two serious adverse events in clinical trials using CARs were discussed. One occurred after infusion of an anti-CD19 CAR in a patient with advanced CLL and the second after adoptive transfer of an anti-HER-2/neu CAR in a patient with metastatic colon cancer. The treatment-related death with the anti-CD19 CAR occurred shortly after the patient received cyclophosphamide for lymphodepletion and infusion of CAR-transduced cells. The patient showed symptoms of acute sepsis, renal failure and resultant shock, and adult respiratory distress syndrome. In contrast, toxicity with the Her2/neu CAR-modified cells may have been a direct effect of infusion of a large number of T cells recognizing the target antigen expressed at low levels on normal cells in the lung.

References

• Rossig, C; et al. (Oct 15 2001). "Targeting of GD2 positive tumor cells by human T lymphocytes engineered to express chimeric T-cell receptor genes". Int J Cancer 94 (2): 228–36. doi:10.1002/ijc.1457. PMID 11668503.