Artificial T cell receptor
Encyclopedia
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 cell
s can be generated for use as adoptive cell therapy. . Phase I clinical studies of this approach show efficacy.
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.
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.
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
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.
-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.
-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.
T cell
T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells , by the presence of a T cell receptor on the cell surface. They are...
. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell
T cell
T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells , by the presence of a T cell receptor on the cell surface. They are...
; with transfer of their coding sequence facilitated by retroviral vectors. In this way, a large number of cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
-specific T cell
T cell
T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells , by the presence of a T cell receptor on the cell surface. They are...
s 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 hybridomaHybridoma
Hybridoma technology is a technology of forming hybrid cell lines by fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis...
14g2a (which recognizes disialoganglioside GD2
GD2
GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans....
). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2
GD2
GD2 is a disialoganglioside expressed on tumors of neuroectodermal origin, including human neuroblastoma and melanoma, with highly restricted expression on normal tissues, principally to the cerebellum and peripheral nerves in humans....
(e.g. neuroblastoma
Neuroblastoma
Neuroblastoma is the most common extracranial solid cancer in childhood and the most common cancer in infancy, with an annual incidence of about 650 cases per year in the US , and 100 cases per year in the UK . Close to 50 percent of neuroblastoma cases occur in children younger than two years old...
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.
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
Helix
A helix is a type of smooth space curve, i.e. a curve in three-dimensional space. It has the property that the tangent line at any point makes a constant angle with a fixed line called the axis. Examples of helixes are coil springs and the handrails of spiral staircases. A "filled-in" helix – for...
usually derived from the original molecule of the signalling endodomain which protrudes into the cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
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
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
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 peptideSignal peptide
A signal peptide is a short peptide chain that directs the transport of a protein.Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals....
directs the nascent protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
into the endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
. 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
Heavy chain
]The immunoglobulin heavy chain is the large polypeptide subunit of an antibody .A typical antibody is composed of two immunoglobulin heavy chains and two Ig light chains. Several different types of heavy chain exist that define the class or isotype of an antibody. These heavy chain types vary...
, 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 antigenAntigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
recognition domain from native TCR alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4
CD4
CD4 is a glycoprotein expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells. It was discovered in the late 1970s and was originally known as leu-3 and T4 before being named CD4 in 1984...
ectodomain to recognize HIV
HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
infected cells) and more exotic recognition components such as a linked cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
(which leads to recognition of cells bearing the cytokine
Cytokine
Cytokines are small cell-signaling protein molecules that are secreted by the glial cells of the nervous system and by numerous cells of the immune system and are a category of signaling molecules used extensively in intercellular communication...
receptor). In fact almost anything that binds a given target with high affinity can be used as an antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
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 CD3CD3
CD3 or CD-3 may be:* CD3 , an antigen, cluster of differentiation protein , part of the T cell receptor complex on a mature T lymphocyte* Ford CD3 platform* MediaMax CD-3, copy protection scheme* MiniCD, a 3-inch CD...
-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
CD3
CD3 or CD-3 may be:* CD3 , an antigen, cluster of differentiation protein , part of the T cell receptor complex on a mature T lymphocyte* Ford CD3 platform* MediaMax CD-3, copy protection scheme* MiniCD, a 3-inch CD...
-zeta transmembrane charged aspartic acid residue
. Different transmembrane domains result in different receptor stability. The CD28
CD28
CD28 is one of the molecules expressed on T cells that provide co-stimulatory signals, which are required for T cell activation. CD28 is the receptor for CD80 and CD86 . When activated by Toll-like receptor ligands, the CD80 expression is upregulated in antigen presenting cells...
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 CD3CD3
CD3 or CD-3 may be:* CD3 , an antigen, cluster of differentiation protein , part of the T cell receptor complex on a mature T lymphocyte* Ford CD3 platform* MediaMax CD-3, copy protection scheme* MiniCD, a 3-inch CD...
-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3
CD3
CD3 or CD-3 may be:* CD3 , an antigen, cluster of differentiation protein , part of the T cell receptor complex on a mature T lymphocyte* Ford CD3 platform* MediaMax CD-3, copy protection scheme* MiniCD, a 3-inch CD...
-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28
CD28
CD28 is one of the molecules expressed on T cells that provide co-stimulatory signals, which are required for T cell activation. CD28 is the receptor for CD80 and CD86 . When activated by Toll-like receptor ligands, the CD80 expression is upregulated in antigen presenting cells...
and OX40 can be used with CD3
CD3
CD3 or CD-3 may be:* CD3 , an antigen, cluster of differentiation protein , part of the T cell receptor complex on a mature T lymphocyte* Ford CD3 platform* MediaMax CD-3, copy protection scheme* MiniCD, a 3-inch CD...
-Zeta to transmit a proliferative / survival signal, or all three can be used together.