Chimeric antigen receptor
Chimeric antigen receptor
Chimeric antigen receptors (CARs), (also known as chimeric immunoreceptors, chimeric T cell receptors, artificial T cell receptors or CAR-T) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell (T 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. The receptors are called chimeric because they are composed of parts from different sources.
A CAR therapy for cancer, using a mechanism called adoptive cell transfer, [1] has been approved by the US Food and Drug Administration for use against acute lymphoblastic leukaemia. [Two] T cells are eliminated from a patient and modified so that they express receptors specific to the patient’s particular cancer. The T cells, which can then recognize and kill the cancer cells, are reintroduced into the patient. Modification of T-cells sourced from donors other than the patient are also under investigation.
Contents
The most common form of CARs are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane- and endodomain. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2).
The variable portions of an immunoglobulin mighty and light chain are fused by a lithe linker to form an scFv. [ citation needed ] This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (cloven). A limber spacer permits the scFv to orient in different directions to enable antigen cording. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signalling endodomain that protrudes into the cell and transmits the desired signal.
Type I proteins are in fact two protein domains linked by a transmembrane alpha helix. The cell membrane lipid bilayer, through which the transmembrane domain passes, isolates the inwards portion (endodomain) from the outer portion (ectodomain). Linking an ectodomain from one protein to an endodomain of another protein makes a molecule that combines the recognition of the former to the signal of the latter.
ScFv/CD3-zeta hybrids result in the transmission of a zeta signal in response to recognition by the scFv of its target. 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.
Ectodomain Edit
The ectodomain is the domain of a membrane protein which is outside the cell, outside the cytoplasm [extracytoplasmic] and exposed to the extracellular space (when not inwards the lumen on intracellular vesicles in the secretory or endocytic pathways). For a cell surface receptor or protein, this will be the domain exposed to the extracellular space.
Signal peptide Edit
A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to glycosylate and anchor in the cell membrane. Any eukaryotic signal peptide sequence usually works. Generally, the signal peptide natively affixed to the amino-terminal most component is used (e.g. in a scFv with orientation light chain – linker – strenuous chain, the native signal of the light-chain is used).
Antigen recognition region Edit
The antigen recognition region is usually a scFv, albeit many alternatives exist. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have elementary 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). Almost anything that ties a given target with high affinity can be used as an antigen recognition region.
Spacer Edit
A spacer region links the antigen tying domain to the transmembrane domain. It should be nimble enough to permit the antigen trussing 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 is determined empirically.
Transmembrane domain Edit
The transmembrane domain 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. [Trio] Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly voiced, stable receptor.
Endodomain Edit
This is the functional 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 three ITAMs. This transmits an activation signal to the T cell after the antigen is tied. CD3-zeta may not provide a fully competent activation signal and 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.
Very first generation CARs typically had the intracellular domain from the CD3 ζ- chain, which is the primary transmitter of signals from endogenous TCRs. 2nd 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 extra signals to the T cell. Preclinical studies indicated that the 2nd generation improves the antitumor activity of T cells. More latest, third generation CARs combine numerous signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to augment potency.
The evolution of CAR therapy is an excellent example of the application of basic research to the clinic. The PI3K tying site used was identified in co-receptor CD28, [Five] while the ITAM motifs were identified as a target of the CD4- and CD8-p56lck complexes. [6]
The introduction of Strep-tag II sequence (an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin [7] ) into specific sites in synthetic CARs or natural T-cell receptors provides engineered T cells with an identification marker for rapid purification, a method for tailoring spacer length of chimeric receptors for optimal function and a functional element for selective antibody-coated, microbead-driven, large-scale expansion. [8] [9] Strep-tag can be used to stimulate the engineered cells, causing them to grow rapidly. Using an antibody that trusses the Strep-tag, the engineered cells can be expanded by 200-fold. Unlike existing methods this technology stimulates only cancer-specific T cells.
SMDC adaptor technology Edit
SMDCs (puny molecule drug conjugates) platform in immuno-oncology is a novel (presently experimental) treatment that makes possible the engineering of a single universal CAR T cell, which trusses with extraordinarily high affinity to a benign molecule designated as FITC. These cells are then used to treat various cancer types when co-administered with bispecific SMDC adaptor molecules. These unique bispecific adaptors are constructed with a FITC molecule and a tumor-homing molecule to precisely bridge the universal CAR T cell with the cancer cells, which causes localized T cell activation. Anti-tumor activity in mice is induced only when both the universal CAR T cells plus the correct antigen-specific adaptor molecules are present. Anti-tumor activity and toxicity can be managed by adjusting the administered adaptor molecule dosing. Treatment of antigenically heterogeneous tumors can be achieved by administration of a combination of the desired antigen-specific adaptors. Thus, several challenges of current CAR T cell therapies, such as:
- the inability to control the rate of cytokine release and tumor lysis
- the absence of an “off switch” that can terminate cytotoxic activity when tumor eradication is accomplish
- a requirement to generate a different CAR T cell for each unique tumor antigen
may be solved or mitigated using this treatment. [Ten] [11] [12]
The use of CARs in the clinic is based on reprogramming the T cell antigen receptor using a vector (for example viral) that is specific for malignant cells. Pre-clinical and clinical trials have focused on optimizing structure and signalling.
The very first generation of CAR-modified T cells (CARTs) showcased success in pre-clinical trials and have entered phase I clinical trials in ovarian cancer, neuroblastoma and various types of leukemia and lymphoma. To date, these clinical trials have shown little evidence of anti-tumor activity, with insufficient activation, persistence and homing to cancer tissue. Some anti-tumor responses have been reported in patients with B cell lymphoma (treated with alfaCD20-CD3zeta CAR-modified T cells) and some neuroblastoma patients (treated with ScFv-CD3zeta CARTs) reported partial response, stable disease and remission.
2nd and third generation CARTs provided enhanced activation signals, proliferation, production of cytokines and effector function in pre-clinical trials. Both 2nd and the third generation CARTs are injecting clinical trials. In a investigate with alfaCD19.4-1BB.CD3zeta CARTs in patients with chronic lymphocyte leukemia, accomplish remission was ongoing ten months after treatment. CARTs cells were found to expand 3-logs in these patients, and to have infiltrated and lysed cancer tissue. A fraction of these cells displayed a memory phenotype for preventive tumor relapses. Albeit these CARTs produced significant therapeutic effect, their activity led to life-threatening tumorlysis three weeks after the very first infusion.
Adverse events were reported that highlight the need for caution while using 2nd and third generation CARTs. One patient died five days after cyclophosphamide chemotherapy followed by infusion of CARTs recognizing the antigen ERBB2 (HER-2/neu). [13] The toxicity led to a clinically significant release of pro-inflammatory cytokines, pulmonary toxicity, multi-organ failure and eventual patient death. This “cytokine storm” (cytokine release syndrome) was thought to be due to CAR T cell cytotoxicity against normal lung epithelial cells, known to express low levels of ERBB2. This and other adverse events highlight the need for caution when employing CARTs, as unlike antibodies against tumor-associated antigens, these cells are not cleared from the assets quickly. Long exposure to CARTs is necessary for good clinical outcome, but is not feasible due to adverse effects, particularly prolonged absence of myelopoiesis. [14] Efforts to circumnavigate this problem with a suicide gene are presently underway. [14]
A two thousand sixteen review indicates that latest clinical trials evaluating active immunization or immunoconjugates to mesothelin in patients with pancreatic adenocarcinoma or mesothelioma have shown responses without toxicity. [15]
The superb promise of cancer immunotherapy is to clear the tumor without the toxicity of conventional treatments. The treatment of cancer with CARTs has several advantages: HLA-independent recognition of antigen, broad applicability for many patients and rapid delivery. Successful application of CARTs will require the identification of a tumor-associated antigen that is voiced only on tumor cells, thereby minimizing toxicity risk, [16] [17]
Early examples Edit
A list of tumors antigens and CARs in in vitro and in vivo trials As of two thousand twelve [update] : [17] [Legitimate]