Chimeric antigen receptor

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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.

Artificial T cell receptors are under investigation as a therapy for cancer, using a technique called adoptive cell transfer.[1] T cells are removed from a patient and modified so that they express receptors specific to the particular form of cancer. The T cells, which can then recognize and kill the cancer cells, are reintroduced into the patient. Phase I clinical studies of this approach have shown efficacy.

Structure[edit]

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.

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.

Ectodomain[edit]

Signal peptide[edit]

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

Antigen recognition region[edit]

The antigen recognition domain is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (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.

Spacer[edit]

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[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[2] . Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly expressed, stable receptor.

Endodomain[edit]

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[edit]

Depiction of first, second, and third generation chimeric antigen receptors with the scFv segments in green and the various TCR signalling components in red, blue and yellow.[3]

"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.

History[edit]

The concept of doctoring T-cells genetically was first developed in the 1980s by Dr. Zelig Eshhar and colleagues at the Weizmann Institute of Science in Rehovot, Israel. By 1989, Eshhar and his colleagues had created the first CAR T cells that worked - in the lab

Clinical studies[edit]

This method is based on the reprogramming by the vector (for example viral), and chimeric antigen receptor, that redirects them against malignant cell and enables their destruction. There has been interest of sciences in the use of CAR-modified T cell as cancer immunotherapy. In the pre-clinical and clinical trial, there has been a focus upon optimizing in field of structural and signaling.

The first generation of CAR-modified T cell showed success in pre-clinical trials and has entered phase I clinical trials. Clinical trials have commenced in ovarian cancer, neuroblastoma and various types of leukemia and lymphoma. The clinical trials showed little evidence of anti-tumor activity with insufficient activation, persistence and homing to cancer tissue. Some anti-tumor responses being have been reported in patient with B-lymphoma (treated with alfaCD20-CD3zeta CARs-modified T cell) and neuroblastoma (treated with ScFv-CD3zeta CARs-modified T cell) have reported partial response, stable diseases and remission.

The second and the third generation CAR-modification T cells are capable of delivering enhancement of activation signal, proliferation, production of cytokines and effector function of CAR-modified T cell in pre-clinical trials. Both the second and the third generation CAR-modified T cell are entering clinical trials now. The first clinical trial has shown some promising results. In a study with alfaCD19.4-1BB.CD3zeta T cells in patients with chronic lymphocyte leukemia complete remission has been ongoing 10 months after treatment. The CAR-modified T cell were found to expand 3-logs in these patients, that infiltrated and lysed cancer tissue. Interestingly, a fraction of these cell displayed a memory phenotype of T cell for preventive tumor relapses. Although these CAR-modified T cell produced significant therapeutic effect, their activity led to life-threatening tumorlysis 3 weeks after the first infusion of CAR-modified T cell.

Recently adverse events were reported which highlight the need for caution while using second and third generation of CAR-modified T cells. One patient died 5 days after cyclophosphamide chemotherapy followed by infusion of CAR-modified T cells recognizing the antigen ERBB2 (HER-2/neu).[4] The toxicity lead to a clinically significant release of pro-inflammatory cytokines, pulmonary toxicity, multi-organ failure and eventual death of the patient. This "cytokine storm" 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 CAR-modified T cells, as unlike antibodies against tumor-associated antigens, these cells are not cleared from the body within a short amount of time.

The great promise of cancer immunotherapy is the cure of tumor without the toxicity of conventional treatments. The pre-clinical and clinical trial of CAR-modified T cells have given hope in cancer therapy. The treatment of cancer with modified T cells has several advantages: HLA-independent recognition of antigen, broad applicability for many patients and the rapid delivery of CAR-modified T cells. The successful application of cell will require the identification of target antigen, that are expressed only on tumor cells, thereby minimizing the risk of toxicity,[5][6]

Actual list of tumors antigens and CARs in in vitro and in vivo trials,:[7][8]

Target antigen Associated malignancy Receptor type CARs generation
α-Folate receptor Ovarian cancer ScFv-FcεRIγCAIX First
CAIX Renal cell carcinoma ScFv-FcεRIγ First
CAIX Renal cell carcinoma ScFv-FcεRIγ Second
CD19 B-cell malignancies ScFv-CD3ζ (EBV) First
CD19 B-cell malignancies, CLL ScFv-CD3ζ First
CD19 B-ALL ScFv-CD28-CD3ζ Second
CD19 ALL CD3ζ(EBV) First
CD19 ALL post-HSCT ScFv-CD28-CD3ζ Second
CD19 Leukemia, lymphoma, CLL ScFv-CD28-CD3ζ vs. CD3ζ First and Second
CD19 B-cell malignancies ScFv-CD28-CD3ζ Second
CD19 B-cell maligniancies post-HSCT ScFv-CD28-CD3ζ Second
CD19 Refractory Follicular Lymphoma ScFv-CD3ζ First
CD19 B-NHL ScFv -CD3ζ First
CD19 B-lineage lymphoid malignancies post-UCBT ScFv-CD28-CD3ζ Second
CD19 CLL, B-NHL ScFv-CD28-CD3ζ Second
CD19 B-cell malignancies, CLL, B-NHL ScFv-CD28-CD3ζ Second
CD19 ALL, lymphoma ScFv-41BB-CD3ζ vs CD3ζ First and Second
CD19 ALL ScFv-41BB-CD3ζ Second
CD19 B-cell malignancies ScFv-CD3ζ (Influenza MP-1) First
CD19 B-cell malignancies ScFv-CD3ζ (VZV) First
CD20 Lymphomas ScFv-CD28-CD3ζ Second
CD20 B-cell malignancies ScFv-CD4-CD3ζ Second
CD20 B-cell lymphomas ScFv-CD3ζ First
CD20 Mantle cell lymphoma ScFv-CD3ζ First
CD20 Mantle cell lymphoma, indolent B-NHL CD3 ζ /CD137/CD28 Third
CD20 indolent B cell lymphomas ScFv-CD28-CD3ζ Second
CD20 Indolent B cell lymphomas ScFv-CD28-41BB-CD3ζ Third
CD22 B-cell malignancies ScFV-CD4-CD3ζ Second
CD30 Lymphomas ScFv-FcεRIγ First
CD30 Hodgkin lymphoma ScFv-CD3ζ (EBV) First
CD33 AML ScFv-CD28-CD3ζ Second
CD33 AML ScFv-41BB-CD3ζ Second
CD44v7/8 Cervical carcinoma ScFv-CD8-CD3ζ Second
CEA Breast cancer ScFv-CD28-CD3ζ Second
CEA Colorectal cancer ScFv-CD3ζ First
CEA Colorectal cancer ScFv-FceRIγ First
CEA Colorectal cancer ScFv-CD3ζ First
CEA Colorectal cancer ScFv-CD28-CD3ζ Second
CEA Colorectal cancer ScFv-CD28-CD3ζ Second
EGP-2 Multiple malignancies scFv-CD3ζ First
EGP-2 Multiple malignancies scFv-FcεRIγ First
EGP-40 Colorectal cancer scFv-FcεRIγ First
erb-B2 Colorectal cancer CD28/4-1BB-CD3ζ Third
erb-B2 Breast and others ScFv-CD28-CD3ζ Second
erb-B2 Breast and others ScFv-CD28-CD3ζ (Influenza) Second
erb-B2 Breast and others ScFv-CD28mut-CD3ζ Second
erb-B2 Prostate cancer ScFv-FcεRIγ First
erb-B 2,3,4 Breast and others Heregulin-CD3ζ Second
erb-B 2,3,4 Breast and others ScFv-CD3ζ First
FBP Ovarian cancer ScFv-FcεRIγ First
FBP Ovarian cancer ScFv-FcεRIγ (alloantigen) First
Fetal acethylcholine receptor Rhabdomyosarcoma ScFv-CD3ζ First
GD2 Neuroblastoma ScFv-CD28 First
GD2 Neuroblastoma ScFv-CD3ζ First
GD2 Neuroblastoma ScFv-CD3ζ First
GD2 Neuroblastoma ScFv-CD28-OX40-CD3ζ Third
GD2 Neuroblastoma ScFv-CD3ζ (VZV) First
GD3 Melanoma ScFv-CD3ξ First
GD3 Melanoma ScFv-CD3ξ First
Her2/neu Medulloblastoma ScFv-CD3ξ First
Her2/neu Lung malignancy ScFv-CD28-CD3ζ Second
Her2/neu Advanced osteosarcoma ScFv-CD28-CD3ζ Second
Her2/neu Glioblastoma ScFv-CD28-CD3ζ Second
IL-13R-a2 Glioma IL-13-CD28-4-1BB-CD3ζ Third
IL-13R-a2 Glioblastoma IL-13-CD3ζ Second
IL-13R-a2 Medulloblastoma IL-13-CD3ζ Second
KDR Tumor neovasculature ScFv-FcεRIγ First
k-light chain B-cell malignancies ScFv-CD3ζ First
k-light chain (B-NHL, CLL) ScFv-CD28-CD3ζ vs CD3ζ Second
LeY Carcinomas ScFv-FcεRIγ First
LeY Epithelial derived tumors ScFv-CD28-CD3ζ Second
L1 cell adhesion molecule Neuroblastoma ScFv-CD3ζ First
MAGE-A1 Melanoma ScFV-CD4-FcεRIγ Second
MAGE-A1 Melanoma ScFV-CD28-FcεRIγ Second
Mesothelin Various tumors ScFv-CD28-CD3ζ Second
Mesothelin Various tumors ScFv-41BB-CD3ζ Second
Mesothelin Various tumors ScFv-CD28-41BB-CD3ζ Third
Murine CMV infected cells Murine CMV Ly49H-CD3ζ Second
MUC1 Breast, Ovary ScFV-CD28-OX40-CD3ζ Third
NKG2D ligands Various tumors NKG2D-CD3ζ First
Oncofetal antigen (h5T4) Various tumors ScFV-CD3ζ (vaccination) First
PSCA Prostate carcinoma ScFv-b2c-CD3ζ Second
PSMA Prostate/tumor vasculature ScFv-CD3ζ First
PSMA Prostate/tumor vasculature ScFv-CD28-CD3ζ Second
PSMA Prostate/tumor vasculature ScFv-CD3ζ First
TAA targeted by mAb IgE Various tumors FceRI-CD28-CD3ζ (+ a-TAA IgE mAb) Third
TAG-72 Adenocarcinomas scFv-CD3ζ First
VEGF-R2 Tumor neovasculature scFv-CD3ζ First

Adoptive transfer of CAR-modified cells as a cancer therapeutic[edit]

Depiction of adoptive cell transfer therapy with CAR-engineered T cells

Adoptive transfer of T cells expressing chimeric antigen receptors is a promising anti-cancer therapeutic as CAR-modified T cells can be engineered to target virtually any tumor associated antigen. There is great potential for this approach to improve patient-specific cancer therapy in a profound way. Following the collection of a patient's T cells, the cells are genetically engineered to express CARs specifically directed towards antigens on the patient's tumor cells, then infused back into the patient.[9] Although adoptive transfer of CAR-modified T-cells is a unique and promising cancer therapeutic, there are significant safety concerns. Clinical trials of this therapy have revealed potential toxic effects of these CARs when healthy tissues express the same target antigens as the tumor cells, leading to outcomes similar to graft-versus-host disease (GVHD). A potential solution to this problem is engineering a suicide gene into the modified T cells. In this way, administration of a prodrug designed to activate the suicide gene during GVHD triggers apoptosis in the suicide gene-activated CAR T cells. This method has been used safely and effectively in hematopoietic stem cell transplantation (HSCT). Adoption of suicide gene therapy to the clinical application of CAR-modified T cell adoptive cell transfer has potential to alleviate GVHD while improving overall anti-tumor efficacy.[3]

See Also[edit]

References[edit]

  1. ^ Pule, M; Finney H; Lawson A (2003). "Artificial T-cell receptors". Cytotherapy 5 (3): 211–26. doi:10.1080/14653240310001488. PMID 12850789. 
  2. ^ Bridgeman, JS; Hawkins RE; Bagley S; Blaylock M; Holland M; Gilham DE (2010). "The Optimal Antigen Response of Chimeric Antigen Receptors Harbouring the CD3zeta Transmembrane Domain Is Dependent Upon Incorporation of the Receptor Into the Endogenous TCR/CD3 Complex". Journal of Immunology 184 (12): 6938–49. doi:10.4049/jimmunol.0901766. PMID 20483753. 
  3. ^ a b Casucci, Monica; Attilio Bondanza (2011). "Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes". Journal of Cancer 2: 378–382. doi:10.7150/jca.2.378. PMC 3133962. PMID 21750689. Retrieved 30 April 2012. 
  4. ^ Morgan, Richard; Yang, J. C. Kitano, M. Dudley, M. E. Laurencot, C. M. Rosenberg, S. A. (2010-02-25). "Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2". Molecular therapy : the journal of the American Society of Gene Therapy 28 (4): 843–51. doi:10.1038/mt.2010.24. PMID 2862534. 
  5. ^ Curran, Kevin J.; Pengram Hollie J; Brentjens Renier J (2012). "The promise and potential". J Gene Med 14 (6): 405–415. doi:10.1002/jgm.2604. PMID 22262649. Retrieved 5 January 2012. 
  6. ^ Lipovska-Bhalla, Grazyna; Gilham David E., Hawkins Robert E., Rothwell Dominic G. (2012). "Target immunotherapy of cancer with CAR T cell: achievements and challenges". Cancer Immunol Immunother 61 (7): 953–962. doi:10.1007/s00262-012-1254-0. PMID 22527245. Retrieved 25 March 2012. 
  7. ^ Lipowska-Bhalla, Grazina; Gilham David E., Hawkins Robert E., Rothwell Dominic G. (2012). "Target immunotherapy of cancer with CAR T cell: achievements and challenges". Cancer Immunol Immunother 61 (7): 953–962. doi:10.1007/s00262-012-1254-0. PMID 22527245. Retrieved 25 March 2012. 
  8. ^ Sadelain, M.; Gilham David E., Hawkins Robert E., Rothwell Dominic G. (2009). "The promise and potential pitfalls of chimeric antigen receptor". Curr Opin Immunol 21 (2): 215–223. doi:10.1016/j.coi.2009.02.009. PMID 19327974. 
  9. ^ Jacobson, Caron; Jerome Ritz (3 November 2011). "Time to put the CAR-T before the horse". Blood 118 (18): 4761–4762. doi:10.1182/blood-2011-09-376137. PMID 22053170. Retrieved 7 May 2012.