Chimeric antigen receptor

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Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because they are fused of parts from different sources. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer therapy.

The basic principle of CAR-T cell design involves recombinant receptors that combine antigen-binding and T-cell activating functions. The general premise of CAR-T cells is to artificially generate T-cells targeted to markers found on cancer cells. Scientists can remove T-cells from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells.[1] Once the T cell has been engineered to become a CAR-T cell, it acts as a "living drug".[2] CAR-T cells create a link between an extracellular ligand recognition domain to an intracellular signalling molecule which in turn activates T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of molecule that is targeted.[3]

CAR-Ts can be derived from either a patient's own blood (autologous) or derived from another healthy donor (allogenic). These T-cells are genetically engineered to express an artificial T cell receptor, through which they are targeted to disease-related antigens.[4] This process is MHC independent and thus the targeting efficiency is greatly increased. These CAR-T cells are programmed to target antigens that are present on the surface of tumors. When they come in contact with the antigens on the tumors, the CAR-T cells are activated via the signal peptide, proliferate and become cytotoxic.[5] The CAR-T cells destroy the cancer cells through mechanisms such as extensive stimulated cell proliferation, increasing the degree to which the cell is toxic to other living cells i.e. cytotoxicity, and by causing the increased production of factors that are secreted from cells in the immune system that have an effect on other cells in the organism. These factors are called cytokines and include interleukins, interferons and growth factors.[6]

CAR-T cells are developed to be specific to an antigen expressed on a tumor that is not expressed on healthy cells. CD19 is expressed on B-cells throughout their development and as a result, CD19 is also expressed on nearly all B-cell malignancies. Additionally, CD19 is only expressed in the B-cell lineage and not in any other lineages or tissues. These malignancies include forms of cancer such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and many different forms of Hodgkin’s lymphoma.[7]

Use in cancer[edit]

The diagram above represents the process of chimeric antigen receptor T-cell therapy (CAR), this is a method of immunotherapy, which is a growing practice in the treatment of cancer. The final result should be a production of equipped T-cells that can recognize and fight the infected cancer cells in the body.
1. T-cells (represented by objects labeled as ’t’) are removed from the patient's blood.
2. Then in a lab setting the gene that encodes for the specific antigen receptors are incorporated into the T-cells.
3. Thus producing the CAR receptors (labeled as c) on the surface of the cells.
4. The newly modified T-cells are then further harvested and grown in the lab.
5. After a certain time period, the engineered T-cells are infused back into the patient.
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 collection of a patient's T cells, the cells are genetically engineered to express CARs specifically directed toward antigens on the patient's tumor cells, then infused back into the patient.[8]

Preparation[edit]

The first step in the introduction of CAR-T cells into the body of a patient is the removal of activated leukocytes from the blood in a process known as leukocyte apheresis. The leukocytes are removed using a blood cell separator. The patient’s autologous peripheral blood mononuclear cells (PBMC) are then separated and collected from the buffy coat that forms.[9] The products of leukocyte apheresis are then transferred into a cell processing center. In the cell processing centre, specific T-cells are activated in a certain environment in which they can actively proliferate. The cells are activated using a type of cytokine called an interleukin, specifically Inter-Leukin 2 (IL-2) as well as anti-CD3 antibodies.[10]

The T-cells are then transfected with CD19 CAR genes by either an integrating gammaretrovirus (RV) or by lentivirus (LV) vectors. These vectors are very safe in modern times due to a partial deletion of the U3 region.[11] The patient undergoes lymphodepletion chemotherapy prior to the introduction of the engineered CD CAR-T cells.[5] The depletion of the number of circulating leukocytes in the patient upregulates the number of cytokines that are produced which help to promote the expansion of the engineered CAR-T cell.[12]

Safety concerns[edit]

CAR-T cells are undoubtedly a major breakthrough in cancer treatment. However, there are still expected toxicities as well as some unexpected toxicities that come in conjunction to CAR-T cells being introduced into the body. These toxicities include cytokine release syndrome (CRS), neurological toxicity, On-target/Off-Tumour Recognition, insertional mutagenesis and anaphylaxis.[5]

CRS is a condition in which the immune system is activated and releases an increased number of inflammatory cytokines. The clinical manifestations of this syndrome include: high fever, fatigue, myalgia, nausea, tachycardia, capillary leakages, cardiac dysfunction, hepatic failure and renal impairment.[13]

The neurological toxicity associated with CAR-T cells have clinical manifestations that include delirium, the partial loss of the ability to speak a coherent language while still having the ability to interpret language (expressive aphasia), obtundation and seizures.[14] During some clinical trials deaths caused by neurotoxicity have occurred. The main cause of death from neurotoxicity is cerebral edema. In a study carried out by Juno Therapeutics, Inc., five patients enrolled in the trial died as a result of cerebral edema. Two of the patients were treated with cyclophosphamide alone and the remaining three were treated with a combination of cyclophosphamide and fludarabine.[15] In another clinical trial sponsored by the Fred Hutchinson Cancer Research Center, there was one reported case of irreversible and fatal neurological toxicity 122 days after the administration of CAR-T cells.[16]

On-target/Off-tumor recognition occurs when the CAR-T cell recognises the correct antigen, but the antigen is expressed on a non-pathogenic tissue. This adverse effect can vary in severity from B-Cell Aplasia to severe toxicity which leads to death.[10]

Anaphylaxis is an expected side effect as the CAR is made with a foreign monoclonal antibody and as a result, invokes an immune response. There is also a potential for insertional mutagenesis that can occur when inserting vector DNA into a host cell. Lentiviral (LV) vectors carry a lower risk than retroviral (RV) vectors. however, both have the potential to be oncogenic.

There hasn’t been much long-term research done into the effects of CAR-T cells as they are a relatively new medicine still in the trial phases so there is still concern for long-term survival as well as pregnancy complications in female patients treated with CAR-T cells.[14]

Early FDA approvals[edit]

The first two FDA approved CAR-T therapies were targeted at CD19 (found on many types of lymphoma cells; mainly B-cell lymphomas).[17] They are approved for relapsed/refractory diffuse large B-cell lymphoma (DLBCL) for axicabtagene ciloleucel and relapsed/refractory B-cell precursor acute lymphoblastic leukemia (ALL) for tisagenlecleucel.[17]

Structure[edit]

CARs are composed of three regions: the ectodomain, the transmembrane domain and the endodomain.

Different components of an artificial TCR

Ectodomain[edit]

The ectodomain is the region of the receptor that is exposed to the extracellular fluid and consists of 3 components: a signalling peptide, an antigen recognition region and a spacer.

A signal peptide directs the nascent protein into the endoplasmic reticulum. The signal protein in CAR is called a single-chain variable fragment (scFv),[18] a type of protein known as a fusion protein or chimeric protein. A fusion protein is a protein that is formed by merging two or more genes that code originally for different proteins but when they are translated in the cell, the translation produces one or more polypeptides with functional properties derived for each of the original genes.[19]

A scFv is a chimeric protein made up of the light and heavy chains of immunoglobins connected with a short linker peptide. The linker consists of hydrophilic residues with stretches of glycine and serine in it for flexibility as well as stretches of glutamate and lysine for added solubility.[20]

Transmembrane domain[edit]

The transmembrane domain is a hydrophobic alpha helix that spans the membrane. The transmembrane domain is essential for the stability of the receptor as a whole. At present, the CD28 transmembrane domain is the most stable of the domains.

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.[21] Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a highly expressed, 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.[18] The most commonly used endodomain component is CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after the antigen is bound. 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.

History[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.[22]

First generation CARs were developed in 1989 by Gideon Gross and Zelig Eshhar[23][24] at Weizmann Institute, Israel.[25] The first generation of CARs are composed of an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains.[4] Extracellular binding domain contains single‐chain variable fragments (scFvs) derived from tumor antigen‐reactive antibodies and usually have high specificity to tumor antigen.[4] All CARs harbor the CD3ζ chain domain as the intracellular signaling domain, which is the primary transmitter of signals. Second generation CARs also contain co‐stimulatory domains, like CD28 and/or 4‐1BB. The involvement of these intracellular signaling domains improve T cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence.[4] Besides co-stimulatory domains, the third‐generation CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to augment T cell activity. Preclinical data shows the third-generation CARs exhibit improved effector functions and in vivo persistence as compared to second‐generation CARs.[4] Recently, the fourth‐generation CARs (also known as TRUCKs or armored CARs), combine the expression of a second‐generation CAR with factors that enhance anti‐tumoral activity (e.g., cytokines, co‐stimulatory ligands).[26]

The evolution of CAR therapy is an excellent example of the application of basic research to the clinic. The PI3K binding site used was identified in co-receptor CD28,[27] while the ITAM motifs were identified as a target of the CD4- and CD8-p56lck complexes.[28]

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[29]) 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.[30][31] Strep-tag can be used to stimulate the engineered cells, causing them to grow rapidly. Using an antibody that binds the Strep-tag, the engineered cells can be expanded by 200-fold. Unlike existing methods this technology stimulates only cancer-specific T cells.[citation needed]

Smart T cell[edit]

Combined with exogenous molecules, some synthetic control devices have been implemented on CAR-T cells and alter the cell activity. Smart T cell is engineered with suicide gene or other synthetic control panels to precisely control therapeutic function over the timing and dosage, there by alleviating cytotoxicity.[32] Several strategies to improve safety and efficacy of CAR-T cells are:

Suicide gene engineering: engineered T cells are incorporated with suicide genes, which can be activated by extracellular molecule and then induce T cell apoptosis. Herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (iCas9) are two types suicide genes have been integrated into CAR-T cells.[32][33][34] In iCas9 system, the suicide gene is composed of the sequence of the mutated FK506-binding protein with high specificity to a small-molecule, AP1903 and a gene encoding human caspase 9 switch. When the release of cytokines by CAR-T cells becomes more pronounced than basic levels, the iCas9 can be dimerized and lead to rapid apoptosis of T cells. Although both suicide genes demonstrate a noticeable function of as a safety switch in clinical trials for cellular therapies, some hinder defects limit the application of this strategy. HSV-TK is derived from virus and may be immunogenic to humans.[32][35] The suicide gene strategies may not act quickly enough to eliminate off-tumor cytotoxicity as well.

Dual-antigen receptor: T cells are engineered to express two tumor-associated antigen receptors at the same time. The dual-antigen receptor of engineered T cell module has been reported to have less intense side effects.[36] The activation of CAR-T cell via TCR-CD3ζ signal transduction pathway is transient and a complementary signal pathway provided by co-stimulatory molecules on antigen presenting cells promotes survival of modified-T cell can ability in controlling tumor.[37] An in vivo study in mice shows the dual-receptor T cells effectively eradicated prostate cancer and achieved complete long-term survival.[38]

ON-switch: ON-switch CAR-T cell split synthetic receptors into two parts: the first part mainly contains an antigen binding domain towards and the other part features two different downstream signaling elements (e.g. CD3ζ and 4-1BB). Upon the presence of an exogenous molecule (rapamycin analogs for example), two physically separated signaling elements fuse together and CAR-T cells exert therapeutic functions.[39] In this mechanism, the engineered T cell shows therapeutic function only in the presence of both tumor antigen and a benign exogenous molecule.

Bifunctional molecules as switches: The bispecific antibodies are developed as an efficacious bridge to target cytotoxic T cells to cancer cells and causes localized T cell activation. In this strategy, the bispecific antibody targets CD3 molecule of T cell and tumor-associated antigen presented on cancer cell surface.[40] The anti-CD20/CD3 bispecific molecule shows high specificity to both malignant B cells and cancer cells in mice.[41] FITC is another bifunctional molecule used in this strategy. FITC can redirect and regulate the activity of the FITC-specific CAR-T cells toward tumor cells with folate receptors.[42]

SMDC adaptor technology[edit]

SMDCs (small molecule drug conjugates) platform in immuno-oncology is a novel (currently experimental) approach that makes possible the engineering of a single universal CAR T cell, which binds 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 controlled by adjusting the administered adaptor molecule dosing. Treatment of antigenically heterogeneous tumors can be achieved by administration of a mixture 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 complete
  • a requirement to generate a different CAR T cell for each unique tumor antigen

may be solved or mitigated using this approach.[43][44][45]

Clinical studies[edit]

The First CAR-T therapy has been approved by FDA is Novartis's tisagenlecleucel, also known as Kymriah.[46] The first launch of Kymriah is in August, 2016. The clinical trial result shows an 83% remission rate of all types of B-cell acute lymphoblastic leukemia after three months post treatment.[46] However, 49% of patients also suffered severe side-effects, such as neurotoxicity and cytokine release syndrome.[46] These side effects have been reported to be responsible for multiple death in late-stage clinical trials with CAR-T therapy. As of August 2017 there were around 200 clinical trials happening globally involving CAR-T cells.[5] Of those trials, around 65% were trials in which haematological malignancies were explored, and 80% of them involved CD19 CAR-T cells targeting the B-cell cancers.[5] Studies had begun by 2016 to explore the viability of other antigens such as CD20.[47]

Early examples[edit]

A list of tumors antigens and CARs in in vitro and in vivo trials

A list of tumors antigens and CARs in in vitro and in vivo trials As of 2012:[48][49]

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 malignancies 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 acetylcholine 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
IL13RA2 Glioma IL-13-CD28-4-1BB-CD3ζ Third
IL13RA2 Glioblastoma IL-13-CD3ζ Second
IL13RA2 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

Armoured CAR-T cells[edit]

CAR-T cells are more effective on liquid tumours and have not shown much promise in treating solid tumours. Ovarian cancer is one of the major killers of women since most cases of ovarian cancer (approximately 70%) are diagnosed at a late stage. Of those diagnosed, approximately 30% are expected to survive for five years. Ovarian cancer is difficult to treat because it is a solid tumour with a microenvironment that suppresses adoptively transferred T-Cells. The hostile microenvironment of the solid tumour is also composed of myeloid-derived suppressor cells (MDSC) and tumour-associated macrophages (TAMs).[50] TAMs and MDSCs promote aspects of tumour growth and development.[51] The tumour microenvironment is also composed of vascular leukocytes (VLC) which promote the progression of the solid tumour.[52] All of these components of the microenvironment of the tumour act to suppress T-Cells.

The armoured CAR-T cell is engineered to secrete potent cytokines such as interleukin 12 (I L-12) as well as expressing tethered or soluble ligands on its membrane to improve the efficacy of the CAR-T cell. The secretion of IL-12 is promising as it is a proinflammatory cytokine known for its ability to improve the cytotoxic capabilities of CD8+ cells, engage and recruit macrophages to prevent the escape of antigen-loss tumour cells.[53] CD19 CAR-T cells secreting IL-12 could eradicate established lymphoma in mice without the need for pre-conditioning through the induction of host immunity.[54] A recent phase II clinical trial was carried out in ovarian cancer patients where they were administered IL-12. This treatment led to stable disease in 50% of the cohort.[55]

See also[edit]

References[edit]

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