Chimeric antigen receptors (CARS)
Websites: ClinicalTrials.gov (lists many active CART trials).
See also Enzymes and CRISP
Introduction to CARs/CARTs and TCRs
A very promising immunotherapy is the use of a chimeric antigen receptor (CAR) on T cells to reprogram the T cells to recognize proteins such as CD19 and CD22 found on cancer cells. This has had considerable success with acute lymphoblastic leukemia (ALL). Patients are infused with their own T cells which have been genetically modified for the CAR. CAR-T cell therapy targetting the B cell antigen CD19 was approved by the FDA for child hood acute lymphoblastic leukimia in 2017 (Hu, “Toward personalized, tumour-speciic, therapeutic vaccines for cancer” 2018). Today, 70-90% of pateints with B-cell acute lymphblastic leukemia (R/R B-ALL) who are treated with CD19 directed CAR T cells can acheive complete remission. (Klichinsky, “A milestone in macrophage-based cancer treatment” BioProcess International, 22(4), April 2024),
Chimeric antigen receptors are protein which graft the specificity of, for example, a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activaiton siganls. The most common form of these moleules are fusions of single-chain variable fragments (scFv) derived from mAbs which recognize a target antigen, fused via a spacer and a trans-membrane domain to a signaling endodomain. Such molecules result in activaiton of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARS have been developed against tumour assoicated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currenlty in clinical trial for the treamtent of various cancers. (Thomas (WO 2016/102965)
T cells require multiple signals for optimal function; CD28 on teh T cell which interacts with B-7 (CD80); CTLA-4 (CD152) with B72 (CD86), TCR with MHC, PD-1 with PD-L1 (B7-H1). (“Next Generation of engineered T cells for immunotherapy” Maus, Cancer Crosslinks, January 18, 2018)
CARs are synthetic receptors that are comprised of extracellular ligand-binding domains fused to intracellular co-stimulatory and activation domains. First-generation CARs lack co-stimulatory signalling domains and have limited efficacy owing to insufficient signalling strenght and durability; thus second-generation and subsequent CAR designs have incorproated one or more co-stimulatory domains to enhance and sustain T cell activaiton signalling. Hou, Nature Reviews, Drug Discovery, 29, July 2021)
CARS are not MHC restricted:
CARs are not MHC restricted but only see surface proteins. (“Next Generation of engineered T cells for immunotherapy” Maus, Cancer Crosslinks, January 18, 2018).
Both chimeric antigen receptor (CAR) and T cell receptor (TCR) engineering can direct T cell cytoxicity towards tumour cells. Conventional CARs derive their antigen-targeting moiety from antibodies and, unlike TCRs, which are restricted to major histocompatibility complex (MHC), recognize peptides in an MHC -independent manner. Although conventional CARS derive their signalling component from the CD3 complex, they have signalling properties distinct from those of TCRs. Despite the fact that the ligand binding comain of CARs frequently has substantially higher binding affinity for its target ligand compared with TCRs, TCRs can respond to a single protien-MHC whereas CARs can require substantially higher anitgen densities to induce a robust T cell response. These unique characteristics of binding a signalling preclude head to head clinical comparisons of T cells engineered with CARs versus TCRs, and the idea choice of receptor may depend on properties of the target antigen, including antigen density and ease of surface presentation. (Hou, Nature Reviews, Drug Discovery, 29, July 2021).
Chimeric antigen receptor (CAR) T cell therapy involves isoalting a patient’s T cells; reprogramming those T cells to produce a specific, targeted receptor (a CAR) on each T cell’s surface; and infusing the patient with the reprogrammed cells. The reprogramming involves introducing genetic material containing a nucleotide sequence encoding for a CAR into the T cell so that the cell produces the CAR on its surface. This CAR allows the T cell to recognize the specific antigen for which it was programmed. U.S. Patent No. 7,446,190 (latter held invalid for lack of written description in Juno therapeutics v. Kite Pharma (Fed. Cir, 2021) was one of the earliest patents on this technology. It claims priority to a provisional applicaiton filed on May 28, 2002 and relates to a nucleic acid polymer encoding a three-part CAR for a T cell.
Structure:
CARs used in two T cell therapies approved to date share a commn basic design. On their extracellular side single-chain variable region termed scFv (which is a fusion of the heavy chain and light chain variable regions of an immunogloublin) recognizes a specific antigen (such as CD19). This scFv is followed by a spacer and transmembrane domain fused to an intracellular co-stimulatory domain, such as CD28 or 4-1 BB, followed by the y-chain or epsilon chian of CD3. Various other modificaitons to the CAR desgin are being investigated to enhance efficacy and/or safety. Kimbrel “Next-generation stem cells — ushering in a new era of cell-based therapies” Nature Reviews: Drug Discovery, 19, July 2020).
1) Intracellular domain: The first portion of the three-part CAR is the intracellular domain of the human CD3 (zeta) chain. It is a signaling domain that, when the T cell binds to an antigen, is actived to create an initial immune response.
First-generation CARs typically had the intracellular domain form the CD#epison chain, which is the primary transmitter of signals from endogenous TCRs. Second generation CARs added intracellular signaling domain from various costimulatoyr protein receptors (e.g., CD28, 41 BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cells. More recent, third-generation CARs combine multiple signlaing domain to further augment potentcy. Chen, (WO/2021/034689)
2) costimulatory region: The second porition is a costimulatory region that includes a specific amino acid sequence that is part of a naturally occurring T cell protein called CD28. When activated, the costimulatory region creates a second signal to augment or prolong the immune response, for example, directing the T cells to multipoly.
3) binding element: the third porition of a CAR is the binding element, which is the portion of teh CAR that determiend what target molecule or antigen the CAR can recognize and bind to. One type of binding element is a single-chain antibody (scFV). A scFV is made by taking two pieces of an antibody, one from the heavy cahin of the antibody’s variable region and one from the light chain of the variable region, and linking them together with a linker sequence.
Engineering Techniques:
The design of the CAR construct is critical to the optimal function and presistence of CAR T cells. Key features including the target binding motif, the linker, the hinge, and the transmembrane and the cytoplasmic signaling domains, can all have a substantial impact on the activation, profieration, target-cell killing and exhaustion of CAR T cells. (Hege, “Context matters in CAR T cell tonic signaling” Nature Medicine, Vo. 27, June 2021).
Chimeric antigen receptors (CARs) are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif; (2) linking/transmembrane motifs and (3) intracellular T-cell signaling motifs. Teh antigen-binding motif of a CAR is commonly fashioned after a single chain fragment variable (scFv), the minimual binding domain of an immunoglobulin (Ig) molecule. Alternate antigen binding motifs, such as receptor ligands (ie, IL-13 has been engineered to bind tumor expressed IL-13 receptor, intact immune receptors , library derived peptides and innate immune system effector molecuels (such as NKG2D) also have been engineered. Alternate cell targets for CAR expression (such as NK or gamma-delta T cells) are also in development. To date the signaling motifs used in CARs always include the CD3-epsilon chain because this core motif is the key signal for T cell activation. (Krueger, WO 2016/201394).
Bispecific CARS (tandem CARS or TanCARs):
Bispecific CARS (tandem CARS or TanCARs) have been developed to target multiple cancer specific markers simultaneously. In a TanCAR, the extracellular domain includes two antigen binding specificities in tandem, joined by a linker. The two binding specificities (scFvs) are thus both linked to a single transmembrane portion; one scFv being juxtaposed to the membrane and the other being in a distal position. For example, Grade (2013, Mol Ther Nucleic Acids 2: e105) describes a TanCAR which includes CD19-specific scFv, followed by a Gly-Ser linker and then a HER2-specific scFv. HER2-scFv was in the jxta-membrane position and the CD19-scFV in the distal position. The Tan CAR was shown to induce dsitinct T cell reactivity against each of the two tumour restricted antigens. Thomas (WO 2016/102965) describes a CAR T cell which expresses two CARs at the cell surface, one specific for CD19 and one specific for CD22. The fact that one CAR binds CD19 and the other binds CD22 is advantageous because some lymphomas and leukaemias become CD19 negative after CD19 targeting so it gives a back-up antigen, should this occur.
Enhancers of CAR-T cells:
CAR-enhancer (CAR-E):
Dana-Farber Research Institute has developed a “CAR-E therapeutic platform” wehre a target antigen is fused with a low-affinity enhancer olecule (“A CAR enhancer increases the activity and persistence of CAR-T cells” Nature Biotechnoloy). For example, a B-cell maturation antgien (BCMA) can be fused with a low-affinity IL-2. The BCMA CAR-E seelctively binds to CAR-T cells, leading to their proliferation, effective clearance of tumor cells and promotion of teh development of memory CAR-T cells with diverse pehnotypes. The BCMA antigen by itself does not affect CAR-T cells, but, together, they ahve a synergy.
Hybrid Receptors
Emerging strategies aim to combine the benefits of MHC independent recognition of antigens by CARS and robust signaling from TCRs. In one strategy, an antibod-TCR (ABTCR) was desigend by fusing a Fab with TCR daomins. In another strategy, TCR fusion constructs (TRuCs) were engineered by fusing an antibody based binding domain to various TCR subunits, which reconstittued complete TCR complexes. A third strategy introduces T cell antigen couplers (TACs) which combine extravellular co-receptor domain. All three CAR-TCR hybrid receptors conferred greater antitumour efficacy than their conventional CAR counterparts in leukaemia, lymphoma and ovarian carcinoma xenograft models, with lower cytokine release levels. (Hou, Nature Reviews, Drug Discovery, 29, July 2021).
Senolytic CAR T cells (Clearing Senescent Cells)
a uPAR-specific CAR comprising an anti-mouse uPAR single-chain variable fragement linked to human CD28 costimulatory and CD3 signalling domains, and transducted human T cells has been shown to efficeinty eliminate uPAR expressing senescent cells. The acculation of senescent cells promotes inflammation and chronic tissue damage, thereby contributing to several ageing assocaited diseases.
CAR NK Therapy
A new type of immunotherapy called CAR NK therapy uses Natural killer cells (NK) cells isolated from umbilical cord blood which have a CAR added to the cells. With the CAR, the cells can recognize an antigen on cancer cells. MD Anderson Cancel cells has been working with this type of therapy.
Although traditional cellular immunothepary strategies are dominated by T cell expressed CARs, NK cells have more recently gained traction as an alternative cell therapy platform. NK cells possess a cytotoxic potential similar to T cells but unlike T cells, NK cells intrinsically lack alloreactivity and do not trigger side effects common to CAR-T adminsitratio such as cytokine release syndrom and immune effecotr cell associated neurotoxicity syndrome. Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)
CAR Macrophages:
Companies: Carisma Therapeutics (oroginally Carma Therapeutics).
Another promising permutation of the CAR concept invovles genetically engineered marcophages. While CART-T has treated leukemia successfully, and the modality is beginning to show promise against some lymphomas and myelomas, solid tumors remain an intractable problem regardelss of indication, target antigen, CAR design and treatment regimen. Tumors devote consdierable energy to recruting macrophages, whereas they generally try to exclude T cells. The reason for this is that T cells can recognize new antigens and thus pose a threat to tumor growth. On the other hand, macrophages can function as immunosuppressive cells when appropriately polarized toa suppressive phenotype, promoting tumor growth and immune escape. The company Carisma provisions the cells that tumors like to recruit with CARs. When a CAR-M binds with a target antgien, the cell is designed to activate as it would during typical antiboy-driven immune resposnes. It will phagocytose the bound cancer cell, induce inflammatory cytokine release, and then present tumor-derived antigens to T cells. The CAR is essentially a sense-and-response system. The receptor binds to a target antigen (eg., HER2) and emits a signal to activate a cascade that leads to further immune cell activaiton and killing of cancer cells. The cells are also polirized to the M1 phenotype so that tumors cannot make them immnosuppressive. (Klichinsky, “A milestone in macrophage-based cancer treatment” BioProcess International, 22(4), April 2024)
Antigens Targeted
BCMA (expressed on MM cells)
EBV Allogeneic CARTS:
HER2 (Human Epidermal Growth Factor 2):
PSCA (Prostate Stem Cell Antigen):
Delta-like ligand 3 (DLL3):
Stull (WO2016138038) discloses chimeric antigen recetpors (CARs) that include a DLL3 binding domain that specifically binds to human DLL3 prtoein which is expressed on tumor initiating cells. When the anti-DLL3 bidning domains are incorproated in a chimeric antigen recetpor expressed on lymphocytes, the resulting DLL3 lymphocytes (e.g., NK cels or T cells) are able to mount an immune response directed to aberrant DLL3 positive cells including cancer stemp cells. The CARS include an extracellular domain that includes a DLL3 binding domain, a transmembrane domain and an intracellular signaling domain that activates certain lymphocytes and generates an immune response directed to DLL3 postive tumor cells.
Applications and Diseases Treated
CARs are synthetic, MHC independent receptors that possess the capability to redirect immune cells such as T cells toward target cells expressing antigens. Given their ability to induce remissionwith otherwise-untreatable tumors, the number of CAR-T cell clinical trials has reisen dramatically over the past decade. Creation of CAR-T cells by gene editing of the TRCR (TRAC) locus is particularly attractive because it allows combination of TCR knockout with high efficiency CAR transgene integration. Ablation of the TCR prevents therapeutic cells form attakcing their host. Reciprocally, effective allogeneic cell therapy in immmuno-oncology requires that the body does not attack the transplanted cells, that is, that the cells persist in the recipient. Adoptively transferred T cells will disply endogenous peptides in class I HLA receptors. Natural human genetic variation within such peptides willl result in donor cells being perceived as foreign. Removal of beta2-microglobulin (B2M), a structural component of all class I HLA complexes, effectively prevents foreign cell antigen display and rejection of donor cells by the adaptive immune system. (Lamothe, “Novel CRISPR-Associated gene-editing systems discovered in metagenomic samples enable efficient and specific genome engeering”, CRISPR Journal, 6(3), 2023)
Large B-cell Lymphoma/ B-cell precursor acute lymphoblastic leukemia (AML/relapsed or refractor mantel cell lymphoma):
–Breyanzi (lisocabtagene maraleucel): is approved for treatment of adults with large C cell lymphoma, including diffuse large B cell lymphoma not otherwise specified (including diffuse large B cell lymphoma arising from indolent lymphoma), high grade B cell lymphoma, primary mediastinal large B cell lymphoma, and follicular lymphoma grade 3B who have refractory disease to first line chemoimmunotherapy or relapse within 12 monhts of the first line chemoimmunotherapy, or refractory disease to frist line chemoimmunotherapy or relpase after first line chemoimmunotherapy and are not eligible for hematopoietic stem cell tranplantation due to comorbidities or age, or relapased or refractory disease after two or mroe lines of systemic therapy.It is also indicated for treatment of adult patients with relpased or refractory chrnoic lymphocytic leukemia or small lymphocytic lymphoma who have received at least two prior lines of therapy, including a Bruton’s tyrosine kinase inhibitor and a B cell lymphoma 2 inhibitor.
–Yescarta (axicabtagene ciloleucel)(Kite, a Gilead Company): is approved for treatment of adults with large C-cell lymphoma that is refractory to first-line chemoimmunotherpay or that relapses within 12 months of the first line chemoimmunotherpy. Also, treatment of adults with relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma not otherwise specified, primary mediatinal large b-cell lympoma, high grade B-cell lymphoma and diffuse alrge B cell lymphoma arising from follicular lymphoma. YESCARTA is a CD19-directed genetically modified autologous T cell immunotherapy.
–Kymriah (tisagenlecleucel):
Kymriah (tisagenlecleucel) is approved for treatments of patients up to 25 years of age with B cell precursor Acute Myeloid Leukemia (AML) that is refractory or in second or later replase; adult pateints with relapsed or refractory (r/r) large B cell lympoma after two or more lines of systemic therapy; including diffuse alrge B cell lymphoma not otherwise specified, high grade B cell lymphoma and diffuse large B cell lympoma arising from follicular lymphoma; and adult pateints with replpsed or refratory follicular lymphoma after two or more lines of systemic therapy.
–Tecartus (brexucabtagene autoleucel) (Kite, a Gilead Company): is approved for adults with replased or refractory mantle cell lymphoma. Also, treatment of adults with relapsed or refractory B cell precurosr acute lymphoblastic leukemia.
Mantle cell lymphoma (MCL) is a rare form of cancerous B-cell non-Hodgkin’s lymphoma which usually occurs in middle or older aged adults.
Metastatic Melonma
Multiple Myeloma (MM)
–Abecma (Idecabtagene vicleucel) (Bristol Myers Squibb and 2seventy bio): is a CAR T cell therapy for the treatment of adult patients with replased or refractory multiple myeloma after two or mroe prior lines of therapy including an immunomodulatory agent, a protease inibitor, and an anti-CD38 monoclonal antibody.
–Carvykti (citacabtagene autoieucel) (Johnson & Johnson): Carvykti is approved for treatment of adult patients with relapsed or refractory multiple myeloma after four or mroe prior lines of therapy, including a protease inhibitor, an immunomodulatory agent, and an anti-CD38 monoclonal antibody. It works by by changing your own T cells to recognize and attack a target on the surface of multiple myeloma cells and certain other healthy cells after a single infusion. The entire process generally takes about 2 to 3 months.
Factors to Consider in Therapy
Target antigen: is a major determinant of safety and efficacy for CART cell therapy. The CAR redirects T cell cytotoxicity towards it cognate antigen, irrespective of the identity of the cell that presents it. Consequently, healthy cells that share target antigen expression are at risk of on-target, off-tumour bystandinger killing. In principle, the ideal tumour antigen should be highly and uniformly expressed on tumour cells but absent on healthy tissue. Somtimes these consequences can be managed. For example, CAR-T cell therapeis targeting the pan B cell marker CD19 have demonstrated impressive clinical resposnes for the treatment of haematological malignancies, but succesful treatment by CD19 CART cells also invariably results in B cell apiasis, which is a predictable consequence of targeting CD19 that can be clinically managed by immunoglobulin transfusion. (Hou, Nature Reviews, Drug Discovery, 29, July 2021)
Cancel cells has been working with this type of therapy.