Overview of Chimeric Antigen Receptors

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Chimeric antigen receptor (CAR) can combine the extracellular antigen recognition domain from antibodies with the immune cell signaling domain to redirect T cell specificity and induce potent antitumor activity. CAR is an artificial transmembrane receptor that connects the extracellular antigen recognition domain, hinge domain (HD), transmembrane domain (TMD), and intracellular signal transduction domain in series.

Molecular Domains and Functions

CAR is mainly composed of the extracellular single-chain variable fragment (scFv) recognizing tumor antigens, TMD, intracellular immunoreceptor tyrosine-based activation motifs (ITAMs) from CD3 zeta chain (CD3ζ) and costimulatory domain (Fig. 1).

Fig.1 Structural and Functional Basis of CAR

scFv

Although CAR has undergone several generations of changes, the scFv from the antibody has been retained, which shows that scFv is a crucial component of CAR. CAR binds to target cell surface antigens through an scFv recognition domain, initiates and determines the intensity of T cell activation, provides specificity in an MHC-independent manner, and can prevent tumor escape through MHC downregulation. scFv is a fusion protein formed by linking the heavy and light chain variable regions of a monoclonal antibody through a short linker peptide. The stability and solubility of the scFv, as well as the exposure of the scFv's binding epitope on the target cell, can affect the function of the CAR. In general, higher affinity scFvs result in greater antitumor activity but also eliminate more normal cells with lower antigen density. How to reduce the on-target/off-tumor effect of CAR-T while maintaining its killing ability is a hot issue in current research. Altering the affinity of CAR scFv is expected to provide an alternative way to optimize CAR-T to overcome on-target/tumor toxicity.

Transmembrane domain

The transmembrane domain (TMD) connects the extracellular and intracellular signaling domain of the CAR, serving as an anchor to the cell membrane. It is typically derived from transmembrane receptor proteins such as CD19, CD28, CD8α, CD3ζ, among others. The CD28-TMD mediates the transmembrane domain-dependent heterodimer association of CAR with the endogenous CD28 receptor. CARs containing the CD3ζ-TMD can form complexes with endogenous T-cell receptor (TCR), which may optimize T-cell activation. The TMD of inducible co-stimulator (ICOS) from the B7 family enhances the interaction between T cells and antigen-presenting target cells by activating phosphoinositide 3-kinase and enhancing calcium mobilization triggered by the TCR. This co-stimulatory function relies on the unique TMD of ICOS, which promotes association with the tyrosine kinase Lck. Reformulating TMD may be a common strategy for all CAR-T cell therapies and is crucial for reducing tonic signaling and prolonging CAR-T cell persistence.

Intracellular domains

Researchers have been developing innovative methods to modify the intracellular domain of CAR-T cell to improve their efficacy in killing cancer cells while reducing toxicity by improving the co-stimulatory and signal transduction domains. Costimulatory domains are usually derived from the CD28 receptor family (CD28, ICOS) or the tumor necrosis factor receptor family (4-1BB, OX40, CD27). These domains can activate both costimulatory molecules and intracellular signals, allowing T cells can continue to proliferate and release cytokines, enhancing their anti-tumor ability. CAR-T cells with a 4-1BB costimulatory domain persist longer and exhaustion happens more slowly than CAR-T cells with a CD28 costimulatory domain. Combining complementary costimulation domains leads to better performance than using only CD28 and 4-1BB co-stimulatory domains, such as ICOS plus 4-1BB, TLR2 (Toll/Interleukin 1 receptor domain of Toll-like receptor 2) plus CD28, and 4- 1BB plus OX40. However, adding a costimulatory domain alone may cause serious side effects and accelerate the aging of CAR-T cells. The signal transduction domain is usually the TCR/CD3ζ chain or the immunoglobulin Fc receptor FcεRIγ chain, which contains ITAMs and plays the role of T cell signal transduction. TCR signaling responses are initiated, controlled, transmitted, and amplified by direct linkage of intermolecular TCR-pMHC conformation and intramolecular TCR-CD3ζ distance to T cell surface binding. The β and γ subunits of FcεRI contain ITAM domains involved in signal transduction.

Hinge domain

The hinge domain (HD) connects the scFv and the TMD. Most CARs derives their HD from the hinge of IgG or the extracellular region of CD8α/CD28. The length of the HD depends on the location and exposure of the target cell antigen epitope. HD plays a role in controlling the pattern of CAR expression, the efficiency of CAR membrane transport, and the definition of CAR signaling thresholds. CAR structure-activity relationship analysis for HD/TMD can adjust the functional strength of CAR-T cells and the response to antigen density, while retaining the antigen specificity of ARD and the signaling characteristics of the signal transduction domain. Several studies have shown that the length of the hinge region is related to the activation of CAR-T cells. Adjusting the length of the hinge region can keep the CAR-T cells and target cells at an optimal distance, and avoid the action of large phosphatases, which can weaken the CAR signal during the antigen-antibody binding process. The optimal length of the hinge region varies for different antigenic epitopes, and the length of the hinge region may need to be adjusted accordingly when targeting neoantigens. Post-translational modifications of CAR HD affect CAR-T cell activity. Hinge length adjustment provides a programmable strategy for limiting antigen sensitivity in CAR targeting membrane-proximal epitopes. It can be used for CAR optimization and increased tumor selectivity.

Evolution of CAR Design

The first-generation CARs consist of single-chain variable fragments (scFvs) for targeted binding through a spacer domain (mainly IgG1 CH1CH2) linked to the transmembrane and intracellular signaling domains of TCR-derived CD3ζ. The second-generation CARs add a costimulatory domain to the first generation, usually from the CD28 family, to enhance the activity and persistence of CAR-T cells. Second-generation CARs provide complete T-cell activation independent of costimulatory ligands on cognate target cells. The third-generation CARs containing two or more co-stimulatory domains (CD28, 4-1BB, OX-40, et al.) to enhance the activity and persistence of CAR-T cells. The fourth-generation CAR-T cell therapy is based on the principle that checkpoint inhibitor therapy can reverse the exhaustion of CAR-T cells in relapsed patients in the short term and shape the tumor environment by inducing the release of transgenic immune modulators. Fourth-generation CAR-T cells, also known as TRUCK, are engineered to secrete genetically modified cytokines, such as interleukin-12, which are designed to reshape the tumor environment to promote therapeutic success. By delivering chemokines or cytokines to tumor tissue using TRUCKs, an environment favorable to normal cells can be attracted and shaped. In addition to immunomodulators, second-generation CAR-T cells also transduce T-cell engagers and some membrane receptors, which are often referred to as next-generation CAR-T cells. Fourth- and next-generation CAR-T cells fall into two categories: cells that utilize secreted molecules and cells that utilize membrane receptors.

Mechanism of Different CAR Cells

The killing mechanisms of CAR-T cells, CAR-natural killer (NK) cells, and CAR-Macrophages (CAR-M) are shown in Fig. 2. (1) Activated CAR-T cells can specifically recognize tumor-associated antigens (TAAs). The cytotoxic activity of CAR-T cells is mediated by perforin and granzyme granule secretion and by the activation of death receptor pathways such as Fas/Fas-L leading to apoptosis and necrosis of cancer cells. Activated CAR-T cells also secrete interferon-γ and tumor necrosis factor-α, which can promote the antitumor cytotoxic activity of natural killer cells. (2) Activation signals and inhibitory receptors expressed on NK cells regulate the activity of CAR-NK cells. Activated CAR-NK cells secrete the cytotoxic proteins perforin and granzyme B, which act synergistically to induce cancer cell necrosis and apoptosis. The death ligands FasL and TRAIL expressed on NK cells bind to the corresponding receptors on cancer cells and induce apoptosis. Furthermore, CAR-NK cells trigger ADCC by recognizing the CD16 Fc receptor of antibody-opsonized cancer cells. In addition, CAR-NK cells secrete IFN-γ and TNFα, promote their activation and stimulate other T lymphocytes, thereby enhancing the anti-tumor immune response. (3) TAAs bind to the CAR receptor on the surface of CAR-M, generating an activation signal that activates the transcription factor (NF-kB) with tumor phagocytosis and the release of pro-inflammatory cytokines. These cytokines can, in turn, activate T-cell-mediated anti-tumor immune reactions.

Fig. 2 The killing mechanism of different CARs (Maalej, 2023)

Targets of CAR Therapies

CAR molecules specifically target tumor cell surface antigens. Potential targets can include not only proteins but also carbohydrates and glycolipid molecules, and the interaction between the CAR and the target results in the creation of immunological synapses, which serve as the foundation for contact-dependent cytotoxicity. CAR-T cells should target the large majority of tumor cells in order to achieve excellent tumour eradication. The majority of CAR-T treatments now available with positive clinical results satisfy the requirements for high coverage, such as CD19 and BCMA. The following CAR targets have undergone substantial research.

Challenges and Prospects

CAR-T cells represent a breakthrough in personalized cancer therapy. Despite their success in refractory B-cell malignancies, the optimal efficacy of CAR-T cell therapy for many other cancers, especially solid tumors, has yet to be achieved. Several factors such as T cell depletion, lack of CAR-T cell persistence, cytokine-related toxicity, and bottlenecks in autologous product manufacturing, hamper the safety, efficacy, and availability of this approach. CARs have some fundamental limitations, such as the difficulty in tumor infiltration by cytotoxic T cells, insufficient recruitment of T cells to tumor sites due to aberrant chemokines secreted by solid tumor cells and an immunosuppressive tumor microenvironment. Adverse events such as on-target off-tumor toxicity and cytokine release syndrome further suppress the therapeutic index. Additionally, other toxicities induced by CAR-T cells, such as tumor lysate syndrome, neurotoxicity, and cytopenia-related adverse events, are also common limitations of this therapy. The direction of our efforts is finding surrogate immune effector cells that can be engineered with CARs for use as anti-tumor cell immunotherapy.

References

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