Chimeric antigen receptors (CARs) are engineered synthetic receptors that redirect lymphocytes, most commonly T-cells, to recognize and eliminate cells expressing specific target antigens. CAR binding to target antigens on the cell surface is independent of MHC receptors, resulting in vigorous T-cell activation and potent anti-tumor responses. Currently, CAR-T cells have shown significant clinical responses in treating certain subtypes of B-cell leukemia or lymphoma, as well as multiple myeloma. However, there are also many limitations that prevent their effectiveness, such as severe toxic side effects, antigen escape, limited tumor infiltration, and suppression by the tumor microenvironment.

To better understand the current research progress of CAR-T cell therapy, we will discuss three key foundational aspects:

★ CAR structure and design principles

★ Limitations and potential risks of CAR-T cell therapy

★ Innovative strategies to overcome difficulties and improve treatment efficacy

CAR Structure and Design Principles

In this article, we will begin by reviewing the basic principles of CAR structure and design. CAR is a modular synthetic receptor consisting of four main parts: an extracellular target antigen-binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains.

01 Antigen Binding Domain

The antigen binding domain is the part of CAR that confers specificity for the target antigen, and is typically derived from the variable heavy chain (VH) and variable light chain (VL) of antibody, connected by a flexible linker to form a single-chain variable fragment (scFv). This part can directly recognize the antigen on the surface of tumor cells, leading to MHC-independent T cell activation.

In addition to recognizing and binding to the target epitope, scFv has several important features that can affect CAR function. For example, the interaction mode between VH and VL chains and relative position of complementary determining regions can affect the affinity and specificity of CAR for its target epitope. The antigen binding affinity of CARs must be high enough to recognize the antigen on tumor cells, induce CAR signal transduction, and activate T cells, but not too high, as to cause activation-induced cell death and trigger toxicity in CAR-expressing cells. To optimize the binding of CAR to its target antigen, other factors must be considered, including: location of the epitope, density of the target antigen, and avoidance of ligand-independent strong signaling.

02 Hinge Region

The hinge or spacer region is defined as the extracellular structural region that extends from the transmembrane domain to the binding unit (scFv). The hinge provides flexibility to overcome steric hindrance, allowing the antigen binding domain to access the target epitope. Differences in length and composition of hinge can affect the function of CAR-T cells in multiple ways, including binding flexibility, CAR expression, signal transduction, epitope recognition, activation output strength, and epitope recognition. In addition, some studies also demonstrated that the length of the spacer is crucial for providing sufficient cell-to-cell distance to promote immunological synapse formation.

The most commonly used hinge regions are derived from the amino acid sequences of CD8, CD28, IgG1, or IgG4. Among these, IgG-derived spacers can lead to CAR-T cell exhaustion, that reduce their persistence in vivo, since they can interact with Fcγ receptors. Currently, some studies are attempting to fix this problem through mutating IgG Fc receptor binding sites.

03 Transmembrane Domain

The main function of the transmembrane domain is to anchor CAR to the T cell membrane. However, other evidence suggest that it might also related to the function of CAR-T cell. The CAR transmembrane domain can affect the expression and stability of CAR, which plays an important role in signal transduction or synapse formation, as well as the dimerization of endogenous signaling molecules.

Most transmembrane domains are derived from natural proteins, including CD3ζ, CD4, CD8α, or CD28. Since the CD3ζ transmembrane domain mediates CAR dimerization and incorporation into endogenous TCR, the CD3ζ transmembrane domain may facilitate T cell activation; however, it may reduce CAR stability at the same time. In addition, since the transmembrane and hinge regions can affect cytokine production and activation-induced cell death (AICD), CAR-T cells with CD8α transmembrane and hinge domains release less TNF and IFN-γ than those with CD28, and have reduced sensitivity to AICD.

In summary, connecting the proximal intracellular domains to the corresponding transmembrane domains can facilitate proper CAR-T cell signal transduction. Moreover, using CD8α or CD28 transmembrane domains can enhance CAR expression and stability.

04 Intracellular Signaling Domain

The most well-studied component of CAR molecular structure is the co-stimulatory domains.

The first generation of CARs, designed in the late 1990s, included CD3ζ or FcRγ signaling domains, but their durability and persistence in vitro were not ideal, and they showed limited or no therapeutic efficacy in clinical trials.

Based on the understanding that co-stimulatory domains play an important role in persistent therapy, the second generation of CARs with a single co-stimulatory domain, concatenated with CD3ζ intracellular signaling domain, was developed. Currently, two most common co-stimulatory domains of FDA-approved CAR-T therapy products are CD28 and 4-1BB (CD137), which are associated with high patient response rates. Clinically, second-generation CAR-T cells have shown strong therapeutic responses in various hematological malignancies such as chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma. For solid tumors, they have been mainly applied in glioblastoma, advanced sarcoma, liver metastases, ovarian cancer, etc. In addition, there are several alternative co-stimulatory domains could be used.

Presumably, the incomplete activation could be due to co-stimulation through only one domain. Therefore, the third generation of CARs, which includes two co-stimulatory domains concatenated with CD3ζ, was developed. However, preclinical studies on the third-generation CARs have shown different and even contradictory results, thus constraining their translational applications.


Optimization of CAR design and whole process of research and development

CAR molecules are crucial to the effectiveness of CAR-T cell therapy, so understanding the composition and structure of CAR molecules, and choosing appropriate CAR design together play an important role in preclinical studies. By optimizing the affinity, or selecting appropriate co-stimulatory domains, CAR-T cells can persist in the body and maintain killing function, thereby further ensuring the effectiveness and safety of this therapy.

Based on years of research experience in tumor immunology field, Cyagen can provide professional and customized CAR molecule design services for CAR-T/other cell therapy researchers. Furthermore, we can provide comprehensive support for CAR-T/other cell therapy research and development, including antibody development, CAR virus preparation, immune cell preparation and phenotypic analysis, as well as the construction of cell and animal models, in vitro/in vivo pharmacological evaluation, etc. You are welcome to contact us at 800-921-8930 or email to animal-service@cyagen.com for further discussion.


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Reference:

[1] Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 Apr 6;11(4):69. doi: 10.1038/s41408-021-00459-7. PMID: 33824268; PMCID: PMC8024391.

 

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