Nanobodies due to their small size, excellent stability, high affinity, and ease of fabrication, have been widely used as antigen-binding domains in CAR-T therapy. Nanobody-based CAR structures have demonstrated good function at more than ten different tumor-specific targets. The resulting nanobody-based CAR-T or CAR-NK cells have shown anti-tumor effects both in vivo and in vitro. The application of nanobodies in CAR-T therapy has been well-proven from the laboratory to the bedside and shows great potential in forming more challenging advanced CAR-T cells.
Chimeric receptors were first proposed by Zelig Eshhar and Gideon Gross in 1989. They replaced the Vα and Vβ regions of the αβ-T cell receptor (TCR) with the VH and VL regions of an antibody, respectively, to construct an artificial chimeric TCR, which can take the form of VH-Cα/VL-Cβ or VL-Cα/VH-Cβ, as shown in Fig 2. This chimeric TCR can bind to target cells in an MHC-independent manner and activate cells through the TCR mechanism.
Fig 1. Schematic diagram of TCR, chimeric TCR and different generations of scFv as CAR structural domains[2].
Currently used CARs consist of four components: an extracellular antigen recognition domain (including single-chain antibody fragments scFv or nanobodies VHH); structural components such as hinges and transmembrane domains; a co-stimulatory signaling domain that provides for maintaining CAR-T cell effector function; and a CD3ζ activation domain.
First-generation CARs contain only the CD3ζ chain signaling domain. While first-generation CAR-T cells can specifically kill targeted tumor cells in vitro and in mouse models, they exhibit lower cytotoxic activity and shorter in vivo survival due to the scarcity of co-stimulatory receptor ligands such as B7 expressed by tumor cells, and the absence of co-stimulatory receptor domains in first-generation CARs, thus lacking clinical efficacy.
Second-generation CARs insert co-stimulatory domains, such as CD28, 4-1BB, or OX40, between the transmembrane sequence and the ITAM domain of CD3ζ. Second-generation CAR-T cells exhibit optimized T cell activation, increased in vitro antigen-dependent proliferation, enhanced in vivo persistence, and more potent antitumor activity. Second-generation CARs with CD28 or 4-1BB co-stimulatory domains have been well established, and three FDA-approved anti-CD19 CAR-T cells are based on this design.
Third-generation CARs incorporate additional co-stimulatory domains to further enhance T cell activation. Typical intracellular domains consist of CD28/4-1BB/CD3ξ or CD28/OX40/CD3ξ. Clinical trials have shown that CAR-T cells carrying third-generation CARs exhibit stronger expansion and longer survival times compared to second-generation CARs, especially in patients with mild disease and low levels of normal B cells[3].
Fourth-generation CARs add the ability to secrete cytokines or antibodies, enhancing their anti-tumor activity by modulating the tumor immune microenvironment. Fourth-generation CAR-T cells combine the direct anti-tumor function of CAR-T cells with the immunomodulatory ability to release cytokines from the tumor site, avoiding the adverse effects of systemic drug administration on cytokines, and hold promise for targeted therapy of solid tumors.
Fifth-generation CARs are designed based on second-generation CARs, with cytokine receptors added to their intracellular domains.
Fig 2. A brief history of the development of cell immunotherapy from the laboratory to the clinic[2].
scFv, as a CAR, readily aggregates on the cell surface, triggering effector cell activation and cytotoxic signaling cascades, leading to T cell depletion. The main reasons for the high self-aggregation tendency of scFv are the exposed free hydrophobic residues on the heavy chain variable region and the unstable folding of the VH/VL region. In contrast, VHH-based CAR-T cells do not have this risk.
Fig 3. Schematic diagram of scFv aggregation on CAR-T surface[1].
Two mechanisms lead to scFv aggregation on CAR-T surface. The first (left) is called VH-VL mismatch, which is the pairing between the VL domain of one CAR and the VH domain of another CAR. The second (right) is called VH-VH pairing, which is the pairing between the VH domain of one CAR and the VH domain of another CAR.
The linker peptide sequence connecting VH and VL in the scFv structure, as well as the backbone sequence of the murine antibody, poses an immunogenic risk, potentially leading to the production of anti-drug antibodies (ADA) in the body. ADA effects may neutralize CAR-T cell function, causing severe side effects, CAR-T cell loss, and even CAR-T therapy failure. In contrast, VHH does not require a linker peptide, and VHH shares 75-90% sequence similarity with human VH (VH3 gene family), thus the risk of immunogenicity is unlikely to occur.
The structure of scFvs limits the potential for constructing more complex CARs. When constructing bispecific CARs, cross-pairing of VH and VL in two independent scFv molecules can lead to reduced affinity, and the size of the inserted gene in multiple scFvs affects viral packaging efficiency. VHHs are more advantageous for constructing the targeting domain of bispecific CARs than scFvs, which are prone to mismatches, and their transfection and viral packaging efficiencies are also superior to scFVs.
In addition to these advantages, VHHs also have a more favorable structure in terms of antigen epitope binding, solubility, and stability. Due to these properties, VHHs have unique potential in developing various forms of CAR-T.
Fig 4. Comprehensive comparison of scFv and VHH. Image source: https://doi.org/10.1186/s40364-021-00332-6
Fig 5. Schematic diagram of CAR-T design using nanobodies as targeting domains.
BCMA is a transmembrane activator and calcium regulator that plays a crucial role in B cell maturation and differentiation into plasma cells. Due to the high BCMA expression levels in malignant plasma cells, they have become an important target for various cancer immunotherapies. Legend Biotech's Cilta-cel (development code name: LCAR-B38M), approved by the FDA in 2022 for the treatment of adult relapsed/refractory multiple myeloma (MM), is China's first FDA-approved cell therapy product and the world's first approved CAR-T cell therapy using a nanobody as its target domain.
Cilta-cel employs a unique bivalent nanobody design. Clinical data show that, with an average follow-up of 12.4 months, the overall response rate (ORR) is as high as 97%, the sustained response rate (sCR) is as high as 67%, the 12-month progression-free survival rate is 77%, and the 12-month overall survival rate is 89%. The approval of Cilta-cel demonstrates the feasibility of developing and manufacturing bispecific/bivalent CAR-T cells using nanobodies.
Besides the approved Cilta-cel which uses nanobodies as CAR-T targeting domains, current research on nanobodies for CAR-T targeting domains covers multiple popular targets, including VEGFR2, HER2, PSMA, TAG-72, GPC2, CD38, CD33, CD7, MUC1, EGFR, CD20, CD105, PD-L1, and EIIIB.
Fig 6. Using nanobodies as CAR-T targets for targeting structural domains[2].
UniCAR T cells are an improved T cell therapy based on CAR technology. Unlike traditional CAR T cells, UniCAR T cells do not bind directly to tumor cells, but are redirected to specific cell surface antigens by binding to a targeting module. Because nanobodies are small molecules, they can penetrate into tumor tissues more easily and are more flexible in preparation and synthesis, making them a good choice for targeting modules. Research data also show that targeting modules based on bivalent VHH can redirect UniCAR-expressing T cells to low-level cancer cells expressing EGFR antigens[4].
Fig 7. Using scFv-based, monovalent VHH and bivalent VHH-based targeting modules to redirect UniCAR-expressing T cells to target specific target antigens[1].
Currently, several CAR-T drugs have been approved for marketing globally, but they are all for hematologic malignancies. Solid tumors, however, have a large patient population and a more complex tumor microenvironment. Fourth-generation CAR-T therapy promises to enhance anti-tumor activity by modulating the tumor immune microenvironment of solid tumors through autocrine antibodies or cytokines. The most advanced product is the autocrine PD1 nanobody MSLN-CAR T-cell product (BZD1901) independently developed by Shanghai Cell Therapy Group, which received clinical approval in March of this year. This product is administered intravenously. The reinfused CAR-T cells are activated upon recognizing tumor antigens, killing tumor cells through cytotoxicity. Simultaneously, the PD1 nanobody is released, binding to PD1 on the surface of T cells, blocking PD-1/PD-L1 signaling, altering the inhibitory immune microenvironment, and exerting a synergistic effect to kill tumor cells.
Nanobodies, as the extracellular antigen recognition domains of CARs, offer advantages not only in molecular size, stability, and structural flexibility, but more importantly, VHHs can be obtained through mature antibody discovery and screening platforms and further used in the design and development of CAR molecules. Therefore, the discovery of high-quality VHH antibodies is a crucial starting point for VHH-based CAR development.
A complete VHH-based CAR development process typically includes target selection, antibody library screening, candidate VHH acquisition, affinity and specificity evaluation, CAR structure design, and validation of candidate CAR molecules. For different tumor targets, it is necessary to combine factors such as antigen expression levels, extracellular structure, epitope location, and antibody affinity to screen for suitable VHH molecules for CAR applications.
In this process, antibody discovery platforms can provide diverse candidate binding molecules for CAR development. For example, through phage display, yeast display, and other antibody screening technologies, VHH candidate molecules with different binding properties and epitope recognition capabilities can be obtained against the target antigen. Further screening using indicators such as affinity, specificity, stability, and expression performance helps to obtain antigen-binding domains more suitable for CAR construction.
AlpVHHs provides CAR molecule development services, bridging VHH antibody discovery with CAR design. Based on antibody development platforms such as phage display technology platform and yeast display tachnology platform, AlpVHHs can discover and screen VHH/scFv candidate molecules targeting client antigens, and further design CAR molecules based on the binding characteristics of candidate antibodies. For projects with multiple potential antigenic epitopes or requiring enhanced tumor targeting capabilities, multi-epitope or multi-specific CAR molecule design can also be performed based on candidate VHHs, providing a candidate molecule basis for subsequent CAR-T cell research.
Therefore, for researchers and biopharmaceutical companies hoping to develop VHH-based CAR-T, CAR development is not simply about linking a VHH to the CAR backbone, but requires systematic optimization from target and epitope selection, VHH discovery and screening, to CAR structure design and candidate molecule evaluation. Combining a mature VHH discovery platform with CAR molecule development can further improve the efficiency of candidate CAR molecule development and support novel cell therapy strategies such as CAR-T, CAR-NK, and multispecific CAR.
If you are conducting VHH-based CAR-T, CAR-NK, bispecific CAR, or multitarget CAR research, you can learn more about the AlpVHHs CAR Molecular Development Service, which provides support for the development of cell therapy candidates from antigen-binding molecule discovery to CAR molecule design.
Reference
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[2].Bao C, Gao Q, Li LL, Han L, Zhang B, Ding Y, Song Z, Zhang R, Zhang J, Wu XH. The Application of Nanobody in CAR-T Therapy. Biomolecules. 2021 Feb 8;11(2):238. doi: 10.3390/biom11020238.
[3]. Ramos, C.A.; Rouce, R.; Robertson, C.S.; Reyna, A.; Narala, N.; Vyas, G.; Mehta, B.; Zhang, H.; Dakhova, O.; Carrum, G.; et al. In Vivo Fate and Activity of Second- versus Third-Generation CD19-Specifific CAR-T Cells in B Cell Non-Hodgkin’s Lymphomas.Mol. Ther. 2018, 26, 2727–2737.
[4]. Albert S, et al. From mono- to bivalent: improving theranostic properties of target modules for redirection of UniCAR T cells against EGFR-expressing tumor cells in vitro and in vivo. Oncotarget. 2018;9(39):25597–616.
