Chimeric antigen receptor (CAR) T-cell therapy leverages genetically engineered T cells equipped with surface-expressed chimeric antigen receptors to specifically recognize tumor-associated antigens and execute precise cytotoxic elimination of malignant cells. In recent years, CAR-T immunotherapy has achieved remarkable clinical success in the treatment of hematological malignancies and has yielded encouraging preliminary outcomes in select solid tumor types. Despite these advances, conventional CAR architectures remain constrained by two critical clinical bottlenecks, namely off-target cytotoxicity and treatment-related adverse toxicities. As an innovative alternative, nanobody (variable domain of heavy-chain-only antibody, VHH)-engineered CAR constructs have emerged as a transformative strategy, owing to their inherent superior properties including ultra-small molecular size, robust structural stability, low immunogenicity, and high antigen specificity. Currently, nanobody-based CAR-T therapy has evolved into a core frontier of CAR-T technological innovation. This review systematically elaborates the iterative evolution of CAR structural design, interprets the fundamental advantages of nanobodies over single-chain variable fragments (scFvs) as CAR extracellular targeting domains, and comprehensively summarizes the latest research progress of nanobody CAR-T therapy targeting 14 pivotal tumor antigens.
1. Evolution and Structural Optimization of CAR-T Therapy
1.1 Iterative Upgrading of Five Generations of CAR Architectures
The conceptual prototype of CAR-T therapy was proposed in the 1980s. The first-generation CAR design consists solely of the intracellular CD3-ζ signaling domain. Although these constructs mediate antigen-specific anti-tumor cytotoxicity in vitro, they exhibit insufficient in vivo persistence and attenuated tumoricidal activity, resulting in limited clinical therapeutic efficacy.
Second-generation CARs integrate a single co-stimulatory domain (e.g., CD28 or 4-1BB) into the intracellular signaling module, which substantially potentiates T-cell proliferation, survival capacity, and anti-tumor immune responses. This design has since become the mainstream paradigm for clinical CAR-T construction.
Third-generation CARs adopt a tandem dual co-stimulatory domain design, which further amplifies intracellular T-cell activation signaling cascades and enhances immune effector functions.
Fourth-generation CARs, also termed T cells redirected for universal cytokine-mediated killing (TRUCKs), integrate targeted tumor cytotoxicity with localized tumor microenvironment cytokine delivery, thereby remodeling the immunosuppressive tumor microenvironment and augmenting systemic anti-tumor immunomodulatory effects.
Fifth-generation CARs are engineered based on the second-generation framework with an additional cytokine receptor domain, aiming to further prolong T-cell persistence in vivo and sustain long-term anti-tumor functional activity.
1.2 Evolution of Targeting Domains: Paradigm Shift from scFvs to Nanobodies
A canonical CAR structure comprises four modular components: an extracellular antigen-binding domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. Among these, the extracellular targeting domain serves as the core functional module that determines the antigen recognition specificity and targeting accuracy of CAR-T cells. Traditional CAR constructs predominantly utilize scFvs as the antigen-binding moiety. However, scFv fragments are prone to spontaneous self-aggregation, which triggers persistent CAR-T cell exhaustion and elicits undesirable immunogenic responses, severely compromising the stability and clinical efficacy of conventional CAR-T products.
Nanobodies (VHHs), derived from heavy-chain-only antibodies naturally present in camelids and cartilaginous fish, have emerged as an optimal replacement for scFv-based targeting modules. Featuring an ultra-small molecular weight of approximately 15 kDa, superior structural stability, negligible aggregation propensity, and minimal intrinsic immunogenicity, VHHs effectively avoid antigen-independent aberrant activation of effector T cells. These unique structural and functional advantages render nanobodies a superior engineering scaffold for next-generation CAR design and optimization.
2. Research Progress of Nanobody CAR-T Therapy Targeting 14 Tumor Antigens
2.1 Solid Tumor Targets
VEGFR2: Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed on tumor vascular endothelial cells and head and neck squamous cell carcinoma cells. Nanobody-based VEGFR2 CAR-T cells can significantly induce the secretion of effector cytokines IL-2 and IFN-γ, and upregulate T-cell activation biomarkers CD69 and CD25 upon co-culture with target cells, demonstrating prominent potential for targeting tumor angiogenesis and inhibiting tumor vascular proliferation.
HER2: Human epidermal growth factor receptor 2 (HER2) is overexpressed in a broad spectrum of malignancies, with high prevalence in breast cancer. Nanobody-engineered HER2 CAR-T cells exhibit superior proliferative capacity, enhanced effector cytokine secretion, and stronger specific anti-tumor cytotoxicity compared with conventional scFv-based CAR-T cells in vitro.
TAG-72: Tumor-associated glycoprotein 72 (TAG-72) is abundantly overexpressed in multiple solid tumors, including pancreatic, breast, colorectal, and ovarian cancers. Nanobody-based TAG-72 CAR-T cells retain potent and stable anti-tumor activity even under supra-physiological antigen concentration conditions, accompanied by significantly reduced immunogenicity risks.
PSMA: Prostate-specific membrane antigen (PSMA) is a well-validated therapeutic and diagnostic biomarker for prostate cancer. Nanobody-based PSMA CAR-T cells undergo robust antigen-dependent clonal expansion and exert prominent cytokine secretion responses when co-cultured with PSMA-positive LNCaP prostate cancer cells.
GPC2: Glypican-2 (GPC2) is overexpressed in approximately 50% of neuroblastoma cases. Preclinical studies have verified that nanobody-based GPC2 CAR-T cells exert potent tumor-suppressive effects and significantly inhibit malignant proliferation both in vitro and in xenograft animal models.
MUC1: Mucin 1 (MUC1) is abnormally overexpressed in breast cancer. Nanobody-based MUC1 CAR-T cells can specifically recognize and target MUC1-positive breast cancer cells, with robust tumor-killing efficacy fully validated in in vitro cellular assays and in vivo animal models.
EGFR: Epidermal growth factor receptor (EGFR) is highly overexpressed in breast cancer, head and neck cancer, and prostate cancer. Notably, bivalent nanobody-based EGFR-targeted CAR modules exhibit stronger cytotoxicity against tumor cells with low EGFR expression levels, overcoming the targeting insufficiency of traditional CAR constructs against low-antigen tumor clones.
CD105: Endoglin (CD105) is specifically enriched on newly formed tumor vascular endothelial cells. Nanobody-based CD105 CAR-T cells effectively suppress tumor angiogenesis and tumor growth in hepatocellular carcinoma cell models and mouse xenograft models, and markedly prolong the survival time of tumor-bearing mice.
Dual-Targeting PD-L1/EIIIB: Researchers have developed a novel dual-specific nanobody CAR-T system targeting both PD-L1 and fibronectin EIIIB domain. This dual-targeted CAR-T modality exerts potent anti-tumor efficacy both in vitro and in vivo, which effectively retards tumor progression and prolongs the survival of tumor-bearing mice. This dual-targeting strategy not only significantly enhances the therapeutic efficacy of CAR-T against solid tumors but also remodels the immunosuppressive tumor microenvironment and activates local systemic anti-tumor immune responses.
2.2 Hematological Malignancy Targets
CD38: Cluster of differentiation 38 (CD38) is highly and specifically expressed in multiple myeloma cells. Nanobody-based CD38 CAR-T cells elicit robust anti-tumor cytotoxicity in in vitro cellular experiments and mouse models. However, off-target toxicity against normal CD38-positive immune cells remains a key limitation, necessitating further structural optimization to improve therapeutic safety.
CD33: Cluster of differentiation 33 (CD33) is a characteristic surface biomarker highly expressed on acute myeloid leukemia (AML) blasts. Nanobody-based CD33 CAR-T cells can specifically kill multiple AML cell lines and significantly prolong the survival of tumor-bearing animals. Nevertheless, potential on-target/off-tumor toxicity against normal CD34-positive hematopoietic progenitor cells requires further strategic mitigation.
CD7: Cluster of differentiation 7 (CD7) is overexpressed in T-cell acute lymphoblastic leukemia and T-cell lymphoma. Of note, CD7 is also endogenously expressed on conventional T cells, leading to potential CAR-T fratricide and self-depletion. Recent phase I clinical data demonstrate that nanobody-based CD7 CAR-T therapy exhibits favorable safety and tolerability profiles, as well as prominent clinical therapeutic potential.
CD20: Cluster of differentiation 20 (CD20) is a pivotal therapeutic target for B-cell malignancies. Nanobody-based CD20 CAR-T cells exert robust and specific anti-tumor activity both in vitro and in vivo. Furthermore, researchers have constructed dual-specific CD20/HER2 CAR-T cells, which achieve efficient targeted killing of tumor cells co-expressing CD20 and HER2 antigens.
BCMA: B-cell maturation antigen (BCMA) is a validated core therapeutic target selectively overexpressed on malignant plasma cells in multiple myeloma. The novel nanobody-based LCAR-B38M CAR-T product is designed to target two distinct epitopes of BCMA simultaneously. In a phase I clinical trial enrolling 57 patients with relapsed/refractory multiple myeloma, the treatment yielded an overall response rate (ORR) of 88%, with 68% of patients achieving complete response (CR), robustly validating the outstanding clinical translational value of nanobody-based CAR-T therapy in hematological malignancies.
3. Conclusion: A New Era of Clinical Translation for Nanobody CAR-T Therapy
CAR-T immunotherapy has revolutionized the treatment paradigm for refractory and relapsed hematological malignancies. However, the inherent defects of traditional scFv-based CAR structures, including suboptimal efficacy, unsatisfactory safety profiles, and poor adaptability to solid tumor immunosuppressive microenvironments, have severely restricted the broader clinical application of CAR-T therapy. The integration of nanobody technology provides a groundbreaking solution to these core challenges. The superior structural stability, ultra-low immunogenicity, and minimal aggregation tendency of nanobodies fundamentally reduce the risk of CAR-T cell exhaustion and abnormal immune activation, laying a solid foundation for the optimization and breakthrough of next-generation CAR-T therapy.
2022 marked a landmark milestone in this field, as ciltacabtagene autoleucel, the world’s first nanobody-based CAR-T therapeutic product, received U.S. FDA approval for the treatment of relapsed/refractory multiple myeloma. This approval formally signifies the official clinical implementation of nanobody CAR-T technology, opening a new chapter for tumor immunotherapy.
Looking ahead, with the in-depth advancement of innovative strategies including dual-epitope/dual-specific CAR design, modular UniCAR platform construction, and tumor microenvironment-adapted dual-targeting engineering, nanobody-based CAR-T therapy is expected to achieve pivotal breakthroughs in solid tumor treatment, safety optimization, and clinical accessibility. Collectively, these technological advances will further expand the application boundary of CAR-T immunotherapy and bring unprecedented opportunities for precision cancer treatment.
