Insight

With in-depth research on antibodies, nanobodies have emerged as promising therapeutic agents. While traditional monoclonal antibodies have achieved certain success in treating various diseases, their inherent limitations including large molecular weight (approximately 150 kDa), poor tissue permeability, high immunogenicity and complicated production processes have hindered their further clinical application.

Derived from heavy-chain antibodies of camelids such as camels and alpacas, nanobodies consist solely of a single variable heavy domain (VHH) with a molecular weight ranging from 10 to 15 kDa. They possess ultrahigh target affinity and can specifically recognize and bind to target antigens with binding potency comparable to natural antibodies. Benefiting from their miniature size, nanobodies exhibit superior tissue penetration capacity, enabling deep infiltration into solid tumors—a capability hardly achievable by conventional antibodies.

Additionally, nanobodies feature low immunogenicity, which minimizes the risk of in vivo immune responses and substantially reduces adverse therapeutic reactions. Their streamlined production workflow also cuts down manufacturing costs and facilitates large-scale industrial preparation. Owing to the above superior properties, nanobodies stand out as ideal candidates for targeted therapy and offer a novel strategy to address the bottlenecks of oligonucleotide delivery.

Nanobody-oligonucleotide conjugates are chimeric molecules formed by covalently or non-covalently linking nanobodies with therapeutically functional oligonucleotides. Endowed with high affinity, exceptional specificity and potent tissue penetrability, nanobodies act as precise navigation carriers that specifically recognize and bind to antigens on the surface of target cells, thereby accurately delivering conjugated oligonucleotides to designated cells.

Subsequently, oligonucleotides exert powerful gene regulatory functions. Upon intracellular delivery, they interact with endogenous nucleic acid sequences to precisely modulate the expression of specific genes and ultimately achieve therapeutic effects.

Linkers play an indispensable role in conjugate construction, which not only maintains stable conjugation between nanobodies and oligonucleotides but also profoundly affects the pharmacodynamic and pharmacokinetic profiles of final conjugates. Parameters including linker length, chemical composition, hydrophobicity and secondary structure jointly determine the structural integrity and biological functionality of conjugates.

Two major categories of linkers have been developed for diversified application demands: cleavable linkers and non-cleavable linkers. Cleavable linkers can undergo specific cleavage under intracellular microenvironments such as acidic conditions or enzymatic catalysis, releasing free oligonucleotides to execute biological functions. In contrast, non-cleavable linkers ensure robust structural stability and preserve conjugate integrity during systemic circulation.

After binding to cell surface antigens, conjugates are internalized via receptor-mediated endocytosis. Following a series of intracellular trafficking processes, oligonucleotides escape from endosomes into the cytoplasm or nucleus to exert therapeutic effects, such as silencing target gene expression through RNA interference and blocking protein translation via binding to messenger RNA.

Multiple chemical strategies have been established to achieve efficient conjugation between nanobodies and oligonucleotides. Electrostatic interaction serves as a mainstream approach: negatively charged oligonucleotides can spontaneously bind to positively charged regions on nanobody surfaces.

To strengthen binding stability, polycationic proteins or amino acids including protamine and polyarginine are commonly introduced. These substances form ionic bonds with phosphate groups of oligonucleotides while interacting with nanobodies, facilitating conjugate formation and promoting endosomal escape of oligonucleotides into the cytoplasm.

Moreover, native chemical ligation reactions are widely adopted in conjugate synthesis. Typical strategies involve utilizing amine side chains of lysine residues or maleimide-thiol coupling reactions. Oligonucleotides modified with active N-hydroxysuccinimide esters can react with primary amines on nanobody surface lysines to form stable amide bonds. Alternatively, cysteine residues can be introduced into nanobodies via chemical reduction or genetic engineering, which further react with maleimide-functionalized oligonucleotides to generate thioether linkages. Nevertheless, these conventional chemical methods tend to produce heterogeneous conjugate mixtures, compromising product homogeneity.

Biological conjugation techniques are developed to overcome the drawbacks of chemical crosslinking and enhance conjugation specificity and precision. Non-natural reactive groups such as azides can be incorporated into nanobodies through genetic code expansion technology. Subsequent click chemistry reactions between modified nanobodies and functionalized oligonucleotides enable site-specific and high-efficiency conjugation.

Besides, strong non-covalent interaction between biotin and streptavidin as well as enzymatic conjugation reactions are also widely applied in the assembly of nanobody-oligonucleotide conjugates.

Nanobody-oligonucleotide conjugates have become a core research direction for nucleic acid delivery towards non-hepatic cells. By targeting cell-specific surface proteins to induce selective endocytic uptake, these conjugates realize precise intracellular nucleic acid delivery.

Plenty of preclinical studies have verified their therapeutic potential: conjugates composed of EGFR-targeted nanobodies and siRNA can effectively downregulate target mRNA levels in EGFR-overexpressing cell lines; engineered chimeric nanobodies targeting HIV co-receptor CXCR4 are capable of inhibiting HIV infection. These findings shed new light on the treatment of intractable diseases, while multiple obstacles remain to be resolved for widespread clinical popularization, including improving intracellular delivery efficiency and preventing in vivo degradation of oligonucleotides during circulation.

Although relevant research is still in the early translational stage, several clinical trials are currently underway. Dyne Therapeutics has launched clinical trials targeting myotonic dystrophy type 1 and Duchenne muscular dystrophy, and its proprietary conjugates have demonstrated favorable efficacy in early-phase studies, capable of ameliorating muscle functions and clinical symptoms of patients. Avidity Biosciences has also advanced multiple antibody-oligonucleotide pipeline candidates for diverse muscular disorders into clinical development, laying a solid foundation for the clinical transformation of this emerging therapeutic modality.

Nanobody-oligonucleotide conjugates exhibit prominent advantages in in vitro detection and clinical diagnosis. Oligonucleotides can be rationally designed with target-complementary sequences, which generate specific detectable signals upon target binding and effectively reduce false-positive results.

In pathogen detection, conjugates assembled by pathogen-specific nanobodies and aptamers achieve detection with high sensitivity and specificity. High-affinity aptamers screened against Acinetobacter baumannii and Vibrio cholerae have been conjugated with corresponding nanobodies, enabling accurate isolation and identification of pathogenic bacteria from clinical specimens via sandwich enzyme-linked aptamer assay.

In super-resolution bioimaging, the DNA-PAINT technology achieves high-resolution cellular imaging relying on nanobody-oligonucleotide conjugates. Nanobodies are conjugated with short docking oligonucleotides to bind to intracellular targets, followed by the introduction of fluorescently labeled imaging strands. Repeated hybridization and dissociation between imaging strands and docking strands generate continuous fluorescent signals, which are further processed to reconstruct high-resolution spatial images of target molecules. This technology supports high-precision visualization of intracellular organelles including mitochondria, chromatin and Golgi apparatus. Furthermore, technologies such as immuno-PCR and proximity extension assay also utilize nanobody-oligonucleotide conjugates to accomplish ultrasensitive antigen detection and protein quantitative analysis.

Nanobody-oligonucleotide conjugates bring unprecedented development opportunities to the biomedical industry. In the future, this versatile platform is expected to facilitate the development of novel vaccines by delivering specific antigens to immune cells to initiate targeted immune responses, serve as high-performance biosensors for biomarker detection in biological samples, and accelerate the development of personalized gene therapies tailored to individual genetic mutations in precision medicine.

Nevertheless, this cutting-edge technology is still in its infancy and confronts multiple challenges. In terms of conjugate design, it is critical to construct stable linkages without impairing the inherent biological functions of nanobodies and oligonucleotides, and further improve conjugation efficiency and site specificity. Ideal linkers are required to maintain high stability in systemic blood circulation while achieving efficient intracellular cleavage to release functional oligonucleotides.

In terms of targeting performance, despite the excellent specificity of nanobodies, antigen expression heterogeneity may still lead to off-target binding, causing toxic effects on healthy tissues and unintended immune system activation. Additionally, enhancing endosomal escape efficiency to deliver oligonucleotides to intracellular action sites remains a core technical bottleneck.

With the continuous iteration and optimization of related technologies, the above technical hurdles will be gradually overcome. It is foreseeable that nanobody-oligonucleotide conjugates will smoothly advance from laboratory research to clinical practical application, providing more efficient and precise therapeutic regimens for patients, and ushering in more innovative breakthroughs in the field of targeted biomedicine.