Insight

First discovered in camels in 1993, nanobodies (Nbs) are single-domain antibodies. Distinct from conventional antibodies, they solely consist of antigen-binding heavy chains with low molecular weight. Different from traditional antibodies, camel-derived single-chain antibodies lack light chains and the heavy chain constant region 1 (CH1), retaining only CH2, CH3 and heavy chain variable regions (VHH). Nbs possess excellent application potential in diagnosis and therapeutics. Benefiting from their unique structural characteristics, they feature superior stability, high binding affinity, low immunogenicity, rapid tissue penetration and favorable blood-brain barrier permeability.

With advances in molecular biology technologies, Nbs can be conjugated with fluorescent labels, cysteine tags, gold nanoparticles and radionuclides. Such labeled Nbs serve as ideal imaging probes for diagnosing cancers, cerebral disorders and infectious diseases. Moreover, remarkable progress has been achieved in Nb-derived therapeutics, especially in the clinical treatment of autoimmune diseases.

Owing to unique physicochemical properties, Nbs support diversified delivery strategies. Intravenous injection is the mainstream systemic delivery route, suitable for diseases such as tumors and systemic infections requiring rapid target distribution. Subcutaneous and intramuscular injections are preferred for sustained-release and local administration scenarios. Nbs exhibit strong tolerance to extreme pH environments and proteases, laying a solid foundation for oral delivery. Inhalable administration delivers Nbs directly to lung tissues via atomization devices, and their excellent mechanical stress resistance effectively prevents structural damage during delivery. In addition, local administration enables precise targeting of specific tissues including brain tissues by virtue of the outstanding penetrability of Nbs.

Surface functionalization and carrier integration further enhance delivery efficiency and spatiotemporal controllability of Nbs, facilitating precise treatment of intractable diseases. In general, common delivery forms of Nbs are classified into direct administration, conjugate delivery and gene fusion delivery.

Direct injection is a straightforward delivery approach for Nbs. Optimized delivery strategies and molecular design have substantially improved the therapeutic efficacy of locally administered Nbs against tumors. In addition to exerting the tissue penetration advantage brought by low molecular weight, intratumoral injection maximizes the therapeutic potential of Nbs. For instance, researchers designed collagen-targeted Nb variants based on the collagen-binding extracellular domain of mouse leukocyte-associated immunoglobulin-like receptor-1, which were further fused with immune activator interleukin-2 (IL-2) to construct precisely localized immunotherapeutic complexes. This intratumoral delivery strategy restricts systemic toxicity of IL-2 and potentiates immunomodulatory effects within the tumor microenvironment.

Targeted delivery can be realized via Nb conjugation strategies, which greatly boost targeting efficiency and therapeutic outcomes through precise regulation of molecular density and integration of functional modules. In relevant studies, hepatocyte growth factor receptor-targeted Nbs were conjugated onto the surface of PEGylated liposomes at varying densities. Experimental results demonstrated that adjustment of conjugation density could alter non-specific interactions between nanoparticles and phagocytes in isolated human blood, enabling the balance between targeting specificity and immune clearance risk.

In the field of tumor microenvironment regulation, dual-targeted liposomal systems modified with anti-PD-L1 nanobodies and transferrin receptor-binding peptide T12 were developed for the treatment of brain metastasis of non-small cell lung cancer. Such liposomes are capable of crossing the blood-brain barrier via elevating intracellular reactive oxygen species levels and inhibiting the EGFR/AKT signaling pathway.

Furthermore, natural extracellular vesicles emerge as novel delivery carriers due to superior biocompatibility. Via protein ligase-mediated covalent conjugation of EGFR-targeted peptides or anti-EGFR Nbs onto vesicle surfaces, low-dose and high-efficiency delivery of paclitaxel was achieved in EGFR-positive lung cancer xenograft models. Compared with conventional therapeutic regimens, these nanocarriers possess higher drug loading capacity, as well as optimized cytotoxicity, structural stability and pharmacokinetic properties, providing innovative tools for precision medicine.

Nanobodies have also shown promising prospects in gene fusion modification and gene therapy applications. Optimization of Nbs and self-amplifying RNA vectors contributes to enhanced anti-tumor efficacy. Anti-PD-1 and anti-PD-L1 Nbs can effectively block the binding of corresponding immune checkpoints in human and murine models, and incorporation of dimerization domains can further strengthen their inhibitory activity.

Semliki Forest virus (SFV) particles loaded with dimeric Nbs exert potent anti-tumor effects superior to traditional vectors, which can trigger intratumoral pro-inflammatory responses and promote immune cell infiltration. Local plasmid electroporation can further amplify such anti-tumor efficacy, confirming great clinical translational potential.

Adeno-associated virus (AAV) is non-pathogenic to humans and has been widely applied as a mature gene therapy vector. Currently, genetically modified AAV2 capsid proteins have been applied in Nb-based gene therapy, and high-affinity anti-human CD4 Nbs significantly improve drug targeting accuracy. Besides, adenovirus-delivered nanobodies have also become an important branch of Nb-based cancer gene therapy.

Derived from non-human species, nanobodies contain heterogeneous framework region (FR) sequences, which are likely to be recognized as foreign proteins by the human immune system, triggering anti-drug antibody responses, resulting in reduced therapeutic efficacy and potential safety hazards. Accordingly, Nb humanization is the core priority to ensure clinical safety.

The core principle of Nb humanization is to modify framework regions to mimic the structural characteristics of human antibody variable heavy chains, among which FR2 is the major divergent region between camelid VHH and human VH domains. Common modification strategies involve site-directed mutagenesis of key amino acid residues within FR2. Nevertheless, immunogenicity is also affected by molecular stability and pre-existing anti-drug antibodies apart from heterogeneous sequences.

Humanization modification needs to strike a balance between antigen-binding activity and biophysical properties. Since FR2 is closely involved in antigen recognition and molecular solubility, conventional humanization strategies may easily impair biological functions of Nbs. Two mainstream optimized strategies have been proposed: one is to screen candidate molecules independent of FR2-mediated binding during Nb library construction, which relies on high-throughput screening capacity of diversified antibody libraries; the other is to predict antigen epitopes via high-resolution structural analysis and computational simulation, so as to conduct rational design and reserve key functional residues. Analysis of Nb-antigen complex structures can accurately identify functional binding sites on FR2 and avoid invalid modification during humanization, providing systematic solutions for developing low-immunogenicity and high-activity therapeutic nanobodies.

Apart from humanization-related safety risks, rapid renal clearance leads to massive renal accumulation of Nbs after administration, which further increases renal radiation exposure. Relevant data indicate that less than 10% of injected Nbs remain circulating in the blood one hour after administration. Therefore, molecular modification is urgently required to prolong blood circulation time and mitigate adverse reactions. Co-administration of positively charged amino acids can competitively inhibit renal reabsorption and reduce renal accumulation. In addition, with the rapid development of radionuclide-conjugated Nbs for targeted tumor therapy, more attention should be paid to radioactive hazards, organ toxicity and immune adverse reactions, and targeted protective measures need to be formulated accordingly.

Post-translational modifications including glycosylation, deamidation and oxidation occurring during production and storage severely affect the biological potency and bioavailability of protein therapeutics, which remains a major bottleneck in biopharmaceutical research and development. Although Nb-based therapeutics are also confronted with such challenges, their compact and simple molecular structures endow them with prominent advantages in production optimization and structural analysis compared with monoclonal antibodies.

Most nanobodies are recombinantly expressed in prokaryotic systems, which avoids common enzymatic glycosylation and heterogeneous modifications occurring in eukaryotic expression of traditional antibodies. When eukaryotic expression is required, Nbs lack the classic Fc modification sites of full-length antibodies, hence targeted genetic engineering modification is necessary to ensure standardized post-translational modification. In terms of modification detection, the small molecular weight (approximately 15 kDa) of Nbs enables direct top-down mass spectrometric analysis without preliminary fragmentation or customized pretreatment, greatly simplifying structural identification and modification characterization.

The oligomeric state of clinical Nb drugs is mainly determined by two core indicators: in vivo circulation half-life and antigen-binding kinetics. Rational oligomerization design is commonly adopted to prolong systemic retention time and enhance antigen-binding capacity of Nbs.

Ozoralizumab and Sonelokimab, two clinically approved trimeric nanobody drugs launched in Japan, adopt human serum albumin-binding nanobodies as the core structural domain, which effectively extend in vivo circulation half-life. Caplacizumab approved by the FDA is a homodimer linked via trialanine repeats; it specifically binds to the A1 domain of von Willebrand factor and inhibits platelet aggregation. Such dimeric structures significantly elevate in vivo antigen-binding efficiency compared with monomeric nanobodies.

Nevertheless, oligomerization may reduce the bioavailability of Nbs in certain application scenarios such as solid tumor targeted therapy, indicating that existing approved oligomeric formulation strategies lack universal applicability. In the research and development of heterobivalent nanobodies targeting distinct epitopes of the same antigen, structural parameters including linker length, spatial orientation and assembly mode are critical to pharmaceutical performance. Computational protein modeling combined with molecular docking simulation can facilitate the rational design of oligomeric Nbs and maintain synergistic binding effects among monomeric units.

Nanobodies possess multiple superior properties including miniature molecular size, excellent water solubility, high antigen affinity and specificity, easy heterologous expression and flexible modification potential, and are playing an increasingly important role in the global biopharmaceutical industry. Research institutions and commercial enterprises worldwide are actively promoting the research and industrial transformation of nanobodies. Despite numerous candidates entering clinical trials, the number of commercially available Nb products remains limited.

This paper systematically summarizes the mainstream delivery strategies and core industrialization bottlenecks of nanobodies, clarifying the current development constraints in this field. It is predictable that with continuous technological breakthroughs, the above-mentioned challenges will be gradually resolved. More cost-effective nanobody-based diagnostic reagents and therapeutic drugs will be launched on the market, effectively making up for the functional defects of traditional monoclonal antibodies and broadening the application boundary of antibody drugs.