Insight

As pivotal biological tools, recombinant antibodies have been widely applied in pathogen detection, toxin neutralization, tumor diagnosis and other fields over the past three decades. Since the approval of rituximab, the first monoclonal antibody (mAb) therapeutic drug, antibody engineering has continuously driven innovations in biopharmaceutical therapy. Conventional immunoglobulin G (IgG) adopts a heterotetrameric structure composed of two heavy chains and two light chains. In contrast, nanobodies (Nbs), originally discovered in camels and sharks, break this structural pattern. Devoid of light chains and the CH1 domain of heavy chains, nanobodies are formed by cloned heavy-chain variable regions only, constituting the smallest functional antigen-binding fragments with a molecular weight of 12–15 kDa.

The production of nanobodies generally starts with constructing immune libraries targeting specific antigens, followed by multiple rounds of screening to obtain high-affinity clones. With the launch of caplacizumab, the first marketed nanobody drug, and numerous candidate nanobodies advancing into clinical trials, nanobodies have emerged as a research hotspot for innovative drug development. Nevertheless, efficient large-scale expression of nanobodies remains challenging. In particular, the frequent presence of atypical disulfide bonds in nanobodies complicates protein folding, which has become a major bottleneck restricting industrial-scale production.

Escherichia coli serves as the gold-standard prokaryotic expression platform and a core workhorse for laboratory-scale recombinant protein production. It features a simple genome, convenient genetic manipulation, low cultivation costs and well-established technical systems accumulated through decades of research. However, the risk of endotoxin residue limits its direct application in therapeutic protein manufacturing.

Given that nanobodies naturally lack Fc domains and have no demand for N-linked glycosylation modification, they do not rely on post-translational modification capabilities of eukaryotic expression systems, making E. coli an ideal platform for their large-scale production. It can effectively mitigate adverse impacts of endotoxins on final products while exerting the high-efficiency and cost-effective advantages of prokaryotic expression systems.

Periplasmic expression is currently the predominant approach for nanobody production. Different from the reductive cytoplasmic environment that easily triggers formation of non-functional aggregates, the oxidative periplasmic space of E. coli provides a favorable microenvironment for nanobody expression. This compartment is equipped with an oxidation-reduction system facilitating correct disulfide bond formation and molecular chaperones that assist protein folding.

Nanobody transmembrane translocation is mainly achieved via three pathways: the Sec pathway mediates post-translational transport guided by N-terminal signal peptides such as outer membrane protein A, alkaline phosphatase A and pectate lyase B; the signal recognition particle (SRP) pathway enables co-translational translocation through SRP-ribosome complexes; the twin-arginine translocation pathway is capable of transporting fully folded intact proteins. In the later expression stage, non-lytic release of periplasmic nanobodies can be realized via osmotic shock to enhance outer membrane permeability selectively.

Although this method has achieved nanobody yields of dozens of milligrams per liter in multiple studies, insufficient periplasmic molecular chaperones still lead to low expression levels for most nanobodies. Studies have verified that co-expression of the pTUM4 plasmid carrying four molecular chaperones can boost the yield of nine types of nanobodies by 2–10 folds, yet it shows no obvious improvement on nanobodies with only a single conserved disulfide bond, proving that this strategy improves production efficiency by rectifying abnormal disulfide bond formation. In addition, cytoplasmic aggregation and low transmembrane transport efficiency remain core constraints for high-yield production.

As an alternative to periplasmic expression, cytoplasmic expression has drawn increasing attention in nanobody research in recent years. Despite the reductive cytoplasmic environment tends to induce nanobody aggregation into non-functional inclusion bodies, this expression mode is indispensable for fusion proteins that fail to secrete via the Sec pathway.

Novel dialysis-dilution devices equipped with dynamic urea gradient and rotary mixing systems have been developed to improve inclusion body refolding efficiency, raising the refolding rate from 13% via conventional methods to 85%. Antigen supplementation strategies and three-step cold dialysis protocols are also adopted to optimize refolding processes.

To facilitate intracellular disulfide bond formation, double mutant strains deficient in thioredoxin and glutathione reduction systems have been constructed, including Origami, Rosetta-gami and Shuffle®T7 strains. Among them, Shuffle®T7 can effectively catalyze disulfide bond isomerization via overexpressing periplasmic disulfide bond isomerase DsbC. Multiple researches have confirmed that fusion expression with thioredoxin or co-expression of sulfhydryl oxidase Brv1p can markedly elevate nanobody yields. Nevertheless, the applicability of mutant strains remains controversial.

Culture media exert remarkable influences on expression efficiency; the yield achieved using EnPresso medium is 5–10 times higher than that of LB medium, while its high cost restricts widespread application. Optimized promoter systems such as T7, lacUV5 and pm/xylS, together with innovative purification strategies including GFP fusion tags and SNAP/SORT labeling systems, can enhance soluble expression, though fusion tags may increase downstream purification difficulty. Furthermore, optimization of cultivation temperature, shaking speed, inducer concentration, as well as sequence modification via molecular evolution and site-directed mutagenesis, have all been proven effective in improving nanobody production.

Lactic acid bacteria, well-recognized safe intestinal symbionts without pathogenicity, are widely used for the production and delivery of therapeutic molecules. They can express nanobodies through surface display or secretory systems, yet the expression level is merely 1–3 μg/mL, far lower than that of E. coli. Bifidobacterium longum presents similar low nanobody expression capacity.

In contrast, Bacillus brevis can achieve a high nanobody yield up to 3 g/L in a 3-L fed-batch fermenter. Its prominent advantages including endotoxin-free property, low endogenous protease activity and easy genetic manipulation endow it with great industrialization potential. Besides, psychrophilic marine bacteria are emerging as distinctive research hosts, whose low-temperature adaptability favors correct nanobody folding.

Yeast serves as a vital eukaryotic platform for nanobody production, realizing correct folding and extracellular secretion of functional nanobodies via secretory pathways. Though most nanobodies lack Fc domains and corresponding N-glycosylation sites, approximately 10% of nanobodies are prone to undergo unintended N-linked oligosaccharide modification in Pichia pastoris and Saccharomyces cerevisiae, resulting in abnormal molecular weight elevation.

Relevant studies indicate that N-glycosylation may strengthen the toxin neutralizing activity of nanobodies but impair their antigen-binding affinity; non-native O-glycosylation also carries potential risks, hence precise regulation is required to avoid induced immunogenicity.

Pichia pastoris has become the mainstream yeast expression host by virtue of its high biosafety and high-density fermentation capacity. Its tightly regulated and highly efficient methanol-inducible alcohol oxidase 1 (AOX1) promoter enables robust target protein expression, whereas accompanied N-glycosylation may weaken antigen-binding bioactivity. Saccharomyces cerevisiae achieves higher productivity with nanobody yield exceeding 100 mg/L in shake-flask culture, while it also faces the same glycosylation interference issue. Up to now, few studies have reported significant applications of Kluyveromyces and Hansenula polymorpha in nanobody production. Further exploration of glycosylation regulation strategies is urgently needed to balance expression efficiency and structural-functional integrity of nanobodies.

Filamentous fungi represented by Aspergillus awamori are promising platforms for nanobody production due to their acknowledged safety and powerful secretory capacity. However, endogenous proteases in these strains tend to degrade target nanobodies, and protein secretion efficiency is restricted by endoplasmic reticulum-associated degradation and cell wall adsorption effects.

Ustilago maydis can achieve nanobody expression through unconventional secretory pathways. Although Trichoderma reesei, a classic industrial expression host, has been successfully applied in Fab antibody fragment production, there is still no relevant report on nanobody biosynthesis. Current researches highlight the core roles of fusion tag optimization and secretory pathway regulation in fungal expression systems, and further efforts are required to balance protein stability and process feasibility.

Compared with traditional cultured insect cells, the baculovirus-insect larva expression system shows overwhelming superiorities including higher yield, improved biosafety, simplified production processes and lower costs. Lepidopteran insect hosts are widely utilized for heterologous protein expression, yet their inherent N-glycosylation patterns differ from those of mammalian cells, which may interfere with the biological functions of glycosylated antibody drugs.

Engineered insect cell lines can reconstruct N-linked glycan structures to optimize glycan profiles of expressed products. This technical route validates the great potential of insect larva expression systems in large-scale nanobody manufacturing, and its high-yield and cost-saving characteristics open up new prospects for biopharmaceutical industrialization.

Mammalian cell expression systems remain the gold standard for functional antibody preparation. Chinese hamster ovary (CHO) cells stand out as the dominant platform for therapeutic antibody production owing to rapid proliferation capability, adaptability to serum-free suspension culture and high productivity.

Human embryonic kidney 293 (HEK293) cells with transient and semi-stable expression systems are more suitable for laboratory-scale research, featuring short experimental cycles and simple operation to facilitate rapid parameter evaluation, while their application in nanobody production is rarely documented.

To endow nanobodies with enhanced biological functions, researchers frequently fuse nanobodies with Fc domains derived from human or murine IgG, so as to extend in vivo half-life, activate complement-dependent cytotoxicity and reinforce anti-tumor efficacy. Such fusion proteins demand precise post-translational modification exclusively provided by mammalian cells, making mammalian expression systems the first choice for complex nanobody molecular design. Nevertheless, extremely high production costs severely limit the large-scale popularization of mammalian cell platforms, despite that murine B cells and other mammalian cell lines are also competent for nanobody expression.

Plant expression systems possess unique advantages including convenient genetic transformation, large-scale production potential and high biosafety, and can accomplish accurate post-translational modification at low production costs, emerging as competitive platforms for nanobody biosynthesis.

Agrobacterium-mediated stable nuclear transformation is the mainstream technical approach, by which nanobodies can account for 1% of total soluble plant proteins. Hosts such as Arabidopsis thaliana seeds, rice seeds and transgenic potatoes have attracted extensive attention due to high expression yield and oral delivery potential. It is noteworthy that glycosylation discrepancies between plant and mammalian cells may cause endoplasmic reticulum retention of nanobody-Fc fusion proteins, which can be resolved via glycoengineering modification to optimize product performance.

Distinctively, this system enables oral administration of nanobodies via edible plant tissues, providing an innovative strategy for the development of low-cost biological preparations.

Characterized by small molecular weight, compact hydrophobic core and outstanding thermostability, nanobodies have become research frontiers in therapeutic antibody development. Different from conventional full-length antibodies, the structural features of nanobodies lacking Fc segments and native N-linked glycans greatly simplify the selection of expression hosts.

Among mainstream expression strategies, Escherichia coli systems are preferred for laboratory research due to easy operation and low cost, yet both periplasmic and cytoplasmic expression modes have inherent limitations. In eukaryotic expression systems, Pichia pastoris gains prominent advantages relying on high-density fermentation and low-cost culture media, whereas its inherent N-glycosylation tends to introduce high-mannose glycan structures and raise immunogenic risks, calling for advanced glycosylation regulation technologies for optimization.

Filamentous fungi own mature recombinant protein production experience but involve more complicated manufacturing processes than prokaryotic and yeast systems. Fusion protein engineering can effectively increase nanobody yield and endow molecules with complement-dependent cytotoxicity and tumor targeting ability, while the exorbitant production cost of mammalian cell systems restricts its extensive application. Although insect cell and plant expression systems have respective merits, they still face challenges such as glycosylation heterogeneity and poor economic efficiency.

In summary, the selection of optimal expression hosts should comprehensively balance expression efficiency, post-translational modification requirements and overall cost-benefit ratio according to specific research and industrial demands.