Nanobody-Drug Conjugates (NDCs)

Nanobodies (Nbs) are heavy-chain-only antibodies derived from the variable region of camelids, with a molecular weight that is only about one-tenth that of heavy-chain antibodies. Nanobodies have strong penetrating ability into solid tumors, high affinity, superior physical and chemical properties, and are not easily denatured in extreme conditions. At the same time, nanobodies can quickly screen and verify antigens, which can speed up the development of new drugs. At present, it has gradually emerged in the development of antibody drugs. Nanobody-drug conjugates (NDCs) developed from nanobodies is a potential optimization direction for improving the efficacy of ADC. BOC Sciences has broad capabilities in the development of NDCs. We have expertise in the design, production and characterization of nanobodies and in conjugating these molecules with a variety of drugs to achieve targeted delivery and enhance therapeutic efficacy.

Why are Nanobodies Useful?

In terms of disease diagnosis and treatment, nanobodies have the advantages of easy penetration and rapid renal clearance. They can be labeled with fluorescent probes, enzyme tracers, biotin and other molecules as tracers. Combining nanobodies with molecular imaging techniques can optimize imaging systems and make them ideal for in vitro and in vivo imaging. In addition, due to the small molecular weight, strong penetrating power, high sensitivity and strong specificity of nanobodies, when they enter the body, they can efficiently penetrate cells to quickly capture antigens and neutralize viruses to achieve therapeutic purposes. Nanobodies have shown excellent application value and prospects in central nervous system diseases, circulatory system diseases, infectious diseases, tumors and inflammatory diseases. Based on the structural characteristics of nanobodies, the advantages of nanobodies are summarized as follows:

Fig. 1. Potential applications of nanobodies in bacterial therapeutics and diagnostics (Biochemical Pharmacology. 2023, 214: 115640).

• Strong specificity, high affinity, and weak immunogenicity to humans: Nanobodies are the smallest unit of heavy chain antibodies that bind to antigens and retain antigen-binding activity; nanobodies are easy to humanize and have low immunogenicity; nanobodies do not have Fc fragments, avoids the complement reaction caused by Fc, and has good biocompatibility.
• Small molecular weight and strong tissue penetration: Nanobodies are approximately 12~15 kDa and have deeper tissue penetration. Nanobodies can penetrate the blood-brain barrier, providing new ideas for the study of brain diseases; highly permeable nanobodies can enter dense tissues, such as tumors; nanobodies are expected to act on intracellular proteins; nanobodies are easily filtered from the glomeruli to achieve faster renal clearance.
• High solubility, strong tolerance, and high stability: Compared with the VH-VL of human antibodies, the hydrophobic amino acids in the FR2 region of the nanobody are mutated into hydrophilic amino acids, which have good solubility and stability, and are not prone to aggregates.
• The structure is simple and suitable for prokaryotic/eukaryotic expression systems: Nanobodies can achieve high-level expression in E. coli and yeast, enabling low-cost industrial production.
• Modifiable: Producing nanobodies through genetic engineering can achieve humanized transformation or the design of multivalent antibodies.

Our NDCs Development Capabilities

In order to improve the efficiency of NDCs, scientists from BOC Sciences usually consider the key points of NDCs development from several factors, such as enhancing the specificity/affinity of Nbs, the selection of conjugated drugs, site-specific conjugation strategies, the location of drugs and the ratio of drugs to Nbs.

• Screening of Cytotoxins

Currently, conjugated drugs used to prepare NDCs are mainly divided into DNA damaging agents and microtubule inhibitors. It is worth mentioning that BOC Sciences has been committed to providing a variety of DNA damaging agents and microtubule inhibitors for research and development purposes for many years. We have strong supply capabilities for these products, focusing on high quality and purity. In addition, our strong R&D team provides custom synthesis and manufacturing services for these products, allowing for tailor-made solutions based on specific research requirements.

• Screening of Linkers

Linker molecular design is a key factor affecting pharmacokinetics/pharmacodynamics. In order to maximize NDC efficiency, the ideal linker should have high stability in human plasma; be self-cleavable in tumor-specific environments; and use hydrophilic linkers to reduce NDCs aggregation. BOC Sciences can provide one-stop linker design and synthesis services for NDC development to ensure optimal conjugation and drug release. Our linkers undergo rigorous characterization and quality control processes to ensure linker quality and purity, providing researchers with reliable and consistent materials for NDC studies.

• Site-Specific Conjugation

Compared with ADC, the simple chemical conjugation strategy of nanobodies and drugs has wider applicability and even surpasses ADC in terms of low production cost and long-term stability. For nanobodies, we generally support lysine conjugation, cysteine conjugation, aspartate conjugation, and glutamate conjugation as chemical conjugation sites to achieve a higher drug to nanobody ratio. In addition, with the progress of protein chemistry in recent years, we also provide homogeneous conjugation support services for drugs and nanobodies, including lysine amide conjugation, insertion of cysteine residues, unnatural amino acids conjugation and enzymatic conjugation.

• Drug to Antibody Ratio

Although theoretically NDC does not show any drug loading advantage over mAbs, the effective accumulation and stability of NDC at the tumor site is better than that of ADC. One study reported that despite the higher drug loading capacity of mAbs compared with Nbs, only 1.56% of ADCs were able to effectively enter the target site. However, the powerful properties of Nbs (high antigen specificity, stability, solubility, and lower immunogenicity) will make them more effective than mAbs in entering target cells. Based on this, BOC Sciences' drug-to-antibody ratio (DAR) and drug distribution analysis services can provide comprehensive support services for NDC. Our DAR services are tailored to meet the unique requirements of each project, ensuring an efficient and precise coupling process. The stability, specificity, and biological activity of the NDCs were thoroughly tested to ensure their suitability for further development and application in targeted drug delivery and therapy.

Advantages of Nanobodies in ADCs

Using nanobodies to develop NDCs has many advantages over traditional ADCs in terms of production cost and specificity, and has a wide range of clinical application scenarios. The main advantages of nanobodies in ADC drugs are:

Fig. 2. Application of nanobody-drug conjugates in fungal treatment (Acta Biomaterialia. 2023, 69: 398-409).

• Higher penetration rate into solid tumors: Through experiments on 3D cell models of tumors, it was found that because nanobodies are very small, the penetration rate into solid tumors is significantly better than that of ADCs.
• Dual-target antibodies are more efficient: Their advantage is that the overall advantages of nanobodies can be grafted onto NDC through two different epitopes, bringing greater benefits in clinical practice.
• Possible site-specific coupling: Since the structure of the nanobody is very simple, it can be easily modified. Cysteine can be added to its C-terminus at a specific site, or a linker can be added to the tail of the nanobody using an enzyme-catalyzed method. Compared with the composite system produced by conventional ADC random coupling, the product of fixed-point coupling is very uniform.
• Microorganisms can be used for expression: NDC drugs can be expressed in yeast instead of CHO. At present, judging from our development experience, the production process of nanobodies expressed in yeast can achieve industrialization.
• The production cost is extremely low and the production cycle is short: Nanobodies are expressed by microorganisms, and it only takes ten days to complete a batch of nanobodies through irrigation and purification, and the production cost is significantly lower than ADC.

Analysis and Characterization

Additionally, BOC Sciences has the ability to thoroughly characterize NDCs, including assessment of their binding affinity, stability, pharmacokinetics, and pharmacodynamics. This allowed us to optimize the conjugation process and ensure the development of efficient and selective NDCs. In addition, we have the ability to perform in vitro analysis and in vivo analysis to evaluate the efficacy and safety of NDCs. This includes cell-based assays, animal models, and pharmacokinetic studies to evaluate the therapeutic potential of NDCs. Overall, BOC Sciences' nanobody-drug conjugate development capabilities enable us to provide comprehensive support for the discovery and development of novel targeted therapeutics, improve efficacy and reduce off-target effects.

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Nanobody-Drug Conjugates (NDCs)

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