Nanobodies: How a 15 kDa Single-Domain Antibody Breaks Through the Limits of Conventional Therapy-Nano-body Support Articles-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Nanobodies: How a 15 kDa Single-Domain Antibody Breaks Through the Limits of Conventional Therapy

In the field of antibody therapeutics, conventional monoclonal antibodies (mAbs) have long held a dominant position. Like precision-guided "biological missiles," they have repeatedly proven their value in the treatment of tumors, autoimmune diseases and infectious diseases. Yet mAbs are not perfect: with a molecular weight of approximately 150 kDa, they face inherent limitations in tissue penetration, solid-tumor penetration and the recognition of certain specialized targets. Nanobodies—"micro-missiles" of only about 15 kDa—have broken through this barrier.

Figure 1. Structural features of conventional IgG antibodies and nanobodies.

What Is a Nanobody?

Nanobodies, formally known as VHH or single-domain antibodies (sdAbs), are derived from a unique class of antibodies that occurs naturally in camelids (alpacas, camels and related species)—the heavy-chain-only antibodies. These antibodies lack light chains, and their antigen-binding capacity is borne entirely by a single heavy-chain variable domain; this domain is the nanobody. Compared with conventional antibodies, nanobodies offer the following advantages:

● Extremely small size. At 15 kDa, the molecular weight is only one tenth that of conventional IgG, which translates into better tissue penetration and faster blood clearance (the latter is both an advantage and a drawback that requires engineering to overcome).

● Exceptional stability. Nanobodies retain conformational stability under high temperature, extreme pH and protease-rich conditions—a property closely linked to their single-domain architecture and to substitutions of hydrophilic amino acids in the FR2 region.

● Longer CDR3 loops. The CDR3 loops of camelid VHHs are significantly longer than those of human VH domains, forming "finger-like extensions" that can reach deep into enzyme active pockets or receptor clefts that conventional antibodies struggle to access.

● Low immunogenicity and ease of humanization. VHH sequences share high homology with human VH genes, so immunogenicity can be further reduced through CDR grafting or resurfacing.

Discovery and Screening of Nanobodies

The discovery strategy determines the upper limits of nanobody diversity and affinity. Current mainstream methods fall into three categories: immune libraries, na?ve libraries and synthetic libraries.

● Immune libraries: the most classic and most efficient strategy. Researchers immunize alpacas or camels with the target antigen; after several weeks, mRNA is extracted from peripheral blood lymphocytes, the VHH gene sequences are amplified by PCR and cloned into phage or yeast display vectors. Because affinity maturation has already occurred in vivo, nanobodies screened from immune libraries typically show high affinity in the nanomolar to picomolar range. However, the limitations of this approach are clear: it requires animal facilities and a long immunization cycle, and it can target only a single antigen.

● Na?ve libraries: derived from non-immunized camelids, these libraries can in principle yield nanobodies against any antigen without animal immunization. However, their initial affinity is usually low, and subsequent affinity maturation steps are often required to reach a therapeutic-grade level.

● Synthetic libraries: by rationally designing amino acid diversity in the CDR loops and grafting this diversity onto an optimized, stable framework, researchers can construct highly controllable nanobody libraries.

Figure 2. Nanobody discovery strategies.

Engineering and Therapeutic Applications of Nanobodies

Because nanobodies are structurally simple, encoded by a single gene and easy to express as fusion proteins, they are readily engineered and modified, and they demonstrate significant advantages in disease treatment.

● Multivalency and multispecificity: two or more nanobodies can be linked through flexible peptide linkers to build bispecific or trispecific molecules. For example, bispecific nanobodies can simultaneously bind an antigen on the surface of tumor cells and CD3 on T cells, thereby redirecting T cells to the tumor site.

● Half-life extension: the blood half-life of natural nanobodies is only a few hours, with extremely rapid renal clearance. Solutions include fusion with an anti-serum albumin nanobody, PEGylation, or fusion with an Fc fragment. For example, after the anti-CTLA-4 nanobody H11 was fused with an anti-serum albumin VHH, its half-life was significantly prolonged and it showed superior antitumor efficacy compared with conventional antibodies in murine models.

● Nanobody–drug conjugates (NDCs): these combine the precise targeting ability of nanobodies with cytotoxic drugs. Compared with conventional antibody–drug conjugates, NDCs offer better tissue penetration, making them particularly suitable for solid tumors. In addition, nanobodies are widely used to engineer the antigen-recognition domains of CAR-T cells, as well as in emerging fields such as targeted protein degradation.

● Diagnostics and imaging: the rapid clearance of nanobodies becomes an advantage here—nanobodies labeled with radionuclides or fluorescent dyes can complete targeted imaging within hours with low background signal, making same-day diagnostic workflows feasible.

Figure 3. Engineering and therapeutic modalities based on nanobodies.

Summary

Although conventional antibodies still dominate mature technology areas such as antibody–drug conjugates, nanobodies have already demonstrated outstanding performance in previously underexplored applications. With the broadening use of nanobodies in next-generation imaging applications and their potential biomedical uses—such as drug conjugation, immunotherapy and drug delivery vehicles—nanobodies will maintain an exceptionally strong trajectory of progress in the coming years.

Figure 4. Workflow of nanobody development based on phage display technology at Tek Biotechnology.

Tek Biotechnology (Tianjin) Co., Ltd. has established a comprehensive nanobody candidate discovery platform built on phage display and yeast display technologies. The company provides high-quality nanobody discovery services based on alpaca, llama and camel, together with downstream antibody expression and validation services (including EC50 assays, BLI/SPR affinity validation, flow cytometry validation and other preliminary antibody developability assessments) and product development services (including ADC drugs, bispecific antibodies, and CAR-T design and construction services), delivering strong technical and data support for the research projects of customers across various sectors.

References

[1] Wang, Y., et al. (2024). Influenza A Virus H7 nanobody recognizes a conserved immunodominant epitope on hemagglutinin head and confers heterosubtypic protection. Nature Communications.

[2] Blueggel M, et al. Small but mighty: the versatility of nanobodies in modern medicine. Nanoscale Horizons. 2025;10(10):2158-2171.

[3] Babamohamadi M, et al. Anti-CTLA-4 nanobody as a promising approach in cancer immunotherapy. Cell Death & Disease. 2024;15:17.

[4] Verma V, et al. Nanoscale warriors against viral invaders: a comprehensive review of Nanobodies as potential antiviral therapeutics. mAbs. 2025;17(1):2486390.

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