Nanobodies (Nbs) were first discovered in camelid blood by Belgian scientists Hamers and colleagues. Unlike conventional antibodies, these unique antibodies lack light chains and consist solely of a heavy-chain antibody (HcAb) with two constant domains (CH2 and CH3). With a molecular weight of approximately 15 kDa, they are aptly named nanobodies.The antigen-binding specificity of HcAbs relies exclusively on their variable heavy-heavy domain (VHH). The recombinant form of this VHH domain is referred to as a single-domain antibody (sdAb) or a nanobody fragment.

I. Advantages of Nanobodies Over Conventional Antibodies：

(1) Superior target-binding specificity;

(2) Enhanced tissue penetration capability;

(3) Greater stability (e.g., heat resistance);

(4) Suitable for large-scale industrial production;

(5) Easier modification and optimization;

(6) More straightforward humanization process.

II. Preparation Methods of Nanobodies

Conventional monoclonal antibodies are typically produced using the hybridoma method, whereas nanobodies are predominantly generated through phage display technology.

Phage display technology (i.e., nanobody technology) involves inserting the DNA sequence of an exogenous protein or peptide into an appropriate location within the P3 protein gene sequence of the M13 phage. This enables the expression of the exogenous gene alongside the phage coat protein, resulting in its display on the phage surface. The advantage of phage display lies in the diversity of variant antibody genes within the nanobody library. Each recombinant phage (commonly the M13 phage) displays different antigen-binding domains on its surface. Compared to the traditional hybridoma method, phage display technology offers significant efficiency advantages in terms of antibody gene display and screening. For instance, the fusion efficiency of hybridomas is generally<0.4%, 1="" 4="" meaning="" only="" about="" fusion="" events="" occur="" per="">

III. Nanobody Production Workflow

The production process of nanobodies mainly consists of four steps: alpaca immunization, phage library construction, antibody library screening, and antibody expression with functional validation.

To establish an immune library, the first step is to immunize young, healthy camelids such as llamas, dromedaries, or alpacas. Typically, over a span of 2 months, the animals receive 4-8 injections of a mixture containing the target antigen and adjuvant. Generally, each injection contains approximately 50-200 μg of immunogen; the exact amount depends primarily on the antigen's molecular weight, but more critically on its immunogenicity and/or toxicity.

Types of immunogens: The preferred recommendation is to use soluble, correctly folded recombinant proteins, though direct DNA immunization is also employed. Typically, up to 10 proteins are mixed to immunize the animals, but more complex mixtures have been used as well (e.g., viral, bacterial, or parasitic proteins, or protein extracts from whole mouse spleen cells or cancer cells).

To increase the likelihood of successfully obtaining nanobodies against specific epitopes, it is advisable to immunize more than one animal. These animals are inbred, and each will generate a unique immune response, yielding a larger panel of nanobodies from which the best-performing ones can be selected.

Some studies have shown that dromedary and Bactrian camels produce a higher proportion of HCAb antibodies compared to classical antibodies than llamas and alpacas. After immunization, 50–100 mL of anticoagulated blood is collected (typically from the jugular vein, though lymph node biopsies also serve as excellent source material) to prepare lymphocytes and extract mRNA. The mRNA is then reverse-transcribed into cDNA, which is used to amplify VHH genes via a two-step nested PCR.

Certain molecules, such as RNA or DNA, lack immunogenicity and cannot stimulate HCAb production. Additionally, some compounds may be too toxic, highly infectious, or hazardous to the animals or the environment. In such cases, constructing a naïve nanobody library or a synthetic nanobody library can be considered.

To successfully screen for high-affinity nanobodies, a naïve antibody library should generally reach a size of 10⁹–10¹⁰, with approximately 80% of the sequences encoding nanobodies. To build such a large and diverse naïve nanobody library, blood must be collected from multiple animals (at least 10 to avoid bias in HCAb production due to allergies or prior infections). Given that each mL of blood contains ~10⁶ lymphocytes—only a fraction of which are B cells, and roughly 50% of those may express HCAbs—around 10 liters of blood are required to construct a library of 10¹⁰ distinct VHH clones.

KMD Bioscience has established a comprehensive and well-developed phage display antibody technology platform. Based on this phage display platform, KMD Bioscience offers key experimental services including antigen design, alpaca immunization, library construction and screening, as well as functional activity validation, providing global scientists with highly specific and high-affinity alpaca VHH antibodies.Additionally, KMD Bioscience possesses extensive experience in antibody engineering and provides end-to-end upstream and downstream antibody services. These include antibody humanization services, human scFv antibody library construction, human Fab antibody library construction, human antibody phage library preparation, customized phospho-specific antibody development, and antibody affinity maturation services, catering to diverse research needs of clients worldwide.

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