Creative Biolabs is a well-recognized leader in single domain antibody (sdAb) library construction and screening services. Our unique services include:

• Construction of immunized sdAb library (camel, llama, alpaca, or even shark repertoire)
• Construction of synthetic sdAb libraries (naïve or camelized human repertoire)
• Screening of single domain antibody libraries
• Large-scale production of recombinant sdAbs

Why are single domain antibodies preferred?

Single domain antibodies represent the smallest antibody that has been proven to have diagnostic and therapeutic usefulness. They are antibody fragments that are engineered from single monomeric variable domains of either camelids' heavy-chain antibody (VHH) or cartilaginous fishes' IgNAR (VNAR). On the other hand, it is also possible to develop single domain antibodies from camelized human antibodies.

In particular, single domain antibodies combine the benefits of conventional antibodies with important features of small molecule drugs. They are able to bind to hidden epitopes that are not accessible to whole antibodies. In addition, in comparison with small molecule drugs that target enzyme active sites and receptor clefts, single domain antibodies have the potential of greater affinity and selectivity, thus promising lower side effects and better efficacy. Furthermore, because of their small size (they are only 1/10 the size of a whole antibody), single domain antibodies can penetrate tissues faster than other antibodies and even break through the blood-brain barrier (BBB), which makes them excellent candidates for central nervous system disease therapies. Single domain antibodies have higher stability during changes in temperature and chemical environments and great potential for gastrointestinal stability and oral availability. Also, thanks to the simple nature of a single-chain peptide, single domain antibodies have exceptional drug format flexibility and allow efficient drug discovery and development.

Fig. 1 Overview of different antibody formats. (Timo W M De Groof, 2019)

Construction of an Immunized Single Domain Antibody Library

In terms of the advanced phage display technology, Creative Biolabs has unparalleled capabilities for the construction of VHH or VNAR based single domain antibody libraries through immunized camel, llama, alpaca, or shark. In comparison with naïve libraries, immune libraries usually produce antibodies of greater affinity, thus avoiding the time-consuming in vitro sdAb affinity maturation effort. Antibodies from naïve libraries frequently require affinity maturation before they are useful due to affinity issues. Furthermore, in comparison with naïve libraries, which usually require a size of 1–10 billion independent clones to be useful, an immune library can be much smaller. Our experience showed that immune libraries with a size of 1–10 million variants could be sufficient to produce excellent antibodies.

Our scientists have extensive experience in cloning the single domain antibody repertoire from immunized plasma cells into our phage display vectors. By reverse transcription and polymerase chain reaction, a library of single domain antibodies containing 10–100 million clones is regularly produced.

Fig. 2 The structure of IgNAR and VNAR. (Nuttall SD, 2012)

Construction of a Synthetic Single Domain Antibody Library

Creative Biolabs also has sufficient expertise in constructing synthetic naïve single domain antibody libraries. By nature, single domain antibodies developed from either VHH or VNAR are more stable and soluble, although their immunogenicity can be higher than single domain antibodies engineered from human antibodies. If the immunogenicity of the single domain antibodies is not a concern, a synthetic VHH or VNAR single domain antibody library can be made by randomizing the CDR1 and CDR3 using trimer codon technology.

In case where the immunogenicity of single domain antibodies is a concern, our services allow the construction of camelized human single domain antibody libraries that are based on the monomerization of human antibody heavy chain framework. The binding region of normal human IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. The monomerization will be accomplished by replacing lipophilic with hydrophilic amino acids at a few proprietary positions. Such synthetic libraries represent a good source of single domain antibodies against self, non-immunogenic, and toxic antigens since the libraries are usually sufficiently vast and diverse.

Screening of a Single Domain Antibody Library

To select interested single domain antibodies with high affinity and specificity, scientists from Creative Biolabs can offer high-quality screening service for phage-displayed sdAb libraries. In particular, we are specialized in isolating single domain antibody inhibitors that can access cavities within molecular targets such as enzyme active sites and receptor clefts. Meanwhile, a method based on heat denaturation of single domain antibodies on the surface of phages has also been developed to select non-aggregating single domain antibodies.

Fig. 3 Procedure of solid phase panning of VHH using phage display. (Dorota Smolarek, 2012)

Single Domain Antibody Production

Creative Biolabs has established novel sdAb production platforms that enable the generation of target-specific single domain antibodies within 8 weeks. We have produced a large number of single domain antibodies with high affinity and specificity against a large number of distinct targets, including cytokines, cell surface receptors, tumor cell markers, viral antigens, and enzymes. Of note, single domain antibodies are perfectly stable polypeptides harboring the full antigen-binding capacity of conventional antibodies. This unique structural and functional property renders these molecules ideal candidates for a new generation of antibody therapeutics.

To meet the special needs of our clients on single domain antibodies, Creative Biolabs has combined our cutting-edge techniques with advanced phage display technology to offer a series of services, including sdAb library construction, screening, and sdAb production. Our scientists are confident in tailoring the most appropriate protocol to develop high-quality single domain antibodies for our clients' research purposes.

Published Data

• Development of Multivalent VHH Antibodies by Alpaca Naïve Phage Display Library

Fig. 4 Biophysics of the VHH antibodies binding to CHIKV E2. (Qianlin Li, 2022)

Chikungunya virus (CHIKV) is a re-emerged mosquito-borne alphavirus that can cause musculoskeletal diseases. High affinity antibodies are needed for the diagnosis and treatment of CHIKV infections. In this study, the researchers used alpaca naïve phage display library to produce 20 VHH antibodies as conjugates of CHIKV E2 glycoprotein. Compared with monovalent antibodies, the multivalent VHH antibodies Nb-2E8 and Nb-3C5 can bind with CHIKV virus E2 glycoprotein with high affinity and specificity. They are potential candidates for diagnostic applications and can better detect CHIKV virions in sera.

FAQ

1. What is a single domain antibody library?

   A single-domain antibody library is a large-scale collection of single-domain antibody variants used to screen the high affinity and specific sdAbs of specific antigens. These libraries are usually constructed by randomly combining different antibody variable (V) gene fragments and can contain millions to billions of different single-domain antibody candidates.

2. How is the single domain antibody library constructed?

   The process of constructing a single-domain antibody library usually involves the following steps. First, the gene sequence of the antibody is extracted from natural sources (such as camels, sharks, or humans) with extensive antigen recognition. Then a large number of random combinations of antibody variable (V) genes are generated by PCR amplification, DNA recombination, and mutation introduction. These V-region gene fragments will be cloned into a suitable phage or yeast display vector to form a large gene pool for screening. Through the display technology, sdAb candidates can be displayed on the cell surface and can be directly screened for specific antigens to identify high affinity and high specificity of sdAbs.

3. What is the screening process for a single domain antibody library?

   Screening of single-domain antibody libraries is usually carried out by phage display or other surface display techniques. First, the phage or yeast pool containing a single-domain antibody library was mixed with the target antigen. These target antigens can be fixed to the solid surface or exist in solution form. Subsequently, the unspecifically bound candidates were removed through a series of washing steps, leaving only the sdAbs with strong interactions with the antigen. Next, the antigen-binding sdAbs are recovered by infecting the host cell (for phage display) or by direct selection (for yeast display). This step needs to be repeated many times to further improve the affinity and specificity of the screened single-domain antibodies. Finally, the selected sdAb candidates will be cloned, expressed, and purified, and their affinity, specificity, and other related characteristics will be evaluated. The successfully identified single domain antibody can then be used for further research or developed as a therapeutic candidate.

4. How to optimize the single domain antibody libraries to improve screening efficiency?

   Increasing the diversity of the library is an important factor in improving the success rate of single-domain antibody screening, which can be achieved by introducing antibody sequences from different species, or by using a variety of random or directed mutation strategies to produce a wider range of amino acid diversity. It is also important to ensure that the sdAbs in the library are folded and displayed correctly. The use of optimized signal sequences and helper proteins can help to improve the correct display of single-domain antibodies on the surface of phage or yeast, thus improving effective binding with antigens. In addition, high-throughput screening can also speed up the process of identifying and isolating sdAbs that highly interact with specific antigens. The quality of the selected single domain antibody can be further improved by setting strict screening conditions, such as using low affinity antigens or screening under competitive conditions.

5. How to evaluate the stability of single-domain antibodies in a single-domain antibody library?

   First of all, the structural stability of single domain antibodies at different temperatures can be evaluated by thermal stability analysis, which is usually carried out by measuring the denaturation temperature (Tm) of sdAbs during heating. The higher the Tm value, the better the thermal stability of the antibody. Secondly, the evaluation of the solubility and activity retention of sdAbs under conditions of extreme pH, high salt concentration, or the presence of a desorption agent is also an important index to judge their stability. These tests help to simulate the extreme conditions that sdAbs may encounter in storage and clinical applications. In addition, long-term storage stability testing, that is, assessing the structural and functional retention of single-domain antibodies under different storage conditions, is also an important aspect of evaluation. Through these comprehensive assessments, we can effectively predict the performance of single-domain antibodies in practical applications.

Resources

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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.