Single Domain Antibody Library Services

Creative Biolabs is the recognized leader in single domain antibody library fields. A wide range of unique services are available, including construction of immunized single domain antibody libraries using llama and camel, construction of synthetic camelised human single domain antibody libraries using DNA synthesis, bio-panning of single domain antibodies libraries and large scale production of recombinant single domain antibodies.

A single domain antibody (sdAb, also called domain antibody or nanobody) is an antibody fragment consisting of a single monomeric variable antibody domain, and lacking the light chain and CH1 domain of the heavy chain. The first single domain antibodies were engineered from heavy chain antibodies found in camelids (dromedaries, camels, llamas and alpacas); these are called VHH fragments. Cartilaginous fishes (for example sharks) also have heavy chain antibodies (IgNAR, immunoglobulin new antigen receptor), from which single domain antibodies called VNAR fragments are obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers by camelising a few key residues.

Single-domain antibodies represent the smallest antibody that was proven of diagnostic and therapeutic usefulness. In particular, they combine the benefits of conventional antibodies with important features of small molecule drugs. Single domain antibodies derived from camelid and fish antibodies are less lipophilic and better soluble in water, owing to their CDR3, which forms an extended loop covering the lipophilic site that normally binds to a light chain. This property permits easier production in bacterial cells compared with scFv and Fab fragments. In addition, single-domain antibodies are able to bind to hidden epitopes that are not accessible to whole antibodies, for example to the active sites of enzymes and receptor clefts. This property has been shown to result from their extended CDR3 loop, which is able to penetrate such sites. Also, 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 effect and better efficacy. Furthermore, because of their small size (they are only 1/10th the size of a whole antibody), single domain antibodies can penetrate tissues faster than other antibodies, and even break through the brain´s blood barrier, which turns them into excellent candidates for central nervous system disease therapies. Single domain antibodies have higher stability to changes in temperature and chemical environments, and have the great potential of gastrointestinal stability and oral availability. Also, due to its simple nature of a single-chain peptide, single domain antibodies have exceptional drug format flexibility and allow efficient drug discovery and development. Of note, single domain antibodies have also been used with industrial purposes in products ranging from personal hygiene (dandruff shampoo and toothpaste) to laundry.

Construction of Immunized single domain antibody library

We have unparallel capabilities for construction of llama and camel single domain antibody libraries that are based on phage display technology. In comparison with naïve libraries, immunized-libraries usually produce antibodies of greater affinity, thus avoiding time-consuming in vitro antibody affinity maturation effort. Antibodies from naïve libraries frequently require affinity maturation before they are useful due to affinity issues. Also, in comparison with naïve libraries, which usually require a size of 1-10 billion independent clones to be useful, an immunized library can be much smaller. Our experience showed that immunized libraries with a size of 1-10 million variants could produce excellent antibodies.

We have extensive experience in cloning the single domain antibody repertoire from plasma cells of immunized llama and camel 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.

Construction of synthetic single domain antibody library

We have extensive experience in constructing synthetic naïve llama and camel single domain antibody libraries. By nature, llama and camel single domain antibodies are the most stable and soluble single domain antibodies, although their immunogenicity can be higher than single domain antibodies derived from human antibodies. If the immunogenicity of the single domain antibodies is not a concern, a synthetic llama or camel single domain antibody library can be made by randomizing the CDR1 and CDR3 using trimer codon technology.

In case the immunogenicity of single domain antibodies is a concern, our services allow construction of camelised human single domain antibody libraries that are based on the framework of human antibody heavy chains. The binding region of common human IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity; monomerization is 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 large and diverse.

Premade single domain antibody library

We have built up a HuSdL® Human Single Domain Antibody Library that allows rapid discovery of large numbers of high-potency camelised human single domain antibodies against any therapeutic targets. In contrast with Nanobody Technology that generates camel-derived nanobodies, our library produces camelised human antibodies that have a human origin, thus the lowest immunogenic potential in humans, especially for long-term and multiple-dose administration. The antibody library was constructed based on camelised human VH3 in FR2. At the same time, the human HCDR3 was elongated and sequence-randomized. The current size of this library is 1.5×10⁹.

This library was preselected based on the thermostability and expressibility [in E. coli.] of the displayed antibodies. Specifically, in the library, the antibody repertoire was heat-treated to remove clones that could not withstand heat-induced aggregation. The genetic codons used to encode the antibody binders are also optimized of bacterial preference.

Screening of single domain antibody library

Our staff scientists have extensive experience in screening phage display single domain antibody 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. Also a method based on heat denaturation of single domain antibodies on the surface of phage was developed to select non-aggregating single domain antibodies.

Engineering and manufacturing of single domain antibody

Once the most potent clones are identified, their DNA sequence is codon-optimized and expressed in either bacteria cells or mammalian cells. We are also able to engineer single domain antibodies in a wide range of formats, including unique multivalent, bivalent and bi-specific molecules.
 

Our single domain antibody library platforms enable the generation of panels of high-affinity single domain antibodies ready for further development in 8 weeks of target access. We have produced a large number of high-affinity, high specificity single domain antibodies again 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 new generation of antibody therapeutics.