Generating Ultra-specific Custom Monoclonal Antibodies in Just 8 Weeks

AbD Serotec provides monoclonal antibody development using the HuCAL^® GOLD recombinant antibody library, which offers many unique advantages over animals for monoclonal antibody generation¹. In addition to being fast - the library’s more than 15 billion antibody specificities can be screened in less than 6 weeks - this phage-display-based technology allows the selection process to be modified to target direct generation of exquisitely specific antibodies.

The Key – Guiding the Selection Procedure
Generating a highly specific antibody using animals can be a long process. Since antibody generation inside the animal cannot be controlled, most antibody projects require the use of multiple animals to generate as many different antibodies as possible which must then be laboriously screened to determine their specificity.

With HuCAL^®, more than 15 billion high quality antibody specificities are ready and waiting to be screened against antigens in vitro. This in vitro antibody selection process is the key to the power of this technology, since it can be guided to find the exact specificity needed. Intelligent blocking and subtraction strategies are used to generate epitope-specific antibodies which may be selected to either distinguish between closely-related antigens or to recognize multiple antigens – or antigens may be denatured, captured, or masked to ensure that the selected antibodies recognize the antigen under specific assay conditions.

Exquisite Specificity

Example 1 – modified amino acids
Initially the HuCAL^® library is incubated with the unmodified form of the peptide to remove the antibodies which recognize it. At this time, other related peptides are often added to improve the final specificity. This depleted library is then screened with a peptide displaying the desired modification (for example phosphorylated or oxidated² amino acids) to generate specific antibodies. Once the antibodies have been tested for specificity by QC ELISA against the target and both related and unrelated peptides, they are produced in larger scale ready for use. The entire process takes just 8 weeks.

Example 2 – highly conserved antigens
The blocking/subtraction strategy described above is also ideal for selecting specific antibodies when the antigen is highly conserved, for example a protein that varies only slightly between species, or has a single amino acid mutation. If both the target and the related protein are available, the library can be simply depleted of unwanted specificities. If one or more of the proteins are unavailable, AbD Serotec assists with the identification of suitable regions for fragment expression (see AgX below) or peptide synthesis.

Example 3 – distinguishing the parts from the whole
The generation of antibodies able to recognize a cleavage product but not the full length antigen demonstrates the power of this technology very well. For example, the HuCAL^® library was used to generate antibodies to two cleavage peptides of 10 amino acids each, which should not recognize the 20 amino acid full length peptide. As in the cases above, the undesired specificity – in this case the full length peptide – was used to deplete the library of the unwanted antibody specificities before screening with each of the target peptides.

Example 4 – one epitope, many antigens
Selecting antibodies that bind to a specific pair or group of antigens is just as simple as finding the antibodies that distinguish between them. In this case, the target antigens can be used alternately to screen the library so that only antibodies recognizing both targets are isolated. This is both faster and more efficient that animal-based antibody generation, which usually relies on creating a peptide antigen representing a common protein sequence.

A Choice of Antibody Formats
Antibodies from the HuCAL^® library are recombinant, which means that they can be very easily manipulated in vitro to create the ideal format for a variety of applications. The antibodies are initially screened in the form of monovalent Fab fragments. Once the desired monovalent antibody specificities are isolated, they can be cloned into a variety of antibody expression vectors offering different formats and protein tags. For most applications, AbD Serotec recommends the use of bivalent Fab fragments. These have the advantage of increased avidity due to the presence of two antigen binding sites, yet are considerably smaller than an Ig molecule, so they diffuse more readily and eliminate the cross-reactivity problems sometimes associated with the Fc region.
The antibodies can be tagged with myc, His, Flag or Strep peptide sequences, or fused directly to recombinant enzymes such as alkaline phosphatase.

AgX Antigen Expression Service
HuCAL^® technology requires only about 0.5 mg of protein to generate antibodies, but even that amount is too high for some researchers. If the DNA sequence is available, peptides are often seen as the easiest choice as antigen, however antibodies generated against peptides are often not ideal for use with the entire protein. The use of antigen fragments expressed as fusion proteins is an excellent alternative to peptides, since the fragments are more likely fold and thus present a more complex antigen for antibody selection³. The AgX service from AbD Serotec includes advice on the selection of the fragment, and full service cloning, expression, and purification of the antigen.

The HuCAL Antibody Library
HuCAL^® (Human Combinatorial Antibody Library) is both naïve and synthetic⁴ and was developed by MorphoSys for the generation of therapeutic antibodies against drug targets. The library was constructed in silico based on sequence information obtained by bioinformatics analysis of the human immune system, and is based on a modular system of 49 framework genes and 6 highly variable CDR fragments to create more than 15 billion different specificities expressed in vitro by phage display.

Summary
HuCAL^® technology for rapid generation of highly specific monoclonal antibodies has many advantages over animal-based technologies. Not only is it considerably faster, it also offers direct selection of exquisitely specific antibodies in a variety of formats. The technology is entirely in vitro, so there are no animals involved at any stage of the procedure. To date, AbD Serotec has generated over 4000 unique antibodies to more than 100 different antigens, with a greater than 90% success rate.

References:
1. Antibodies for proteomic research: comparison of traditional immunization with recombinant antibody technology.
Ohara et al, Proteomics, 2006, 6: 2638-2646
2. Establishment of specific antibodies that recognize C106-oxidized DJ-1
Ooe et al, Neurosci Lett, 2006, 404 (1-2): 166-169
3. From EST to IHC: Human antibody pipeline for target research.
Frisch et al, J. Immunol Methods, 2003, 75:203-212
4. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.
Knappik et al, J Mol Biol, 2000, 296:57-86