The hybridoma methodology for developing monoclonal antibodies has been a lab staple for more than 30 years, and it still works great for immunohistochemistry and various cell biology techniques. During the past decade, however, recombinant technologies for antibody development, combinatorial libraries, and antibody engineering have gained a strong following, especially for high-end antibody applications, including therapeutic drug development.
While recombinant methods such as phage display require fancier footwork, if you?re working with an antigen that is highly toxic or nonimmunogenic, such as some self-tumor or virus antigens, recombinant techniques may be the way to go, according to Louis Weiner at Fox Chase Cancer Center in Philadelphia. ?For many years, scientists tried to get around problems by changing species, and devising novel hybridoma strategies to work something out,? he says. ?But with some highly conserved antigens this turns out to be very difficult with respect to monoclonal antibodies.?
Whether you?re looking...
Hybridomas are hardly an endangered species
First developed in 1975 by Köhler and Milstein,
The importance of choosing the right technique hinges on what exactly you need your antibody to do. If you?re looking for a high yield of antibodies, the hybridoma method may be the right choice. However, some of its drawbacks lie in the long generation time and incomplete epitope identification.
• Straightforward methodology (infect mouse or other animal with antigen, extract B-cells, and clone)
• High yield
• Highly specific antibodies
• Long generation time (5?15 months)
• Not all epitopes are identified
• Not all antigens are solved
• No antibodies for nonimmunogenic or highly toxic antigens
• Functional screens only after clone selection and culture
• Antibodies may need to be humanized
Why recombinant technology is up and coming
Labs looking at structural antibody characteristics or highly targeted binding affinity, especially in drug development, primarily use recombinant antibody methods. Some researchers, including Dennis Burton at the Scripps Research Institute, use combinatorial antibodies to study how viruses such as HIV and herpes interact with the immune system. Others at Scripps, including Carlos Barbas, are looking for combinatorial antibodies that perform a discreet function, or display certain protein folding.
Whether to incorporate recombinant techniques into your lab depends on the end goal. ?If there?s a random reagent we need in the lab and we?re not willing to invest much time in preparing it, we?ll outsource to the core facility to make antibodies,? says Barbas. ?But if you have a protein and want to explore every epitope on that protein for biological function, you might go both ways so that you can get a larger diversity of antibodies against all possible epitopes on the protein of interest.?
• Shorter generation time (from 10 days to 2 months)
• Solves difficult antigens
• Greater epitope diversity
• More control over whole-system/in vitro advantages
• Functional screens can be paired with antibody identification
• Can increase antibody affinity
• Minimizes the use of animals in the lab
• Technically intensive
• Lower antibody yield
• Licensing antibodies and proteins from outsourced companies can be expensive
• Many result in nonspecific binding
From Isolation to Expression
Several different recombinant techniques can be used to develop and refine a particular antibody. While many industry and academic groups have developed their own techniques, phage display dominates the arena (see below). Expression systems in insect and mammalian cells (see next page) are conceptually similar, but other molecular evolution-type programs are being used to refine and isolate antibodies.
Cambridge Antibody Technologies and others using large-scale antibody screening of massive antibody libraries often combine methods, such as phage display and ribosome display, to further refine antibody affinity. As long as interests don?t compete, some labs lend their libraries or engage in collaboration.
However, relatively few antibody libraries are commercially available, because intellectual property rights have encumbered the technology.
In phage display, genes of antibodies, either isolated from immunized individuals, naive individuals, or existing libraries, are inserted into the phage DNA. The portion of the antibody molecule that encodes for the antibody-variable regions and antigen binding, known as the scFvs, is linked to the phage protein coats. The phage infects Escherichia coli, in which the phage single-stranded DNA is replicated and phage particles are assembled and secreted into the culture without bursting the bacterial cell. Phages are incubated with the desired antigen, and those that bind are isolated, expanded, and purified. This aspect makes it easy to screen large numbers of clones.
?With phage display you can make larger libraries than with hybridomas,? says Sachdev Sidhu, in the department of protein engineering at Genentech. ?It is also the dominant technology for naïve selections ? trying to get a first antibody. For that you need large libraries to get a naïve binder.? While you can get large libraries and strong binding affinity between epitope and antigen, some epitopes that don?t bind strongly enough may be washed away. Using phage technology, antibody fragments can be generated for some carbohydrate groups on cell surfaces or tumor cell markers.
This cell-free display technology is based on the formation of a stable antibody-ribosome-mRNA complex. Thus it is similar to phage display in that the antibody protein is physically linked to its encoding sequence. Antibody genes are transcribed, creating a population of mRNA molecules, each encoding for a different antibody gene. The mRNA molecules are then incubated with bacterial ribosomes that translate the mRNA into proteins, but stall when they reach an unnatural base at the 3? end. Each complex displays a different antibody and when run through an affinity column with the desired antigen, some bind and those that don?t are washed away. This type of display is entirely in vitro and creates much larger libraries than other methods, all without cloning.
?In general we use phage and ribosomal display to combine libraries,? says Lutz Germatis, at Cambridge Antibody Technologies. Because they are looking for very specific antibody characteristics, researchers can run both displays to get maximum matching. However, working with RNA can be technically challenging, requiring exact technique.
Yeast display uses a mating factor protein (Aga2p) to display ScFvs on the surface of Saccharomyces cerevisiae. Biotinylated antigens can be used to isolate cells of interest. Moreover, with fluorescently labeled antigens, flow cytometry can give real-time read out of binding and accelerate the refinement of phenotypic traits.
?Half of our projects we do on yeast,? says James Marks, at the University of California, San Francisco. ?With yeast it?s easier to get biochemical information. We prefer it for affinity maturing antibodies, and make a fair number of immune libraries from immunized humans or rodents or other species, and put them straight on yeast.? This process yields many different epitopes with strong expression, but it requires knowledge of yeast genetics and flow cytometry techniques. It also has a lower transformation efficiency than phage and requires a large amount of DNA to create a library. However, according to some experts, yeast display is becoming more established in the field of antibody derivation and design.
Baculovirus has become the most popular of insect cell-expression systems, because it produces large amounts of active proteins; early reports indicated 1?500 mg of recombinant protein per liter of infected insect cells.
Some drawbacks of this method include the need for careful culturing, as the insect cells require much oxygen. In addition, timing is a key component of this system; the expression of the antibody protein is controlled by a late viral promoter and peaks just when the cells are starting to die because of the viral infection. Stable transformation has several alternative forms, including cotransfecting an antibiotic with the promoter. According to a recent review led by John Kappler
Mammalian cell display
Expression in mammalian cells is advantageous because mammalian systems immediately recognize the antibodies. Virus infection and direct incorporation of DNA into the cell are the two methods by which expression occurs. The human 293 cell line and the Chinese Hamster Ovary cell line are commonly used in mammalian cell antibody expression. However, this method requires a bit of genetic engineering, and growing mammalian cells is time consuming, not producing the large yields that may be desired.
References1. G. Köhler and C. Milstein, ?Continuous cultures of fused cells secreting antibody of predefined specificity,? Nature, 256:495, 1975. 2. R. Verma et al., ?Antibody Engineering: comparison of bacterial, yeast, insect and mammalian expression systems,? J Immunol Methods, 216:165?81, 1998. 3. F. Crawford et al., ?Use of baculovirus MHC/peptide display libraries to characterize T-cell receptor ligands,? Immunol Rev, 210:156?70, 2006.
1. C.F. Barbas, III et al., eds. Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001, 736 pages, $135.
A useful manual covering phage display techniques, using antibody libraries, peptide libraries, and other methods of panning for antibodies using phage. Barbas, one of the editors, also teaches a course each fall at Cold Spring Harbor Laboratory. Students get a complete overview of phage display technology and make their own antibody libraries in two weeks.
See www.cshl.edu for more information.
2. R. Kontermann, S. Dubel, eds., Antibody Engineering, Springer Lab Manuals, 2001, 792 pages, $179.
Step-by-step protocols from experts in the field on deriving antibodies from hybridomas and then using the various expression techniques, including phage display, yeast, insect, and mammalian models. Includes multiple sections on purifying antibody fragment samples, affinity maturation, screening, and epitope mapping.
3. S. Sidhu, ed., Phage Display in Biotechnology and Drug Development, CRC Press, 2005, 768 pages, $152.
Covers the use of phage display for many different applications, from epitope mapping to protein analysis. Examines many different phage display techniques, library construction, and antibody analysis.
4. www.antibodyresource.com ? An online clearinghouse for everything from custom antibody suppliers and recombinant equipment suppliers to antibody educational resources and goings-on in the biotech industry.