I'm looking for the production of a monoclonal antibody to block a GPI-linked protein who have demonstrated to block cancer growth inducing apoptosis when we silenced it by siRNA. We have thought to produce such an antibody by an hybridoma tecnique but I've learned that there is another technique useful to my scope that is the phage display technique. I'm not clear what are the main differences between the two methods in terms of abilities and characteristics of the produced antibodies? For the production of a functional antibody to be used in a new immunotherapeutic approach for colon cancer treatment, what is to be preferred and why?
In monoclonal antibody production, you inject an antigen into a mouse and rely on the immune system of the mouse to find the combination of v- j- and d- segments that recombine to form functional antibodies that recognize your antigen. You immortalize the B-cells of the mouse by hybridization with a myeloma cell and start screening until you find a panel of hybridoma cells producing murine antibodies with more or less the desired characteristics. You have very limited influence on epitope selection, crossreactivity and so on, beyond the amount of effort you put into screening, maybe a few dozen to a few hundred clones. It can be difficult to get antibodies to evolutionary highly conserved antibodies and epitopes, in particular antibodies that crossreact with the mouse homolog of your protein. If you when want to develop these antibodies for therapy you will have to humanize them, may have to do some stability engineering and so on.
If you go for an in-vitro selection method such as phage display, you first decide from what kind of library you wish to start: You can generate an scFv (or Fab) library from spleen cells of your immunized mouse, utilizing the pre-amplification of cognate antibody sequence and affinity maturation by the murine immune system or start from a naive human library or a synthetic or semisynthetic human library, e.g. the HuCAL library, going directly for antibodies with a human sequence. In a phage display library, you can typically start from a diversity of 10 to the ninth power, in ribosome display a diversity of 10 to the 13th different antibody sequences can be sampled. In different rounds of selection, you can vary the antigen: If you alternate between panning against the human and the murine homolog, you select for epitopes that are common to the two (good for in-vivo testing with tumor models), if your antigen is member of a family of related proteins, you can specifically select for non-crossreacting antibodies by pre-panning on the other family members.
Starting from an scFv library, you also select for antibody variable domains that are reasonable stable and well folding, as only those sequences that can fold in E.coli will survive the selection procedure. If eventually you want to go towards scFvs as a targeting moiety to deliver a toxic payload to tumor cells, this stability selection will be helpful. A Fab library is less stringent in respect to stability selection and may pick up binders that will need further improvement.
By including error-prone PCR amplification or mutator strains of host bacteria combined with stringent off-rate selection, you can do affinity- maturation in phage display selection.
For further reading on different display technologies, antibody engineering, alternative binder frameworks and library generation, check out the Plückthun homepage: You can find quite a number of reviews and articles on the topic for download here:
http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-2-0
There is some information about this in a review I wrote on developing antibodies for biomarkers. Here's a link https://www.researchgate.net/publication/47395880_Developing_recombinant_antibodies_for_biomarker_detection?ev=prf_pub
Article Developing recombinant antibodies for biomarker detection
In monoclonal antibody production, you inject an antigen into a mouse and rely on the immune system of the mouse to find the combination of v- j- and d- segments that recombine to form functional antibodies that recognize your antigen. You immortalize the B-cells of the mouse by hybridization with a myeloma cell and start screening until you find a panel of hybridoma cells producing murine antibodies with more or less the desired characteristics. You have very limited influence on epitope selection, crossreactivity and so on, beyond the amount of effort you put into screening, maybe a few dozen to a few hundred clones. It can be difficult to get antibodies to evolutionary highly conserved antibodies and epitopes, in particular antibodies that crossreact with the mouse homolog of your protein. If you when want to develop these antibodies for therapy you will have to humanize them, may have to do some stability engineering and so on.
If you go for an in-vitro selection method such as phage display, you first decide from what kind of library you wish to start: You can generate an scFv (or Fab) library from spleen cells of your immunized mouse, utilizing the pre-amplification of cognate antibody sequence and affinity maturation by the murine immune system or start from a naive human library or a synthetic or semisynthetic human library, e.g. the HuCAL library, going directly for antibodies with a human sequence. In a phage display library, you can typically start from a diversity of 10 to the ninth power, in ribosome display a diversity of 10 to the 13th different antibody sequences can be sampled. In different rounds of selection, you can vary the antigen: If you alternate between panning against the human and the murine homolog, you select for epitopes that are common to the two (good for in-vivo testing with tumor models), if your antigen is member of a family of related proteins, you can specifically select for non-crossreacting antibodies by pre-panning on the other family members.
Starting from an scFv library, you also select for antibody variable domains that are reasonable stable and well folding, as only those sequences that can fold in E.coli will survive the selection procedure. If eventually you want to go towards scFvs as a targeting moiety to deliver a toxic payload to tumor cells, this stability selection will be helpful. A Fab library is less stringent in respect to stability selection and may pick up binders that will need further improvement.
By including error-prone PCR amplification or mutator strains of host bacteria combined with stringent off-rate selection, you can do affinity- maturation in phage display selection.
For further reading on different display technologies, antibody engineering, alternative binder frameworks and library generation, check out the Plückthun homepage: You can find quite a number of reviews and articles on the topic for download here:
http://www.bioc.uzh.ch/plueckthun/index.php?pid=3-2-0
Hybridomas are more or less randomly selected from the B cell population. Not all stages in the B cell development a accessible. (see: Jahn S et al.: Development of specific IgG-producing human hybridomas by fusions of lymphocytes from actively immunized persons. Hybridoma. 1989 Oct;8(5):529-33.http://www.ncbi.nlm.nih.gov/pubmed/2680900 ). ... and only a very limited number of B cells is specific for the antigen of choice.
In scFv libraries is the chance much higher to pick an combination of genes coding the Fv wich is antigen specific. First due to the much higher number you can screen and second due to the fact, that you are picking all genes instead of a more or less defined B cell subset.
for my opinion, phage display will show the positive result more than mAb produced from mouse. The both peptide and antibody obtain from phage .. will random bind to the target... but phage look easier and faster than produce from mouse....
Good luck
Only one comment: in hybridoma technology you really produce the binders. With phage display you select a binder (e.g. from naive antibody gene libraries or immune library), but you do not produce the binders. After selection you can produce the binder as scFv or Fab (depending on the library type) in E.coli or as scFv-Fc or IgG in mammalia cells (OK, I'am a nit-picker regarding selection and production ;-)).
To my personal opinion and personal experience ( 20 years in an academic lab), hybridoma technology remains the method of choice in your particular case. Phage-displayed technologies may show an advantage if for some reason immunization would not be feasible. I realize that phage-displayed methods look easier and faster (and fancier) but when you have experience with hybridoma technology and evaluate the result at the very end of your process (i.e. the real application; cfr also remark of Michael). I do dare to compete ;-). In over 95 % of the cases we succeeded in getting high affinity monoclonals representing diverse epitopes within 6-9 months ready for use.
Please note that the majority of therapeutic MA's (approved as well as in the pipeline) have been and are being generated by classical hybridomatechnology (sometimes combined with transgenic mice expressing human antibodies, if intended for use in humans)
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies