Updated: February 8, 2021
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Overview I Customized sABs I Guidelines and considerations I FAQs I References
Overview
It is well known that the majority of monoclonal antibodies are expensive, have very limited applications and frequently do not work effectively. Fortunately, due to pioneering work by a Chicago-led team, a major effort is underway to economically produce the next generation of affinity reagents. The core element of the established technology platform is a high throughput pipeline to generate customized synthetic antibodies (sABs) using novel phage display libraries and selection strategies. Their attributes provide for the ability to generate sABs that not only have high affinity and specificity to their target antigen, but also can be engineered to: 1) target specific regions on the surface of the antigen, 2) recognize specific conformational or oligomeric states, 3) induce conformational changes, and 4) capture and stabilize multi-protein complexes. Additionally, they are recombinant proteins, whose DNA can be archived and stored. These expanded functions provide the tools to investigate systems at a level that cannot be even contemplated using traditional monoclonal approaches (click here for references).
Due to a CBC Lever Award, CBC researchers have special access to this powerful technology.
The Center for Production of Affinity Reagents for Human Transcription Factors: Chicago Synthetic Antibody Pipeline (CSAP) was established in May 2012 with a CBC Lever Award. The principal investigators on the Lever Award are Anthony Kossiakoff and Geoffrey Greene (UChicago), Brian Kay (UIC) and Jason Brickner (NU). The CSAP headquarters are at the University of Chicago. The CBC Lever was awarded in conjunction with a NIH U54 grant, titled “Recombinant Antibody Network (RAN),” which, in addition to Chicago participants, also involves scientists from the University of California, San Francisco and the University of Toronto. The goal of the NIH grant is to generate renewable, high quality affinity reagents against all human transcription factors. The Lever Award enables use of the NIH RAN infrastructure to generate affinity reagents for the CBC community against a variety of targets, including membrane and soluble proteins, protein complexes and functional RNA. Investigators will also have access to the synthetic antibodies that have already been (or will be) generated from the NIH project. These targets include human transcription factors and epigenetic regulatory proteins.
CBC researchers can benefit from the CSAC by:
- Production of customized sABs
A high-throughput sAB generation system is now operational for human transcription factors and epigenetic regulation proteins. Investigators (or groups of investigators) can nominate protein antigens as targets for sAB generation using existing technology.
Additional information
- Northwestern Recombinant Protein Production Core (rPPC)
Researchers can request support from the Northwestern rPPC. The Core will help design constructs and provide expressed and purified material. The Core will also provide limited amounts of the sABs that have been generated and validated in the pipeline. Some of the costs will be defrayed. - For general information on the methods used to produce sABs, visit the RAN website.
- For general guidelines and considerations for proposed customized sAB generation, click here.
- For Frequently Asked Questions, click here.
Production of customized sABs
Single or groups of investigators can nominate protein antigens for sAB generation using CSAP technology. The protein production facility at Northwestern, the Recombinant Protein Production Core (rPPC), can be used for antigen and sAB production at subsidized costs. Candidate protein antigens should fit within the existing pipeline methodology.
For specific information on the quantity and qualities of protein antigens that are appropriate for sAB generation see the FAQ section. With questions, contact Elena Davydova (edavydov@uchicago.edu, 773-834-5327).
Investigators should prepare a one-page proposal containing a description of the project and justification of how access to high-performance affinity reagents will enable their research.
Applications should be submitted via email to Elena Davydova (edavydov@uchicago.edu).
The application should be assembled into ONE PDF DOCUMENT including: 1) a cover sheet listing the protein antigen’s name and the names(s) and contact information of the investigator(s) and 2) the one-page proposal. The pdf file must be named with the protein antigen’s name and the principal investigator(s) last name(s).
Projects will be accepted based on ‘do-ability’ using the existing technology.
Guidelines and considerations for sAB generation pipeline
- The amount of the target protein antigen needed will vary depending on its size. A general rule is that about 0.5 mgs of purified antigen will be required for selections and primary validation.
- Research applications should involve the folded form of the antigen: full length proteins or stable domains (no peptides). sABs will not be generated for simply western blot analysis as an end product.
- sABs that are generated will recognize the provided form of the antigen, that is, the investigator should also provide all of the cofactors, ligands, etc. whose presence in the cell might alter the conformational state of the protein so the phage display selections can be done on the functional form of the protein.
- Bacterial expression is preferable since it is essential that the antigens have a covalent tag; preferably, the antigen should be biotinylated.
- To be most effective and to service the largest number of investigators, there is a preference for target antigens that can be formatted to run in the high throughput mode.
- Priority will be given to projects where the generated reagents will impact the largest community or exploit the reagents produced for the NIH projects involving human transcription factors and epigenetic regulation proteins.
- Projects that involve transcription factors or proteins that regulate epigenetic processes have a high priority since they fit within on-going NIH funded projects.
FAQs
- What are the best targets?
3-D epitopes of stable proteins or domains. - Can peptide fragments be used as antigens?
Peptides do not make good antigens. Many commercial antibodies fail for this reason. Natively unfolded proteins or protein segments are also problematic. - Are there any biochemical features of the antigen that are especially problematic?
Highly basic proteins (PIs > 10) can be problematic because they attract the phage coat, but this can be addressed. - Can this approach identify post-translational modifications?
Identification of post-translational modifications takes extra effort. Heavy glycosylation is problematic because of the heterogeneity of the sugars. Glycosylation at a few sites can be handled. Phospho-specific sABs can be generated if the protein undergoes a conformational change as a result of phosphorylation. - Can this approach discriminate between isoforms?
Discrimination of isoforms is possible depending on the extent of the differences. Changes in a couple of amino acids are probably too subtle to detect except with a lot of work. However, splice variants should work as long as they meet the other criteria. - Why must the protein have a covalent tag and which tag is preferable?
The phage display binding selections are most successful if there is a rigorous washing step during the process. Non-covalent tags like His-tag or FLAG-tag are too weak to withstand the washing conditions. The preferred tag is a biotinylated AviTag, which binds very tightly to commercial streptavidin magnetic beads.
References
Generating conformation-specific synthetic antibodies to trap proteins in selected functional states. Paduch M, Koide A, Uysal S, Rizk SS, Koide S, Kossiakoff AA. Methods. 2013 Mar 15;60(1):3-14. (PubMed)
Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins. Rizk SS, Paduch M, Heithaus JH, Duguid EM, Sandstrom A, Kossiakoff AA. Nat Struct Mol Biol. 2011 Apr;18(4):437-42. (PubMed)
A portable RNA sequence whose recognition by a synthetic antibody facilitates structural determination. Koldobskaya Y, Duguid EM, Shechner DM, Suslov NB, Ye J, Sidhu SS, Bartel DP, Koide S, Kossiakoff AA, Piccirilli JA. Nat Struct Mol Biol. 2011 Jan;18(1):100-6. (PubMed)
High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. Fellouse FA, Esaki K, Birtalan S, Raptis D, Cancasci VJ, Koide A, Jhurani P, Vasser M, Wiesmann C, Kossiakoff AA, Koide S, Sidhu SS. J Mol Biol. 2007 Nov 2;373(4):924-40. (PubMed)
Engineering of recombinant crystallization chaperones. Koide S. Curr Opin Struct Biol. 2009 Aug;19(4):449-57. Review. (PubMed)