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Reply to Critics: How to Obtain or Produce Effective and Replicable Antibodies in the Most Animal-Friendly Way

Prof. Stephen F. Eisenman, Northwestern University

The Northwestern University Committee on Animal Resources (CAR) has generously taken the time to read and respond to my “Report Concerning the Procurement and Use of Research Antibodies” published on Altweb. Their “Statement on Recombinant Antibodies” is found here:

http://www.research.northwestern.edu/committees/car/documents/CAR_Statement_on_Recombinant_Antibodies.pdf

Notwithstanding their criticisms of my report, they have also kindly agreed to support an investigation, undertaken by the office of the Northwestern University Vice President for Research, into the feasibility of establishing on campus a core facility for the production of recombinant antibodies.

The text below is my reply to CAR’s statement.

 Purpose of Original Report:

The purpose of shifting from monoclonal antibodies (mAabs) to recombinant antibodies (rAbs) is to advance science and adhere to the principles of the 3Rs. As I wrote in my original paper, recombinant antibodies permit scientists to improve the quality and reproducibility of their biomedical research. Commercial, monoclonal antibodies too often show poor specificity or fail to recognize their targets. This failure has been discussed in several recent publications in prestigious journals including Nature, PEDS, and Molecular and Cellular Proteomics.[1]  In addition, because rAbs are produced entirely in vitro, there is no possibility that guiding 3Rs principles (Replacement, Reduction and Refinement) will be contravened, or that a supply chain may be disrupted when a provider is found to be in violation of university rules or the Animal Welfare Act, as was recently the case with Santa Cruz Biotechnology.[2] The shift I have proposed would take place gradually and only be completed when all existing research needs are successfully accommodated.

Cost:

CAR is rightfully mindful of economics. The ever-diminishing Federal largesse and the ever-increasing cost and complexity of research magnifies the need for frugality. The rAb facility I proposed at $500,000 would use existing infrastructure. Naturally, a larger and more automated one with greater productivity would cost more. Several individuals and institutions with whom I have been in contact have taken the leap I am proposing and can provide detailed information regarding design, output and cost.[3] They sometimes work collaboratively with other labs, and sometimes on a fee-for-services basis. The Toronto Recombinant Antibody Centre (TRAC) is a stand-alone facility that provides antibodies locally and to collaborators across Canada and internationally. It employs two faculty-investigators allowing a great breadth of work, from target identification to antibody production – as well as validation in cell-based assays.[4]

There is no question that the establishment of an rAb lab at NU or in collaboration with another institution is expensive. But three factors must be considered against this: 1) grant support; 2) validation costs; 3) lost money arising from nonreplicable research:

  1. Grant opportunities exist, for example from the Alternatives Research and Development Fund and the NIH. The former has supported recombinant research and facilities in the past – it is one of their main activities. The latter recently supported a “Protein Expression Shared Resource” at the Wistar Institute, National Cancer Center in Philadelphia. It provides its researchers “expert technical assistance in recombinant DNA plasmid engineering, protein expression in bacteria and baculovirus-infected insect cells, purification of recombinant proteins, and production of high-titer retroviruses (e.g. lentiviruses) for delivery of shRNA and cDNAs to mammalian cells.”[5] The NIH also supports other core facilities. Though the value of these grants may be modest compared to the total costs, they might be able to be leveraged to bring in other foundation support. In addition, it may be possible to establish a public-private consortium of industry, academia, government, and NGOs to fund the development of commonly used affinity reagents that would then be available to all researchers.
  2. As indicated, current mAb methods require extensive and time-consuming validation. These costs are often not figured in to research expense and would be obviated by a gradual shift to rAbs. (When validation does not occur, faulty or nonreplicable research may result.) And while rAbs also require validation, they only need to be validated once – each new batch would arrive, as it were, pre-validated, subject only to routine review to ensure that transport or faulty storage has not compromised binding activity.[6]
    Although as CAR notes, disputes over intellectual property have slowed rAb development and adoption, they will in time be overcome.  The creation of rAbs is now almost routine, and simple development kits may even be purchased. Moreover, as more universities begin working with rAbs, more libraries will enter the public domain further breaking down the walls of copyright. [7]
  3. As indicated in my initial proposal, and as has been extensively documented, the cost of unreliable antibodies is high, perhaps $350 million in the U.S. alone and twice that globally. The expense of rAbs thus pales in comparison, and anyway costs are headed downwards. Bradbury and Pluckthun note: “Production costs for recombinant binding reagents — currently similar to those for monoclonals — should decrease as technologies improve, demand increases and processes become automated.” And once a development and production pipeline is established, rarely used rAbs will be far more economical than they are now. More importantly, the lack of biomedical reproducibility is attributable in significant measure to commercial, animal-derived antibodies that show poor specificity or fail to recognize their targets. [8]

Technical Challenges:

CAR has described a number of impediments to the switch from mAbs to rAbs. Some of this argumentation is obscure, but in general recombinant antibodies bind to a wide variety of targets and can be used for the same purposes as animal-derived monoclonal antibodies. And because the antibody selection occurs in vitro, it is possible to precisely match antibodies and targets. More specifically, recombinant methods can generate antibodies against specific protein conformations and post-translational modifications.[9] In addition, contrary to CAR’s assertion, rAbs can target carbohydrates and lipids. Mao et al write: “Our study demonstrated that high-affinity human antibodies against tumor-associated carbohydrate antigens could be selected from a phage library constructed from the PBLs of various cancer patients.”[10] And Fukuda states: “Phage display has also been used in single-chain antibody technology by inserting an immunoglobulin's variable region sequence into the phage. A single-chain antibody library can then be screened with a carbohydrate antigen as the target, resulting in a recombinant anti-carbohydrate antibody with high affinity to the antigen.”[11]

Finally, CAR’s response contains an extended discussion of “humanized” mAbs. Unless I misunderstand, this point is not germane to the discussion here since humanized antibodies are not used as research reagents.

Ethical Considerations:

The ascites method of antibody generation has for some time been recognized as a painful and even cruel procedure. Its use has been severely restricted or ended in most EU countries, Australia and many other places. While it is becoming more rare in the US, it is still legal and employed by a number of mAb producers including Molecular Diagnostics Services, Maine Biotechnology Services, and Pierce Antibodies. CAR should insist upon a blanket prohibition of the procurement and use of ascites antibodies at Northwestern. CAR’s assertion that “where a non-animal alternative is available, it should be used,” is welcome. That is indeed the very rationale for my original report and this reply: rAbs are precisely such a non-animal approach and should be used routinely. It must be noted however, that U.S. law does not require non-animal models be used whenever they are available.  The Animal Welfare Act simply states in Section 13(a)(3)(B) "that the principal investigator consider alternatives to any procedure likely to produce pain or distress in an experimental animal".  That is why an active and engaged CAR and NU-IACUC are so critical to the university: maintaining the highest level of scientific achievement and the broadest level of public and governmental support requires the university to aspire to the fullest possible adherence to 3Rs protocols both within the NU research community and among those who sell to the university.

I appreciate the serious consideration given by CAR, NU-IACUC and the NU Vice President for Research to the issue of restricting purchasing from Rockland Immunochemical and more recently, Santa Cruz Biotechnology. In its search for alternative suppliers, I encourage the university to turn first to non-animal alternatives available from a number of universities and private companies, such as the University of Geneva, the University of Toronto, AbD Serotec, and Aptagen.[12]

Conclusion:

My original antibodies report was not primarily intended to promote the establishment of a core rAb facility at NU. It was to encourage a process whereby the research community could “start the conversion from mAbs to rAbs.”  The proposed methods were: “1) for researchers to choose recombinant over monoclonal versions of the same antibody if the former already exists; 2) for the schools or central administration to dedicate a sum of money to allow selected researchers to use recombinant antibodies, even when they must be made from scratch; and 3) begin planning for an rAb facility at NU or work in collaboration with other universities to establish one.”  Given the profound issues of replicability and growing concerns about the ethics of animal based research, conversion is inevitable. The goal of my report and this Reply to CAR is to encourage the university to be a leader in the process of conversion, and to ensure that all faculty research needs are satisfied fully, economically and without any possible interruption.


[1] ; Bradbury and Plückthun, “Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents,” Protein Engineering Design and Selection, vol. 28, issue 10, pp. 303-305. Also see: Katherine Groff, Jeffrey Brown, Amy J. Clippinger, “Modern affinity reagents: Recombinant antibodies and aptamers,” Biotechnology Advances, (in press).  

[2] As the result of my original report and subsequent investigations by CAR and the Office of the Vice President, Santa Cruz was removed from the Northwestern Preferred Vendor list, and notice of the allegations of animal abuse was sent to all university investigators.

[3] For example: Dr. Pierre Cosson (Pierre.Cosson@unige.ch) at the University of Geneva, Dr. Christine Misquitta (christine.misquitta@utoronto.ca) at the University of Toronto, and Dr. Andrew Bradubury (amb@lanl.gov) at the Los Alamos National Laboratory. 

[4] Chrsitine Miquitta, email communication, May 18, 2015.

[6] Andrew M. Bradbury and Andreas Pluckthun, “Antibodies: Validate Recombinants Once,” Nature, vol. 520, April 2015, p. 293.

[7] C.B. Shoemaker, “When will rAbs replace mAbs in labs?” The Veterinary Journal, vol. 170, September 2005, pp. 151-2.

[8]  Baker, passim. Also see Berglund et al: “only 49% (2,726 out of 5,436) of commercial, animal-derived antibodies could be validated to recognize only their targets.”

[9] C.R. Geyer et al, “Recombinant antibodies and in vitro selection technologies,” Methods of Molecular Biology, vol. 901, 2012, pp.11-32.

[10] S. Mao et al, “Phage-display library selection of high-affinity human single-chain antibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx,” Proceedings of the National Academy of Sciences,” vol. 96, pp. 6953-8, 1999.

[11] Also see: E. Kobatake  et al, E1, “Targeted delivery using immunoliposomes with a lipid-modified antibody-binding protein,” Applied Biochemical Biotechnology,  vol. 163, no. 2, January 2011, pp. 296-303

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