The Future Of Antibodies: The Antibody Reproducibility Problem
Posted:  Tuesday, March 29, 2016           By:  The GenLogica Team

This post is a part of our Future of Antibodies series where the GenLogica Team explores new products, research, and initiatives to drive antibody research forward. Our goal is to provide analysis of these topics and drive conversation about their role in immunoassay research.

 

The Reproducibility Problem & Antibodies

Life sciences has a reproducibility problem (Ref. 1, 2, 3). Though complex, the immunoassay research community is now strongly voicing their concerns with how the antibody market contributes to these issues (1). Research groups and industry leaders are now proposing different solutions to solve the problem of antibodies reproducibility.

Herein GenLogica will review the proposed solutions and identify benefits for each. We also will analyze the chances for each of these proposals to succeed so that antibodies don’t hinder life sciences research in the future. Finally, we will propose immediate solutions that are simple and effective to address antibody reproducibility.

 

Standardization of antibody citations

One of the most prevalent causes for antibody irreproducibility is the omission of basic information provided by authors in the manuscripts they submit to peer review journals. Often the author will only acknowledge the source of the antibody. Without the basic information that characterizes the antibody researchers will not be able to find the product and reproduce previously published experiments.

Antibody identify can be provided by the following four parameters:

  • Commercial Source:  Supplier name (lab or researcher’s name if from private source)
  • Catalog Number:  All commercial antibodies are indexed by an established system of identification
  • Lot Number:  Especially desirable for polyclonal antibodies as they often suffer lot-to-lot inconsistencies
  • Clone ID:  If the antibody is monoclonal

Publishers need to ensure they adhere to this stricter information standard, even at a bare minimum. Without this information researchers are at a disadvantage for being able to reproduce key experiments.

Contribution to better reproducibility

  • Accurate match to previously used antibodies
  • Can compare and replicate exact experimental conditions
  • Streamlines product ordering process

 

Solution:  Standardization of available antibody sources

Monoclonal Antibody Banks

Using the Administrative Procedures Act to formally request regulatory amendments, the American Anti-Vivisection Society (AAVS) asked the National Institutes of Health (NIH) to ban the production and use of monoclonal antibodies (MAbs) resulting from the mouse ascites method. The NIH gave serious consideration to the petition from the AAVS and prepared a substantive response denying the AAVS request. However, the NIH recognized the validity, reliability, and availability of many non-animal methods for producing MAbs and strongly encouraged their use. 

NIH core and center grants support Hybridoma Monoclonal Antibody facilities. One of the principal objectives is the development of alternative methodologies for MAb production. Presently 13 NIH Institutes and Centers support 64 projects in 20 states totaling over $10M. Examples are The Developmental Studies Hybridoma Bank, NeuroMabs, and AbMiner. MAbs are being made commercially available to researchers in increasing numbers. However, some of them do not have the necessary affinity, specificity, or additional characteristics for a particular application which leads to wasted time and effort by researchers (4).

Recombinant Antibodies

Recombinant antibodies are developed by using bacteria, viruses, and yeast versus more traditional animal models such as mice or rabbits. The technologies used to develop recombinant antibodies are varied, but provide the same intrinsic benefits:

  • Access:  Immortalized high affinity antibody chains (CDRs) by accessing mature (germ-line) Ig-genes
  • Inventory:  Production of large antibody stocks via fermentation that are uniform in quality
  • Quality:  Improving antibody affinity through optimization of the host, mutagenesis and in-silico modeling
  • Ethical:  Avoids the need for animal utilization

In a Nature article (Reproducibility:  Standardize antibodies used in research) the two authors, along with over 100 co-signatories, propose that antibodies currently in the market be sequenced so that they then can be developed using recombinant antibody techniques. This is proposed due to the problems faced in the antibody market, such as lot-to-lot consistency, and in theory will lead to a defined set of antibody products always available as their protein sequences are captured in stored plasmids.

However, for this approach to be successful it must overcome a variety of challenges

  • Industry Opposition:  The research antibody market was $2.2 Billion in 2014 and is growing, which means suppliers will significantly resist initiatives that could reduce revenues and profits.
  • Intellectual Property:  A large number of antibody products may be protected by established IP that will prevent them from being accessible via recombinant techniques.
  • Number of Available Antibodies:  According to antibody aggregate sites, such as CiteAb, there are over 2.5 Million antibodies available. The effort involved to sequence even a reasonable fraction of these products is vast.
  • Antibody Validation Conditions:  Even the best antibodies don’t work under all possible experimental conditions. Researchers often have to search for antibodies because they have discovered that the most popular or highest quality choices failed in their system. For the initiative to be successful, it would have to take this into account, and create libraries of individual antibody targets and database their performance in various systems in order to give researchers access to an appropriate amount of options to encapsulate different experimental conditions.
  • Resources:  The effort to standardize antibodies using recombinant techniques will require a significant input of money and time. Unless a private enterprise develops a sustainable business model to offer this as a solution, the onus will be on public research funds. Currently, research funding is going through a significant “crunch” and finding the large number of scientific groups and money necessary to fund them may be challenging.

Contribution to better reproducibility

  • Easy to maintain same antibody clones, ensuring lot-to-lot consistency
  • Possible to improve affinity and stability via in-vitro mutagenesis
  • Recombinant antibodies negate need for animals

 

Solution: Embryonic Stem Cell Systems

Since the first application 25 years ago, monoclonal antibodies produced in transgenic animals now represent a rapidly growing market hundreds of billions dollars. Their success is based on an established technology with DNA insertion (BACs or YACs containing Ig-loci) into germline (by oocyte microinjection or transfection of ES cells) followed by expression of human antibody repertoires. For optimal antibody production, the endogenous Ig-loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection.

One limitation of this technology is that mammalian antigens are conserved throughout mammalian evolution and induce weak or no immunogenic response in mice. Making monoclonal antibodies (MAbs) in different transgenic species could compensate for this weakness. For example, high-affinity human Ab have been obtained from several different rodents lines; OmniRat and OmniMouse, Kymouse and Harbour Mouse. Rabbits and chickens (DT40) have also been used to produce human MAbs. Novel antibodies with common light chains have been obtained from MeMo mouse and OmniFlic lines.

A very effective approach of engineering Ig-loci in ES cells is using BACs followed by real-time PCR screening to identify the successfully replaced mouse with human variable Ig-regions. Given the fact the industry standard for manufacturing cell line development is 6–9 months, the Veloci platform has demonstrated gram quantity production of purified material weeks after lead selection. Such rapid identification, in vivo testing, and scale-up capacity would be critical for cost reduction in the future, and so this technology, or convergent derivatives, should be implemented for the development of research grade antibody libraries and recombinant production.

Contribution to better reproducibility

  • Provides access to the same antibody gene, ensuring lot-to-lot consistency
  • Increases affinity and specificity by exploring targets from low immunogenic species
  • Decreased development time versus transgenics


Solution: Intrabodies

Intracellular antibodies (intrabodies) are an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, siRNA, shRNA, miRNA, CRISPR, TALEN and small molecule inhibitors. Intrabodies are typically seen as an experimental tool to reveal the function of proteins by interfering with their function. This approach is also reported to have therapeutic potential against viral infections, brain diseases or cancer. 

Intrabodies can be generated by cloning the respective cDNA from an existing hybridoma clone, or more conveniently new scFvs/Fabs can be selected from in vitro display techniques such as phage display. Although intrabodies have been known for more than a quarter of a century (5, 6) and have demonstrated very encouraging results, their use in research is not as widespread as expected.

The technology to generate research antibodies from phage display in large numbers is now robust and reliable (7, 8, 9, 10, 11), and international research consortia like the “Affinomics” initiative in the EU and similar initiatives in the US have generated several thousand antibodies covering hundreds of intrabody targets. Along with their V-region genes, this provides a vast resource of scFv DNA for future intrabody development, meaning this strategy allows a novel and systematic approach to identify protein functions of uncharacterized proteins. 

Contribution to better reproducibility

  • Bind targets in live cells, thus avoiding cell fixation and potential errors from antigen retrieval
  • Interfere with function which eliminates all errors during antibody permeability
  • Reduces cross-reactivity derived from targeting different isoforms based on epitope sequence

 

Solution: Next generation Immunohistochemistry

Immunohistochemistry (IHC) allows detection of a single protein to over a dozen proteins using various methods (12, 13). Two papers have taken the detection of proteins on slides to a new level of quantification and multiplexing by using mass spectroscopy (MS) (14, 15). The novelty of this MS-driven-IHC, or next-gen IHC (ng-IHC), is that it solves the two major problems of IHC:

  • Lack of reproducibility in the scientific literature (16)
  • Failure in the clinical trials.

The ability to multiplex within a pixel allows instant quality assessment of multiple antibodies to the same protein. That is, three or more antibodies against the same protein are validated for specificity due to the low chance of them showing the same spurious cross-reactivity. In this way, ng-IHC solves the problem of antibody variability in the way of technology implementation. The ng-IHC technology could be the key to moving in-situ measurement to the levels of accuracy and reproducibility required for both novel scientific discoveries and robust, high-dimensional clinical tests.

Contribution to better reproducibility

  • Use of multiple antibodies highlights “good” antibodies versus “poor” antibodies
  • Multiple signals avoids errors versus obtaining signal from single antibody in spate sections
  • Method is quantifiable versus use of binary enzymatic signal

 

Solution:  Alternatives to Antibodies / Non-Ig replacements

There are inherent characteristics of antibodies that limit their efficacy. For example, the generation of antibodies depends on animal immunization, which rules out toxic, low-immunogenic, or otherwise incompatible targets. Antibodies are temperature sensitive, undergo irreversible denaturation, experience lot-to-lot variability, and have a limited shelf life which results in reproducibility issues.

In 1990 a revolutionary method presented an in-vitro generation of high-affinity molecules against selected targets, which became known as Aptamers. Using this technology, an alternative to antibodies (Peptide Aptamers) was developed six years later (17). Advances in the technology of scaffold design circumvent some of the limitations of classical immunoglobulins as biomedical tools. These include:

  • Antibody-based single-domain (dAbs)S
  • Single-chain variable fragment (scFv)
  • Antigen-binding fragment (Fab)
  • Avibody
  • Minibody
  • CH2D domain
  • Fcab
  • bi-specific T-cell engager (BiTE) molecules
  • Non-immunoglobin protein structures (e.g. Knottins, Fibronectin type III domains, Bromodomains)

While it took millions of years for nature to construct modern antibodies, it took just two decades for researchers to develop more than fifty different alternative protein scaffold designs (18). Antibody fragments designed on the basis of naturally occurring heavy-chain-only antibodies, like nanobodies derived from Camelidae and VNARs from sharks, look especially promising. It is to be seen which approach will eventually gain the upper hand, but most likely some degree of technological convergence (18, 19) and segregation into the fields where these technologies will prove most effective.

Contribution to better reproducibility

  • Has benefits of recombinant antibodies, but potentially higher affinity from additional mutagenesis
  • Unique target specificity from natural design
  • Higher stability and penetration due to smaller size

 

Our Thoughts

Why do antibodies contribute so heavily to reproducibility problems within the life sciences? Polyclonal antibodies (PAbs) have provided many benefits to research, but are a limited resource and their characteristics such as unpredictable affinity/specificity and lot-to-lot consistency contribute to reproducibility issues.  Monoclonal antibodies (MAbs) improve upon many of the issues that are experienced with PAbs, but still have shortcomings. Ultimately, even the “best” antibody will not be able to be universally utilized, leading to the current glut of antibodies on the market.

As far as new technologies, history has shown these take time to mature and be adopted. The greater the promise of the technology the longer the development timeline and the greater potential for complications. In-vitro technologies have demonstrated potential to meet the demands of large scale antibody development. In the last two decades the development of phage display was driven mainly by the opportunity to make human antibodies for therapeutic applications. Now the technology is yielding valuable research tools which are now appearing in the market. The generation and application of standardized antibody gene libraries is key to the success of a universal antibody pipeline.

 

Technology milestones of antibody generation. Shown are seven technologies in antibody generation (grey arrows). ES cells present significant advantage in obtaining stably integrated Ig-genes. Screening could be accelerated using arrays. Numerous non-Ig scaffolds could be used for genetically engineered Ab-like binders. Synthetic peptidic mimics like Aptamers could shortened significantly the generation time as compared to library generated antibodies. Expressing the genes for the genetically engineered antibodies (e.g. single chain Fv) in living cells provides key advantages.

 

The antibody market is too vast for the solutions proposed above to singlehandedly solve the reproducibility problems experienced by researchers using Ab. It will take a concerted effort to converge the various technologies and initiatives in such a way that the issues with Ab reproducibility are addressed on multiple fronts. Ultimately one technology or initiative may coalesce as the most popular solution, but for the foreseeable future multiple approaches will be needed.

However, there are some immediate steps that can be undertaken:

  • Standardize Antibody Citation Information Requirements
    • There is little reason that this solution cannot be enacted quickly. All journals should adhere to a universe template for antibody information within their papers. This will allow researchers to quickly source the appropriate products to reproduce results.
  • Centralize Currently Available Antibody Products
    • High quality, originally manufactured antibody products should be accessible for search and exploration by the scientific community.

GenLogica’s mission is to immediately address problems with antibody reproducibility. Our solution employs two main features:

  • Advanced Antibody Search
    • Provides detailed, personalized search, finding products that exactly match required experimental conditions
    • Independent quality assessment by scoring antibody performance across different applications
    • Only list originally manufactured products (see the GenLogica blog post about antibody resellers)
  • Supplier Match
    • Researches can submit antibody products they currently use and receive detailed report on whether they are buying from the original manufacturer)

 

Diagram detailing how GenLogica offers immediate antibody solutions by (1) simplifying redundant distribution channels caused by supplier cross-selling (orange arrow on the left) and (2) filters antibodies by quality using application scores (asterisk), ensuring researchers find the best antibody.

 

These solutions are not currently provided by the antibody market for a variety for reasons that include:

  • Antibody makers don’t have access to other company data
  • Cross selling tactics and deception
  • More antibody “suppliers” with poor data and product characterization

GenLogica is committed to the providing immediate solutions to the antibody market, and our team will endeavor to further enhance and empower scientific discovery.

 

The GenLogica Team
 

If you would like to get in touch with us please visit our Contact page.

 

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