Workshop Report
The NIH Protein Capture Common Fund Workshop: Renewable Protein Capture Reagents
October 20-21, 2010
Purpose and Workshop Overview
The purpose of the NIH Common Fund Workshop: Renewable Protein Capture
Reagents, October 20-21, 2010, Hilton Washington DC/Rockville Hotel
& Executive Meeting Center was to gather information on the state of
the art in technology for production of a comprehensive public resource
of affinity reagents for the human proteome (http://nihroadmap.nih.gov/proteincapture/).
The NIH Common Fund is exploring the technical and practical ability
to produce a comprehensive resource of renewable affinity reagents
against the human proteome. As a beginning, in 2010 and 2011, common
fund initiatives will solicit production of affinity reagents against
human transcription factors. Although monoclonal antibodies are
currently a mature methodology with clear downstream applications
already developed, they have drawbacks that may be a practical barrier
to creating a more comprehensive set of protein capture reagents. For
example, the high cost of development and the quantity/type of
downstream validations that are applied to ensure wider utility of the
reagents.
In addition to monoclonal antibodies, there is a range of alternative
technologies such as recombinant antibodies, engineered proteins, and
aptamers, which are at various stages of development, and present
encouraging opportunities towards generating a comprehensive set of
protein capture reagents. Given the combined considerations of cost and
range of end use, it may be that multiple approaches will be needed to
generate a comprehensive resource.
Central questions remain in what are the most promising approaches for
the generation of a complete set of protein affinity capture reagents
for wide use by the research community; what are the limitations and
advantages of each approach; what type of affinity reagent can be
productively encouraged to move forward at a "production level" what
are timelines; what is the best way to stimulate each; what are the
advantages or disadvantages of "waiting" until one or more of these are
ready for production?
The concept of a "comprehensive" set of capture reagents begs the
question of how many human proteins should be considered to represent
the proteome in a way that is meaningful for biomedical research. How
will the resource address the issues of splice variants and
post-translational modifications? Clearly, these considerations could
have a large effect of the ultimate cost in addition to the biological
and technical issues involved in producing “comprehensive” reagents. The
intent of the workshop was to solicit practical advice about regarding
these issues and how to balance costs and utility in a framework with
reasonable milestones for a comprehensive resource.
The NIH is aware that there are other similar efforts, at various
scales and with various aims. The workshop participants (see attached
participant list) were asked to provide feedback on how other similar
and parallel efforts should be integrated or considered in a federal
resource. In addition, participants were asked to provide discussions
of the balance of intellectual property constraints and resource
openness to the scientific community in the context private and public
approaches.
Executive Summary
The Workshop agenda organized the proceedings into plenary and
breakout sessions. The opening session consisted of presentations in
plenary session (see attached agenda) which introduced general
discussions in topical areas of: (a) Current Technologies, (b)
Alternative/Future and (c) Coordination and Resource Distribution.
Presenters offered discussions and a range of opinions based on special
knowledge and interests which included existing widely applied
polyclonal and monoclonal immunoglobulin technologies, developing
recombinant engineered protein affinity technologies, aptamers, and
advanced chemical methodologies. These discussions served as starting
content for the break-out sessions which divided the participants into
three topics: (1) What are the prime considerations in developing a
scalable protein capture resource now? (2) Technologies for the near
future: What is needed for significantly improving throughput and
reducing cost within 3 to 5 years, and (3) Strategic issues:
Integrating with other worldwide efforts, maximizing resource
accessibility and downstream utility.
- The consensus of the workshop was that there are least two
approaches ready to scale up, to some level, the production of renewable
affinity reagents: traditional monoclonal and recombinant antibodies.
The Human Protein Atlas, lead by Dr. Mathias Uhlen, has put most of its
effort in the production of polyclonal antibodies and recently reached
the goal of producing reagents for about 10,000 proteins. However, a
small fraction of these are monoclonal. The Structural Genomics
Consortium, lead by Dr. Aled Edwards, is another significant effort that
was recently started and it aims at the production of monoclonal
antibodies. The existence of these parallel efforts will provide
opportunities for coordination to avoid duplication and cooperation on
targets, standards and dissemination as the NIH effort is initiated.
- Workshop participants thought that reagent quality was paramount
and were unanimous in urging the extensive experimental standardization
of protein capture reagents including the determination of affinity
(Kd) and standard performance validation in each of the several
important areas of application including ELISA, immunofluroescence,
immuniprecipitation, etc. This extensive and more costly
characterization raised concern that the projected budget for the
production effort of about $5-7K per reagent ($10M over five years for
reagents for 1000-2000 human transcription factors) would not be
adequate to support the necessary effort including validation, startup
equipment purchases and incremental technical development through the
project. Reagents for the production of antigen targets for the effort
may be already available through other open and publically funded
initiatives such as those mentioned above or the Arizona State plasmid
repository (http://dnasu.asu.edu/DNASU/Home.jsp) where many clones including those generated within the NIGMS Protein Structure have been deposited (http://psimr.asu.edu/).
- The workshop participants highlighted several technologies
including aptamers and alternate recombinant display approaches which
offered prospects of scalable development over 3-5 years. More focused
technical developments and enhancements of production pipeline
operations might also be worthwhile investment. These opportunities
justified potential plans for an investment in several awards totaling
$15M over three years.
- Participants in the workshop supported the availability of
protein capture reagents usable in standard assays at a minimal cost
without intellectual property constraints and with nonexclusive
licensing arrangements which would facilitate a combined profit/
non-profit distribution scheme. Under such an arrangement, private
sector partners would be able to add value through multiple modes of
distribution or through products or services which leverage basic
reagent production.
Resume of Break-out Discussions
Sessions:
- i. What are the prime considerations in developing a resource now
- ii. Technologies for the near future: a survey
- iii. Strategic issues: Integrating with other worldwide efforts; maximizing resource accessibility and downstream utility
i. What are the prime considerations in developing a resource now?
The discussion focused on considerations relating to the direct and
immediate development of a protein capture reagent resource. The
consensus among meeting participants was that based on renewability,
cost, throughput, consistency of efficiency and applicability in the
hands of many researchers that monoclonal antibody and recombinant
binder technologies are ready for scaling for production. The program
would envision cost control and the use of the reagents by academic or
private sector researchers should be unconstrained by burdensome
intellectual property restrictions. Whether in a centralized or
distributed format, the effort should be managed from beginning to end,
from antigen selection to product catalogue and the process be as
transparent and integrated as possible. The research community should
recognize the NIH supported resource as a uniquely useful and reliable
brand based on performance and reinforced by a carefully chosen name,
term and logo. In the case of a monoclonal antibody reagent resource
careful attention to quality assurance and quality control will be the
essential for the success of the program. Information on the quality
and characterization data of the affinity binder reagents should be
easily and readily available. Characterization of the reagents by the
primary producer and also by independent core testing facilities should
be organized in a single data document together with clear "how to use"
methodology included or offered in easily accessed secondary sources, as
done in focused public efforts to develop and distribute antibodies of
interest to specific research communities (e.g. http://antibodies.cancer.gov).
In addition to the initial characterization of the antigen a standard
set of criteria for the characterization such as those already been
adopted elsewhere (e.g. MIAPAR, http://www.nature.com/nbt/journal/v28/n7/full/nbt0710-650.html)
should be applied. Performance within the several broad areas of
application of such reagents including enzyme-linked immunofuorescence
(ELISA), Western blotting, immunohistochemistry and immunoprecipitation
should be specified. Where the possible application in more specialized
assays, such as ChIP-seq or flow-cytometry, should be included in the
product specification data sheet. While recombinant binders have so far
seen fewer downstream applications it was thought that they represent a
clearly viable approach for scalable developments. Given the
availability of antigens for development a choice based on the relative
merits of these two approaches which revolve around issues of
availability and costs of screening technologies and other practical
matters should be left to the process of application and peer review of
responses to a formal funding opportunity announcement. Responses to
such an announcement should be judged by standards which include the
clear goals and milestones for quality, performance and cost with
improvements in efficiency and output over the life of the effort.
Distribution of the affinity reagent products of this effort should be
centralized and stable over the long term with the Developmental Studies
Hybridoma Bank (DSHB, http://dshb.biology.uiowa.edu/) or the Mammalian Gene Collection (MGC, http://mgc.nci.nih.gov/) considered useful models.
ii. Technologies for the near future: a survey focused on a
discussion of technologies which might be capable of large scale
production in the near future (3-5 years). There was a consensus that
single-chain variable fragment fusion proteins and other immunoglobulin
display derivatives were ready for testing during that timeframe.
Perhaps also aptamers might be considered in this case. Other
technologies including combinatorially engineered scaffolds such as
Affybodies and fibronectins, imprinted polymers and chemical reagents
were considered but not discussed extensively. There was the sense that
these technologies might not be at the same level of development as the
recombinant antibodies but might have good value in specific
applications. Combinatorially engineered protein scaffolds as
alternatives to the immunoglobulin fold were considered valuable,
particularly in the context of well adapted production platforms such as
yeast or phage. As with these approaches, every candidate technology
should have demonstrated initial proof of concept. Discussants agreed
that whatever class of affinity reagent chosen that convincing proof of
these technologies would require demonstration of sensitivity and
specificity according to common standards varying with the assay (e.g.
ELISA, immunoblot, immunohistochemistry, immunoprecipitation, etc.).
Initial performance baseline should be set at minimum to the level of
existing technologies. The ultimate proof of utility at pilot scale
would require deploying the technology in a monitored pipeline. This
scheme should be established according to preselected milestones and
operable determine of costs, quality and throughput throughout the
process. The product of developments should be judged quantitatively by
affinity (Kd), sensitivity, specificity, cost and more generally based
on renewability, throughput and scalability of the process, ease of use
and utility tied to satisfactory performance in user’s hands and
generalizability of the reagent technology to other protein classes
including post-translationally modified proteins. High affinities in
the nanomolar range have often been coupled with “high quality”
reagents. Although a clear cut off here can’t be determined, because it
is dependent on the application, the minimum is that all reagents are
well characterized by their affinities and specificities and validated
for their intended application. It should be mandatory that release of
the quality controlled product reagents at the end of the process should
be combined with the fully documented validation experiments
documenting the quality of the reagent. Additionally, data, methods and
technology related to the reagents should be broadly disseminated
through publications, presentations and the web. Successful applicants
for these scale-up projects should include plans for the distribution
of the target antigens employed during the development and the capture
reagents which are produced by this pilot activity. These projects
should be funded and administered as cooperative agreements to
facilitate the production and ultimate distribution of the products.
Monitoring of progress by NIH program staff should include a mid-course
review with the specific inclusion of adjustments of budget and scope
within the individual projects in order to maximize the impact of the
overall initiative. Among the potential approaches there is still a
great deal to be learned about which may be the most attractive for
further focused effort. The program should take an opportunity to
select a range of approaches with the object of capturing the best of
potentially scalable technologies. Longer term research and development
efforts that can not show scalability to proteome scale within 3-5
years should be outside the scope of the present effort.
iii. Strategic issues: Integrating with other efforts, maximizing
resource accessibility and downstream utility focused on the integration
of the proposed NIH resource with ongoing and planned activities
planned in Europe (http://www.proteinatlas.org/, http://www.antibody-factory.de and http://www.proteomebinders.org/)
and Asia and on the promotion of resource dissemination and
community-wide access and impact. The NIH aims to create a community
resource leveraged through consensus and coordination with other efforts
minimizing costs and maximizing timely impact. Division by agreement
among the cooperating partners of the target human proteome by protein
class, systematic collections, biological sub-proteome [sic] or end use
of the reagent would be an advantage. This will also provide the
opportunity to prioritize targets during the effort to focus the effort
on the most valuable targets at the various stages of the effort. This
coordination may provide for overlap where this might be an advantage,
for example where some reagent technologies are more suitable for
different classes of protein target, but avoiding wasteful redundancy.
However, it should be noted that the need for multiple reagents for the
same target has been recognized and that at this stage some overlap was
identified as beneficial. The establishment of a common approach to
intellectual property addressing the interests of stakeholders and
consistent with laws and regulations in participating countries will be
essential. The governing principle should remain minimal intellectual
property constraint, so that these reagents can be as widely used as
possible, and that creative scientists are enabled to perform
experiments and to devise creative downstream applications for the
reagents. Similarly, a mixture of public resources and private sector
entities would be an advantage for dissemination of data, methods and
materials. Examples of such public-private partnerships in several
public sector research efforts suggest the practicality of this approach
(http://mgc.nci.nih.gov/). All
the products from the sponsored effort would be available to users at
minimal cost and with a minimum of complication and formality through a
comprehensive public resource. Nonexclusive licensing of technology
developed by the research network to private sector entities would
provide an alternative source of reagents and also derived technology.
Participants advised NIH staff to take strong measures to ensure this,
including obtaining a Determination of Exceptional Circumstance. The
effort and product of the NIH supported effort should be identified to
the research community as a uniquely useful and reliable brand. A
related name, term and logo should be adopted to facilitate marketing
and advertisement. One or several web-based portals should provide a
ready source for information about the reagents and resources available
for distribution (see for example http://www.antibodypedia.org, http://dshb.biology.uiowa.edu/ and http://www.sbkb.org/).
The methods and technology developed should be compiled and offered
for dissemination for academic research purposes and with appropriate
licensing for commercial use. This information portal should provide
results from validation testing performed prior to the release of the
reagent and also provide a forum for ongoing evaluation and reporting of
the performance of the materials by the users. The information
portal(s) should provide the community with an opportunity to provide
input on the operation and management of the activity and to make
requests for the preparation of certain reagents which are of particular
value and interest in their individual research endeavors.
Acknowledgement of the use of the reagents should be required in
publications by customers. The web resource should provide pointers to
publications produced by the research network and also to those by
resource users where the reagents were used in the work.
The importance of establishing a rational priority among protein
target pursued within the production effort (i.e. human transcription
factors) and targets within other classes of proteins which might be
selected by pilot studies in earlier developmental stages. Care should
be taken to distinguish targets which are ready for production and
reagent preparation from those which require extra effort. The early
establishment of pipeline operations requires attention to availability
of appropriate target materials for operation.
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