Chicken Immunoglobulinbinding proteins: a novel facilitating technology for immunotechnology Michael

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Chicken Immunoglobulin-binding proteinsa novel
facilitating technology for immunotechnologyMic
hael D. Boyle, Ph.D.Von Liebig Chair of
Bio-medical SciencesJuniata College,Huntingdon,
Pennsylvania.In collaboration withBioCheck
Laboratoriesa division of Laboratory Resources,
Inc. BioCheck LaboratoriesToledo, Ohio
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Immunoglobulin binding proteinsa facilitating
technology
Reporter systems based on detecting Fc region
allows a single reagent to be used for
immunotechnological applications that also
utilize the specificity of the antibody combining
region
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Types of immunoassays

• Radio immunoassay
• Enzyme linked immunoassay
• Immunofluorescence
• Fluorescent activated cell sorting
• Affinity purification
• Western blotting
• Immunoelectron microscopy

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Sources of Antibody

• Protein A Protein G
• Rabbit
• Goat
• Human
• Mouse
• Chicken - -

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Why care about chicken antibodies?

• Easy to produce
• Do not require bleeding (egg)
• One chicken can produce a gram of antibody in a
  month
• Antibodies to highly conserved human proteins can
  be produced
• Therapeutic antibodies are safe and well
  tolerated in the majority of people.

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If chicken antibodies are so useful, why are more
companies not using this system?

• Lack of support technology for measuring and
  purifying antibodies.
• Historically mouse monoclonal antibodies were
  developed rather than the more stable rat
  monoclonal antibodies because of the availability
  of the screening and purification reagent protein
  A.

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Our unique proprietary solution

• Identification of a protein A equivalent molecule
  for chicken antibodies
• The molecule can be produced rapidly and
  economically by recombinant genetic engineering
  strategies
• A variety of different tracer forms can be
  generated for broad applications in
  immunotechnology
• Protein A and protein G provide a model for
  marketing and profitability of such a product.

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Technology edge

• Phase I studies have identified first bacterial
  chicken IgY binding protein

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Cloning, Sequencing and Expressing Recombinant
Protein
ATGGCAATTTGTTGGCGTCTTGCTTAGGCGCTGCTTTCTTCTCTTCCTTC
TTAGCTTCAGGTTTTGGAGCTGGAGCTGGTTTAGGTTGAGGTTTTGGCTT
AGGTTCTGGCTTAGGCTCAGGTTTTGGCTCTGGCTTAGGCTCAGGTTTTG
GTTCTGGCTTAGGCTCAGGTTTTGGTTCTGGCTTAGGCTCAGGTTTTGGT
TCTGGCTTAGGCTCAGGTTTTGGTTCTGGCTTAGGCTCAGGTTTAACCTC
TTCGTACACATAAACCACACGAAGATCGCCTTCTAGAGTTGTACCAGTTT
CACTAGGTGAGCCCTCTTGAAGCTTCTTGAACTTGTAGACCTTACCATCT
TTTTCAATCTTATCAAGACGAAGTGAGGTAGTATCGTACGCTTCACCCGG
CAAGCCTTGTGATACCGCAACGGTTTCACCTAAAACATTACCTTCTGTAT
CCACATAAGATACAAGAACGTTAGCATTAAACGGAACAAATTCCCAGTAA
CCGATTAACATGACATCGCCATCACCAGGTGCAACAAAGTTTGATGAAAC
CTTTTCTTCTTTTTCAGAAGATAGAATATTATACCAACCTGCAAATCTCC
ATTCACCAGTTGCAGTCCTTACATTGCTATAGGTTGGCAATTCAATCTTG
TCACCTTTTTTAACAGTTGCAATGTCTGGTAACTTAACAGCATTAAGAAG
TTCTTCTGGTAACAAACGACCTTGATTGCTGCCAAACATGTAAACCACTT
GATTAGTTTCATTAACAAGCGGTTTTGTAATGCCATACTCCCAGTCTTTT
GCCGCTTTAAGCTGTGCTTCTGAAACTACTTCTTCAATTTCAGAAGGCTC
AATTTTTTCTGCAATAGCCTTGCGAACAGAAGCCAGATCTGTTCCAGGAT
ATTGCGCTGCTAAGTTATCAAGCTTAGCATAAGCCTCAGCTCTAG
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Summary of Phase I studies

• Purified an IgY-binding protein
• Developed a tracer form of the protein
• Cloned and sequenced the gene for the protein
• Demonstrated reactivity with chicken, turkey, and
  duck antibodies

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Intellectual property position

• The functional activity is unique, non-obvious
  and useful.
• Utility has been demonstrated
• The sequence defines the activity and affords a
  strong IP position
• Identifying the domain or subsequence that binds
  with functional activity would strengthen
  position.

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Target applications

• Diagnostic tests in humans and animals
• Therapeutic antibodies
• Chicken monoclonal antibody technology
  development
• Research applications in neurobiology and
  development
• Disease surveillance and vaccine efficacy studies
  for the poultry industry

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Market opportunities

• Increased national and international surveillance
  for avian influenza provides timely opportunities
  for
• newly developed immunoassay technology
  designed specifically for avian samples.

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Current market opportunity

• Monitoring health in chickens used in food
  production
• Recent changes in regulation for food exports
• Bioterrorism concerns
• Potential for flu pandemic

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Costs related to avian influenza outbreaks

• The recent outbreak in Italy resulted in 112
  million in compensation for destroyed birds, but
  it was estimated that the indirect costs of the
  outbreak topped 400 million.
• A US department of agriculture budgetary item
  increase of 12.7 million has been proposed for
  increased poultry testing in fiscal year 2005.
• The estimated dollars lost to influenza in humans
  in the United States rises to over 100 billion
  annually. Predicting a potential pandemic from
  bird influenza screening-priceless.

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Competitive technologies

• AGID test, is inexpensive with respect to
  reagents but is labor intensive, highly
  subjective and requires 36 hours to obtain
  results.
• ELISA test is more sensitive, rapid (providing
  results in 3 to 5 hours) and capable of being
  automated. The major drawback with this assay is
  cost. The retail price for five 96 sample ELISA
  plates and reagents to perform the test is 635.

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Investment to date

• Phase I award from Life Sciences Greenhouse of
  Central Pennsylvania with match form Juniata
  College and Biocheck Laboratories Inc. 145,338.
• NIH SBIR phase II award to Biocheck Lab and
  Juniata College on a related project.(50,000)

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Current focus of investment

• Identification of functional protein
• Generation of a recombinant form of the protein
• Proof of concept studies
• Securing the basis for a strong intellectual
  property position

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Phase II research focus

• To secure optimal patent protection requires
  subcloning studies and identification of binding
  domains
• Demonstration that assay system can successfully
  detect avian antibodies to avian influenza with
  sufficient sensitivity, specificity and
  reproducibility to exceed the capability of
  current assays

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Phase II research budget

• The expected budget and timeline for the proposed
  phase II studies would be equivalent to the Phase
  I investment of
• 140,000 and anticipated to be complete within
  a 6-9 months.

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The wild card

• All of the focus to date has been on technology
• Is there a market?
• How can the technology transfer be most
  effectively achieved?
• Who has the expertise to write a business plan?
• Is there a sufficient technology base around
  which to form a company?
• In the absence of a company structure can
  sustaining funding be obtained?

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The trump card

• Immunotechnology is at the core of most
  diagnostic testing in humans and animals.
• Many false positives in diagnostic testing due to
  Fc receptor binding would be eliminated if
  chicken antibodies were used.
• Therapeutic antibodies have great potential
  particularly to treat the consequences of a
  bioterrorist attack.
• Chicken monoclonal antibody technology has been
  developed but is stalled because of absence of
  the type of reagents we are developing.
• The low cost of antibody production in chickens
  and the ability to produce antibodies to highly
  conserved mammalian proteins offers profitable
  opportunities for custom antibody production.

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Complementing Technologies

• Over the past two decades our laboratory has
• Identified, purified and cloned protein G.
• Identified, purified and cloned a human
  IgA-binding protein.
• Identified, purified and cloned IgG subclass
  specific proteins.
• Identified, purified and cloned a human
  IgE-binding protein.
• Identified and cloned a series of albumin binding
  protein.
• Identified a rat IgG-binding protein
• Identified a mouse IgA-binding protein.
• Identified and cloned a human plasmin binding
  protein.
• Developed a unique immunoproteomic platform in
  which these reagents can be used for high
  throughput proteomics

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Where do we go from here?