UNITED STATES PATENT AND TRADEMARK OFFICE
`
`________________________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`________________________________
`
`FLUIDIGM CORP.,
`
`Petitioner,
`
`v.
`
`THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV.,
`
`Patent Owner.
`
`________________________________
`
`Case IPR2017-00013
`
`Patent 7,563,584
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`________________________________
`
`Declaration of Dr. Tom Huxford
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`Page 1 of 60
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`Fluidigm
`Exhibit 1002
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`
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`________________________________
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`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`________________________________
`
`FLUIDIGM CORP.,
`
`Petitioner,
`
`v.
`
`THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV.,
`
`Patent Owner.
`
`________________________________
`
`Case IPR2017-00013
`
`Patent 7,563,584
`
`________________________________
`
`Declaration of Dr. Tom Huxford
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`Page 1 of 60
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`Fluidigm
`Exhibit 1002
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`I.
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`INTRODUCTION AND QUALIFICATIONS
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`1.
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`I am over 18 years of age. I have personal knowledge of the facts and
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`opinions stated in this Declaration and could testify competently to them if asked
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`to do so.
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`2.
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`I am a currently a Professor in the Department of Chemistry &
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`Biochemistry and a Professor at the Molecular Biology Institute both at San Diego
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`State University (SDSU) in San Diego, California. I am also the Director of the
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`Macromolecular X-ray Crystallography Facility at SDSU.
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`3.
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`Courses I have taught while at SDSU cover undergraduate to graduate
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`level topics in chemistry, biochemistry and molecular biology. The topics
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`discussed in the courses I have taught include antibody structure and preparation,
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`conjugation to small molecule fluorophores, detection of antibodies using labels,
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`activation of proteins via phosphorylation or proteolysis, and cell signaling
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`pathways.
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`4.
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`I received a B.S. degree in Honors Biochemistry with University
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`Honors from Brigham Young University in Provo, Utah in 1995. I also received a
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`Ph.D. in Chemistry (Biochemistry) from the University of California San Diego in
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`La Jolla, California in 2001. A primary area of research during my Ph.D. studies
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`was examining the activation state of transcription factor NF-κB through x-ray
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`crystallography and in vitro biochemistry with my dissertation being “Inactivation
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`of Transcription factor NF-κB through Its Association with the IκBα Inhibitor
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`Protein.” Immediately following my Ph.D., I continued on as a Postdoctoral
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`Fellow at the University of California using structural and biophysical approaches
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`to understand regulatory aspects of the NF-κB signal transduction pathway.
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`5.
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`I have personally performed numerous research investigations that
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`required the use of antibodies to detect cellular signaling proteins, including
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`detecting the activation state of those proteins. For example, as discussed above,
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`my 2001 dissertation for my Ph.D. was examining the inactivation of transcription
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`factor NF-κB due to an association with the IκBα inhibitor protein. NF-κB is a
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`transcription factor protein that controls survival gene expression in response to
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`potentially toxic cell stimuli. The series of biochemical steps that link the original
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`stimulus to activation of the transcription factor are known collectively as the NF-
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`κB signaling pathway. As part of my experiments to perform this analysis, which
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`were performed prior to 2001, I used antibodies specific to each of the
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`phosphorylated and unphosphorylated forms of the NF-κB inhibitor protein IκBα
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`in order to detect if and when NF-κB became inactive in the presence of IκBα.
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`6. My research in NF-κB signaling has continued to the study of kinase
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`enzymes that function upstream of NF-κB. These include the IκB Kinase (IKK)
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`and other kinases involved in cell survival.
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`7.
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`In addition to my research, I was actively involved in the cellular
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`biology scientific community from the mid-1990s to the present day, writing
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`primary research articles and literature reviews, reviewing journal articles, and
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`speaking at seminars. A sampling of these activities that may be relevant to this
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`proceeding is provided below.
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`
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`Representative research articles:
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` ●
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` Huxford T., Huang D.-B., Malek S. & Ghosh G. (1998). The crystal structure
`of the IκBα:NF-κB complex reveals mechanisms of NF-κB inactivation. Cell
`95 759-770.
`
` ●
`
` Malek S., Huang D.-B., Chen Y., Huxford T., Ghosh S. & Ghosh G. (2003). X-
`ray crystal structure of an IκBβ:NF-κB p65 homodimer complex. J. Biol.
`Chem. 278 23094-23100.
`
` ●
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` Shaul J.D., Farina A. & Huxford T. (2008). The human IKKβ subunit kinase
`domain displays CK2-like phosphorylation specificity. Biochem. Biophys. Res.
`Commun. 374 592-597.
`
` ●
`
` Wojciak J.M., Zhu N., Schuerenberg K.T., Moreno K., Shestowsky W.S.,
`Hiraiwa M., Sabbadini R. & Huxford T. (2009). The crystal structure of
`sphingosine-1-phosphate in complex with a Fab fragment reveals metal
`bridging of an antibody and its antigen. Proc. Natl. Acad. Sci. USA 106 17717-
`17722.
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` ●
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` Fleming J.K., Wojciak J.M., Campbell, M.A. & Huxford T. (2011).
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`Biochemical and structural characterization of lysophosphatidic acid binding by
`a humanized monoclonal antibody. J. Mol. Biol. 408 462-476.
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` ●
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` Polley S., Huang D.-B., Hauenstein A.V., Fusco A.J., Zhong X., Vu D.,
`Schröfelbauer B., Kim Y., Hoffmann A., Verma I.M., Ghosh G. & Huxford T.
`(2013). A structural basis for IκB Kinase 2 activation via oligomerization-
`dependent trans auto-phosphorylation. PLoS Biology 11 e1001581.
`
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`Representative review articles/Book Chapters
`
` ●
`
` Huxford T., Malek S. & Ghosh G. (1999). Structure and mechanism in NF-
`κB/IκB signaling. Cold Spring Harbor Symp. Quant. Biol. 64 533-540.
`
` ●
`
` Huxford T. & Ghosh, G. (2003). The structural biology of NF-κB. In, Nuclear
`Factor κB: Regulation and Role in Disease. Edited by Rudi Beyaert R. Klüwer
`publishers, Netherlands 179-200.
`
` ●
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` Huxford T. & Ghosh G. (2009). A structural guide to proteins of the NF-κB
`signaling module. Cold Spring Harbor Perspectives in Biology. 1 a000075.
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` ●
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` Huxford T., Hoffmann A. & Ghosh G. (2011). Understanding the logic of
`IκB:NF-κB regulation in structural terms. In, Current Topics Microbiology and
`Immunology: NF-κB in Health and Disease. Edited by Michael Karin.
`Springer-Verlag, Heidelberg, Germany 1-24.
`
`
`Representative invited seminars/Poster presentations
`
` ●
`
` “Specificity in the interaction of transcription factor NF-κB and the IκB
`inhibitor protein.” December 7, 2001. The Arizona Cancer Center, University
`of Arizona, Tucson, AZ
`
` ●
`
` “Regulation of transcription factor NF-κB by IκB inhibitor proteins.” Martin D.
`Kamen Prize Lecture. September 25, 2003. University of California, San
`Diego, La Jolla, CA
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` ●
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` “Biochemistry of the NF-κB signaling pathway.” Department of Chemistry &
`Biochemistry Seminar, February 20, 2009, University of California Los
`Angeles, Los Angeles, CA
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`● “Antibody recognition of a signaling lipid.” Department of Chemistry Seminar,
`April 23, 2009, University of California Irvine, Irvine, CA
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` ●
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` “IκB Kinase oligomerization and activation of NF-κB” Department of
`Chemistry Seminar, February 3, 2012, University of Kentucky, Lexington, KY
`
` ●
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` “A structure-based mechanism for IκB kinase activation.” Molecular Biology
`Institute Seminar, February 19, 2013, University of Oregon, Eugene, OR
`
` ●
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` “Activation and specificity of an engineered monomeric IKK2.” Keystone
`Symposium—The NF-κB System in Health and Disease. February 23-28,
`2014, Keystone, CO
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`
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`8.
`
`I understand that my curriculum vitae with a more detailed description
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`of my background is being submitted as Exhibit 1003.
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`9.
`
`I have been asked by the Petitioners to provide my opinions about the
`
`technical issues addressed below with respect to U.S. Patent No. 7,563,584 to
`
`Perez et al., entitled “Methods and Compositions for Detecting the Activation State
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`of Multiple Proteins in Single Cells” (“the `584 patent”).
`
`10.
`
`I am being compensated for my time spent on this matter at my
`
`standard hourly consulting rate of $180/hour, but I have no financial interest in the
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`outcome of this or any related proceeding. My compensation is not dependent
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`upon the opinions that I am providing in this declaration.
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`II. DOCUMENTS REVIEWED
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`11.
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`In conducting my analysis and formulating my opinions, I have
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`reviewed the following documents, which I understand will be given the specific
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`exhibit numbers referenced below in these Proceedings:
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`Exhibit Description
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`1001
`
`1004
`
`1005
`
`1006
`
`1007
`
`1008
`
`U.S. Patent No. 7,563,584 to Perez et al., entitled “Methods and
`Compositions for Detecting the Activation State of Multiple Proteins in
`Single Cells” (“the `584 patent”)
`
`Intracellular
`Fleisher, Thomas A., et al., Detection of
`Phosphorylated STAT-1 by Flow Cytometry, Clinical
`Immunology, Vol. 90, No. 3, pp. 425–430, March 1999
`(“Fleisher”)
`
`PCT Application No. WO 99/44067 to Darzynkiewicz, et. al., entitled
`“Flow Cytometric Detection of Conformations of pRB in Single Cells”,
`publication date of Sept. 2, 1999 (“Darzynkiewicz”)
`
`Yen, Andrew, et al., Retinoic Acid Induced Mitogen-activated
`Protein (MAP)/Extracellular Signal-regulated Kinase (ERK)
`Kinase-dependent MAP Kinase Activation Needed to Elicit HL-
`60 Cell Differentiation and Growth Arrest, Cancer Research, Vol.
`58, pp. 3163-3172, July 15, 1998 (“Yen”)
`
`Belloc, F., et al., Flow Cytometry Detection of Caspase 3
`Activation in Preapoptotic Leukemic Cells, Cytometry, Vol. 40,
`pp. 151-160, June 2000 (“Belloc”)
`
`Dai, Y., et al., Pharmacological Inhibitors of the Mitogen
`activated Protein Kinase (MAPK) Kinase/MAPK Cascade
`Interact Synergistically with UCN-01 to Induce Mitochondrial
`Dysfunction and Apoptosis in Human Leukemia Cells, Cancer
`Research, Vol. 61, pp. 5106-5115, July 1, 2001 (“Dai”)
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`1009
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`1010
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`1011
`
`1012
`
`1013
`
`
`
`1014
`
`Morgan, M., et al., Cell-cycle–dependent activation of mitogen-
`activated protein kinase kinase (MEK-1/2) in myeloid leukemia
`cell lines and induction of growth inhibition and apoptosis by
`inhibitors of RAS signaling, Blood, Vol. 97, No. 6, pp 1823-1834,
`March 15, 2001 (“Morgan”)
`
`Wang X., et al., Requirement for ERK Activation in Cisplatin-
`induced Apoptosis, Journal of Biological Chemistry, Vol. 275,
`No. 50, pp. 39435-39443, December 15, 2000 (“Wang”)
`
`Baumgarth, N., et al., A Practical Approach to Multicolor Flow
`Cytometry for immunophenotyping, Journal of Immunological
`Methods, Vol. 243, pp. 77-97, September 2000 (“Baumgarth”)
`
`Szöllősi, J., et al., Application of Fluorescence Resonance Energy
`Transfer in the Clinical Laboratory: Routine and Research,
`Cytometry, Vol. 34, pp. 159-179, August 1998 (“Szöllősi”)
`
`Horn., I., et al., Selection of phage-displayed Fab antibodies on
`the active conformation of Ras yields high affinity conformation-
`specific antibody preventing the binding of c-Raf to Ras,
`Federation of European Biochemical Societies Letters, Vol. 463,
`pp. 115-120, Dec. 10, 1999 (“Horn”)
`
`Pasinelli, P., et al., Caspase-1 and -3 are sequentially activated in
`motor neuron death in Cu, Zn superoxide dismutase-mediated
`familial amyotrophic
`lateral scelrosis, Proceedings of
`the
`National Academy of Sciences., Vol. 97, No. 25, pp. 13901-
`13906, December 5, 2000 (“Pasinelli”)
`
`III.
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` PERSON OF ORDINARY SKILL IN THE ART
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`12.
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`I understand that a “person of ordinary skill in the art” is a
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`hypothetical person who is presumed to have known the relevant art at the time of
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`the invention. I further understand that the relevant timeframe for assessing the
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``584 patent is prior to July 10, 2001, the date on which the earliest provisional
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`patent application cited by `584 patent was filed. If I refer to the time of the
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`invention in this declaration, I am referring to this timeframe.
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`13. Given my experience in the fields of biochemistry and molecular
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`biology, I can provide opinions about the understanding and qualifications of a
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`person of ordinary skill in the art concerning the technology at issue in this
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`proceeding. A person of ordinary skill in the art in the field of the `584 patent, as
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`of the time of the invention, was typically a person who had a Ph.D. in the areas of
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`chemistry, biochemistry, cell biology or molecular biology including five or more
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`years of experience in dealing with antibodies, protein labeling, protein interaction,
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`and protein detection.
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`IV. STATE OF TECHNOLOGY AT THE TIME OF THE INVENTION
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`14. The broad significance of protein phosphorylation to cellular
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`physiology began to be appreciated by at least the 1960s and the study of
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`mechanisms by which proteins are regulated by phosphorylation continued up until
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`the time of the invention. In general, researchers were interested in determining
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`how a class of enzymes known as protein kinases directs the attachment of
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`phosphate to specific proteins resulting in their switching between states of
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`inactive and active forms that function to control cell regulation. This knowledge
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`was particularly important in understanding the regulation of metabolism, immune
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`responses, and cell division. Proper control of protein phosphorylation is central to
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`avoiding diverse diseases including many types of cancer.
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`15. By at least the early 1990s it was clear that the activities of many
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`protein kinases were themselves regulated by phosphorylation. From this
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`knowledge emerged the notion of “protein phosphorylation cascades” in which
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`activation of some upstream protein kinase could lead in turn to activation of
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`another downstream protein kinase and so on through a “signaling pathway.”
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`Similarly, other types of proteins, such as caspases, were also found to undergo
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`activation through a cascade of cleavage reactions and could also play a part in
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`signaling pathways. Thus, by the 1990s researchers had become interested in the
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`interaction and correlation between activation states of proteins to better
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`understand these signaling pathways.
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`16. A predominant method to detect a specific protein at the time of
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`invention, and well before the time of the invention, was by using an antibody
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`specific for the protein of interest. An antibody is specific for a protein in a
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`particular assay if the antibody has a higher binding affinity for the protein of
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`interest relative to its binding affinity for other proteins. One then needs to detect
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`the antibody-protein complex, which could be done more easily than detecting the
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`protein directly. At the time of the invention, the detection of the antibody-protein
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`complex was done either through direct labeling of the antibody or by the use of a
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`secondary antibody.
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`17. The fact that a single protein can exist in different states does not
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`change this fundamental approach. One of skill in the art that desired to detect a
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`particular state of a protein would require an antibody that has high binding affinity
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`for the state of the protein of interest and low binding affinity for other states of the
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`protein of interest as well as low binding affinity to other proteins.
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`18. By the time of the invention, the process of making an antibody with
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`high binding affinity to a protein was well-understood and routinely performed.
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`For example, the process to create an antibody to detect a protein found in human
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`cells starts by inoculating a mammal (e.g., rabbit, goat, mouse) with an antigen that
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`corresponds to the specific protein. This means that the injected antigen would be
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`composed of a sequence of amino acids that were found on the specific protein.
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`The mammal’s immune system would then produce antibodies that bind to the
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`amino acid sequence found on the antigen. These antibodies would then be
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`purified from the mammal’s blood serum using standard and well-known
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`purification techniques. Once purified, these antibodies could then be used to
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`detect proteins having the target amino acid sequence.
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`19.
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`It is common for protein kinases that undergo phosphorylation to
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`present certain amino acid sequences as antibody binding targets that were not
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`present prior to phosphorylation of the protein kinase. Because of this, antibodies
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`can be produced that would be specific for a phosphorylated form of a protein
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`kinase but not the un-phosphorylated form of the same protein kinase. The same
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`holds true for caspases that undergo cleavage in that different amino acid
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`sequences are presented for cleaved caspases compared to the same caspase prior
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`to cleavage (an uncleaved caspase is sometimes referred to as a pro-caspase).
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`20. However, since some antibodies can react with proteins other than the
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`target protein or to different forms of the same target protein, one of skill in the art
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`had to optimize the use of an antibody for work within the parameters of the assay
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`he or she was studying. For example, the antibody used should preferably bind
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`only to the particular target protein, or state of the target protein, in the assay being
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`studied. It is also acceptable to one of skill in the art if some other proteins exist in
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`the assay that the antibody might bind so long as those other proteins are in low
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`enough concentrations to not substantially impact the data obtained relative to the
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`protein of interest. The optimization of antibodies to bind to a target protein was
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`well-known at the time of the invention as well as techniques to establish a proper
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`assay, including selecting antibodies and proteins that would avoid any binding
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`affinity problems.
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`21. Hence, the primary factor in determining if an antibody could be
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`produced that was specific to a certain form of a protein was if there was a
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`sequence of amino acids that were presented on the form of the protein of interest
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`but not on other forms of that protein. Once such a sequence was known, the
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`production of the antibody itself and the use of the antibody in an assay only
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`required routine and well understood tasks to one of skill in the art.
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`22. At the time of the invention, those of skill in the art were aware of
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`many protein kinases where the phosphorylated form presented a sequence of
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`amino acids not found on the unphosphorylated form of the same protein kinases.
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`Indeed, at the time of the invention the study of the phosphorylated state of many
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`protein kinases and cleaved form of caspases was so widespread and demand for
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`antibodies specific to the phosphorylated or cleaved forms these proteins was
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`sufficiently high that numerous private companies were in the business of selling
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`these antibodies including Cell Signaling Technologies, New England BioLabs,
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`Sigma-Aldrich, and Santa Cruz Biotechnology Inc.
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`23. For specific examples, below are excerpts from scientific studies
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`published by the time of the invention that used antibodies obtained commercially
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`to analyze activated forms of proteins:
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`The following antibodies were used as primary antibodies: phospho-
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`p44/42 MAPK (Thr202/Tyr204) antibody (1:1000; rabbit polyclonal;
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`NEB [New England Biolabs], Beverly, MA); p44/42 MAPK antibody
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`(1:1000; rabbit polyclonal; NEB); phospho-p38 MAPK (Thr180/Tyr182)
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`antibody
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`(1:1000;
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`rabbit polyclonal; NEB); phospho-SAPK/JNK
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`(Thr183/Tyr185) antibody (1:1000; rabbit polyclonal; Cell Signaling
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`Technology, Beverly, MA); SAPK/JNK antibody (1:1000; rabbit
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`polyclonal; Cell Signaling Technology); anti-phospho-CREB (1:1000;
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`rabbit polyclonal; Upstate Biotechnology, Lake Placid, NY); phospho-
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`cdc2 (Tyr15) antibody (1:1000; rabbit polyclonal; Cell Signaling
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`Technology);
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`anti-p21Cip/WAF1
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`(1:500; mouse monoclonal;
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`Transduction Laboratories, Lexington, KY); anti-p27kip1 (1:500; mouse
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`monoclonal; PharMingen, San Diego, CA); MAP kinase phosphatase-1
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`(M-18; 1:200; rabbit polyclonal; Santa Cruz Biotechnology Inc., Santa
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`Cruz, CA); MAP kinase phosphatase-3 (C-20; 1:100; goat polyclonal;
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`Santa Cruz Biotechnology Inc.);
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` rabbit polyclonal; Santa Cruz
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`Biotechnology Inc.); antihuman/mouse XIAP (1:500; rabbit polyclonal;
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`R&D System, Minneapolis, MN); anti-caspase-3 (1:1000; rabbit
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`polyclonal; PharMingen); cleaved-caspase-3 (Mr 17,000) antibody
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`(1:1000; rabbit polyclonal; Cell Signaling Technology); anti-caspase-9
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`(1:1000; rabbit polyclonal; PharMingen); anti-PARP (1:2500; mouse
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`monoclonal; Calbiochem); and cleaved PARP (Mr 89,000) antibody
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`(1:1000; rabbit polyclonal; Cell Signaling Technology).
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`Ex. 1008, Dai at 5107-5108.
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`Antibodies against H-, K-, and N-RAS, ERK-1/2, MEK-1/2,
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`monophospho- and diphospho-ERK-1/2 (PP-ERK-1/2) were from Santa
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`Cruz Biotechnology
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`Inc
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`(Santa Cruz, CA) and Sigma-Aldrich
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`(Deisenhofen, Germany). Diphospho-MEK-1/2 (PP-MEK-1/2), CREB-1,
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`and phospho-CREB-1 antibodies were from New England Biolabs
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`(Frankfurt, Germany).
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`Ex. 1009, Morgan at 1823-1824.
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`[T]he anti-phospho-ERK and anti-phospho-JNK rabbit polyclonal
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`antibodies were purchased from Promega (Madison, WI). Anti-phospho-
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`p38 and anti-phospho-MEK1/2 (Ser217/221) antibodies were purchased
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`from New England Biolabs, Inc. (Beverly, MA), and the anti-caspase-3
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`antibody was from Transduction Laboratories (Lexington, KY).
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`Ex. 1010, Wang at 39436.
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`[R]abbit anti-human phosphorylated STAT-1 (New England Biolabs,
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`Beverly, MA)
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`Ex. 1004, Fleisher at 426.
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`The antibody used to detect activated ERK1 and ERK2 (V667I rabbit
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`polyclonal antibody; Promega, Inc., Madison, WI)
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`Ex. 1006, Yen at 3165.
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`[P]olyclonal phycoerythrin (PE) – conjugated anti–active caspase 3
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`antibodies (PharMingen–Becton Dickinson, Le Pont de Claix, France).
`
`Ex. 1007, Belloc at 153.
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`24. One of skill in the art would have known about the availability and
`
`applicability of these antibodies at the time of the invention and would have been
`
`able to utilize them in their own assays to analyze the phosphorylated or cleaved
`
`proteins targeted by these antibodies. The “p44/p42” antibody description refers to
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`ERK 1/2, as “p44” is an alternative name for ERK 1 and “p42” is an alternative
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`name for ERK 2 as known to those of skill in the art. In addition, ERK 1/2 is
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`commonly treated as a single protein, as is MEK 1/2, with respect to activation
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`state specific antibodies to those of skill in the art.
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`25. Antibodies were used to aid in the detection of specific proteins
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`because at the time of the invention, and still today, it is easier and cheaper to
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`directly detect an antibody-protein complex than a protein alone. One common
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`method was to attach a chemical label covalently to the antibody using
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`straightforward and predictable chemistry. The general process to covalently attach
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`a label to an antibody is the same regardless of what antigen a particular antibody
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`is specific for. This means that the chemistry to attach a label to an antibody that is
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`specific to a phosphorylated form of a protein is generally the same process as to
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`attach a label to an antibody that is specific to an un-phosphorylated form of the
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`same protein.
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`26. The labels attached to the antibodies permit detection using a variety
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`of well-known mechanisms, one of which is fluorescence. For fluorescence
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`detection, the label would be a fluorophore that emits light at a one particular
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`wavelength when it is excited by light of a different, higher energy wavelength. In
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`general, a laser emits light at a specific wavelength within the excitation range of
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`the fluorophore which is directed at the sample containing the labeled antibodies
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`and the intensity of light emitted at the fluorophore’s emission wavelength could
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`be in turn be detected which correlates to the amount of labeled antibody in the
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`sample. If there is no light detected in the fluorophore’s emission range it means
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`the sample does not contain the labeled antibody.
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`27. Another common labeling technique at the time of the invention was
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`based on the application of FRET (Förster resonance energy transfer or commonly
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`stated in journals as fluorescence resonance energy transfer). FRET is a type of
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`fluorescence detection that involves two different fluorophores, a donor and an
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`acceptor. The donor fluorophore’s emission wavelength overlaps the acceptor
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`fluorophore’s excitation wavelength. When the donor and acceptor fluorophores
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`are in very close physical proximity the emission of the donor fluorophore will
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`substantially excite the acceptor fluorophore. To detect a FRET label, a laser’s
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`wavelength would be selected to excite the donor fluorophore, whose emission
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`light would excite the acceptor fluorophore (which also has the effect of quenching
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`the donor fluorophore) if the two fluorophores are in very close physical proximity.
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`A device could then detect for the amount of light emitted at the donor’s emission
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`wavelength, the amount of light detected at the acceptor’s emission wavelength, or
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`the ratio between the two.
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`28. FRET had at least two common applications at the time of the
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`invention. One was to determine if a molecule labeled with an acceptor
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`fluorophore was in close proximity with a molecule labeled with a donor
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`fluorophore. Another application is in the use of labels known as “tandem labels.”
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`29. A tandem label is a label composed of donor fluorophores and
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`acceptor fluorophores that are designed to remain in close physical contact with
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`each other. One purpose of tandem labels was to increase the range of emission
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`wavelengths available to fluorophores with a common excitation wavelength,
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`which can permit the detection of more labels using a single excitation source. As
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`explained in Szöllősi:
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`The detection of multiple fluorescent biomarkers on a single cell by flow
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`cytometry provides a powerful tool for cell analysis. Therefore, there is
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`always a need for more fluorophores that can be used simultaneously in
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`immunofluorescence applications. Because most clinical flow cytometers
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`utilize single-laser excitation, conventional low molecular weight
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`fluorescent dyes having small Stokes shift cannot be used for the
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`detection of more than two fluorescent parameters on a single cell. The
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`introduction of the phycobiliprotein-based tandem dyes has significantly
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`enhanced the capabilities of these single-laser flow cytometers for
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`performing multiparametric analysis (5, 60,86,115).
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`…The large Stokes shift associated with the fluorescence resonance
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`energy transfer of these tandems produces emission that can easily be
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`resolved from direct PE or fluorescein emissions. The PE-TR tandem
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`emits at 613 nm, the APC-PE and Cy5-PE at 660–670 nm, and the Cy7-
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`PE at 780 nm, respectively (60,86,115). The largest Stokes shift of 300
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`nm is provided by the Cy7 PE conjugate that can be efficiently excited at
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`488 nm and emits at 780 nm. The Cy5-PE and Cy7-PE tandems are very
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`bright fluorescent reagents and can be used with fluorescein and PE with
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`excitation from a single laser light (488 nm) providing a useful
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`fluorophore set for four color immunofluorescence (86).
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`Ex. 1012, Szöllősi at 167-168.
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`Another motivation to use tandem labels is to achieve an emission
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`wavelength
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`that avoids cellular autofluorescence which can create
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`background noise:
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`The other motivation in the development of the red excitable tandem
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`dyes was the challenge to avoid fluorescence near or at cellular
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`autofluorescence, which is often a limiting factor in signal detection.
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`Competition with autofluorescence is much less of a problem when the
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`dye selected is in the red and far-red region.
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` Ex. 1012, Szöllősi at 168.
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`30. With either fluorescence or FRET labels, one of skill in the art could
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`use more than one label in a particular assay, such as two different fluorescent
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`labels, two different FRET labels, or one fluorescent label and one FRET label.
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`Doing so only requires choosing labels that have different emission wavelengths
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`that could be separately detected by an optical detector.
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`31. To detect and analyze proteins within a cell, there were at least two
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`known techniques at the time of the invention. The first is simply to lyse the cell
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`so that the contents of the cell, including intracellular proteins, become exposed
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`and could then be directly contacted by antibodies forming antibody-protein
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`complexes. This technique destroys the cell, so another known technique that
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`could keep a cell intact was through fixation and permeablization of a cell.
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`Fixation of cells uses chemical agents to stabilize a cell’s cellular and sub-cellular
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`structures. Permeabilization then weakens cell membranes so that antibodies can
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`pass through the membrane to the interior of the cell, where the antibodies can then
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`contact intracellular proteins forming antibody-protein complexes.
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`32.
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`In the study of intact cells, one popular detection method was with a
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`flow cytometer with fluorescence capabilities. When this type of device is used to
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`analyze and sort cells it is known as a fluorescence-activated cell sorting (FACS)
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`device, which was a common application of flow cytometry at the time of the
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`invention. A FACS instrument can sort individual cells from populations of cells
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`on the basis of as many parameters as can be simultaneously detected by
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`fluorescence. For example, at the time of the invention flow cytometers could
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`separate cells on the basis of eleven different fluorescent labels, using various
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`fluorescent dyes (e.g., fluorophores) and lasers as illustrated in the graphic below:
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`400
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`500
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`600
`700
`Wavelength (nm)
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`Excitation
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`Emission
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`800
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`Bandpass
`filter
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`Ex. 1011, Baumgarth at 79.
`Ex. 1011, Baumgarth at 79.
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`33. As seen in the figure above, the various known distinguishable labels
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`are shown on the left side with the graphic depicting the excitation and emission
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`wavelengths. For example, one label could be FITC and second label is PE. Id. at
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`83. Both labels are excitable by the same 488nm Argon laser but emit light at
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`different detectable emission wavelengths of 525nm and 575nm respectively. As
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`an alternative, the labels could include the tandem dyes Cy5PE or Cy5.5PE, which
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`are excitable by the same 488nm Argon laser but emit light at even greater shifted
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`emission wavelengths of 665nm and 720nm respectively As stated in the study
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`referenced above, the fluorophores, lasers, and filters used in the figure above were
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`in “routine use” at the time. Id. at 79.
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`34. A listing of combinations of fluorescent dyes that were known by the
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`time of the invention to be simultaneously distinguishable is shown in Table 1.
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`Reproduced below is the portion of Table 1 for a four parameter experiment, where
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`four different fluorescent dyes are listed that can all be simultaneously resolved.
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`Id. at 83. As shown on Table 1, it was known that FITC, PE, CyPE5, Cy5.5PE or
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`Cy7PE could be simultaneously detected with a flow cytometer using only a single
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`488nm Argon laser.
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`35. Therefore, at the time of the invention the importance of detecting the
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`activation state of proteins in cellular signaling pathways was known, antibodies
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`specific to the activated form of proteins within these pathways were known,
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`labeling of antibodies was known and the detection of cells having multiple
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`different labeled antibodies using flow cytometry was known.
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`V. OVERVIEW OF THE `584 PATENT
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`36. The `584 patent states “the invention relates to simultaneously
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`detecting the activation states of multiple proteins in single cells using flow
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`cytometry.” Ex. 1001 at 1:17-20.
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`37. However, the `584 patent acknowledges that protein investigation
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`performed prior to the time of the invention already analyzed “a few proteins at a
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`time.” Id. at 3:43-45 (“Biochemical investigation of protein expression and
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`function have traditionally focused on one or a few proteins at a time.”)
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`38. The `584 patent states, “In particular, the invention provides
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`activatable prot
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