`
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`_______________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_______________
`
` ILLUMINA, INC.,
`Petitioner,
`
`v.
`
` THE TRUSTEES OF COLUMBIA UNIVERSITY
`IN THE CITY OF NEW YORK
`Patent Owner.
`_______________
`
`Case IPR2020-00988 (U.S. Patent 10,407,458)
`Case IPR2020-01065 (U.S. Patent 10,407,459)
`Case IPR2020-01177 (U.S. Patent 10,435,742)
`Case IPR2020-01125 (U.S. Patent 10,457,984)
`Case IPR2020-01323 (U.S. Patent 10,428,380)
`
`DECLARATION OF KENNETH A. JOHNSON, PH.D.
`
`
`
`
`
`
`
`
`
`
`
`
`
`Columbia Ex. 2071
`Illumina, Inc. v. The Trustees of
`Columbia University in the City of
`New York
`IPR2020-00988, -01065, -01177,
`-01125, -01323
`
`

`

`
`
`Table of Contents
`
`I.
`
`Introduction and Qualifications ....................................................................... 1
`
`II. Materials Considered ....................................................................................... 5
`
`III. The Person of Ordinary Skill in the Art .......................................................... 5
`
`IV.
`
`Illumina’s Challenges Fail ............................................................................... 6
`
`V.
`
`There Was No Expectation Of Success In Achieving The
`Claimed Invention ........................................................................................... 9
`
`A.
`
`B.
`
`C.
`
`Polymerase incorporation of 3'-O-capped nucleotides
`was expected to be rare and unpredictable .......................................... 11
`
`Hovinen and Kwiatkowski provided no expectation of
`incorporation for the MOM embodiment ............................................ 18
`
`There was no expectation of incorporation for the
`claimed adenine nucleotides ................................................................ 26
`
`VI. There Was No Motivation To Arrive At The Claimed Invention ................. 28
`
`A.
`
`There was no interest in the MOM capping group for
`SBS ...................................................................................................... 28
`
`B.
`
`Illumina’s Hiatt theory fails ................................................................ 30
`
`1.
`
`Hiatt’s technology is different than SBS .................................. 30
`
`a.
`
`SBS requires template-dependent
`polymerases .................................................................... 32
`
`b.
`
`SBS requires high-fidelity polymerases ......................... 35
`
`2.
`
`There was no reason to select the MOM capping
`group from Hiatt’s laundry list ................................................. 40
`
`a.
`
`b.
`
`No reason to narrow Hiatt’s capping groups
`to alkyl ethers .................................................................. 40
`
`No reason to narrow Hiatt’s capping groups
`to small capping groups .................................................. 42
`
`
`
`ii
`
`

`

`
`
`
`
`c.
`
`No other reason to narrow Hiatt’s capping
`groups ............................................................................. 50
`
`3.
`
`Hiatt’s data supporting incorporation of capped
`nucleotides are highly suspect .................................................. 59
`
`C.
`
`A POSA would not have been motivated to use the
`MOM capping group because there was no expectation
`that it would be efficient enough to sequence twenty base
`pairs ..................................................................................................... 61
`
`iii
`
`

`

`
`
`I.
`
`INTRODUCTION AND QUALIFICATIONS
`
`1.
`
`I have been retained on behalf of The Trustees of Columbia University
`
`in the City of New York (“Columbia”) in connection with the challenges by
`
`Illumina, Inc. (“Illumina”) to the claims of Columbia’s U.S. Patent Nos. 10,407,458;
`
`10,407,459; 10,435,742; 10,457,984; and 10,428,380 (the “patents-at-issue”).
`
`2.
`
`I am being compensated for my time consulting in this matter at the rate
`
`of $700 per hour. I have no financial interest in the outcome of this proceeding and
`
`my compensation is in no way contingent upon my opinions or the outcome of this
`
`proceeding.
`
`3.
`
`I am the Roger Williams Centennial Professor of Biochemistry at the
`
`University of Texas at Austin and the President and founder of KinTek Corporation,
`
`a company noted internationally for its manufacture of instruments and software that
`
`I designed for advanced kinetic analysis.
`
`4.
`
`I earned a Bachelor of Science in Chemistry with Honors and Highest
`
`Distinction from the University of Iowa in 1971. I earned a Ph.D. in Molecular
`
`Biology from the University of Wisconsin in 1975 for work done with Professor
`
`Gary Borisy.
`
`5.
`
`From 1975 to 1979 I was a postdoctoral scholar working with Dr.
`
`Edwin W. Taylor at the University of Chicago Department of Biophysics and
`
`
`
`1
`
`

`

`
`
`Theoretical Biology. During this time I was supported by fellowships from the
`
`National Institutes of Health and the Muscular Dystrophy Association.
`
`6.
`
`Starting as an Assistant Professor in the Department of Biochemistry
`
`and Biophysics at The Pennsylvania State University in March 1979, I advanced to
`
`the rank of Paul Berg Professor of Biochemistry before leaving in August 1998.
`
`7.
`
`Since August of 1998, I have been the Roger Williams Centennial
`
`Professor of Biochemistry at The University of Texas at Austin initially in the
`
`Department of Chemistry and Biochemistry–the Biochemistry division was
`
`subsequently reorganized into the Department of Molecular Biosciences.
`
`8.
`
`In 1987 I founded KinTek Corporation to manufacture and market
`
`instruments that I designed to perform single turnover and transient-state kinetic
`
`analysis. I also designed and worked closely with computer programmers to develop
`
`a novel approach for modeling and fitting kinetic data that is now adopted
`
`worldwide.
`
`9.
`
`As a Principal Investigator, I have authored more than 195 original
`
`publications and review articles in peer-reviewed journals including Science,
`
`Nature, The Proceedings of the National Academy of Sciences, Journal of Biological
`
`Chemistry, Biochemistry, Journal of Molecular Biology, Journal of Cell Biology,
`
`Antimicrobial Agents and Chemotherapy, Nucleic Acids Research, Journal of
`
`
`
`2
`
`

`

`
`
`Physical Chemistry, and Journal of the American Chemical Society. My
`
`publications have been cited more than 19,000 times.
`
`10.
`
`I have been invited to present lectures on 188 occasions at universities,
`
`biotechnology and pharmaceutical companies and international conferences.
`
`11.
`
`I received the Pfizer Award in Enzyme Chemistry and the Penn State
`
`Faculty Scholar Award, and I am a Fellow of The American Association for the
`
`Advancement of Science and a Fellow of the Biophysical Society. From 1983-1989
`
`I was an Established Investigator of the American Heart Association. I was elected
`
`to organize Gordon Conferences in two fields and to organize the Enzyme
`
`Mechanisms Conference.
`
`12. Since 1985 I have pioneered in the development and application of
`
`accurate methods
`
`to quantify DNA polymerase
`
`fidelity and establish
`
`structure/function relationships governing nucleotide selectivity. I have published
`
`more than 80 papers and review articles on DNA polymerase mechanisms and
`
`nucleotide selectivity involving studies on several enzymes: Klenow fragment of
`
`DNA polymerase I, T7 DNA polymerase, HIV reverse transcriptase, Taq
`
`polymerase, human mitochondrial DNA polymerase, dengue virus polymerase,
`
`hepatitis C viral RNA-dependent RNA polymerase, and the SARS CoV-2 RNA-
`
`dependent RNA polymerase.
`
`
`
`3
`
`

`

`
`
`13.
`
`In my work on DNA polymerases, I have provided novel insights into
`
`understanding nucleotide recognition and fidelity and the structure/function
`
`relationships governing discrimination of polymerases against nucleotide analogues.
`
`This work has included analysis of the evolution of resistance to nucleoside
`
`analogues by HIV reverse transcriptase and the toxic side effects of these nucleoside
`
`analogues due to their incorporation by the human mitochondrial DNA polymerase.
`
`My recent work has established the kinetic and structural basis for inhibition of the
`
`SARS-CoV-2 RNA-dependent RNA polymerase by Remdesevir, the only direct-
`
`acting antiviral drug currently approved by the FDA for treatment of COVID-19.
`
`14.
`
`I have served on the Editorial Board of the Journal of Cell Biology and
`
`the Journal of Biological Chemistry, and I have served as a peer-reviewer on
`
`numerous papers for journals including Nature, Science, Journal of Biological
`
`Chemistry, Biochemistry, Analytical Biochemistry, Biophysical Journal, Journal of
`
`the American Chemical Society, Plos One, Journal of Molecular Biology, Journal of
`
`Physical Chemistry, and Nucleic Acids Research.
`
`15.
`
`I have published a critically acclaimed textbook on modern kinetic
`
`analysis of enzyme mechanisms. This book highlights the novel approaches that I
`
`have developed for quantifying enzyme specificity, including examples of my work
`
`on DNA polymerase fidelity in which we established a new paradigm for
`
`understanding the role of conformational dynamics in enzyme specificity.
`
`
`
`4
`
`

`

`
`
`16. From 1994 to 2001 I was a consultant for Applied Biosystems. In this
`
`capacity, I advised the research team on the application of knowledge gained from
`
`DNA polymerase kinetics to optimize their four-color Sanger sequencing methods.
`
`17. Based on my extensive experience in DNA polymerase kinetics,
`
`nucleotide selectivity and DNA sequencing methods, and because I was active in the
`
`field from 1985 through 2000, I am qualified to judge what a person of ordinary skill
`
`in the art would have known and understood in October of 2000.
`
`18. My curriculum vitae describes in greater detail my professional
`
`experience and qualifications. Ex. 2073.
`
`II. MATERIALS CONSIDERED
`
`19. Appended to my Declaration is a list of documents that I have
`
`considered in connection with this Declaration.
`
`III. THE PERSON OF ORDINARY SKILL IN THE ART
`
`20.
`
`I understand that the claims of the patent-at-issue must be assessed from
`
`the perspective of a hypothetical person of ordinary skill in the relevant art (“POSA”)
`
`before the date the invention described in the claims was made. For the purposes of
`
`
`
`5
`
`

`

`
`
`this Declaration, I have been asked to assume the invention was made by October 6,
`
`2000.1
`
`21.
`
`I understand that a POSA has knowledge of the relevant art, as
`
`described in published and issued patents and in scientific publications, and
`
`possesses ordinary creativity and a basic skill set. For the purposes of this
`
`proceeding, I rely upon Illumina’s criteria for defining a POSA, which I have
`
`reproduced below:
`
`[A] POSA would have been a member of a team of
`
`scientists developing nucleotide analogues, researching
`
`DNA polymerases, and/or addressing DNA sequencing
`
`techniques. Such a person would have held a doctoral
`
`degree in chemistry, molecular biology, or a closely
`
`related discipline, and had at least five years of practical
`
`academic or industrial laboratory experience.
`
`IV.
`
`ILLUMINA’S CHALLENGES FAIL
`
`22.
`
`I have reviewed Illumina’s Petitions, which allege that Columbia’s
`
`patents-at-issue are invalid for obviousness on the premise that it would have been
`
`
`
` My positions herein relate to a POSA prior to Columbia’s invention, i.e., a POSA
`
` 1
`
`without the benefit of the Columbia inventors’ insights set forth in Columbia’s
`
`patents-at-issue.
`
`
`
`6
`
`

`

`
`
`obvious for a POSA to make an embodiment of Columbia’s claims where R is a
`
`methoxymethyl capping group (the “MOM embodiment”), as depicted below:
`
`
`
`23.
`
`I have been informed that an invention is obvious if a POSA would
`
`have been motivated to arrive at the claimed invention with a reasonable expectation
`
`of success. It is my opinion that Illumina’s challenges should be rejected because a
`
`POSA, without using the Columbia patents for guidance, would not have reasonably
`
`expected success in achieving the claimed MOM embodiment, nor would the POSA
`
`have been motivated to arrive at that embodiment.
`
`24. First, I offer a note on nomenclature and the enzymology of DNA
`
`replication. Numerous terms are used to describe the process by which a DNA
`
`polymerase catalyzes formation of a DNA strand that is complementary to a DNA
`
`template according to well established canonical base-pairing rules. For example,
`
`in the table below I show that a high-fidelity DNA polymerase will incorporate dTTP
`
`whenever it encounters an A in the template strand, while T codes for dATP, G codes
`
`for dCTP and C codes for dGTP.
`
`
`
`7
`
`

`

`
`
`
`
`Template
`base
`A
`T
`G
`C
`
`Complementary
`nucleotide
`dTTP
`dATP
`dCTP
`dGTP
`
`Herein I will use the following terms interchangeably to represent the process by
`
`which a DNA polymerase creates a new DNA strand that is complementary to a
`
`DNA template strand: copy, replicate, replication, duplicate, duplication. SBS relies
`
`upon accurate DNA replication to read out the sequence of the template strand by
`
`identifying the complementary nucleotide that is incorporated during each round of
`
`template-directed DNA polymerization. Some enzymes replicate DNA with low-
`
`fidelity in that they are sloppy in their selection of the correct nucleotide. See, e.g.,
`
`Ex. 2085 (Goodman & Tippin) at 166-167. Although these enzymes are required
`
`biologically for processes such as DNA repair, they are of no utility for SBS because
`
`of their high error rate. The term “DNA polymerization” generally refers to the
`
`process synthesis of a DNA strand that is complementary to a temple. In a few
`
`instances reference will be made to a process of DNA synthesis that is independent
`
`of a DNA template. Although this reaction is still catalyzed by a DNA polymerase
`
`(an enzyme that synthesizes a DNA polymer), it should be obvious that an enzyme
`
`that synthesizes DNA without requiring a template strand is of no utility to SBS.
`
`
`
`8
`
`

`

`
`
`V. THERE WAS NO EXPECTATION OF SUCCESS IN ACHIEVING
`THE CLAIMED INVENTION
`
`25.
`
`I have reviewed the claims of the patents-at-issue, which I understand
`
`detail the claimed inventions at issue in these proceedings. Each of the claims of the
`
`patents-at-issue require that the modified nucleotides be capable of being
`
`“incorporated at the end of a growing strand of DNA during a DNA polymerase
`
`reaction.” As an example, below I’ve reproduced claim 1 of the ’458 patent:
`
`A guanine deoxyribonucleotide analogue having the
`
`structure:
`
`
`
`wherein R (a) represents a small, chemically cleavable,
`
`chemical group capping the oxygen at the 3′ position of
`
`the deoxyribose of the deoxyribonucleotide analogue, (b)
`
`does not interfere with recognition of the analogue as a
`
`substrate by a DNA polymerase, (c) is stable during a
`
`DNA polymerase reaction, (d) does not contain a ketone
`
`group, and (e) is not a -CH2CH=CH2 group;
`
`wherein OR is not a methoxy group or an ester group;
`
`
`
`9
`
`

`

`
`
`wherein the covalent bond between the 3′-oxygen and R is
`
`stable during a DNA polymerase reaction;
`
`wherein tag represents a detectable fluorescent moiety;
`
`wherein Y represents a chemically cleavable, chemical
`
`linker which (a) does not interfere with recognition of the
`
`analogue as a substrate by a DNA polymerase and (b) is
`
`stable during a DNA polymerase reaction; and
`
`wherein the guanine deoxyribonucleotide analogue:
`
`i) is recognized as a substrate by a DNA polymerase,
`
`ii) is incorporated at the end of a growing strand of DNA
`
`during a DNA polymerase reaction,
`
`iii) produces a 3′-OH group on the deoxyribose upon
`
`cleavage of R,
`
`iv) no longer includes a tag on the base upon cleavage of
`
`Y, and
`
`v) is capable of forming hydrogen bonds with cytosine or
`
`a cytosine nucleotide analogue.
`
`Ex. 1001 (Columbia’s U.S. Patent No. 10,407,458) at claim 1.
`
`26. As I explain below, it is my opinion that the Columbia inventors were
`
`the first group to recognize the particular chemical and structural features that dictate
`
`whether a capping group could be useful for SBS, and those features are found in
`
`
`
`10
`
`

`

`
`
`the claims of the patents-at-issue, including that the capping group has to be small,
`
`that it cannot be a methoxy, and that it cannot contain an ester or ketone.
`
`27.
`
`It is my opinion that a POSA, prior to Columbia’s invention, would not
`
`have expected the MOM embodiment to achieve polymerase incorporation, for the
`
`reasons I detail below, as required by the claims.
`
`A.
`
`Polymerase incorporation of 3'-O-capped nucleotides
`was expected to be rare and unpredictable
`
`28.
`
`I understand that Illumina’s expert, Dr. Romesberg, has taken the
`
`position that a POSA would have reasonably expected that the MOM embodiment
`
`would achieve polymerase incorporation in an SBS reaction. I disagree, and I
`
`believe that Dr. Romesberg has failed to properly consider the state of the art as of
`
`October 6, 2000, and in particular, has overlooked the prior art’s reported
`
`unpredictability and high failure rate of achieving polymerase-catalyzed
`
`incorporation of nucleotides modified at the 3'-OH position of the nucleotide sugar.
`
`29. High-fidelity DNA polymerases exhibit precise mechanisms for
`
`identifying and incorporating natural nucleotides into strands of DNA, meaning that
`
`they have evolved to reject non-natural (i.e., modified) nucleotides. Ex. 2078
`
`(Johnson 1993). As of the priority date, only a few 3'-O-capped nucleotides were
`
`believed to have been incorporated to any degree, such as the 2-nitrobenzyl and
`
`
`
`11
`
`

`

`
`
`methyl capping groups. See, e.g., Ex. 1039 (Metzker) at 4263 (Table 2) (reporting
`
`incorporation of 2-nitrobenzyl and methyl capping groups).2
`
`30. Relevant to these proceedings, polymerases are particularly sensitive to
`
`modifications made to the 3'-OH on the nucleotide sugar. Prior to 2000, there were
`
`several papers published showing that even the use of a 3'-H in place of the 3'-OH
`
`(i.e., dideoxy nucleoside triphosphate (“ddNTP”)) requires that the polymerase be
`
`engineered with an amino acid substitution to better accommodate this exceedingly
`
`small change, as first noted by Tabor and Richardson. Ex. 2059 (Tabor and
`
`Richardson) at 6343. This paper clearly shows that the 3'-OH is a key touchpoint
`
`for the enzyme to recognize and align an incoming nucleotide for reaction in order
`
`to achieve efficient and accurate duplication of a DNA template strand. Indeed, the
`
`year 2000 marked the end of a decade of research by several large pharmaceutical
`
`companies to find chain-terminating nucleoside analogues for treatment of HIV
`
`infections to build upon the initial use of AZT (3'azido thymidine) to find more
`
`effective and less toxic analogues. The search was to find analogues that would be
`
`
`
` After the priority date, and therefore unknown by the POSA, Metzker in 2007
`
` 2
`
`retracted his data on the 2-nitrobenzyl capping group, acknowledging that there
`
`was an error in his 1994 publication and that the 2-nitrobenzyl capped nucleotide
`
`was inactive in polymerase incorporation assays. See Ex. 1087 (Wu) at 16463.
`
`
`
`12
`
`

`

`
`
`accommodated by HIV reverse transcriptase (HIVRT) and therefore incorporated to
`
`poison replication of the viral RNA, while at the same time excluded by human
`
`polymerases to reduce toxic side effects. In all this time, with the exception of AZT,
`
`not a single 3'-OH substituent received FDA approval for treatment of HIV
`
`infections other than ddNTPs. Ex. 2080 (Sluis-Cremer) at 1409 (Figure 1); Ex. 2088
`
`(Tan) at 117-132. Although analogues in which the 3’OH was replaced by fluorine
`
`were shown to be effective, fluorine is smaller than oxygen. Ex. 2088 (Tan) at 123.
`
`These data, covering a topic that was prominent in the literature and the popular
`
`press, would have reinforced the opinion by a POSA, prior to Columbia’s invention,
`
`that there would not have been an expectation that a larger blocking group on the 3'-
`
`OH would be accommodated by a polymerase useful for SBS unless data had existed
`
`for a particular capping group and polymerase demonstrating otherwise.
`
`31. Other data further suggest the unpredictability and high failure rate of
`
`achieving incorporation with 3'-O-capped nucleotides, including Metzker et al.,
`
`“Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-
`
`triphosphates,” Nucleic Acids Research, 22:4259-67 (1994) (“Metzker”). See Ex.
`
`1039 (Metzker). Metzker disclosed a hypothetical SBS method, which he and his
`
`colleagues called “Base Addition Sequencing Scheme (BASS).” Ex. 1039
`
`(Metzker) at 4259.
`
`
`
`13
`
`

`

`
`
`32. Metzker synthesized eight 3'-O-capped nucleotides and tested their
`
`usefulness for Sanger sequencing and/or SBS with further modifications.3 Ex. 1039
`
`(Metzker) at 4259. Metzker examined whether any of eight polymerases (AMV-
`
`RT, M-MuLV-RT, Klenow fragment, Sequenase, Bst DNA polymerase, AmpliTaq
`
`DNA polymerase, rTth DNA polymerase, VentR(exo-) DNA polymerase) could
`
`incorporate any of eight different 3'-O-capped nucleotides (3'-O-methyl-dATP, 3'-
`
`O-acyl-dATP,
`
`3'-O-allyl-dATP,
`
`3'-O-tetrahydropyran-dATP,
`
`3'-O-(4-
`
`nitrobenzoyl)-dATP, 3'-O-(2-aminobenzoyl)-dATP, 3'-O-(2-nitrobenzyl)-dATP,
`
`and 3'-O-methyl-dTTP). Ex. 1039 (Metzker) at 4263 (Table 2).
`
`33. Metzker reported the results of his assays in Table 2. Metzker
`
`characterized the results of each assay as “-,” meaning that “no activity was
`
`detected,” as “Termination,” meaning that “the reaction containing the test
`
`compound mimicked the migration pattern of the ddNTP control,” or as “Inhibition,”
`
`meaning that “the presence of the test compound prevented the polymerase from
`
`incorporating the natural nucleotides.” Ex. 1039 (Metzker ) at 4263. A POSA would
`
`understand that “Termination”
`
`indicated
`
`incorporation of
`
`the 3'-O-capped
`
`nucleotide.
`
`
`
` SBS requires nucleotides modified to have (i) a capping group and (ii) a label.
`
` 3
`
`Metzker’s nucleotides had a capping group but no label.
`
`
`
`14
`
`

`

`
`
`34. Metzker further characterized the results of his assays with an asterisk
`
`(“*”) to indicate where “activity was incomplete at a final concentration of 250 uM.”
`
`Ex. 1039 (Metzker) at 4263.
`
`35. Out of the sixty-four polymerase/nucleotide combinations tested,
`
`Metzker reported some level of incorporation (reported as Termination or
`
`Termination*), only in ten instances, and only in six of those ten instances was the
`
`incorporation complete (reported as Termination):
`
`
`
`Ex. 1039 (Metzker) at 4263 (Table 2) (highlighting added, red indicates no
`
`incorporation, green indicates successful incorporation, yellow indicates incomplete
`
`incorporation). Stated as percentages, Metzker’s Table 2 shows that only 15% of
`
`the polymerase/nucleotide combinations achieved any level of incorporation. That
`
`amounts to an 85% failure rate. That less than 15% of the polymerase/nucleotide
`
`
`
`15
`
`

`

`
`
`combinations achieved any level of incorporation would have demonstrated to a
`
`POSA the difficulty in achieving incorporation with 3'-O-capped nucleotides.
`
`36. Additionally, Metzker’s assays would have suggested to a POSA
`
`unpredictability as to which polymerase/nucleotide combinations could achieve
`
`incorporation. I note that Metzker reports that “no consistent activity pattern
`
`between the polymerases and the analogues could be discerned,” Ex. 1039 (Metzker)
`
`at 4265, which is certainly consistent with assessment of the results in Metzker’s
`
`Table 2. For example, Table 2 reports that AMV-RT polymerase incorporated an
`
`adenine nucleotide with a methyl capping group (Compound 1), but it failed to
`
`incorporate a thymine nucleotide with the same capping group (Compound 8):
`
`Ex. 1039 (Metzker) at 4263 (Table 2). The opposite was true for the Bst and
`
`AmpliTaq polymerases, which incorporated thymine nucleotides with a methyl
`
`
`
`
`
`16
`
`

`

`
`
`capping group (Compound 8), but were incapable of incorporating adenine
`
`nucleotides with the same capping group (Compound 1):
`
`
`
`Ex. 1039 (Metzker) at 4263 (Table 2).
`
`37. Dr. Romesberg speculated in his deposition that observation of
`
`Inhibition was positive evidence that the analogue was binding to the enzyme in a
`
`manner that could be optimized with altered solution conditions so that it would be
`
`able to be incorporated. Ex. 2072 (Dr. Romesberg Deposition Transcript) at 253:15-
`
`255:12. I disagree. Enzyme inhibitors are classified as either competitive,
`
`noncompetitive or uncompetitive and no data is provided by Metzker to ascertain the
`
`nature of the inhibition. Only competitive inhibitors are thought to bind to the same
`
`active site on the enzyme but even then, such inhibitory binding can arise by the
`
`analogue binding at the active site in an orientation that precludes chemical reaction
`
`or interferes with proper base pairing with the DNA template.
`
`
`
`17
`
`

`

`
`
`38. The only reasonable conclusion supported by Metzker’s data is that a
`
`POSA would have believed that polymerase incorporation of 3'-O-capped
`
`nucleotides was unpredictable. Before the Columbia invention, because of the
`
`absence of an understanding of the spatial and chemical constraints in the
`
`polymerase active site around the 3'-O of a nucleotide, a POSA was unable to
`
`reasonably predict whether a 3'-O-capped nucleotide would be incorporated by a
`
`polymerase, without conducting an assay, as Metzker did with the 2-nitrobenzyl.
`
`Consistent with this unpredictability, I note that Dower repeatedly disclosed the need
`
`to conduct “screening” assays to determine whether a polymerase could incorporate
`
`any given 3'-O-capped nucleotides. Ex. 1030 (Dower) at 18:21-32 (“Screening will
`
`be performed to determine appropriate polymerase and monomer analog
`
`combinations”), 17:43-48 (“Each of these analogs can be tested for compatibility
`
`with a particular polymerase by testing whether such polymerase is capable of
`
`incorporating the labeled analog”), 19:15-20 (“A test using a primer with template
`
`which directs the addition of the nucleotide analog to be incorporated will determine
`
`whether the combination is workable”). Such assays were needed precisely because
`
`the prior art suggested a high failure rate and unpredictability.
`
`B. Hovinen and Kwiatkowski provided no expectation of
`incorporation for the MOM embodiment
`
`39. Given the prior art’s reported high failure rate and unpredictability of
`
`achieving incorporation with 3'-O-capped nucleotides, it is my opinion that prior to
`
`
`
`18
`
`

`

`
`
`Columbia’s invention, a POSA would not have expected the MOM embodiment to
`
`achieve polymerase incorporation, in line with the field’s high failure rate. I
`
`understand that Illumina relies on Hovinen and Kwiatkowski to argue that a POSA
`
`would have had such an expectation. I disagree.
`
`40. First, I take issue with Illumina’s characterization of the modified
`
`nucleotides in Hovinen and Kwiatkowski as containing “substituted MOM” capping
`
`groups. Neither Hovinen nor Kwiatkowski refer to their capping groups as
`
`“substituted MOMs,” and a POSA would not consider them to disclose a 3'-O-MOM
`
`because Hovinen’s and Kwiatkowski’s capping groups are not chemically or
`
`functionally related to MOM. Below I depict the contrasting structures of the three
`
`capping groups:
`
`
`
`MOM embodiment
`(thymine version):
`
`
`
`
`
`
`
`19
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`

`

`
`
`
`
`
`
`Hovinen:
`
`
`
`
`
`
`
`
`
`Kwiatkowski:
`
`
`
`
`
`
`
`
`
`41.
`
`In addition, Hovinen’s capping groups are functionally different than
`
`MOM. Hovinen makes clear that the feature most critical to his capping groups is
`
`the “long flexible acetal arm.” Ex. 1060 (Hovinen) at 212. A POSA would not
`
`understand a MOM capping group (i.e., –O–CH2–O–CH2–) to have that function.
`
`Thus, a POSA would not have considered Hovinen as disclosing a 3'-O-MOM or a
`
`“substituted” MOM, but would instead understand Hovinen to disclose a long
`
`flexible acetal arm.
`
`42.
`
`I also disagree with Illumina’s speculation that a POSA would have
`
`believed that polymerase incorporation of a “substituted” capping group would lead
`
`to an expectation of incorporation of the “unsubstituted” capping group. Such
`
`
`
`20
`
`

`

`
`
`speculation is inconsistent with the prior art and counter to well established
`
`principles in enzymology. For example, in 1959 Koshland proposed an “induced-
`
`fit mechanism” to explain how an enzyme can exclude a substrate that is smaller
`
`than the favored substrate. Ex. 2081 (Koshland) at 252. The ability to discriminate
`
`against a smaller substrate is a common feature of enzyme catalysis. Moreover, an
`
`induced-fit mechanism was known to be an important contributor to polymerase
`
`specificity prior to October 2000. Ex. 2078 (Johnson 1993).
`
`43. For example, Metzker reported that an adenine nucleotide with an allyl
`
`capping group was incorporated (inefficiently, as indicated by Termination*) by the
`
`VentR (exo-) polymerase. Ex. 1039 (Metzker) at 4263 (Table 2). According to
`
`Illumina’s nomenclature, the allyl capping group is a substituted methyl capping
`
`group because one of the hydrogens on the methyl group is replaced by another
`
`chemical group. The methyl portion of the capping group is highlighted below:
`
`
`See Ex. 1039 (Metzker) at 4260 (Figure 2). According to Illumina’s theory, because
`
`the VentR (exo-) polymerase incorporated an adenine nucleotide with a substituted
`
`methyl capping group (i.e., the allyl capping group), it would also be expected to
`
`incorporate an adenine nucleotide with the unsubstituted methyl capping group.
`
`
`
`21
`
`

`

`
`
`Metzker reported that the opposite was true. Specifically, Metzker reported that
`
`VentR (exo-) did not incorporate the unsubstituted methyl capping group:
`
`Nucleotide
`
`Incorporated by VentR
`(exo-) polymerase?
`
`
`
`Yes (inefficiently)
`
`No
`
`
`
`
`
`Ex. 1039 (Metzker) at Table 2 (compounds 3 and 1, VentR (exo-) assay).
`
`44.
`
`In a second example disproving Illumina’s speculation, Metzker
`
`reported that the Bst polymerase incorporated an adenine nucleotide with a
`
`substituted methyl capping group (i.e., a 2-nitrobenzyl capping group, which using
`
`Illumina’s nomenclature is a “substituted” methyl because one of the hydrogens in
`
`methyl is replaced by another chemical group) but did not incorporate the
`
`unsubstituted methyl capping group:
`
`
`
`22
`
`

`

`
`
`
`
`Nucleotide
`
`Incorporated by
`Bst polymerase?
`
`Yes
`
`No
`
`
`
`
`
`Ex. 1039 (Metzker) at Table 2 (compounds 7 and 1, Bst assay).
`
`45.
`
`In a third example, Metzker reported that the Amplitaq polymerase
`
`incorporated an adenine nucleotide with a substituted methyl capping group but did
`
`not incorporate the unsubstituted methyl capping group:
`
`Nucleotide
`
`Incorporated by
`Amplitaq polymerase?
`
`
`
`Yes (inefficiently)
`
`No
`
`
`
`
`
`Ex. 1039 (Metzker) at Table 2 (compounds 7 and 1, Amplitaq assay).
`
`
`
`
`
`23
`
`

`

`
`
`46.
`
`In a fourth example, Metzker reported that VentR (exo-) polymerase
`
`incorporated an adenine nucleotide with a substituted methyl capping group, but did
`
`not incorporate the unsubstituted methyl capping group:
`
`Nucleotide
`
`
`
`Incorporated by
`VentR (exo-)
`polymerase?
`
`Yes (inefficiently)
`
`No
`
`
`
`
`
`
`Ex. 1039 (Metzker) at Table 2 (compounds 7 and 1, VentR (exo-) assay).
`
`47.
`
`In a fifth example, Canard showed that exo- T7 DNA polymerase (also
`
`known as Sequenase) incorporated a nucleotide with a substituted acyl capping
`
`group (under Illumina’s nomenclature it is “substituted” because one of the
`
`hydrogens on the acyl group is replaced by another chemical group), but Metzker
`
`showed the same polymerase did not incorporate the unsubstituted acyl capping
`
`group:
`
`
`
`24
`
`

`

`
`
`Nucleotide
`
`Incorporated by
`Sequenase polymerase?
`
`Yes
`
`No
`
`
`
`
`
`Ex. 2007 (Canard) at 4 (Figure 4 reporting incorporation of Compound 3); Ex. 1039
`
`(Metzker) at Table 2 (Table 2 reporting no incorporation with Compound 2 in the
`
`Sequenase assay).
`
`48.
`
`In a sixth example, Canard showed that AMV-RT incorporated a
`
`nucleotide with a substituted acyl capping group, but Metzker showed the same
`
`polymerase did not incorporate the unsubstituted acyl capping group:
`
`
`
`25
`
`

`

`
`
`Nucleotide
`
`Incorporated by
`AMV-RT polymerase?
`
`Yes
`
`No
`
`
`
`
`
`
`Ex. 2007 (Canard) at 3 (reporting incorporation of Compound 3 (3'-fluorescein
`
`derivative of dTTP with AMV-RT); Ex. 1039 (Metzker) at Table 2 (Table 2
`
`reporting no incorporation with Compound 2 in AMV-RT assay).
`
`49. The above examples, which I do not intend to be exhaustive, do not
`
`support Illumina’s speculative theory that incorporation of a nucleotide with a
`
`“subs