Columbia Ex. 2076
`Illumina, Inc. v. The Trustees
`of Columbia University in the
`City of New York
`IPR2020-00988, -01065,
`-01177, -01125, -01323
`
`

`

`
`
`A.
`
`ROQUCSES 1.0... cceceeeeesstesseneeeeeeeeeeeeeeeeeeeeeneenaneaansaaesaaeeeaeeeseeeeeaaeenanssesssseessseensees 3
`
`B. Further cited document...............c:cceceeeeeeceeeeeeeeeeeeeeeeeeeeneeeeeeeneeeseeeeeeeeneeeeee 3
`
`C. General comments and outline of arguments.............csseesseesssceseeeeeeeenees 8
`
`1.
`General COMMENTS 0.0... eee e cee eecceee eee ee eeccee sense eeceeeeeecncneeeeeeesnisueeeeeees 8
`I]. Outline Of APQUMENES 00.0... cece ceceeeeesseecaeeeeeeeeeeeceeeeeccessssessseeeaaeeeeeneeeesees 9
`
`D. Conceptual disclosure of SBS versus putting SBS into practice........ 13
`
`The concept of SBS was knownat the effective date of the opposed
`|.
`patent and plausibly established....................ccccccsssessseeeceeeeceeeeeescesstestsssseasaeeees 13
`Il. The applicable case law .............cccceccceescssseeseeeeeeeeeceeceeescessttssseeaaaeeees 18
`
`E.
`
`Insufficiency Of GiSCIOSUIE ........... ccc cscccesseceeeeeeeeeeeeeneeeesneeesceeseeeseeeenseeennas 20
`
`1.
` Germeral COMMENTS «2.0.0... cece cee eeeecee eee ee eeeeeaeeeeeeeceeeceeeesccareeseeeeseneeeeees 20
`ll. Difficulties in putting the invention into practice with allyl and MOM as 37--
`©) ao) ke)(-\e1he [80] 0]6:rn 22
`1.
`Allyl aS 3°-OH protective Qroup.............c ccc ccsesceeeeceeceeesscessetststeeaseeeees 24
`1.1
`Finding a suitable DNA polymerase wasnotroutine .................... 24
`1.2
`Finding suitable cleavage conditions for allyl was not routine ...... 30
`1.2.1 The cleavage conditions set forth in the patent for allyl are not
`Suitable for SBS ooo... ee ceeeccecccceeececceeeeeeeececceeeeeeseecceeeeeeesneeseeeeseeneaeeteeeeees 30
`1.2.2 SBS-compatible cleavage conditions for allyl were not part of the
`COMMON general KNOWlEdGE.............ccccceeeesceseeeeeeeeeeseececessttststeeaseeeees 32
`oC
`S10] 00100:| 35
`2. MOM a3’°-OH protective Group 2.0... ccccccccceccsseesseeeeeeeseeeeeeeeees 37
`Difficulties in putting the invention into practice with 3’-OH protective
`Il.
`groups being as small as allyl and MOM ..................ccccccssssessecceceeeeeeeeeees senna 39
`1.
`Requirementoffitting into the active site of a DNA polymerase.......... 40
`2. Requirement of cleavability with high yields under mild conditions......49
`3. Requirement of having chemical (electronic) properties not to be
`cleaved by the nucleophilic group in the active site of a DNA polymerase .52
`
`F.
`
`INVENTIVE STOP 0.0... ececcceeeeeeeeeseeessneeaaeeesceeeeeeeeeeeeeeeenaaennnesenseeeeseeseeesaeeeeneennnas53
`
`D4 (Tsien) is the closest prior art and the problem underlying the claimed
`|.
`invention amountto providing an alternative SBS ...00... cece eeeeeees 53
`Il. D15 (Sternple) as closest prior art oo... cccecceeceeeeseesstssttseeasaeeees 59
`
`G.
`
`H.
`
`Added subject Matter ...........ccccccccccccccss:ees:s:ss::secssseeeseeeeeseeessneenssesenesnes 60
`
`CONCIUSION 0.0... cccceeeeceeceeceeneeeeeneeeeeeuseneeseeneeaeeeeuseesesaeeeueaeeneuaeeseueeeeeseesens 62
`
`

`

`
`
`Requests
`
`We maintain the request to revoke EP 3 034 627 B71 in its entirety on the
`basis of Articles 100 (a), 100 (b) and 100 (c) EPC.
`
`Our requestfor oral proceedings is maintained as well.
`
`Further cited documents
`
`In addition to the documents on file, we herewith file the following
`documentsin order to address the issuesraised in the preliminary opinion
`in connection with the question of insufficiency of disclosure:
`
`D26:
`
`R. E. lreland et al., J. Org. Chem., 1986, vol. 51, no. 5, pp. 635-
`648
`
`D27:
`
`A. Kamal et al., Tetrahedron Letters, 1999, vol. 40, pp. 371-372
`
`D28:
`
`H. Ruparel et al., PNAS, 2005, vol. 102, no. 17, pp. 5932-5937
`
`D29:
`
`WO 2004/018497 A2
`
`D30:
`
`D31:
`
`D32:
`
`A. F. Gardner et al., Nucleic Acids Research, 2002, vol. 30, no. 2,
`pp. 605-613
`
`F. Guibé, Tetrahedron, 1998, vol. 54, pp. 2967-3042
`
`S. S. Flack, Tetrahedron Letters, 1995, vol. 36, no. 19, pp. 3409-
`3412
`
`D33:
`
`J. Ju et al., PNAS, 2006, vol. 103, no. 52, pp. 19635-19640
`
`D34:
`
`J. Guo et al., PNAS, 2008, vol. 105, no. 27, pp. 9145-9150
`
`D35:
`
`Declaration by F. Romesberg dated July 1, 2020
`
`D36:
`
`Declaration by J. Ju dated May 26, 2017
`
`D37:
`
`Declaration by J. Kuriyan dated June 1, 2020
`
`D38:
`
`D39:
`
`K. Davies, The $ 1,000 Genome’, 2010, Free Press, New York,
`Chapter 5
`
`T. W. Greene, P. G. M. Wuts, Protective Groups in Organic
`Synthesis, Third Edition, Chapter Il, 1999, John Wiley & Sons, Inc.
`
`

`

`
`
`D40:
`
`Declaration by S. M. Menchen dated June 1, 2020
`
`D41:
`
`D42:
`
`S. Lemaire-Audoire et al., Tetrahedron Letters, 1994, vol. 35, no.
`47, pp. 8783-8786
`
`S. Lemaire-Audoire et al., Journal of Molecular Catalysis A:
`Chemical 116, 1997, pp. 247-258
`
`D43:
`
`V. M. Swamy, Synlett, 1997, pp. 513-514
`
`D44:
`
`C.-H. Lee et al., Proc. Natl. Acad. Sci. USA, 1981, vol. 78, no. 5,
`pp. 2838-2842
`
`D45:
`
`L. Levine et al., Biochemistry, 1963, vol. 2, no. 1, pp. 168-175
`
`D46:
`
`D47:
`
`D48:
`
`E. J. Corey et al., Tetrahedron Letters, 1975, no. 38, pp. 3269-
`3270
`
`E. J. Corey et al., Tetrahedron Letters, 1975, no. 31, pp. 2643-
`2646
`
`Grant application by Dr. Romesberg and Dr. Ju issued in May
`2006
`
`D49:
`
`EP 1 530 578 B1
`
`DSO:
`
`Submission of September 23, 2016 by Patentee
`prosecution
`
`during
`
`DS1:
`
`Thesis by Q. Meng, Columbia University, 2006
`
`DS2:
`
`Declaration by S. Peisayovich dated 23 March 2021
`
`DS3:
`
`Patentee’s responsein IPR proceedings dated September 9, 2020
`
`DS4:
`
`WO 98/53300 A2
`
`DS55:
`
`Bentley et al., Nature, 2008, vol. 456, no. 6, pp. 53-59
`
`D26 and D27 are namedin paragraph [0007] of the contested patent as
`evidence of cleavage conditions for MOM and allyl which allegedly are
`compatible with SBS requirements. They are thus both known to the
`Patentee and relied upon by the Patentee for their sufficiency of disclosure
`arguments.
`
`D40 is introduced as evidencethat the cleavage conditions set forth in
`the contested patentfor allyl (i.e., D27) are not suitable for SBS. D40
`
`

`

`
`
`is an expert declaration that has been submitted by the Patentee in the
`context of IPR proceedings relating to US patent 9,868,985, claiming
`priority to the same patent application as the opposed patent, and is thus
`known to the Patentee.
`
`D28 is evidence that extensive research efforts were required after the
`relevant date of the contested patent to identify a polymerase, namely
`a mutant 9°N polymerase, and cleavage conditions which would allow
`allyl to be used as a 3’-OH protective group for SBS. It is co-authored by
`Dr. Ju and is thus known to the Patentee. These research efforts were
`necessary because the polymerase and cleavage conditions in the
`contested patent were not functional.
`
`D29 and D30are introduced as evidence that the only polymerase which
`D28 discloses to be functional with allyl, namely a particular mutant 9°N
`polymerase, was developed after the relevant date of the contested
`patent. D51 and D48is further evidence which shows that polymerases
`other than the 9°N polymerase which were knownat relevant date of the
`contested patent were not suitable for effective SBS when using allyl
`as a 3’-OH protective group. D51 is a PhD thesis from the group of Dr. Ju
`at Columbia. D48 is a grant application by Dr. Romesberg and was
`supported by Dr.
`Ju. Further, both were previously cited in US
`proceedings in which Columbia is participating. These documents are
`thus known to the Patentee.
`
`D31 and D32 are both references which D28 refers to when explaining
`why the cleavage conditions disclosed in these documents were not
`suitable for allyl. D41 and D42 are introduced as evidence that the
`cleavage conditions which were used in D28 for allyl required extensive
`research activities and were not part of the common general knowledge.
`These cleavage conditions differ from those mentioned in the patent, i.e.,
`D27 (Kamal).
`
`D33 is another post-published publication by the group of Dr. Ju and thus
`known to the Patentee.
`It
`is
`introduced as evidence that
`the
`
`photocleavable linker as shownin the various figures of EP’627 had to be
`replaced by an allyllinker in order to implement efficient SBS. D50 is a
`submission by the Patentee during examination of the contested patent
`which comments on D33.
`
`D34 is another post-published publication by the group of Dr. Ju. It shows
`that, based on the findings of D28 (Ruparel) and D33 (Ju), only after
`switching to azidomethyl as a 3’-OH protective group SBS beyond 20
`nucleotides became possible.
`
`

`

`
`
`D35 and D37 were submitted in US Inter Partes review proceedings
`relating to US patents 10,407,458; 10,407,459; 10,428,380, 10,435,742;
`and 10,457,984. D36 was submitted on behalf of the present Patentee
`during examination proceedings before the USPTO relating to US
`15/167,917. D35-D37 are thus all known to the Patentee. They are
`introduced in response to D19 (Ju) to show that the application as filed
`fails to provide guidance for the “as small as” feature. These documents
`further show that different than alleged by the Patentee, azidomethyl
`could not be identified without an undue burden as a 3’-OH protective
`group which would meet the “as small as” feature.
`
`D49 is introduced as evidence establishing azidomethyl as a 3’--OH
`protective group for SBS methods required inventive activity.
`
`D38 is introduced to show that it was self-evident that any SBS method
`had to be able to sequence DNA molecules of at least 20 nucleotides.
`
`D39 is introduced as evidence of cleavage conditions for protective
`groups such as propargyl and methylthiomethyl (MTM) which the skilled
`person was aware of from organic chemistry. D43-D47 are introduced as
`evidence that these cleavage conditions are not suitable for SBS.
`
`D52 is introduced as evidence that other than alleged by the Patentee
`azidomethyl does notfit into the active site of rat DNA polymerase of
`Figure 1 of the contested patent.
`
`D53 is introduced as evidence that also the Patentee took the position
`that one cannot tell
`if a given protecting group such as allyl can be
`incorporated by any polymerase without data showing it could work. The
`document was submitted in US Inter Partes review proceedings relating
`to US patent 10,407,458.
`
`D54is filed to show that D4 was a knownreference to those working in
`the field of SBS. This patent wascited in the IPRs between the parties, so
`Columbia should be awareof this patent.
`
`D55 is a publication by the Opponent in which SBS using azidomethyl as
`3’-OH protective group was reported.
`
`We note that we cite declarations and statements from US proceedings
`such as D35-D37, D40 and D53 with respectto the technical explanations
`and factual statements made therein. We understand that the OD will
`
`need to evaluate this evidence and these statements applying the legal
`standards of the EPC.
`
`

`

`
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`Further, we herewith file Annex 1 summarizing the “Developments in DNA
`sequencing by synthesis (SBS) regarding the 3’-OH protective groups to
`achieve
`efficient
`sequencing” with
`special
`consideration
`of
`the
`developments before and after the effective date of the opposed patent
`EP’627. We refer to this Annex 1
`in this submission where appropriate.
`
`All of the newly cited documents are filed in direct reaction to the
`Responseto the Opposition of April 16, 2020 and the Preliminary Opinion
`of the OD of August 6, 2020 in order to address the question of
`insufficiency of disclosure and in particular the Patentee’s allegation that
`the alleged invention wasthe first implementation of an efficient SBS
`method and that in view of this alleged breakthrough, they would be
`entitled to enjoy protection not only for allyl and MOM as 3’-OH protective
`groups but also for 3’-OH protective groups which are “as small as’allyl
`and MOM.
`
`The documents submitted herewith show that the allegation underlying
`Patentee’s arguments, namely that by using allyl and MOM as 3-OH
`protective group they provided the first efficient SBS method is wrong. The
`documentsfurther establish that the skilled person could not rely on their
`common general knowledge to identify working 3’-OH protective groups
`which are “as small as’ allyl and MOM without an undue burden.
`
`The documents show that new polymerases outside the common
`general knowledge had to be developed when using, é.g., allyl as a 3’-
`OH protective group. The documents further show that the cleavage
`conditions which are disclosed by the contested patent for allyl are not
`suitable for SBS. They also show that suitable cleavage conditions were
`not available for 3’-OH protective groups such as methylthiomethyl which
`has been proposed by the Patentee as a 3’-OH protective group whichis
`allegedly covered by the claims. The documents also establish that
`azidomethyl which has been advanced by the Patentee as another 3’--OH
`group whichis allegedly covered by the claims does not meet the features
`of the claims.
`
`The documents are thus prima facie relevant for the assessment of the
`opposition ground of lack of sufficiency of disclosure.
`
`We further note that the documents filed herewith are known to the
`
`Patentee from parallel US proceedingsinvolving the sameparties.
`
`We thus respectfully request
`admitted into the proceedings.
`
`that
`
`the documents filed herewith be
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`27
`
`28
`
`29
`
`30
`
`31
`
`General comments and outline of arguments
`
`In this submission we will focus on the issues raised in the Responseto
`Opposition of April 16, 2020 and the Preliminary Opinion of the OD of
`August 6, 2020. We emphasize that we maintain all arguments set forth
`with our Grounds of Opposition and reservethe right to comment on these
`in the oral hearing.
`
`General comments
`
`In its introduction to the technology of SBS in its Response to Opposition
`of April 16, 2020, the Patenteecriticizes D4 (Tsien) and D13 (Dower) as
`being theoretical and prophetic.
`
`Yet, Patentee’s own contested patent is equally theoretical and prophetic
`as D4 (Tsien) and D13 (Dower) and devoid of any worked examples. The
`Patentee would not make SBS work for another five years after its priority
`date, in 2006.
`
`Critically, to finally make SBS work in 2006 required the Patentee to make
`several changes to its prophetic approach described in the contested
`patent. As just one example, discussed in more detail below, it took the
`Patentee years to find a polymerase that would sufficiently incorporate a
`3’ protected nucleotide, and the one they eventually found only became
`available after the claimed priority date of the Opposed patent. None of
`these necessary changesare reflected in the contested patent.'
`
`While Patentee criticized the prior art as showing a 100% failure rate for
`finding a protecting group that would work for SBS, the Patentee fared no
`better in the years immediately following the priority date of the contested
`patent.
`It was only after the Patentee started copying the Opponent’s
`patented 3’ protecting group — not disclosed in the contested patent — that
`it achieved real success at making SBS work. However, by that time the
`Opponent had launched a commercial SBS product founded on that, and
`several of its other critical inventions that together made SBS actually
`work well. This achievement earned the Opponent a publication of its
`working method in the prestigious journal, Nature,
`in 2008, an unlikely
`honor had the patentee actually been the one to make SBS workas it has
`claimedin itsfilings.2
`
`1 We refer to the discussion in section C.II.1.1.
`2 See D55, p. 53, right paragraph showsthat azidomethyl was used as 3’-OH
`protective group.
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`32
`
`33
`
`34
`
`35
`
`Patentee’s claim that it was the one that made SBS work beyond what
`was knownin the art is disingenuous. And any suggestion that the 2001
`priority application contained the details necessary to do sois inconsistent
`with what is known about Patentee’s subsequentstruggles.
`
`Outline of arguments
`
`We understand the Patentee to argue that the contested patent allegedly
`identifies for the first time the precise characteristics which modified
`nucleotides have to fulfil in order for SBS to efficiently work.*
`
`Considering the claims these characteristics apparently pertain to the
`properties and positioning of labels and 3’-OH protective groups in the
`modified nucleotides, namely:
`
`(i)
`
`(ii)
`
`(iii)
`
`The function of labels and protective groups should be split
`and attached to different parts of the nucleotides.
`
`Cleavable labels should be attached to the base of the
`nucleotides.
`
`Chemically cleavable protective groups should be attached to
`the 3’-OH group.
`
`Critical issues of the present proceedingsclearly pertain to the properties
`of the 3’-OH protective groups.* The 3’-OH protective groups must be:
`
`(i)
`
`(ii)
`
`(iii)
`
`recognized as substrates by DNA polymerases. This
`requirement pertains to the steric and chemical properties
`of the 3’-OH protective groups;
`
`cleavable under mild conditions. This requirement pertains
`to cleavage conditions and thus (indirectly) also reflects the
`chemical properties of the 3’-OH protective group;®
`
`cleavable with high yield. This requirement again pertains
`to cleavage conditions and thus (indirectly) also relates to
`the chemical properties of the 3’-OH protective group;®
`
`3 See Response to Opposition of April 16, 2020, p. 5, 4" paragraph.
`4 See Preliminary Opinion of August 6, 2020; items14.4 and 17.2.
`5 The requirement reflects that cleavage conditions do not negatively affect the DNA
`during the sequencing reaction.
`§ This requirementreflect that, unless each sequencing cycle is performed with high
`yield, it is not possible to sequence fragments having the minimal required length of
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`(iv)
`
`compatible with the nucleophilic group in the active site
`of DNA polymerases. This requirement also relates to the
`chemical properties, in particular the electronic properties
`of the 3’-OH protective group.
`
`Wewill set forth that in section D.I that the contested patent makes no
`conceptual contribution over the art whatsoever in so far as the
`contested patent alleges
`to have identified the above functional
`characteristics that labels and 3’-OH protective groups needtofulfil.
`
`The functional properties of labels and 3’-OH protective groups hadall
`been already advancedbyprior art references such as D4 (Tsien). The
`conceptual requirements of cleavage conditions were known aswell. We
`again refer to section D.I and the discussion of D4 (Tsien).
`
`As noted above, the Patentee argues that the claimed invention would
`provide the first reduction to practice for efficient SBS by identifying
`the MOM and allyl group as suitable 3’-OH protective groups. According
`to the Patentee it would thus bejustified that the claims also pertain to 3’-
`OH protective groups which have the same size as the MOM and allyl
`group and, by consequence, also provide for efficient SBS.
`
`Wewill address in sections E and F that by selecting these specific 3’°-OH
`protective groups, i.e., allyl and MOM, the claimed SBS methods do not
`meet the requirements of Art. 83 and 56 EPC.
`
`is apparent from the functional
`it
`Regarding sufficiency of disclosure,
`requirements which are mentioned in above paragraph 35 that for
`efficient SBSit is not sufficient to know the chemical and steric properties
`of the 3’-OH protective group.
`
`For example, one must also know the cleavage conditions under which
`these groups can be deprotected and such cleavage conditions must be
`compatible with the requirements for SBS given that the claims pertain
`to such methods. Cleavability of protective group perse is not sufficient
`for SBS.
`
`As will be set forth in section E.1I.1.2, the application as filed fails to
`disclose chemical cleavage conditions for allyl as 3’-OH protective
`group which would be mild and provide a high yield.
`
`36
`
`37
`
`38
`
`39
`
`40
`
`A
`
`42
`
`approximately 20 nucleotides. Sequenced fragments of shorter length cannot be
`reliably aligned to reference sequences. We point to D38, Chapter 5, p. 109, last
`sentence.
`
`10
`
`

`

`
`
`MAIWALD
`INTELLECTUAL
`PROPERTY
`
`43
`
`44
`
`45
`
`46
`
`47
`
`48
`
`49
`
`50
`
`51
`
`52
`
`The cleavage conditions which are disclosed by the contested patent for
`allyl are not suitable for SBS. Cleavage conditions for allyl which would
`be compatible with SBS were also not part of the common general
`knowledge. Post-published evidence such as D28 (Ruparel) confirms
`that suitable cleavage conditions for, e.g., allyl were identified only
`several years after thefiling date.
`
`Similar considerations apply with regard to MOM as 3’-OH protective
`group.
`
`Just naming allyl and MOM as 3’-OH protective groups was thus not
`sufficient to make SBS work because these 3’-OH protective are not
`necessarily linked to SBS-compatible cleavage conditions.
`
`At the filing date of the patent, a skilled person could thus not practice the
`claimed invention without an undue burden at least as far as allyl is
`concerned, i.e., one of the only two specifically disclosed 3’-OH protective
`groups because suitable cleavage conditions were missing.
`
`For these reasons alone, it was not justified to extend the scope of the
`claims to alternatives just because these alternatives allegedly meet the
`size requirementofallyl and MOM.
`
`The application as filed also did not provide the skilled person with any
`guidance howto identify 3’--OH protective groups different than MOM or
`allyl which would have the above-mentioned chemical and steric
`properties and for which suitable cleavage conditions would be known.
`The skilled person could also not identify such other 3’-OH protective
`groups and their cleavage conditions by relying on common general
`knowledge. Werefer to section E.Ill.
`
`The requirements of Art. 83 EPC are thus not met.
`
`Other than preliminarily found by the OD, the contribution of the claimed
`invention can thus not be seen in the provision of a generally applicable
`functional SBS method in which the modified nucleotides can beefficiently
`incorporated and deprotected.’
`
`As noted above, we reject Patentee’s argumentthat the alleged invention
`would provide a new conceptual approach for SBS.
`
`To the extent that making SBS work was within the general skill of people
`in the art in 2001, those generally skilled artisans were equally likely to
`
`? See Preliminary Opinion of August 6, 2020, item 17.2.
`
`11
`
`

`

`
`
`get there from D4 (Tsien) or D13 (Prober) as they were from the contested
`Patent.
`
`We respectfully submit that the alleged invention does not go beyond an
`obvious alternative over the prior art. We refer to section F.
`
`The requirements of Art. 56 EPC are thus not met.
`
`We further respectfully submit that, when assessing the requirements of
`Art. 123(2) and 76(1) EPC, the OD hasincorrectly limited its analysis to
`finding a linguistic basis for the individual features but has ignored the
`technical context in which these features are disclosed and whether the
`claimed feature combination pertains to technical subject matter which
`was does notreflect the technical context of this disclosure.
`
`the application as filed discloses that any 3’-OH
`that
`We submit
`protective group needs to have the above-mentioned chemical and
`steric properties.
`
`However, the “as small as” feature pertains only to the steric properties of
`the reference groups,
`i.e, MOM and allyl, but omits the chemical
`properties. The claimed subject matter is thus is based on undisclosed
`generalization.
`
`The requirements of Art. 123(2) and 76 (1) EPC are thus not met.
`
`53
`
`54
`
`55
`
`56
`
`57
`
`58
`
`12
`
`

`

`
`
`D.
`
`Conceptual disclosure of SBS versus putting SBS into practice
`
`I.
`
`The concept of SBS was knownattheeffective date of the opposed
`patent and plausibly established
`
`59 As explained in section C.1.4 of the Grounds of Opposition, the concept
`of DNA sequencing by synthesis (SBS) was knownin theart at the
`effective date of the opposed patent EP’627.
`
`60 As evidencein this regard, reference is again made to D4 (Tsien), which
`discloses:®
`
`the present invention provides a method for
`“In one aspect,
`determining the deoxyribonucleotide sequence of a single stranded
`DNA subject molecule. This method involves synthesizing,
`in the
`presence of a multitude of identical copies of the subject DNA, the
`DNA molecule which is complementary to it. This synthesis is
`carried out using deoxyribonucleotide triphosphates (dNTP) in a
`stepwise serial manner so as to simultaneously build up numerous
`copies of the complementary molecule, dNTP by dNTP. As each
`dNTP is added to the growing complementary molecules,
`it is
`identified by way of an appropriate label (i.e., reporter group). By
`noting the identity of the bases present in this complementary
`molecule and using standard rules of DNA complementation, one
`can
`translate
`from the
`complementary molecule
`to
`the
`corresponding original subject molecule and thus obtain the
`deoxyribonucleotide sequence of the subject molecule.” (Emphasis
`added)
`
`61 D4 (Tsien) further discloses that each dNTP is modified to contain a
`cleavable protective group at its 3’-OH position:®
`
`“Blocking Groups and Methods for Incorporation
`
`The coupling reaction generally employs 3'hydroxyl-blocked dNTPs
`to prevent inadvertent extra additions.” (Emphasis in underlining
`already in D4)
`
`62 D4 (Tsien) then discusses various aspects of the 3’-OH protective group
`and the label on, e.g., p. 24-27. In this context, D4 considers incorporating
`the label into the 3’-OH protective group.'°
`
`8 D4, p. 6, |. 34-p. 7, |. 14; claim 1.
`° D4, p. 20, Il. 25-27; p. 23, Il. 28-31.
`10 D4, p. 21, 1. 4-8.
`
`13
`
`

`

`
`
`63 However, D4 (Tsien) makes explicitly clear that incorporating the label into
`the 3’-OH protective group is only one option and that the label can as
`well be distinct from the 3’-OH protective group. For this case D4 (Tsien)
`points out that the label can be attached to the baseof the nucleotide:"'
`
`“While the above-described approaches to labeling focus on
`incorporating the label into the 3'-hydroxyl blocking group, there are
`a numberofalternatives - particularly the formation of a 3'-blocked
`dNTP analogue containing a label such as a fluorescent group
`coupled to a remote position such as the base. This dNTP can be
`incorporated and the
`fluorescence measured and removed
`according to the methods described below.” (Emphasis added)
`
`64
`
`introduction to labelling in D4 (Tsien) only explicitly
`the first
`In fact,
`mentions
`the base as
`a potential
`location for
`the
`label, while
`acknowledging that others can exist.'2
`
`“When they are each tagged or labeled with different reporter
`groups, such as different fluorescent groups, they are represented
`as dA'TP, dC"TP, dG"'TP and dT"'TP. As will be explained in more
`detail below,
`the fact
`that
`the indication of labeling appears
`associated with the "nucleoside base part" of these abbreviations
`does not imply that this is the sole place where labeling can occur.”
`
`65 Additionally, D4 (Tsien) singles out labeling of the nucleobase portion as
`having been “reported to exhibit enzymatic competence” and “show
`enzymatic incorporation” of the labeled nucleotides.'®
`
`66 D4 (Tsien) thus clearly teaches that the label and 3’-OH protective group
`can be split and attached to different positions of the nucleotide.
`
`the
`regarding
`guidance
`provides
`also
`(Tsien)
`67 D4
`characteristics of the cleavable 3’-OH protective group: '4
`
`functional
`
`“The criteria for the successful use of 3’-blocking groups include
`
`(1) the ability of a polymerase enzyme to accurately and efficiently
`incorporate the dNTPscarrying the 3’-blocking groups into the
`cDNA chain,
`
`11 D4, paragraph bridging p. 27-28.
`12D4, p. 10, |. 7-14. And see Fig. 2 using that same nomenclature that had been
`defined at p. 10.
`13 D4, p. 28, |. 5-18.
`14 D4, p. 20, |. 28 - p. 21, |. 3.
`
`14
`
`

`

`
`
`(2) the availability of mild conditions for rapid and quantitative
`deblocking, and
`
`(3) the ability of a polymerase enzyme to reinitiate the cDNA
`synthesis subsequentto the deblocking stage.”
`
`68 The functional characteristics which the 3’-OH protective group should
`fulfil are further emphasized by D4 (Tsien) when commenting on
`deprotection of the blocked 3’-OH group,
`i.e.,
`the required cleavage
`conditions:'®
`
`“Deblocking Methods
`
`After successfully incorporating a 3'-blocked nucleotide into the
`DNA chain, the sequencing -scheme requires the blocking group to
`be removed to yield a viable 3-OH site for continued chain
`synthesis. The deblocking method should:
`
`(a) proceed rapidly,
`
`(b) yield a viable 3'-OHfunction in high yield, and
`
`(c) not interfere with future enzyme function or denature the
`DNAstrand.
`
`(d) the exact deblocking chemistry selected will, of course,
`dependfo a large extent upon the blocking group employed.”
`
`68 This shows that D4 (Tsien) did not consider cleavability of 3’-OH
`protective group per se to be sufficient for SBS. Cleavage of 3’-OH
`protective group had to be possible under conditions which are mild,
`which are rapid, and which are quantitative. These cleavage conditions
`reflect requirements which are inherent to the concept of SBS such as
`that cleavage conditions must, e.g., not destroy the DNA‘'® and proceed
`with a high yield.”
`
`7 D4 (Tsien) does not contain any experimental data showing that the
`claimed SBS method works. However, D4 (Tsien) relies on plausible
`
`18 D4, p. 23, |. 27 - p. 24, |. 5.
`16 As used herein “destroying” also includes denaturing of the DNA, i.e., destroying
`the double-stranded conformation of the template and the growingstrand.
`17 This requirementreflects that, unless each sequencing cycle is performed with
`high yield, it is not possible to sequence fragments having the minimal required
`length of approximately 20 nucleotides. These minimal sequence lengths are
`required for subsequentaligning to reference sequences which underlies the
`massively parallel sequencing approach of SBS.
`
`15
`
`

`

`
`
`71
`
`72
`
`73
`
`74
`
`75
`
`scientific rationales, which proved valid as evidenced by later
`publications.
`
`In this regard it is important to note that D4 (Tsien) not only teaches the
`relevance of the compatibility of a polymerase enzyme with the 3’-OH
`protective groups of
`the dNTPs, but also the requirement of mild
`conditions for rapid and quantitative deblocking without interfering with
`future enzyme function or denaturing the DNA strands.'* Both aspects are
`indeed decisive for putting SBSinto practice.
`
`While D4 (Tsien) does not call out “small” as required criteria for a
`protecting group,
`it does provide several examples of protecting groups
`that are small. And in one context, refers to “lower (1-4 carbon)” groupsof
`a certain type being better.
`
`The importance of the 3’-protective group being sufficiently small to
`allow for the incorporation of the 3’-OH blocked dNTP by a DNA
`polymerase was described by D4 (Tsien)'® (explaining a desire to avoid
`“large and bulky” 3’-OH substituents) and confirmed by D13 (Dower) and
`D5 (Welch) as summarized in the herewith submitted Annex 1 under items
`A.l.2 and A.I.3. That D4 (Tsien) disclosed small groups, and that D13
`(Dower) and D5 (Welch) later explained the mechanisms behind the
`choice of smaller groups (in the case of D5 (Welch), explaining it in terms
`of the limited space shownin the active site of polymerases by crystal
`structure) would have hada skilled person looking at the smaller groups
`of D4 (Tsien) by the late 1990s. This was not a newinsight provided by
`the contested patent.
`
`rapid and quantitative
`The importance of mild conditions for
`deblocking was confirmed by D13 (Dower) and D14 (Metzker) as
`summarized in Annex 1 under items A.II.2 and A.IL.3.
`
`Moreover, D4 (Tsien) suggests allyl ethers as 3’-OH protective groups.”
`Notably, Tsien cites to D9 (Gigg and Warren) for the allyl ether protecting
`group and that reference specifically shows, unsurprisingly, the chemical
`structure for the same three carbon allyl group as is used in the contested
`patent. The suitability of ether protective groups in view of their electronic
`properties was confirmed by D8 (Canard), and 3’-O-ally-dNTPs were
`shown to be incorporated by a DNA polymerase by D14 (Metzker) as
`summarized in Annex 1 under items A.IIl.1 and A.III.2.
`
`18 D4, p. 20, |. 28- p. 21, |. 3; p. 23, |. 32-p. 24, 1.2.
`19 D4, p. 26, 31-35.
`20 D4, p. 24, |. 24—p. 25, |. 4.
`
`16
`
`

`

`
`
`76
`
`Moreover, it was D8 (Canard) who ran experimentsin the mid-1990s, after
`D4 (Tsien) had been filed, to find that ester (ketone) groups were not good
`candidates for SBS because those groups could be cleaved by
`polymerases. Again, this was an insight available to the skilled person
`well ahead of the 2001 priority date, and not an insight uniquely provided
`by the contested Patent.
`
`7
`In (Tsien) advances exactly the same_functionalsummary, D4
`
`
`requirements for the chemical and steric properties of the 3’-OH
`protective group and the cleavage conditions that according to the
`Patentee were allegedly first identified by the contested Patent.
`
`17
`
`

`

`