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`——
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`'_Vossius&F’artner GDR PCB 86 0767 81634 Mflnchen Germany.-1
`
`To the
`
`European Patent Office
`
`Munich
`
`'
`
`'
`
` L
`
`PATENTANWALTE
`EUROPEAN PATENT ATTORNEYS
`EUROPEAN TRADEMARK ATTORNEYS
`Dr. VOLKER VOSSIUS. Dipl.-Chem.
`(bis 1992; danach in anderer Kanzlei)
`Dr. PAUL TAUCHNER, Dipl.-Chem.
`r.
`.
`.
`up .-
`em.
`at g:EETT|EERRAHIfqlj:J!EHM»l~3Nh:. <D::>---Phvs-
`Dr. GERHARD HERMANN. Dipl.-Phys.
`JOSEF SCHMIDT. Dip|.—lng.
`Dr, HANS-RAINER JAENICHEN. Dipl.-Biol.
`Dr. ALEXA VON UEXKULL, M.Sc.
`Dr. RUDOLF WEINBERGER, Dipl.-Chem.
`Dr. WOLFGANG BUBLAK, Dipl.-Chem.
`
`’3fE§oi’§:.LMBC‘l2§;.‘éL?;';L"§’ (Biol ,
`EUROPEAN PATENT ATTORNEY
`Dr. RENATE BARTH, Dipl.-Chem.
`RECHTSANWALTE
`HELGA TREMMEL
`BARBARA GUGGENMOS, Dipl.-Chem.
`SIEBERTSTRASSE 4
`81675 MUNCHEN
`GERMANY
`TELEFON: +49-89—41304—O
`FAX G3: +49-89-41304-111
`FAX G4: +49-89-41304-101
`
`
`
`EP-B-0 117 440
`(84 10 0836.0)
`Appeal Case T 0945/97 - 3.3.4.
`ENZO BIOCHEM, INC.
`Our Ref.: 8 808 EP
`
`November 3, 1997
`Uex/Ba/ALR/ne
`
`In the following we submit the grounds in support of the formal appeal dated
`
`September 3, 1997.
`
`‘.
`
`In its Decision the Opposition -Division has revoked the patent alleging that
`neither the subject matter of the claims according to the main request nor to
`the auxiliary requests is allowable under Art. 123(2) EPC,
`is sufficiently
`
`disclosed (Art. 100(b) and Art. 83 EPC), and is novel (Art. 100(a) and Art.
`
`54 EPCl.
`
`We cannot agree thereto for the following reasons:
`
`1.
`
`THE SUBJECT MATTER OF THE PATENT
`
`The subject matter of
`
`the patent provides a method and an
`
`arrangement
`
`for
`
`the detection of polynucleotide
`
`sequences
`
`whereby detection is
`
`effected by
`
`fixing
`
`a
`
`single-stranded
`
`polynucleotide to a solid support which is or is contained within a
`
`system, forming an entity with a labelled polynucleotide probe
`
`BAYERISCHE VEREINSBANK NR. 32920888, BLZ 700202 70 ~ DEUTSCHE BANK A.G. MUNCHEN, NFL65/57 342. BLZ 70070010
`POSTBANK MUNCHEN, NR. 129600-809. BLZ 700 10080. V.A.T. NO. DE 130 751 524
`
`Page 1 Of 15
`
`HOLOGIC EXHIBIT 1016
`
`Hologic V. Enzo
`
`Page 1 of 15
`
`HOLOGIC EXHIBIT 1016
`Hologic v. Enzo
`
`

`
`and generating and detecting the signal originating from the label,
`
`whereby the system is transparent or translucent and non—porous
`
`and the signal is a soluble signal.
`
`THE SUBJECT MATTER OF THE CLAIMS SET DOES NOT GO
`
`BEYOND THE DESCRIPTION AS ORIGINALLY FILED (ART. 123(2)
`
`EPC)
`
`2.1
`
`The terms
`
`"transparentztranslucent system" and "non—porous
`
`substrate or system"
`
`We are of Qthe opinion that
`
`the terms "transparent/translucent
`
`system" and "non-porous substrate or system" are disclosed in
`the specification as originally filed.
`In order to avoid unnecessary
`
`repetitions we want to refer the Board of Appeal to our reply of
`
`February 12, 1996 to the Communication pursuant to Art. 101 (2)
`
`and Rule 58(1) to (4) EPC dated August 2, 1995, where we
`
`extensively discussed why, according to our opinion, the features
`"non-porous substrate or system" and "translucent or transparent
`
`system" are unambiguously derivable from the specification or
`
`contained within the specification as self—evident features, even if
`
`these features are not
`
`literally mentioned in the specification as
`
`originally filed.
`
`In this context we gave a short summary of the prior art (see item
`
`2.1.1)
`
`thereby
`
`providing
`
`a
`
`series
`
`of
`
`documents
`
`partly
`
`incorporated within the description demonstrating that
`
`the
`
`objected to features of claim 1 are self—evident features implicitly
`
`contained within the disclosure as originally filed. We further
`
`referred in item 2.1.2 the Opposition Division to the specific
`disclosure in the description (see pages 50-58) from which the
`
`features
`
`"non—porous
`
`substrate
`
`or
`
`system"
`
`and
`
`"transparent/translucent system"
`
`are clearly derivable as the
`
`support or the system described would not function would it be
`
`porous
`
`or non-transparent/non-translucent.
`
`It
`
`is
`
`furthermore
`
`stated that
`
`the
`
`embodiments
`
`specifically described in
`
`the
`
`application (see the reference to pages 50 to 58) referring to the
`
`later and more difficult embodiments of ELISA as they have been
`
`Page 2 of 15
`
`Page 2 of 15
`
`

`
`developed inthe prior art
`
`for e.g. antigen/antibody reactions,
`
`certainly communicate the older and better known established
`
`ELISA detection utilizing beads and other solid supports within a
`
`distinct and separate system (see the paragraph bridging pages
`
`10 and 11).‘
`
`In summary, our comments provided in the reply to the EPO
`
`demonstrate that
`
`the embodiments of claim 1 wherein the
`
`support
`
`is
`
`the system or
`
`the support
`
`is contained within a
`
`system, the system thereby being transparent or translucent and
`
`non-porous are self-evident and comprised by or derivable from
`
`the description as originally filed.
`
`2.2
`
`The terms "soluble signal", "non—porous system or support" and
`
`"transparent or translucent system" as obiected to in the Decision
`
`Revoking the European Patent
`
`In the following we,want to specifically refer to the statements of
`
`the Opposition Division in the Decision revoking the European
`
`Patent whereby we will demonstrate that the decision and the
`grounds forthe decision are not justified in view of the disclosure
`
`of the application as originally filed.
`
`2.2.1
`
`Soluble Signal
`
`2.2.1.1
`
`The Opposition
`
`Division
`
`first
`
`discussed
`
`a meaningful
`
`interpretation of the term "so|uble signal" as this expression was
`
`seen as being unclear
`
`(which is, however, not a ground for
`
`opposition).
`
`In view of the proprietor's submission of December
`
`28, 1994 it thereby referred to spectrophotometric and ELISA
`techniques ‘involving enzyme-linked reagents which produce a
`
`color change in a
`
`substrate or precipitate and to Table II
`
`disclosing chromogens which produce an insoluble product.
`
`In
`
`view of this the feature "soluble signal" was interpreted in a
`
`broader sense as "a signal that can be detected in solution".
`
`However,
`
`in view of the fact that radioactive signals which are
`
`detectable in solution are excluded from the disclosure of the
`
`Page 3 of 15
`
`Page 3 of 15
`
`

`
`application as
`
`filed,
`
`the Opposition Division
`
`came to the
`
`conclusion that the expression "soluble signal" describes a novel
`
`class of signals which were not disclosed in the application as
`
`filed. Thus, the use of such an expression allegedly violates Art.
`
`123(2) EPC.
`
`2.2.1.2
`
`The term "solub|e signal" per se,
`
`in the context of claim 1 and in
`
`view of the description unambiguously implicates to the skilled
`
`person that a soluble signal per se is soluble in a fluid in contrast
`
`to
`
`the Opposition Division's
`
`interpretation
`
`that
`
`insoluble
`
`precipitates or fixed signalling agents generate a ''soluble signal".
`
`There
`
`are numerous
`
`locations
`
`throughout
`
`the
`
`specification
`
`indicating the generation of soluble signals being measured while
`
`being dissolved in a fluid. We want to refer the Board of Appeal
`to representative disclosure in the specification as e.g. on page
`21,
`lines
`to 26, already referred to in the statement of
`
`December 28, 1994, item 3.3.1.2, relating to spectrophotometric
`
`and ELISA techniques. The
`
`reference to spectrophotometric
`
`techniques including the passage of lines 13 to 21 referring to the
`measurement
`of
`an
`enzymatically
`generated
`product
`for
`
`Quantitative determination and the passage on page 53, lines 1-3
`
`mentioned in the above statement referring to an enzymatically
`
`generated product measured by spectrophotometry clearly show
`
`that by the term "soluble signal" the measurement of a signal in a
`
`solution is comprised.
`
`We cannot share the Opposition Division's opinion that the term
`
`"so|uble signal" comprises signals generated by the chromogen
`
`products of Tables I and II and also radioactive signals detectable
`
`e.g. by a scintillation counter. Tables I and II substantially relate
`
`to insolubleproducts which are visually evaluated while being
`
`bound
`
`to a support
`
`usually
`
`not
`
`allowing
`
`a
`
`quantitative
`
`determination as is e.g.
`
`a significant property of
`
`the soluble
`
`signals. Such precipitates do not need the detection in solution
`
`although detection is possible by e.g. submersing the support into
`
`a clear fluid. This, however, cannot be equalled to a soluble
`
`signal measured in the fluid whereas a precipitate remains an
`
`Page 4 of 15
`
`Page 4 of 15
`
`

`
`insoluble signal. Also the arguments of the Opposition Division
`
`relating to
`
`radioactive labels cannot hold. The radioactively
`
`labelled o|igo- or polynucleotide is fixed to the membrane and
`
`represents therefore an insoluble signal not comparable to signals
`
`being soluble in a fluid.
`
`2.2.1.3
`
`Thus, according to our opinion, the term "so|ub|e signal" is not
`
`only
`
`unequivocally
`
`derivable
`
`from
`
`the
`
`description
`
`but
`
`is
`
`furthermore
`
`clearly
`
`delimited
`
`from other
`
`kinds of
`
`signals
`
`mentioned in the description, as these signals are insolubly
`
`precipitated or fixed signals. Such kinds of signals do not fulfil
`
`the requirements of a soluble signal. Therefore, no novel class of
`
`soluble signals is described, but
`
`the signals comprised by the
`
`term "soluble signal" are clearly derivable from the description.
`
`2.2.2
`
`Non—Porous’gSystem or Support
`
`2.2.2.1
`
`The Opposition Division is of the opinion that the term "non-
`
`porous" cannot be derived from the application as filed neither in
`
`connection with the term "support" nor with the term "system".
`
`The Opposition Division argues that despite the presence of the
`
`word "system" in items 71 and 101-108 and original claims 34 to
`
`37 no meaningful information in connection with the term "non—
`
`porous" could be derived. Also the use of a soluble signal does
`
`not imply the use of a non—porous system in view of the different
`
`embodiments which can be represented by a system.
`
`With respect to the term "non-porous support", the Opposition
`
`Division has
`
`acknowledged that
`
`"non—porous
`
`supports"
`
`are
`
`disclosed in the specification. However,
`
`it emphasizes that since
`
`the specification as filed does not attach any importance to this
`
`feature, a generalization as in claim 1 seems to be unjustified.
`
`2.2.2.2
`
`We cannot agree to the Opposition Division's arguments. The
`
`term "non-porous" is not literally mentioned in the specification.
`
`Such literal disclosure is, however, not required. We are of the
`
`opinion that the specification discloses embodiments of claim 1
`
`which allow the conclusion that
`
`the feature "non—porous"
`
`in
`
`Page 5 of 15
`
`Page 5 of 15
`
`

`
`associationlwith system or
`
`substrate is comprised by the
`
`application so that claiming this feature does not
`
`result
`
`in a
`
`violation of Art. 123(2) EPC.
`
`Suitable disclosures supporting the terms "non-porous substrate"
`or "non-porious system" are inter alia found in item 101 and
`
`following items and on page 50 of the invention as originally
`
`filed.
`
`Item 101 discloses a system comprising besides other
`
`components a transparent substrate to which the DNA probe to
`
`be detected is
`fixed. The system allows a photometrically
`detectable chemical reaction. From this it may be followed that a
`
`soluble signal
`
`is generated. On page 50, a transparent substrate
`
`with the DNA material bound thereto with arrays of depressions
`
`or wells allowing the photometrical detection of a soluble signal is
`disclosed. This means that a fluid with a soluble signal is present
`in the depressions or wells.
`
`The cited locations in the description are embodiments supporting
`claim 1. While item 101 literally mentions the term "system", this
`
`is not the case on page 50. In this case the support is the system
`
`and it is just a matter of designation to designate the transparent
`
`substrate (see page 50,
`
`lines 10 and 11) in claim 1 as a support
`
`or a system.
`
`In order fora feature to be allowable in a claim not violating Art.
`123(2) EPC¢ it does not have to be literally mentioned in the
`
`indirectly but clearly
`feature is
`this
`long as
`description ‘as
`derivable. This is the case here. Although the term "non-porous"
`is not literally comprised in the description it is self-evident to the
`
`skilled person that nothing else than a non-porous support or
`system can; be comprised by the embodiments disclosed and
`
`claimed in claim 1. A support or system (as stated above, this is
`
`just a matter of designation) to which DNA is bound, being a
`
`depression or a well and allowing the determination of the DNA
`whereby washing steps and substrate reactions (see page 50,
`
`lines 22 and 29-31) are performed in the support/system must be
`
`non-porous.
`
`Page 6 of 15
`
`Page 6 of 15
`
`

`
`Furthermore,
`
`a system comprising a substrate (support) and
`
`allowing the photometric detection of a chemical reaction in a
`
`fluid also requires that the system is non-porous.
`
`Thus,
`
`the term "non-porous" in connection with "system" or
`
`"support" is a logical consequence of the diverse embodiments
`
`comprised by claim 1 for the system or the support.
`
`2.2.2.3
`
`The Opposition Division alleges that
`
`the term "system" may
`
`comprise further definitions so that the attribute "non—porous"
`
`does not seem to apply. The Opposition Division mentions e.g. "a
`
`biochemical detection system". However, the meaning of a term
`
`used has to be interpreted based on the disclosure in the whole
`
`specification. We are of the opinion that
`
`there is a suitable
`
`disclosure
`
`for
`
`the
`
`term
`
`"non-porous"
`
`in
`
`connection with
`
`"support" or "system". The wording of claim 1 makes clear that
`
`the term "system" being or containing a solid support is used in a
`limited sense and cannot be interpreted as being any system even
`if further systems are contained in the specification.
`In the sense
`
`the term "system" in connection with the term "non—porous" is
`
`used in claim 1, it is disclosed in the specification. The addition of
`
`the attributes "transparent or translucent" (see item 2.2.3) or
`
`"non-porous" is just a logical consequence which is obvious for
`
`the person skilled in the art.
`
`2.2.3
`
`Transparent or Translucent System
`
`2.2.3.1
`
`The Opposition Division objects to the term "transparent or
`
`translucent system" as the terms "transparent" and "translucent"
`in
`connection with the term "system"
`have
`allegedly no
`counterpart in the specification. The Opposition Division states
`
`that the qualifiers "transparent or translucent" are never attached
`
`to the term "system".
`
`2.2.3.2
`
`The Opposition Division has acknowledged the term "transparent
`
`or
`
`translucent
`
`substrate"
`
`being
`
`disclosed
`
`throughout
`
`the
`
`specification. This means that at
`
`least part of claim 1 with
`
`respect
`
`to the term "transparent or
`
`translucent"
`
`should be
`
`Page 7 of 15
`
`Page 7 of 15
`
`

`
`o
`
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`
`i.
`
`8
`
`nl
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`o
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`o
`0
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`o
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`I
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`
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`
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`
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`
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`
`'00 not
`
`II
`
`n
`
`acknowledged as being allowable. Suitable disclosure to support
`
`the term "transparent or translucent system" may inter alia be
`
`found on page 50 disclosing a transparent glass substrate with an
`
`array of depressions or wells
`
`in which the reaction for
`
`the
`
`detection of nucleic acids is performed. As already stated above
`
`with respect to this embodiment of claim 1
`
`it
`
`is only a matter of
`
`designation whether
`
`the
`
`transparent
`
`substrate
`
`is
`
`called a
`
`"system" or a "support" in claim 1.
`
`The term "transparent system" is also obvious from item 101 as
`
`a
`further embodiment of claim 1. The determination of
`a
`photometrically detectable chemical reaction is possible only if
`light can fall through the system onto the solution. Therefore, the
`"system" should be transparent or translucent.
`
`.
`
`2.2.4.
`
`Summary
`
`In view of our above arguments and the cited disclosure of the
`
`invention as originally filed it can be concluded that the various
`
`embodiments of claim 1 are disclosed or implicitly contained in
`
`the specification as originally filed. The features objected to are a
`
`logical consequence for the functioning of the method of claim 1
`and are implicitly contained within the embodiments described in
`
`.
`
`the specification. We are therefore of
`the opinion that
`the
`features objected to do not go beyond the description as
`
`originally filed.
`
`3.
`
`SUFFICIENCY OF DISCLOSURE (ART. 100(b) and 83 EPC)
`
`The Opposition Division considers claim 29 as being insufficiently
`
`disclosed within the meaning of Art. 83 EPC.
`
`According to our opinion the Opposition Division's objections are
`
`not justified. The skilled person is able to recognize the deficiency
`
`of claim 29 and to compensate this deficiency by applying his/her
`general knowledge so that
`the method of claim 29 can be
`
`performed resulting in the detection of selected polynucleotides.
`
`Therefore,
`
`the method of claim 29 supplemented by general
`
`Page 8 of 15
`
`Page 8 of 15
`
`

`
`0
`C5000 00 0
`000
`O
`
`00
`I
`
`C
`
`knowledgegas is a prerequisite under Art. 83 EPC, can be
`
`performed.
`
`'
`
`NOVELTY (ART. 100(a) and 54 EPC)
`
`The Opposition Division objects
`
`to claims
`
`1
`
`and 26 being
`
`allegedly anticipated by documents 04, O5 and O7 (erroneously
`
`assigned as ‘(D4, D5 and D7) and D2.
`
`4.1
`
`Documents 04, O5 and 07 describe the detection of nucleic acids
`
`by in situ hybridization. The visualization of
`
`the hybridizing
`
`polynucleotides
`
`in ’ document 04 occurs
`
`inter alia by using
`
`rhodamine or an avidin-biotin peroxidase complex and viewing the
`
`labelled DNA through a microscope.
`
`The system of document 05 is similar to that of 04. On page
`
`4384,
`
`left-hand column,
`
`third paragraph,
`
`the generation of an
`
`insoluble precipitate generated by peroxidase is described.
`
`describes the preparation and use of modified
`Document
`nucleotides.’ This document also relates to the generation of
`
`insoluble products.
`
`As has been analyzed above,
`
`these documents disclose the
`
`generation of insoluble products. However, as already outlined in
`
`insoluble products are according to our
`item 2.2.1.2, above,
`opinion not comprised by the term "soluble signal" generated by
`
`the signalling moiety. Therefore,
`
`the subject matter of claims 1
`
`and 26 is not anticipated by these documents.
`
`4.2
`
`Furthermore, we are of the opinion that document D2 cited by
`
`the Opposition Division as anticipating claims 1 and 26 is not
`
`relevant for the evaluation of novelty as outlined below.
`
`According to our opinion, the procedures described in D2 do not
`
`enable the skilled artisan to practice the invention of the opposed
`
`patent. Furthermore,
`
`the procedures of D2 are inoperative and
`
`would not be useful for the detection of a polynucleotide in a
`
`Page 9 of 15
`
`Page 9 of 15
`
`

`
`0
`
`O
`
`D
`dun
`
`0
`000
`
`0 Col
`no
`
`C0
`
`I
`
`10
`
`soluble signalling format using a transparent or translucent, non-
`
`porous system.
`
`D2 does not enable the practice of the invention claimed in the
`
`opposed patent because it does not teach or suggest the use of a
`
`non—porous,.'transparent or translucent system. D2 only describes
`
`supports on which the target polynucleotide is bound and
`
`nowhere does it describe the system in which the supports are
`
`contained. Even if the support is considered to be equivalent to
`
`the substrates claimed in
`
`the opposed patent, D2 is not
`
`anticipating.
`
`In addition to failing to teach or suggest the system or support
`
`claimed in the opposed patent, D2 fails to teach the claimed
`essential feature of generating a soluble signal. D2 discloses on
`
`page 8, lines 3-15:
`
`For example, the following reactions illustrate how a light response is
`elicited using three different light-emitting catalysts as the light labels,
`namely bacterial luciferase, firefly luciferase, and peroxidase:
`
`NADH2 + FMN + RCHO + 0, ‘“‘''‘‘““=*'““» NAD + FMN + RCOOH + H20 + hv
`
`luciferin + ATP + O, ““”""“"”““"oXyluciferin -4- AMP + C0, + PPi + hv
`MW
`.
`
`H,O2 + luminol mm’ oxyluminol + H20 + N, + hv
`
`But the three reactions above are merely copied from another 1978 publication,
`
`Methods in Engymology, Volume LV|l, pages 410, 108 and 96, respectively. A copy of
`pages 410, 108 and 96 and their respective title pages are attached to this declaration
`
`as Exhibit 1 The three above-quoted equations are designated in Exhibit 1 as circled
`
`numbers, 1, 2 and 3, respectively.
`
`Page 10 of 15
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`Page 10 of 15
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`11'
`
`Each of those reactions copied in D2 from Methods in Engymology are
`
`presented in connection with assays for measuring protein or enzymes - but not in any
`
`case for detecting nucleic acids or polynucleotides. The first equation is cited as an
`
`oxidative chemiluminescent reaction for luminol in a short article that is titled "The
`
`Chemiluminescence of Luminol and Related Hydrazides.' The other two equations
`
`concern the assay of modulator protein and the measurement of enzymes, (cyclic
`
`nucleotide phosphodiesterases), respectively. Detecting nucleic acids is not even
`
`disclosed in the three Methods in Engymolggy articles (Exhibit 1). D2 fails to establish
`
`or demonstrate or even present a rationale as to how (or even why) these reactions
`could be usefully applied to nucleic acids and their detection.
`
`in addition to failing to disclose or suggest claimed elements in the opposed
`
`patent, D2 is not operable and thus, the document does not constitute a disclosure that
`
`would permit the skilled artisan to cany out successfully the procedures outlined in the
`
`document. D2 lacks any disclosure or suggestion regarding the practical immobilization
`
`of a polynucleotide to a solid support -- an essential feature required to practice the
`
`procedure of D2, and, moreover, to practice the claimed invention in the opposed
`
`patent. Nowhere is it established in D2 that a polynucleotide can be immobilized on a
`
`support and retain its ability to hybridize to another polynucleotide.
`
`The only disclosure in D2 concerning immobilization of a polynucleotide: to a
`
`solid support is as follows:
`
`Immobilization of the sample single-stranded polynucleotides can be
`accomplished by any suitable method which does not inactivate a
`significant number of bases in the polynucleotide sequence, since a
`representative intact sequence must be available for base pairing with the
`reagent strands. The single-stranded polynucleotide segment can be
`attached or immobilized .
`.
`.
`
`Later in the same paragraph, D2 alleges:
`
`Page 11 of15
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`Page 11 of 15
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`12
`
`Methods in Enzymology, Vol. XXXIV, Part 8, 4553-475, 1974). Arse:
`derivatized forms of polynucleotides, for example, one carrying a terminal
`variety of supports (Mosbach, K., et al., Me , vo|_
`XUV. 859-885. 1975)- Oligoribonucleotides may be immobilized on
`boronate derivates of various supports (Schott, H., et al., Biochemistgg,
`12, 932, 1973).
`
`None of the three above-cited disclosures would permit, however, the
`A.
`immobilization of a polynucleotide to a solid support that could be used in a
`transparent or translucent, and non-porous system. The supports used in the
`three above-cited disclosures have not been shown to be at all useful in the
`assay procedures of D2. Vlflth respect to Weissback, the DNA material was
`
`or for that matter, in a fonnat for generating a soluble signal. The carbodiimide
`treatment disclosed in Weissback would modify residues to the point of rendering
`hybridization altogether unlikely if not impossible. This consequence was vividly.
`disclosed long before
`D2
`or the present invention in the opposed
`patent.
`in the 1972 textbook Qrganic Chemistry of Nucleic Acids, Part B
`
`[Plenum Press, London and New York], the authors discuss reactions with
`
`carbodiimide on pages 331-332 (Exhibit 2):
`
`Single-stranded polynucleotides with no intramolecular hydrogen
`bonds between the bases (polyuridylic acid, for example), react
`smoothly with the carbodiimide Cll [304, 308]; the reaction velocity
`in this case is somewhat
`lower than for uridine (Table 5.7).
`Virtually no reaction takes place with doub|e—stranded complexes of
`polyribonucleotides and DNA [308]. .
`.
`
`The authors later disclose that carbodiimide treatment for the purpose of
`
`immobilization is incompatible with subsequent hybridization reactions:
`
`Page 12 of 15
`
`Page 12 of 15
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`13
`
`Because the course of the reaction with the carbodiimide Cll is so
`
`strongly dependent on secondary structure, and because of
`restriction of nuclease action after modification, the reaction with
`this carbodiimide can be used to identify polynucleotide segments
`in which separation of the double-stranded complex takes place
`during partial denaturation of DNA [312]. After treatment of DNA
`with the carbodiimide Cll, followed by treatment with pancreatic
`DNase
`and
`phosphodiesterase
`from snake
`venom,
`long
`oligonucleotides arising from "defective" segments of the polymer
`can be isolated.
`
`B.
`
`In Mosbach, the only immobilization taught is for coenzymes on a
`
`chromatographic column -- and not for polynucleotides as alleged by D2.
`
`In fact,
`
`l
`
`the title of the Mosbach article is "Immobilized Coenzymes.“ As outlined in the
`
`beginning of Mosbach's article:
`
`The aspects of affinity chromatography have been covered in a
`recent volume of this series and are not dealt with here.
`In this
`
`volume the main emphasis is on their use as active coenzymes
`together with a brief account of
`their application in basic
`enzymology. The methodological part will be centered around the
`various adenine nucleotides, NAD+, NADP+, ATP, ADP, whereas
`work on other coenzymes will be treated only in a summary
`fashion. The various aspects will be treated as outlined below: (1)
`synthesis of a number of adenine nucleotide coenzymes,
`(2)
`coupling to matrices,
`(3) coenzymic activity,
`(4) application in
`enzyme technology and analysis, (5) application in enzymological
`and protein studies, (6) other immobilized coenzymes, (7) general
`discussion.
`
`Absolutely nowhere in Mosbach's article is there any disclosure or
`
`suggestion that nucleic acid or polynucleotides could be immobilized to carry out
`
`successfully the procedure in D2.
`
`C.
`
`in Schott, there is disclosed a borate column for distinguishing molecules
`
`containing a cis—OH group from all others. Molecules with a cis—OH group will
`
`complex with the diborate moiety in the borate column and be retained.
`
`In its
`
`uncharged state (with no amino acid attached), transfer RNA (tFtNA) contains a
`
`cis—OH group and will bind to the borate column; whereas aminoacyl-tRNA
`
`Page 13 of15
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`Page 13 of 15
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`on
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`(having no cis-OH group) will not bind to the borate column. Hence, Schott et al.
`
`merely describes a chromatographic procedure for distinguishing or separating
`
`charged tRNA from uncharged tRNA. As in the case of Weissback, Schott's
`
`chemistry cannot be successfully applied in a transparent or translucent, and
`
`non-porous system for nucleic acid detection.
`
`In particular, borate columns are
`
`known to be unreliable because they have been found to be leaky. Furthennore,
`
`D2 fails to show that tRNA bound to the borate column as in Schott will hybridize
`
`to another nucleic acid, such as a probe.
`
`Even if immobilization could be achieved to carry out the procedure in D2, no
`
`signal could be generated that would be detectable. The supports disclosed in D2 and
`
`in the underlying cited articles (Weissback, Mossbach and Schott) would immerse the
`
`target nucleic acid and any labeled probe hybridized thereto. Accordingly, generation
`
`of the signal by exposure to a light emitting reactant as required by D2 would not be
`
`possible, nor would the detection of any resulting signal be possible through the solid
`
`support in which the polynucleotides are immersed.
`
`A telling point regarding the inoperability of D2 is evidenced by the fact that
`Michael J. Heller, the leading inventor named on the document, subsequently filed in
`
`1985 a different patent application disclosure involving nucleic acid immobilization, that
`
`application having issued as U.S. Patent No. 4,824,776. To obtain that patent, Heller
`
`included additional disclosure concerning nucleic acid immobilization and he also
`
`claimed additional steps in his generic method claim. Thus, Heller's later issued U.S.
`patent required additional teachings on nucleic acid immobilization.
`
`Page 14 of 15
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`Page 14 of 15
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`CO
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`5.
`
`REQUESTS
`
`For the reasons given above and for all
`
`the reasons brought
`
`before the Opposition Division, the patent should be maintained
`
`on the basis of the main request or the auxiliary requests.
`
`If the
`
`Board of Appeal cannot follow our argumentation with regard to
`
`the pending requests, Patentee reserves the right
`
`to submit
`
`further auxiliary requests.
`
`,,4.
`
`L- our//1
`
`Dr. Alexa von Uexkijll
`
`European Patent Attorney
`
`Encl.
`
`Exhibit 1: Roswell, D.F. and White, E.H., Matthews, J.C. and Cormier, M.J.,
`and Fertel, R. and Weiss, B., Methods in Enzymol. Vol. LVII,
`Bioluminescence
`and
`Chemiluminescence,
`DeLuca, M.A.
`(Ed.)(1978), 409-410, 107-108, and 94-96, respectively
`
`et al., Organic Chemistry of Nucleic Acids, Part
`Exhibit 2: Kochetkov,
`B, Kochetkov, N.K. and Budovskii, E.l. (Eds.)(1972), 331-332
`
`Two copies of this letter for Opponents l and II
`
`Page 15 of15
`
`Page 15 of 15