Ex Parte DE HENAU et al

Patent Trial and Appeal Board

Nov 20, 2012

12045183 (P.T.A.B. Nov. 20, 2012)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte HILDE DE HENAU, JOACHIM VAN CROMBRUGGEN,
GEERT JANNES, GERD HABERHAUSEN, and
THOMAS EMRICH
__________

Appeal 2011-012469
Application 12/045,183
Technology Center 1600
__________

Before DONALD E. ADAMS, MELANIE L. McCOLLUM, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to
oligonucleotide probes for identification of Enterococcus species. The
Examiner rejected the claims as obvious. We have jurisdiction under 35
U.S.C. § 6(b). We affirm.

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Statement of the Case
Background
“The present invention relates to a method for the specific detection
and/or identification of Enterococcus species, in particular Enterococcus
faecalis and/or Enterococcus faecium, using new nucleic acid sequences
derived from the ITS (Internal Transcribed Spacer) region” (Spec. 1, ll. 11-
13).
The Claims
Claims 5-7, 15, and 16 are on appeal. Claim 5 is representative and
reads as follows:
5. A set of two or three polynucleotide probes for the
identification of E. faecalis and/or E. faecium, said probes
hybridizing to the same target sequence in adjacent locations on
said target sequence, said probes hybridizing specifically to SEQ
ID NO 1 or SEQ ID NO 2 or homologues, or to their RNA form
wherein T is replaced by U, or to their complementary form,
wherein there are no more than 25 nucleotides between said
probes along said target sequence.

The issues

A. The Examiner rejected claims 5-7 and 16 under 35 U.S.C. § 103(a) as
obvious over Tyrell,
1
Geert,
2
Nitsche,
3
and Genbank
4
(Ans. 4-8).

1
Tyrrell et al., Species Identification of Enterococci via Intergenic
Ribosomal PCR, 35 J. CLIN. MICROBIOLOGY 1054-1060 (1997).
2
Geert et al., EP 1,091,004 A2, published Apr. 11, 2001.
3
Nitsche et al., Different Real-Time PCR Formats Compared for
the Quantitative Detection of Human Cytomegalovirus DNA, 45
CLINICAL CHEMISTRY 1932-1937 (1999).
4
Genbank Accession No. X87186 [gi:1848164] 1-2 (1997).
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B. The Examiner rejected claim 15 under 35 U.S.C. § 103(a) as obvious
over Tyrell, Geert, Nitsche, Genbank, and Stratagene
5
(Ans. 8-9).
A. 35 U.S.C. § 103(a) over Tyrell, Geert, Nitsche, and Genbank
The Examiner finds that Tyrell teaches “the identification of
Enterococcus species using intergenic ribosomal PCR” (Ans. 5). The
Examiner finds that “Tyrell identified the species of Enterococcus by PCR
amplifying the ITS and determining the size, number and or restriction
profiles of the resulting amplification products” (Ans. 5). The Examiner
finds that “Geert taught a modification of the method wherein after
amplification of the ITS region, a probe was hybridized to the amplification
product to allow the identification of the bacterial species (see abstract).
Geert taught that specific primer sets could be derived from the ITS to allow
for species- or group-specific amplification of the ITS” (Ans. 5).
The Examiner finds that “Nitshce [sic] taught a real-time PCR
technique for detecting a particular target sequence during the PCR reaction
itself. In one embodiment, a pair of probes designed to hybridize adjacent
one another on the target sequence enable detection during the PCR reaction
by FRET” (Ans. 6). The Examiner finds that “Nitsche taught probes that
hybridized within 25 nucleotides of one another” (Ans. 6). The Examiner
finds that Genbank “disclosed the ITS sequence of Enterococcus faecalis,
including the sequence of SEQ ID NO 1 . . . In addition, the sequences of
SEQ ID NOs 34 and 69 (which represent isolated nucleic acid molecules
meeting the limitations of claims 5-7 and 16) are also found within the
Genbank sequence” (Ans. 4-5).

5
Stratagene Catalog 39 (1988).
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The issue with respect to this rejection is: Does the evidence of record
support the Examiner‟s conclusion that the combination of Tyrell, Geert,
Nitsche, and Genbank render claim 5 obvious?
Findings of Fact
1. Tyrell teaches that “[s]pecies identification of enterococci can
determine whether a possible outbreak of vancomycin- resistant enterococci
(VRE) is caused by a species such as Enterococcus faecium, is due to an
intrinsically vancomycin-resistant species such as Enterococcus gallinarum,
or is caused by an unusual enterococcal species not previously recognized as
being vancomycin resistant” (Tyrell 1054, col. 1).
2. Tyrell teaches “examining the 16S-23S rDNA (genes coding
for rRNA) intergenic region, referred to as the internally transcribed spacer
region (ITS). It was suggested that this segment of DNA would be under
minimal selective pressure . . . and therefore may allow for species
identification due to enhanced variability between species within a genus”
(Tyrell 1054, col. 2).
3. Tyrell teaches that:
ITS-PCR was able to identify to the species level a
collection of phenotypically aberrant clinical enterococcal
isolates. ITS-PCR identified a biochemically active
enterococcus, strain W185, as E. durans and not E.
raffinosus. ITS-PCR further proved its ability to identify
enterococcal species by determining that W73 and W74
were nonmotile E. casseliflavus VanA strains and not E.
mundtii VanA strains as was originally determined
phenotypically. . . . ITS-PCR also determined that W148
was E. hirae and not E. durans. These two species may be
difficult to distinguish from each other phenotypically;
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however, ITS-PCR resolves this problem readily, as was
evident with the strains examined in this study

(Tyrell 1059, col. 1).
4. Geert teaches that the “the spacer region situated between the
16S rRNA and the 23S rRNA gene, also referred to as the internal
transcribed spacer (ITS), is an advantageous target region for probe
development for detection of pathogens of bacterial origin” (Geert 1 ¶ 0005).
5. Geert teaches a method comprising “amplifying the 16S-23S
rRNA spacer region, or a part of it, with at least one suitable primer pair . . .
hybridizing the polynucleic acids . . . with at least one and preferably more
than one of the spacer probes . . . identification of the micro-organism(s)
present in the sample from the differential hybridization signals” (Geert
abstract).
6. Nitsche teaches that “[n]ew systems based on fluorescence
signal detection during real-time PCR combine numerous advantages. These
assays produce rapid results, allow precise quantification, and require no
post-PCR handling” (Nitsche 1936, col. 2).
7. Nitsche teaches that “FRET is only effective when energy
donor and acceptor molecules are in close proximity, i.e., within a distance
of ˂40 Å. This condition is fulfilled in both hybridization probe sets AD/AA
and BD/AA, in which the distances are 1 and 7 bp” (Nitsche 1937, col. 1).

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8. Figure 1 of Nitsche is reproduced below:

“Fig. 1. Schematic representation of the exonuclease probe format and the
hybridization probe format for online PCR detection. The exonuclease probe
format (A) utilizes a nonextendible dual-labeled oligonucleotide that
hybridizes internally to the flanking primers and is cleaved by the
polymerase during the polymerization step. The hybridization probe format
(B) utilizes two single-labeled, nonextendible oligonucleotides that produce
a fluorescence signal following adjacent hybridization by FRET” (Nitsche,
Figure 1 legend, 1933).
9. Genbank Accession No. X87186 teaches the “E. Faecalis 16S-
23S rRNA spacer DNA & tRNA-Ala gene” (Genbank X87186, subheading
“DEFINITION”).
10. Genbank Accession No. X87186 teaches a sequence which
encompasses SEQ ID NO: 1 (see Spec. 23, Table 1) where SEQ ID NO: 1 is
100% identical to nucleotides 177-240 of the Genbank Sequence (see
Genbank X87186).
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11. Genbank Accession No. X87186 teaches a sequence which
encompasses SEQ ID NO: 34 (see Spec. 23, Table 1) where SEQ ID NO: 34
is 100% identical to nucleotides 187-212 of the Genbank Sequence (see
Genbank X87186).
12. Genbank Accession No. X87186 teaches a sequence which
encompasses SEQ ID NO: 69 (see Spec. 23, Table 1) where SEQ ID NO: 69
is 100% identical to nucleotides 216-240 of the Genbank Sequence (see
Genbank X87186).
13. The Specification teaches that “[p]referably a primer of the
invention is about 5 to about 50 nuc1eotides long, preferably about 15 to
about 25. Its specific length and sequence is to be chosen depending on the
conditions used such as temperature and ionic strength” (Spec. 7, ll. 27-29).
14. The Specification teaches that the “amplification method used
can be either polymerase chain reaction . . . ligase chain reaction . . . nucleic
acid sequence-based amplification . . . strand displacement amplification . . .
or any other suitable method to amplify nucleic acid molecules known in the
art” (Spec. 8, ll. 4-10).
15. The Specification teaches that:
Polynucleotides of the invention may differ in sequence
from any of the polynucleotides specified in Table 1, or
from any of their homologues, either by addition to or
removal from any of their respective extremities of one or
several nucleotides, or by changing one or more nucleotides
within said sequences, or a combination of both, provided
that the equivalents then obtained still hybridize with the
target sequence as the corresponding unmodified
polynucleotides.

(Spec. 8, ll. 14-19).
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16. The Specification teaches that “[a]mong all detection formats
known in the art of real time PCR, the FRET-hybridization probe format has
been proven to be highly sensitive, exact and reliable (WO 97/46707; WO
97/46712; WO 97/46714)” (Spec. 12, ll. 23-25).
17. The Specification teaches that the “method used in the
examples is a method for the detection of Enterococci . . . using the
HybProbe system consisting of two Fluorescein-labeled probes of SEQ IDs
NO 36 and 56, acting as sensor, and one LC-Red-labeled probe of SEQ ID
NO 73 as anchor, in combination with a Enterococcus-genus primer pair of
SEQ IDs NO 5 and 18” (Spec. 28, ll. 4-8).
Principles of Law
An “obviousness finding was appropriate where the prior art
„contained detailed enabling methodology for practicing the claimed
invention, a suggestion to modify the prior art to practice the claimed
invention, and evidence suggesting that it would be successful.”‟ In re
Kubin, 561 F.3d 1351, 1360 (Fed. Cir. 2009) (citing In re O’Farrell, 853
F.2d 894, 902 (Fed. Cir. 1988). The court commented that “[r]esponding to
concerns about uncertainty in the prior art influencing the purported success
of the claimed combination, this court [in O‟Farrell] stated: „[o]bviousness
does not require absolute predictability of success … all that is required is a
reasonable expectation of success.”‟ Kubin, 561 F.3d at 1360 (citing In re
O’Farrell, 853 F.2d at 903-904).

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Analysis
As in Kubin, the issue is whether the claimed primer and probe
sequences, as used in the FRET based Real-Time PCR method of Nitsche
(FF 6-8), are obvious in light of Tyrell, Geert, and Genbank.
In the instant case, Appellants‟ own Specification recognizes that the
primers for the Real-Time PCR and FRET assay may be preferably selected
from any oligonucleotides of 15 to 25 nucleotides in length which hybridize
to form an appropriate size amplification product for detection (FF 13-14,
16). The Specification clearly does not suggest any criticality to the
particular primers exemplified (FF 15), nor does the Specification provide
any comparative data or other evidence to show any unexpected results for
the particular primers of SEQ ID NO: 34 and 69 relative to other primers
selected from the Enterococcus ITS sequence (FF 17).
Tyrell teaches the use of the ITS region for PCR identification of
Enterococcus resulting in efficient and accurate sequence specific detection
of Enterococcus faecium (FF 1-3). Geert also teaches that the ITS region is
an advantageous target region for species probe detection (FF 4-5). Nitsche
teaches accurate detection with FRET based systems in which the probes
closely located to one another, within only 1-7 bases (FF 6-8). Genbank
teaches that the ITS sequence of Enterococcus faecium comprising SEQ ID
NOs: 1, 34, and 69 was known in the art (FF 9-12).
In comparing the obviousness of the instant primers and probes to the
NAIL nucleic acid at issue in Kubin, we note that the instant obviousness
case is better than that in Kubin. Unlike in Kubin where the NAIL sequence
was unknown, the complete and small region of ITS sequence from which
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the instant primers and probes were selected was disclosed in Genbank and
was suggested as a target for primer and probe selection for the particular
bacterial species of Enterococcus faecium in the prior art (FF 1-12). The
method of using the primers and probes was well known in the art (FF 6-8
and 16). Thus, we conclude that it is more than reasonable to find that
selection of particular FRET primer and probe sequences from a known
sequence “would have had a resoundingly „reasonable expectation of
success‟ in deriving the claimed invention in light of the teachings of the
prior art.” Kubin, 561 F.3d at 1360.
Appellants contend that “Tyrell fails to teach or suggest, for example,
a set of two or three probes that hybridize within 25 nucleotides of one
another to SEQ ID NOs:1 or 2, or homologues, or to their RNA form
wherein T is replaced by U, or their complementary form, as required by
claim 5” (Ans. 11). Appellants similarly argue that the other cited art does
not anticipate the instant claims (see App. Br. 12-15).
We are not persuaded. Appellants have provided no reason why the
combined teachings of the references would have failed to suggest the
claimed primers and probes. In this context, it is the combined suggestion of
Tyrell and Geert for ITS region E. faecalis primers and probes along with
the primer requirements of the FRET amplification method of Nitsche and
the express ITS sequence of E. faecalis taught by Genbank which renders
the complete set of primers and probes selected from the known sequence as
obvious equivalents. See In re Keller, 642 F.2d 413, 425 (CCPA 1981)
(“The test for obviousness is not whether the features of a secondary
reference may be bodily incorporated into the structure of the primary
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reference; nor is it that the claimed invention must be expressly suggested in
any one or all of the references. Rather, the test is what the combined
teachings of the references would have suggested to those of ordinary skill
in the art”.); see also In re Merck & Co., Inc., 800 F.2d 1091, 1097 (Fed.
Cir. 1986) (“Non-obviousness cannot be established by attacking references
individually where the rejection is based upon the teachings of a
combination of references. . . . [The reference] must be read, not in isolation,
but for what it fairly teaches in combination with the prior art as a whole.”).
Appellants contend that “[o]ne of ordinary skill in the art will
appreciate that the presently claimed invention provides new nucleic acid
sequences derived from a particular region of the ITS of Enterococcus
species, which can be used, for the detection and/or identification of
Enterococcus species, in particular of E. faecalis and/or E. faecium” (App.
Br. 15).
We are not persuaded. We agree with the Examiner that the ordinary
practitioner would have reasonably selected FRET PCR primers from the
known Enterococcus faecium ITS sequences disclosed by the prior art (FF 6-
12) and that Appellants‟ particular primers would have been obvious and
equivalents or alternatives. This is a situation where the prior art “provides a
„reasonable expectation of success' for obtaining a polynucleotide within the
scope of [the] claim … which, „[f]or obviousness under § 103 [is] all that is
required.‟ O’Farrell, 853 F.2d at 903.” Kubin, 561 F.3d at 1361.
Appellants contend that they “discovered the claimed probe sets that
make it possible to identify, for example, E. faecalis. Such a discovery
overcame difficulties in obtaining probe sets wherein, for example, the
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probes do not cross-react with each other and the probes have the necessary
specificity” (App. Br. 16).
We are not persuaded. We have reviewed the evidence in the
Specification and are not persuaded that unexpected results have been
demonstrated in Table 3, the only table to show data for SEQ ID NOs: 34
and 69, nor that the evidence was compared to the closest prior art. That is, it
is not apparent how the data shows “unexpected” results. To be sure, the
data shows “+++” for these two primers, but there is no persuasive evidence
to support a conclusion that the data shows unexpected results. See In re
Klosak, 455 F.2d 1077, 1080 (CCPA 1972) (“[I]t is not enough to show that
results are obtained which differ from those obtained in the prior art: that
difference must be shown to be an unexpected difference”).
Conclusion of Law
The evidence of record supports the Examiner‟s conclusion that the
combination of Tyrell, Geert, Nitsche, and Genbank render claim 5 obvious.
B. 35 U.S.C. § 103(a) over Tyrell, Geert, Nitsche, Genbank, and
Stratagene
The Examiner finds that the “teachings of Tyrell, Geert, Nitsche and
Genbank have been discussed. In addition, the PCR reaction mixture of
Nitsche can be considered a hybridization buffer” (Ans. 8). The Examiner
finds that “Stratagene catalog teaches a motivation to combine reagents into
kit format” (Ans. 9). The Examiner finds it obvious to “package the
reagents suggested by the combined teachings of Tyrell, Geert, Nitsche and
Genbank in the form of a kit, since the Stratagene catalog teaches . . . the kit
format saves money and resources for everyone by dramatically reducing
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waste” and that the “other service provided in a kit is quality control” (Ans.
9).
The Examiner provides sound fact-based reasoning for combining
Stratagene with Tyrell, Geert, Nitsche, and Genbank. We adopt the fact
finding and analysis of the Examiner as our own. Appellants argue the
underlying obviousness rejection over Tyrell, Geert, Nitsche, and Genbank,
but Appellants do not identify any material defect in the Examiner‟s
reasoning for combining Stratagene with Tyrell, Geert, Nitsche, and
Genbank. Since Appellants only argue the underlying rejection of Tyrell,
Geert, Nitsche, and Genbank which we affirmed above, we affirm this
rejection for the reasons stated by the Examiner.
SUMMARY
In summary, we affirm the rejection of claim 5 under 35 U.S.C. §
103(a) as obvious over Tyrell, Geert, Nitsche, and Genbank. Pursuant to 37
C.F.R. § 41.37(c)(1), we also affirm the rejection of claims 6, 7, and 16 as
these claims were not argued separately.
We affirm the rejection of claim 15 under 35 U.S.C. § 103(a) as
obvious over Tyrell, Geert, Nitsche, Genbank, and Stratagene.

No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

AFFIRMED
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