Ex Parte Lorentsen et al

Patent Trial and Appeal Board

Apr 25, 2013

10553869 (P.T.A.B. Apr. 25, 2013)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
10/553,869 10/21/2005 Rikke Hoegh Lorentsen 66611.000013 1881
151 7590 04/25/2013
HOFFMANN-LA ROCHE INC.
PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY, NJ 07110
EXAMINER
SWOPE, SHERIDAN
ART UNIT PAPER NUMBER
1652
MAIL DATE DELIVERY MODE
04/25/2013 PAPER
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte RIKKE HOEGH LORENTSEN and
CHARLOTTE HARKJAER FYNBO
__________

Appeal 2011-011431
Application 10/553,869
Technology Center 1600
__________

Before FRANCISCO C. PRATS, MELANIE L. McCOLLUM, and
JEFFREY N. FREDMAN, Administrative Patent Judges.

PRATS, Administrative Patent Judge.

DECISION ON APPEAL
This appeal under 35 U.S.C. § 134 involves claims to methods of
using the protease Granzyme B to cleave polypeptides of interest from their
fusion partners. The Examiner entered rejections for obviousness.
We have jurisdiction under 35 U.S.C. § 6(b). We reverse.

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STATEMENT OF THE CASE
Claims 1, 4, 6, 8-11, 13-17, 40, 41, and 43-51 stand rejected and
appealed (App. Br. 1).1 Claim 1 illustrates the appealed subject matter and
reads as follows:
1. A method for the preparation of a polypeptide of interest in
authentic form, said method comprising the steps of:
(i) providing a fusion protein comprising, from its
N-terminal to its C-terminal, (a) a fusion partner,
(b) a Granzyme B protease recognition site comprising a
Granzyme B protease cleavage site that is cleavable by
human Granzyme B protease, and wherein the
recognition site comprises an amino acid sequence of the
general formula
P4 P3 P2 P1 ↓ (SEQ ID NO: 59)
wherein
P4 is amino acid I or V,
P3 is amino acid E, Q or M,
P2 is X, where X denotes any amino acid,
P1 is amino acid D, and
↓ is said Granzyme B protease cleavage site, and
(c) the polypeptide of interest, wherein said cleavage site
is adjacent to the polypeptide of interest, and
(ii) cleaving the fusion protein with Granzyme B protease at
said cleavage site to yield said polypeptide of interest in
authentic form.

The following rejections are before us for review:
(1) Claims 1, 9-11, 16, 17, 40, 44-46, 50, and 51, under 35 U.S.C.
§ 103(a) as obvious over Azad,2 Harris,3 and Casciola-Rosen4 (Ans. 6-7);

1 Appeal Brief entered April 5, 2011.
2 Ahmed A. Azad et al., Large-scale production and characterization of
recombinant human immunodeficiency virus type 1 Nef, 75 JOURNAL OF
GENERAL VIROLOGY 651-655 (1994).
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(2) Claims 4, 6, and 41, under 35 U.S.C. § 103(a) as obvious over
Azad, Harris, Casciola-Rosen, and Boutin5 (Ans. 7-8);
(3) Claims 13-17 and 47-49, under 35 U.S.C. § 103(a) as obvious
over Azad, Harris, and Casciola-Rosen, combined with Sigma6 or
Pharmacia7 (Ans. 8-9); and
(4) Claims 8 and 43, under 35 U.S.C. § 103(a) as obvious over Wan,8
Bleackley,9 and Harris (Ans. 9-10).
OBVIOUSNESS –
AZAD, HARRIS, AND CASCIOLA-ROSEN
In rejecting independent claims 1 and 40 as obvious, the Examiner
cited Azad as describing “a GST-nef27 fusion protein . . ., wherein nef27
contains Met-Gly at the N-terminus” (Ans. 6 (citation omitted)). The
Examiner conceded, however, that Azad did not teach “a GST-Granzyme B
cleavage motif-nef27 fusion protein or using Granzyme B to release
authentic nef27 from such a fusion protein” (id.).

3 Jennifer L. Harris, Definition and Redesign of the Extended Substrate
Specificity of Granzyme B, 273 J. BIOL. CHEM. 27364-27373 (1998).
4 Livia Casciola-Rosen et al., Cleavage by Granzyme B Is Strongly
Predictive of Autoantigen Status: Implications for Initiation of
Autoimmunity, 190 J. EXP. MED. 815-825 (1999).
5 Jean A. Boutin, Myristoylation, 9 CELL SIGNAL 15-35 (1997).
6 Sigma THROMBIN CleanCleaveTM KIT, Technical Bulletin No. MB-445
(1998).
7 AFFINITY CHROMATOGRAPHY PRINCIPLES & METHODS, p. 9 (Pharmacia
Laboratory Separation Division, Uppsala, Sweden (1986)
8 Min Wan et al., Autoprocessing: an essential step for the activation of
HIV-1 protease, 316 BIOCHEM. J. 569-573 (1996).
9 R. Chris Bleackley et al., Isolation of two cDNA sequences which encode
cytotoxic cell proteases, 234 FEBS LETTERS 153-159 (1988).
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To address those deficiencies, the Examiner cited Harris as teaching
the use of Granzyme B “to cleave fusion proteins comprising the motif
IEADXG, wherein X is any amino acid (Fig 5A&D)” (id.).
Based on the references’ teachings, the Examiner concluded that an
ordinary artisan would have considered it obvious to “modify the fusion
protein of Azad et al to incorporate the motif IEAD between the GST fusion
partner and nef27” (id.). The Examiner reasoned that the fusion protein
suggested by the references “would have the structure:
GST-(IEAD↓ [MG)nef27]
wherein, (IEADMG) is the Granzyme B cleavage motif, [MGnef27] is
authentic nef27, ↓ is the Granzyme B cleavage site, and ‘MG’ is comprised
within both the Granzyme B cleavage motif and authentic nef27” (id. at 6-
7). The Examiner further reasoned that “[m]otivation to make the fusion
protein and cleave it with Granzyme B derives from the desire to produce
authentic nef27, which is critical for development of AIDS” (id. at 7).
The Examiner relied on Casciola-Rosen as evidence that human
Granzyme B was known to cleave the motif IEAD↓X (see id.).
As stated in In re Oetiker, 977 F.2d 1443, 1445 (Fed. Cir. 1992):
[T]he examiner bears the initial burden . . . of presenting a
prima facie case of unpatentability. . . .
After evidence or argument is submitted by the applicant
in response, patentability is determined on the totality of the
record, by a preponderance of evidence with due consideration
to persuasiveness of argument.

In KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 415 (2007), while the
Supreme Court emphasized “an expansive and flexible approach” to the
obviousness question, the Court also reaffirmed the importance of
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determining “whether there was an apparent reason to combine the known
elements in the fashion claimed by the patent at issue.” Id. at 418.
Thus, even post-KSR, “[o]bviousness requires more than a mere
showing that the prior art includes separate references covering each
separate limitation in a claim under examination.” Unigene Laboratories,
Inc. v. Apotex, Inc., 655 F.3d 1352, 1360 (Fed. Cir. 2011).
Instead, “[i]n determining whether obviousness is established by
combining the teachings of the prior art, the test is what the combined
teachings of the references would have suggested to those of ordinary skill
in the art.” In re GPAC Inc., 57 F.3d 1573, 1581 (Fed. Cir. 1995) (internal
quotations omitted).
We are not persuaded that a preponderance of the evidence supports
the Examiner’s prima facie case of obviousness. In particular, we are not
persuaded that the cited references would have suggested using Granzyme B
as the fusion protein-cleaving protease in Azad’s process.
As the Examiner found, Azad discloses producing the HIV-1 protein
Nef27 as a fusion protein with a glutathione transferase fusion partner (see
Azad 651). As Azad explains, expression levels of “E. coli-derived
glutathione S-transferase-Nef fusion proteins were very high and a major
portion was soluble. Large-scale production of E. coli-derived Nef27 . . .
was carried out by growing recombinant cells in a fermenter under fed-batch
conditions followed by affinity purification on glutathione-Sepharose before
and after thrombin cleavage” (id. (abstract)).
Azad thus uses the protease thrombin, rather than Granzyme B, as its
cleaving enzyme.
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Harris discloses a study that used combinatorial methods to determine
the proteolytic specificity of Granzyme B (see Harris 27364). In particular,
in assessing the P3, P1', and P2' specificity of Granzyme B, Harris used the
phage display technique to assess Granzyme B’s capacity to cleave 8000
different protein sequences (see id. at 27368-69).
As Harris explains, in its phage display assay, fusion proteins
including the M13 phage coat protein pIII and a histidine affinity tag anchor
were prepared, with a variable linker between the two fusion partners
allowing assessment of Granzyme B’s capacity to cleave the different
sequences (see id.). As Harris further explains, Granzyme B cleaved linkers
with at least twenty different sequences (see id. at 27369).
We note that Harris describes the optimal six amino acid cleavage
sequence of Granzyme B (see id. at 27364 (abstract); see also id. at 27371
(Fig. 5)). We are not persuaded, however, that the Examiner has adequately
explained why the proteolytic properties of Granzyme B described in Harris
would have suggested to an ordinary artisan that Granzyme B would have a
broader applicability, to the extent that the artisan would have been
prompted to use Granzyme B in large scale methods of producing fusion
proteins, such as the GST-Nef27 fusion protein described in Azad.
The Examiner argues:
In the instant case, the relevant rationales for supporting
the obviousness rejection are (a) combining the prior art
references will, more likely than not, lead to the predicated [sic,
predicted] result of a method of producing authentic nef27 by
cleaving the fusion protein GST-IEAD↓[MG-nef27] or HIS6x-
IEAD↓[MG-nef27] with Granzyme B, (b) simple substitution
of one known element for another to obtain the above described
predictable result, (c) use of a known technique to improve the
method of Azad et al by incorporating the teachings of Harris et
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al and Casciola-Rosen et al, and (g) based on knowledge of the
skilled artisan, there would be motivation to combine the
teachings of Azad et al, Harris et al, and Casciola-Rosen et al,
1999 to produce authentic nef27.

(Ans. 19.)
We are not persuaded. The Examiner’s first rationale cited above
presupposes that an ordinary artisan would have been prompted by the
references to substitute Granzyme B for Azad’s thrombin. The Examiner’s
final rationale cited above finds such an impetus for practicing the claimed
process in the knowledge of a skilled artisan.
As noted above, however, the Examiner has not pointed to any clear
or specific evidence of record, in Harris or elsewhere, suggesting that an
ordinary artisan would have considered a protease having the properties of
Granzyme B described in Harris to be suitable in a large scale process of
cleaving fusion proteins, such as that described by Azad. In particular, the
Examiner directs us to no general or specific teaching of the properties
desirable in enzymes used for cleaving fusion proteins in processes such as
that described in Azad, nor has the Examiner explained why an ordinary
artisan would have recognized Granzyme B as having such properties.
Similarly, the Examiner points to no clear or specific evidence of
record suggesting that, given its properties as described in Harris, an
ordinary artisan would have considered Granzyme B to have been a
functional equivalent of Azad’s thrombin. Nor has the Examiner pointed to
any clear or specific evidence suggesting that Granzyme B would have been
an improvement over thrombin.
In sum, for the reasons discussed, we are not persuaded that the
preponderance of the evidence supports the Examiner’s prima facie case of
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obviousness as to claims 1 and 40. We therefore reverse the Examiner’s
obviousness rejection of those claims, as well as their dependent claims
9-11, 16, 17, 44-46, 50, and 51, over Azad, Harris, and Casciola-Rosen.
OBVIOUSNESS –
AZAD, HARRIS, CASCIOLA-ROSEN, AND BOUTIN
The Examiner also rejected claims 4, 6, and 41, each of which
depends from either claim 1 or claim 40, as obvious over Azad, Harris,
Casciola-Rosen, and Boutin (Ans. 7-8). The Examiner cited Boutin as
evidence that an ordinary artisan would have considered it obvious to
substitute the enzyme calcineurin B for the Nef27 in Azad’s process, based
on sequence identity at the N-terminus (see id. at 8). However, as Boutin
does not remedy the shortcomings discussed above of Azad, Harris, and
Casciola-Rosen as to claims 1 and 40, we reverse this rejection as well.
OBVIOUSNESS –
AZAD, HARRIS, CASCIOLA-ROSEN, SIGMA AND PHARMACIA
The Examiner also rejected claims 13-17 and 47-49, each of which
depends directly or ultimately from either claim 1 or claim 40, as obvious
over Azad, Harris, and Casciola-Rosen, combined with Sigma or Pharmacia
(Ans. 8-9). The Examiner cited Sigma and Pharmacia as evidence that an
ordinary artisan would have considered it obvious to immobilize Granzyme
B when using it in Azad’s process (see id.). Again, however, as Sigma and
Pharmacia do not remedy the shortcomings discussed above of Azad, Harris,
and Casciola-Rosen as to claims 1 and 40, we also reverse this rejection.

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OBVIOUSNESS –
WAN, BLEACKLEY, AND HARRIS
The Examiner also rejected claims 8 and 43, which ultimately depend
from claims 1 and 40, respectively, as obvious over Wan, Bleackley, and
Harris (Ans. 9-10). The Examiner cited Wan as describing a method
whereby a fusion protein including the HIV-1 protease “proteolytically
autoprocesses, thereby producing the active authentic HIV-l protease” (id. at
9).
The Examiner conceded that Wan did not describe a method whereby
Granzyme B autoprocessed in the manner taught in Wan, and cited
Bleackley as evidence that Granzyme B “is synthesized as a prepro-form,
which must be cleaved, to remove the first 20 amino acids, for activation”
(id.).
Based on these teachings, the Examiner concluded that an ordinary
artisan would have considered it obvious “to adapt the method of Wan et al
to make a method for producing authentic Granzyme B protease, wherein
the method use[s] a fusion protein comprising Granzyme B protease that
proteolytically autoprocesses” (id.). The Examiner reasoned that, in that
“adapted method, the motif GAEE↓II2 in Granzyme B would be replaced
with the motif IEAD↓IG2 which, as taught by Harris et al, is a motif cleaved
by Granzyme B” (id.). The Examiner further reasoned that “[m]otivation to
make such a method is provided by the desire to produce the active
Granzyme B protease and to screen for inhibitors of auto-processing” (id. at
10).
We reverse this rejection as well.
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We note, as the Examiner pointed out, that Wan discloses that
“autoprocessing of the direct upstream sequence of the protease domain is an
essential step for the activation of recombinant HIV-I protease in the E. coli
expression system” (Wan 569 (abstract)). Based on this finding, Wan
explains that the presence of a fusion partner improves production of the
HIV-1 protease in an E. coli expression system (see id. (“Expression of
HIV-1 protease as fusion proteins revealed that the existence of a fusion
portion increased the accumulation of expressed protease by affecting its
homotypic dimer formation”)).
The Examiner does not, however, point to any teaching in either
Bleackley or Harris suggesting that a similar improvement would occur by
producing Granzyme B as an autoprocessed fusion protein in an E. coli
expression system. Moreover, the Examiner does not point to any clear or
specific evidence of record to suggest that an ordinary artisan would have
been prompted by the cited references to study autoprocessing of Granzyme
B. We therefore reverse the Examiner’s obviousness rejection of claims 8
and 43 over Wan, Bleackley, and Harris.
SUMMARY
We reverse the Examiner’s obviousness rejection of claims 1, 9-11,
16, 17, 40, 44-46, 50, and 51 over Azad, Harris, and Casciola-Rosen.
We also reverse the Examiner’s obviousness rejection of claims 4, 6,
and 41, under 35 U.S.C. § 103(a) as obvious over Azad, Harris, Casciola-
Rosen, and Boutin.
We also reverse the Examiner’s obviousness rejection of claims 13-17
and 47-49 over Azad, Harris, and Casciola-Rosen, combined with Sigma or
Pharmacia.
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We also reverse the Examiner’s obviousness rejection of claims 8 and
43 over Wan, Bleackley, and Harris.

REVERSED

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