Ex parte Kleid et al.

Board of Patent Appeals and Interferences

Jan 30, 2001

08482321 (B.P.A.I. Jan. 30, 2001)

The opinion in support of the decision being entered today was not written for
publication and is not binding precedent of the Board.

Paper No. 54

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte DENNIS G. KLEID, DANIEL G. YANSURA,
HERBERT L. HEYNEKER and MIOZZARI F. GIUSEPPE
__________

Appeal No. 1999-1157
Application No. 08/482,321
__________

ON BRIEF
__________

Before WILLIAM F. SMITH, ADAMS and MILLS, Administrative Patent Judges.

ADAMS, Administrative Patent Judge.

DECISION ON APPEAL1
This is a decision on the appeal under 35 U.S.C. § 134 from the examiner’s
final rejection of claims 1, 9, 10, and 35-37, which are all the claims pending in the
application.
Claim 1 is illustrative of the subject matter on appeal and is reproduced
below:
1. The method of producing a polypeptide product by the expression in
bacteria of a structural gene coding therefor which comprises:
(a) providing a bacterial inoculant transformed with a replicable plasmidic
expression vehicle having a sequence of double-stranded DNA

1 This application has a filing date of June 6, 1995. However, we note that this
application has an effective filing date of March 24, 1980.
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comprising, in phase from a first 5’ to a second 3’ end of the coding
strand thereof, the elements:
(i) a bacterial trp promoter-operator system from a bacterial trp
operon;
(ii) nucleotides coding for a ribosome binding site for translation
of element (iv);
(iii) nucleotides coding for a translation start signal for translation
of element (iv); and
(iv) a structural gene encoding the amino acid sequence of a
heterologous polypeptide;
said sequence comprising neither any trp attenuation
capability nor nucleotides coding for the trp E ribosome
binding site, provided however, that if the heterologous
polypeptide includes any polypeptide also includes
polypeptide sequence not encoded by the trp operon;
(b) placing the transformed inoculant in a fermentation vessel and
growing the same to a predetermined level in a suitable nutrient
media containing additive tryptophan sufficient in quantity to
repress said promoter-operator system; and
(c) depriving said bacteria of said additive while maintaining the viability
of said bacteria so as to derepress said system and occasion the
expression of the product encoded by said structural gene.
The references relied upon by the examiner are:

Cohen et al. (Cohen) 4,237,224 Dec. 2, 1980

Bertrand et al. (Bertrand), “Regulation of Transcription Termination in the Leader
Region of the Tryptophan Operon of Escherichia coli Involves Tryptophan or its
Metabolic Product,” J. Mol. Biol., Vol. 103, pp. 339-349 (1976)

Miozzari et al. (Miozzari (R)), “Translation of the Leader Region of the Escherichia
coli Tryptophan Operon,” J. Bacteriology, Vol. 133, No. 3, pp. 1457-1466 (1978)

Miozzari et al. (Miozzari (S)), “Tryptophan Biosynthesis in Saccharomyces
cerevisiae: Control of the Flux Through the Pathway,” J. Bacteriology, Vol. 134, No.
1, pp. 48-59 (1978)

Goeddel et al. (Goeddel), “Direct expression in Escherichia coli of a DNA sequence
coding for human growth hormone,” Nature, Vol. 281, pp. 544-548 (1979)

The reference relied upon by the Board is:
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Backman et al. (Backman), “Construction of plasmids carrying the cI gene of
bacteriophace ?,” Proc. Natl. Acad. Sci. USA, Vol. 73, No. 11, pp. 4174-4178
(1976)
GROUNDS OF REJECTION

Claims 1, 9, 10 and 35-37 stand rejected under 35 U.S.C. § 103 as being
unpatentable over Cohen in view of Bertrand and Goeddel.
Claims 1, 9, 10 and 35-37 stand rejected under 35 U.S.C. § 103 as being
unpatentable over Cohen in view of Miozzari (R), Miozzari (S) and Goeddel.
We reverse.
DISCUSSION
In reaching our decision in this appeal, we have given careful consideration
to the appellants’ specification and claims, and to the respective positions
articulated by the appellants and the examiner. We make reference to the
examiner’s Answer2 for the examiner’s reasoning in support of the rejections. We
further reference appellants’ Brief3 for the appellants’ arguments in favor of
patentability.

2 Paper No. 53, mailed January 3, 1997.
3 Paper No. 52, received November 13, 1996.
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At the outset we note that this application was filed on June 6, 1995.
However, through a number of continuation applications, this application has an
effective filing date of March 24, 1980. Accordingly, we look back over 20 years to
evaluate the obviousness rejections of record from the perspective of a person of
ordinary skill in the art at the time this invention was made.
Initially, we note that appellants agree with the examiner with regard to the
teachings in Goeddel and the disclosure of Cohen. Appellants state (Brief, page 7)
that “[t]he Examiner cites Goeddel et al solely for the proposition that it teaches the
expression of chemically synthesized genes for human insulin in E. coli. As limited
to this teaching, Appellants concur with the Examiner.” Appellants also “agree
[Answer, page 8] with the Examiner that Cohen et al. teach ‘the formation of
plasmids containing promoters and eukaryotic genes used to transform cells and
produce proteins’. Appellants also agree with the Examiner that Cohen et al. teach
‘the use of trp operons with various deletions’.”
Cohen in view of Bertrand and Goeddel:
The examiner argues (Answer, page 9) that “Bertrand et al. disclose
attenuator-deleted trp operons, and the step of inducing transcription from the trp
operons by trp starvation.” However, we note the limitation of appellants’ claim 1
section (iv) which states in part “a structural gene … comprising 6 amino acids of
the trp leader peptide, [and] … at least about the distal third of the trp E
polypeptide….” The examiner does not explain, and we do not find, where Bertrand
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meets this limitation of appellants’ claimed invention. Cohen and Goeddel fail to
make up for this deficiency in Bertrand.
The initial burden of presenting a prima facie case of obviousness rests on
the examiner. In re Oetiker, 977 F.2d 1443, 1445, 24 USPQ2d 1443, 1444 (Fed.
Cir. 1992). In satisfying this initial burden, every limitation positively recited in a
claim must be given effect in order to determine what subject matter that claim
defines. In re Wilder, 429 F.29 447, 450, 166 USPQ 545, 548 (CCPA 1970). Here
the examiner failed to meet her burden of establishing a prima facie case of
obviousness. Accordingly, we reverse the rejection of claims 1, 9, 10 and 35-37
under 35 U.S.C. § 103 as obvious over Cohen in view of Bertrand and Goeddel.
Cohen in view of Miozzari (R), Miozzari (S) and Goeddel:
Miozzari (R), however, teach (Miozzari (R), figure 1) two constructs
trp?LE1417, and trp?LE1413. Both of these constructs meet the limitation of
appellants’ claim 1 section (iv) with respect to “a structural gene … comprising 6
amino acids of the trp leader peptide, [and] … at least about the distal third of the
trp E polypeptide….” In fact, we note that appellants’ specification (page 10, lines
28-34) discloses:
Two particularly useful plasmids from which the attenuator
region has been deleted are the plasmids pGM1 and pGM3, G.F.
Miozzari et al, J. Bacteriology 133, 1457 (1978). These respectively
carry the deletions trp?LE1413 and trp?LE1417 and express (under
the control of the trp promoter-operator) a polypeptide comprising
aproximately the first six amino acids of the trp leader and distal
regions of the E polypeptide. In the most preferred case, pGM1, only
about the last third of the E polypeptide is expressed whereas pGM2
expresses almost the distal one half of the E polypeptide codons.
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As appellants’ argue (Brief, page 9):

[T]he ‘process’ of Cohen et al referenced by the Examiner is
heterologous gene expression under the control of the lac promoter.
The essential issue with regard to the outstanding rejections,
therefore, is whether it would have been obvious to one of ordinary
skill in the art on 24 March 1980 to substitute the attenuator- and trpE
ribosome binding site-deleted trp promoter for the lac promoter
actually employed in the ‘process’ of Cohen et al.

Much of appellants’, as well as the examiner’s, arguments are directed to an
increase in expression resulting from the use of the trp promoter mutants relative to
the lac promoter. However, we note that nothing in the claims require an increase in
expression. The examiner argues (Answer, page 8) that it would have been obvious
to substitute the lac promoter with the trp promoter as taught by Miozzari. We note
that, where the prior art recognizes two components to be equivalent, an express
suggestion to substitute one for another need not be present in order to render such
substitution obvious. In re Fout, 675 F.2d 297, 301, 213 USPQ 532, 536 (CCPA
1982). Therefore, it appears that the examiner has made out a plausible prima
facie case of obviousness.
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However, a conclusion of prima facie obviousness, does not end a
patentability determination under 35 U.S.C. § 103. As stated in In re Hedges, 783
F.2d 1038, 1039, 228 USPQ 685, 686 (Fed. Cir. 1986):
If a prima facie case is made in the first instance, and if the applicant
comes forward with reasonable rebuttal, whether buttressed by
experiment, prior art references, or argument, the entire merits of the
matter are to be reweighed. In re Piasecki, 745 F.2d 1468, 1472,
223 USPQ 785, 788 (Fed. Cir. 1984).

Here, appellants provide evidence of unexpected results. Specifically, appellants
argue (Brief, page 6) that “the surprisingly high yields of polypeptides obtained
using the claimed invention turned a laboratory phenomenon into an industrially [sic]
process [of] producing recombinant polypeptides.”
In response to appellants’ arguments concerning the lack of motivation to
combine Miozzari with Cohen, the “Examiner strongly disagrees [Answer, page 6]
that the record does not contain a motivation for the combination. … Miozzari et al.
show that deletions of a sequence between the operator and the trp E coding region
can increase the expression of structural genes.”
With respect to the examiner’s arguments concerning increased expression
due to the trp promoter, appellants argue (Brief, bridging paragraph, pages 10-11)
that:
Appellants cannot stress strongly enough that Bertrand et al’s and
Miozzari et al’s demonstration of increased expression from an
attenuator-deleted trp promoter as compared to an intact trp promoter
does not, in any way, suggest that an attenuator-deleted trp promoter
would provide increased expression as compared to the lac
promoter employed by Cohen et al. It is essential for the Examiner to
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keep in mind that the issue is whether there would have been
motivation to substitute the attenuator-deleted trp promoter for Cohen
et al’s lac promoter, not for an intact trp promoter. Thus, while the
ordinarily skilled artisan might possibly have been motivated to
substitute the attenuator-deleted trp promoter for an intact trp
promoter in order to achieve increased gene expression … he or she
simply would not have known whether substitution of the lac promoter
of Cohen et al with the attenuator-deleted trp promoter would have
provided increased gene expression. In other words, as of the filing
date of the present application, there simply was no way to compare
the efficiencies of the attenuator-deleted trp promoter and the lac
promoter employed by Cohen et al.

We agree with appellants. The examiner has provided no evidence
suggesting that the trp promoter mutants of appellants’ claimed invention would
provide an increase in the expression of a heterologous gene relative to the lac
promoter of Cohen. As appellants explain, each of the examiner’s arguments
concerning an increased level of expression resulting from a trp promoter mutant
are relative to the wild-type trp promoter.
Appellants provide the Kleid Declaration4 to support their argument
concerning unexpected results. Kleid explains (Declaration, page 5) that “[t]he
yields obtained by the method of the present invention were unpredictable and
unexpected at the time this invention was made … [t]he relative heterologous
protein yields from small-scale fermentations are apparent from Figures 3, 7 and 11
of the present application.”
Figure 7, of the specification, compares the level of expression of a
heterologous gene, human growth hormone (HGH), resulting from the use of the lac
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promoter or the trp promoter. According to the specification (pages 22-23) figure 7
is a photograph of a stained gel wherein:
Lanes 1 and 7 contain protein markers of various known sizes;
lane 2 is a control … Lane 3 segregates protein from E. [c]oli
294/pHGH 107 [a lac promoter construct] grown in LB media [which
contains tryptophan, resulting in reduced expression from the trp
promoter due to the trp repressor/operator interactions that occur in
the presence of tryptophan]; Lane 4 segregates protein from E. [c]oli
294/pHGH 107 grown in M9 media [which lacks tryptophan]; Lane 5
segregates protein from E. [c]oli 294/pHGH 207 [a trp
promoter/operator construct] grown in LB media; and Lane 6
segretages protein from E. [c]oli 294/pHGH 207 grown in M9. The
dense band in Lane 6 is human growth hormone, as shown by
comparison to the similar bands in Lanes 2-4. As predicted by the
invention, the organism E. [c]oli 294/pHGH 207 when grown in
tryptophan-rich LB media produces less human growth hormone by
reason of tryptophan repressor/operator interactions, and when grown
in M9 media produces considerably more HGH than E. [c]oli
294/pHGH 107 owing to the induction of the stronger tryptophan
promoter-operator system vs the lac promoter-operator system in
pHGH 107.

Kleid explains (Declaration, page 7) that figure 7 “demonstrates the unexpectedly
better results obtained with the present trp system (Lane 6, yield estimate about
10%) as compared with the original lac system (Lane 3, yield estimate about 2%).”
In response to appellants’ presentation of unexpected results, the examiner
argues (Answer, page 9-10) that:
[a]ppellants’ arguments regarding unexpectedly large yields are not
convincing because no support is found in the specification, as
originally filed. … Because the repressor protein is directly regulated
by the level of tryptophan available one can up regulate transcription
by decreasing tryptophan, an analogous mechanism is not available
in the lac promoter-operator system. The examiner’s position is
supported in the decision of In re O’Farrell (7 USPQ 2d 1673, CAFC

4 Executed April 17, 1989, attached to appellants’ Brief as Exhibit A.
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1988) where the court found that the prior art was required only to
“reveal reasonable expectation of success in producing the claimed
invention”. One would have expected a stronger attenuator deleted
tryptophan promoter-operator system because intracellular tryptophan
regulates repressor affinity for operator as well as trp E and trp D by
feedback inhibition-thus two points of regulation exist that do not exist
in an analogous way in the lac-operon system.

We are not persuaded by the examiner’s arguments. With regard to the
examiner’s argument concerning two points of regulation, appellants’ specification
(page 4) explains that “[i]n wild-type E. coli, the tryptophan operon is under at least
three distinct forms of control.” Two of which are regulated by tryptophan. With
regard to the point of control not regulated by tryptophan, the specification (page 4)
explains that this “control is effected by a process known as attenuation under the
control of the ‘attenuator region’ of the gene, a region within the trp leader
sequence.” Appellants’ claimed invention specifically excludes this “attenuator
region,” see claim 1 “said sequence comprising neither any trp attenuation
capability nor nucleotides coding for the trp E ribosome binding site.” With regard
to the two points of control regulated by tryptophan, the specification discloses
(page 4) that one is “by a process of feedback inhibition, tryptophan binds to a
complex of the trp E and trp D enzymes, prohibiting their participation in the
pathway [of tryptophan] synthesis.” Therefore, since appellants’ construct is not
involved in tryptophan synthesis this point of regulation does not appear to be a
relevant point of control for the instant invention.
With regard to the second point of control regulated by tryptophan,
appellants’ specification (page 4) explains “tryptophan acts as a corepressor and
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binds to its aporepressor to form an active repressor complex which, in turn, binds
to the operator, closing down the pathway in its entirety.” It is this point of control
that is relevant to appellants’ construct as demonstrated by the results obtained in
figure 7 of the specification.
We do not agree with the examiner’s position (Answer, page 9) that an
“analogous mechanism [to tryptophan regulation of the trp promoter-operator] is not
available in the lac promoter-operator system.” As explained by appellants5 during
prosecution of the 07/773,740, of which this case is a File Wrapper Continuation:
The attenuator-deleted operon is bereft of the attenuator control
system (it’s deleted), so control only operates through the trp
repressor. This situation is no different than the lac repressor
controlling the expression of the lac promoted protein where, IPTG is
used to derepress lac and promote expression, just as trp deprivation
promotes expression from the trp promoter.”

5 Paper No. 34, received May 26, 1982, at page 3.
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Thus as explained by appellants the lac promoter-operator is regulated by the lac
repressor and this repression of gene expression from the lac promoter/operator is
relieved by IPTG.
This, however, raises the question of what effect IPTG would have had on
appellants’ comparison of the lac promoter-operator with the trp promoter-operator
as explained in the specification (pages 22 and 23), figure 7 and the Kleid
Declaration. This comparison tests the expression of the lac construct in the
presence or absence of tryptophan (which regulates the trp promoter-operator), not
IPTG (which regulates the lac promoter-operator). Appellants’ specification,
however, resolves this issue. As explained in appellants’ specification (page 23) E.
coli 294 cells were used to compare the expression of the lac and trp constructs.
The specification (page 16) makes reference to Backman with regard to E. coli K-
12 strain 294.
Backman use a lac promoter-operator construct to drive expression of the ?
repressor. Backman teach in Table 2 (page 4176) that “Strain 294 makes wild-type
lac repressor levels.” However, Backman also teach (page 4176, column 2) in
reference to Table 2 that “[a] strain bearing a wild-type lac operon does not make
enough lac repressor to repress significantly the synthesis of ? repressor from the
lac promoters….”
Therefore, it does not appear that IPTG would have significantly affected
appellants evidence of unexpected results. Absent a reasoned statement by the
examiner as to why appellants’ results are not unexpected, we find that the examiner
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failed to meet her burden of establishing a prima facie case of obviousness under
35 U.S.C. § 103, in view of all the facts in evidence, including appellants’ evidence
of unexpected results. In re Piasecki, 745 F.2d 1468, 1472, 223 USPQ 785, 788
(Fed. Cir. 1984).
Accordingly, we reverse the examiner’s rejection of claims 1, 9, 10 and 34-
37 under 35 U.S.C. § 103 over Cohen in view of Miozzari (R), Miozzari (S) and
Goeddel.
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Other Issues:
A number of 1449 forms are present in this administrative file with no
indication of being reviewed by the examiner. Upon return of this application, the
examiner should review the 1449 forms and take appropriate action.
REVERSED

)
WILLIAM F. SMITH )
Administrative Patent Judge )
)
)
) BOARD OF PATENT
DONALD E. ADAMS )
Administrative Patent Judge ) APPEALS AND
)
) INTERFERENCES
)
DEMETRA J. MILLS )
Administrative Patent Judge )

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