Ex Parte Nusse et al

Patent Trial and Appeal Board

Jun 20, 2013

12480550 (P.T.A.B. Jun. 20, 2013)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte ROELAND NUSSE and KARL H. WILLERT
__________

Appeal 2012-002890
Application 12/480,550
Technology Center 1600
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Before LORA M. GREEN, JEFFREY N. FREDMAN, and
SHERIDAN K. SNEDDEN, Administrative Patent Judges.

FREDMAN, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal1 under 35 U.S.C. § 134 involving claims to methods
enhancing or maintaining stem cell growth in a culture medium. The
Examiner rejected the claims as failing to comply with the enablement
requirement. We have jurisdiction under 35 U.S.C. § 6(b). We reverse.

1 Appellants identify the Real Part in Interest as the Board of
Trustees of the Leland Stanford Junior University (see App. Br. 1).
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Statement of the Case
Background
“Wnt proteins form a family of highly conserved secreted signaling
molecules that regulate cell-to-cell interactions during embryogenesis”
(Spec. 1 ¶ 01). According to the Specification, “Wnt signaling is involved in
numerous events in animal development, including the proliferation of stem
cells and the specification of the neural crest” (Spec. 1 ¶ 04). The
Specification teaches that “purified Wnt compositions find use in a variety
of therapeutic methods, including the maintenance and growth of stem cells”
(Spec. 2 ¶ 08).
The Claims
Claims 6, 13, and 15-23 are on appeal. Independent claims 6 and 19
are representative and read as follows:
6. A method of enhancing embryonic stem cell self-renewal
and proliferation the method comprising:
contacting mammalian embryonic stem cells with a
culture medium to which an effective dose of purified,
biologically active, substantially homogeneous mammalian
Wnt protein comprising a lipid moiety has been added;
wherein the stem cell self-renewal and proliferation is
enhanced.

19. A method for maintenance of stem cells in in vitro
culture, the method comprising:
contacting mammalian stem cells with a culture medium
to which an effective dose of purified, biologically active,
substantially homogeneous mammalian Wnt protein comprising
a lipid moiety has been added;
wherein the stem cells are maintained in in vitro culture.

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The issues
A. The Examiner rejected claims 6 and 13-18 under 35 U.S.C. § 112,
first paragraph as failing to comply with the enablement requirement (Ans.
4-10).
B. The Examiner rejected claims 19-23 under 35 U.S.C. § 112, first
paragraph as enabling for hematopoietic stem cells but failing to enable the
invention commensurate in scope with the claims (Ans. 10-14).
Because both of these rejections rely on overlapping sets of facts and
turn on the same question of enablement and scope of enablement of claims
6 and 19, we will consider these rejections together.
The Examiner finds that:
given that the state of the art recognizes many diverse
functions of Wnt proteins, and that Wnt signaling and
expression appears to differentiate ES cells . . . and further,
in view of the state of the art of culturing ES cells, with
regard to different culture condition requirements with
regard to the particular animal species of ES cell, as well as
the breadth of the claims, which encompass any ES cell
from any species, and further, in view of the lack of specific
guidance provided by the specification with regard to
utilizing the claimed method for ES cells, it would have
required the skilled artisan to practice undue
experimentation to make and use at the claimed invention

(Ans. 10).
The issue with respect to this rejection is: Does the evidence of
record support the Examiner’s conclusion that undue experimentation would
have been required to enable the full scope of claims 1 and 19?

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Findings of Fact
Breadth of the Claims
1. Claim 6 is drawn to enhancing proliferation of any “mammalian
embryonic stem cells with a culture medium” using a Wnt protein
comprising a lipid moiety (see Claim 6). Claim 19 is drawn to maintaining
stem cells in culture using a Wnt protein comprising a lipid moiety (see
Claim 19).
Presence of Working Examples
2. The Specification teaches “[p]urified Wnt3A causes self-
renewal of hematopoietic stem cells (HSC) in vitro. . . . Single HSCs
responded well to the Wnt3A protein in the presence of limiting doses of
Steel factor. Over a period of 7 days, the frequency of cells proliferating
was 5.8 fold greater compared to control conditions” (Spec. 23 ¶ 81).
Amount of Direction or Guidance Presented
3. The Specification teaches the sequences of a number of
different human Wnt sequences (see Spec. 6 ¶ 20).
4. The Specification teaches that “[a]ctive Wnt molecules,
including the product of the mouse Wnt3A gene, were isolated . . . We have
successfully applied similar purification methods to a variety of other Wnts,
including DWnt8 (Figure 1A), mouse Wnt5A and Drosophila Wingless”
(Spec. 21-22 ¶¶ 74-76).

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State of the Prior Art and Unpredictability of the Art
5. Thomson2 teaches that “[w]ell-characterized ES and EG cells
have been derived only from rodents. . . . Pluripotent cell lines have been
derived from preimplantation embryos of several non-rodent species . . . but
the developmental potentials of these cell lines remain poorly characterized”
(Thomson 7844, col. 1).
6. Thomson teaches “the isolation of an ES cell line (R278.5)
from a rhesus monkey blastocyst. This cloned cell line remains
undifferentiated and continues to proliferate for > 1 year in culture,
maintains a normal XY karyotype, and maintains the potential to
differentiate into trophoblast and to derivatives of embryonic endoderm,
mesoderm, and ectoderm” (Thomson 7844, col. 2).
7. Thomson teaches that
ICM-derived cells from spare in vitro fertilized human
embryos were cultured with LIF in the absence of feeder
layers, and, although alkaline phosphatase positive cells
proliferated, they failed to survive beyond two passages.
These results suggest that soluble LIF alone will not prevent
the differentiation of human ES cells, just as it fails to
prevent the differentiation of rhesus ES cells. The growth of
rhesus monkey ES cells in culture conditions that support
feeder-dependent human EC cells suggests that similar
conditions may support human ES cells.

(Thomson 7848, col. 1.)

2 Thomson et al., Isolation of a primate embryonic stem cell line,
92 PROC. NAT’L. ACAD. SCI. USA 7844-7848 (1995).
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8. Lim3 teaches that “[d]espite many years of using mouse
embryonic fibroblast cells as the feeder support of human ES cells, it is still
not exactly clear what these cells provide for their clients” (Lim 1188, col.
1).
9. Lim teaches that “[o]ur results have also unexpectedly found
many known intracellular proteins, demonstrating the potential of the
proteomics approach in revealing the complexity of proteins in CM. Further
analysis using serum-containing medium in the presence of ES cells, and
using other cell types for feeder layers will be required” (Lim 1203, col. 1).
10. Prowse4 teaches that the “pathways involved in the maintenance
of human embryonic stem (hES) cells remain largely unknown, although
some signaling pathways have been identified in mouse embryonic stem
(mES) cells. Fibroblast feeder layers are used to maintain the
undifferentiated growth of hES cells” (Prowse, abstract).
11. Prowse teaches that:
To date, there is minimal information on factors
regulating self-renewal of hES cells. In comparison there are
three pathways in mouse ES (mES) cells involving the
transcription factors nanog . . . Oct 4, and Stat3 . . . that are
known to be involved in the self renewal of mES cells. hES
cells, however, are difficult to maintain in an
undifferentiated state without the presence of feeder layers .
. . suggesting that the feeder layers may provide factors

3 Lim et al., Proteome analysis of conditioned medium from mouse
embryonic fibroblast feeder layers which support the growth of
human embryonic stem cells, 2 PROTEOMICS 1187-1203 (2002).
4 Prowse et al., A proteome analysis of conditioned media from
human neonatal fibroblasts used in the maintenance of human
embryonic stem cells, 5 PROTEOMICS 978-989 (2005).
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and/or signals (secreted or released) that are necessary for
maintenance of hES cells.

(Prowse 979, col. 1.)
12. Prowse teaches that “[r]ecently the activation of the Wnt
signaling pathway through the inhibition of glycogen synthase kinase-3 has
been shown to maintain pluripotency in both mES and hES cells and
maintain the expression of the pluripotent transcription factors
Oct-3/4 and nanog” (Prowse 979, col. 1).
13. Nusse5 teaches that the “Wnts comprise a large family of
protein ligands that affect diverse processes such as embryonic induction,
generation of cell polarity, and the specification of cell fate” (Nusse 523, col.
2).
14. Nusse teaches that “[a]lthough recent reports indeed implicate
Wnt signaling in keeping ES cells pluripotent, contradicting results have
been published. Consequently, further research is necessary to define the
exact role of Wnt signaling in ES cell maintenance” (Nusse 526, col. 1).
15. Nusse teaches that there are “lines of evidence, mostly coming
from mouse ES cell research, that Wnt signaling components are involved in
ES cell control. . . . Other evidence includes that activation of Wnt signaling,
by genetically eliminating the function of the negative regulator APC,
promotes the undifferentiated phenotype of mouse ES cells” (Nusse 526,
col. 1).

5 Nusse, R., Wnt signaling and stem cell control, 18 CELL
RESEARCH 523-527 (2008).
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16. Dravid6 teaches that “adding Wnt3a increased the percentage
of, and level in, cells incorporating BrdU (Fig. 7G), as compared with the
basal ESC medium group (Fig. 7F). Therefore, Wnt can directly enhance
hESC cycling and proliferation with little effect on preventing apoptosis”
(Dravid 1498, col. 2).
17. Dravid teaches that “we also present several lines of evidence
demonstrating that Wnt is not sufficient to maintain undifferentiated hESC
colonies. Wnt activation can stimulate hESC proliferation but does not
suffice to maintain or expand undifferentiated (and pluripotent) H1 and H9
hESCs for more than one passage” (Dravid 1498, col. 2).
18. Dravid teaches that
Based on our results as well as previous studies, we
favor a new model for the role of Wnt/β-catenin signaling in
hESCs. Wnt, either made endogenously or provided
exogenously, acts on hESCs and enhances cell proliferation
as on many other cell types. The enhancement of cell
proliferation may lead to one of two possible outcomes,
dependent on the presence or absence of other signals. In the
presence of supportive feeder cells or CM (containing the
antidifferentiation factor), endogenous or feeder-derived
Wnt works together with other survival/proliferation factors,
resulting in self-renewing proliferation of undifferentiated
hESCs.

(Dravid 1500, col. 1.)

6 Dravid et al., Defining the Role of Wnt/β-Catenin Signaling in the
Survival, Proliferation, and Self-Renewal of Human Embryonic
Stem Cells, 23 STEM CELLS 1489-1501 (2005).
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19. Lange7 teaches that “the function of single Wnts seems to
depend on the type and developmental stage of the target cells rather than on
the Wnt molecule itself, thereby ascribing multiple roles at multiple times to
the same Wnt in different regions of the nervous system” (Lange 78, col. 1).
20. Brittan8 teaches that the “Wnt family of signalling proteins is
important during embryonic development and organogenesis in a large
number of species” (Brittan 502, col. 1).
21. Brittan teaches that it “is clear that the Wnt/β-catenin signaling
pathway and downstream molecules such as APC, Tcf-4, Fkh-6, Cdx-1, and
Cdx-2 are vital for normal gastrointestinal epithelial function” (Brittan 506,
col. 1).
22. Brittan teaches that “[f]uture studies will be devoted to
increasing our knowledge of the intestinal stem cell and its regulation”
(Brittan 506, col. 2).
23. Yin9 teaches that “[o]ur results told that Wnt-3a-myc protein
inhibited proliferation and maintained monocellular state of adult neural
stem cells in the presence of EGF” (Yin 853, col. 2).
Quantity of Experimentation
24. The Examiner finds that “given that the state of the art
recognizes many diverse functions of Wnt proteins, and that Wnt signaling

7 Lange et al., Wnt Signal Pathways and Neural Stem Cell
Differentiation, 3 NEURODEGENERATIVE DISEASES 76-86 (2006).
8 Brittan et al., Gastrointestinal stem cells, 197 J. PATHOLOGY 492-
509 (2002).
9 Yin et al., Wnt-3a protein promote neuronal differentiation of
neural stem cells derived from adult mouse spinal cord, 29
NEUROLOGY RESEARCH 847-854 (2007).
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and expression has different effects on different types of stem cells, such as
ES, NSCs, and gastrointestinal stem cells, it would not be predictable that
any Wnt protein would be capable of maintaining stem cells in an in vitro
culture” (Ans. 13).
25. Appellants contend that “[w]hile one of skill in the art may
usefully combine wnt with other agents, feeder layer cells and the like in cell
culture methods, such experimentation is not undue, given that one of skill
in the art is now provided with the means to obtain purified compositions of
biologically active wnt” (App. Br. 7).
Skill in the Art
26. The Examiner makes no finding regarding the skill in the art.
27. Appellants contend that the “routine level of skill in the field of
stem cells may be represented by a scientist with a Ph.D. degree and two
years of post-doctoral training” (id.).
Principles of Law
Factors to be considered in determining whether a
disclosure would require undue experimentation ... include
(1) the quantity of experimentation necessary, (2) the
amount of direction or guidance presented, (3) the presence
or absence of working examples, (4) the nature of the
invention, (5) the state of the prior art, (6) the relative skill
of those in the art, (7) the predictability or unpredictability
of the art, and (8) the breadth of the claims.

In re Wands, 858 F.2d 731, 737 (Fed. Cir. 1988).
Analysis
The analytical framework for determining whether claims fail to
satisfy the enablement requirement balances the Wands factors to determine
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if undue experimentation would have been required to perform the
reasonable scope of the claimed method at the time of filing of the
Specification.
While claims 6 and 19 are broadly drawn to the use of any
mammalian Wnt protein and any mammalian embryonic stem cells, the
Specification teaches a large number of different sequences of human Wnt
genes (FF 3) and also teaches purification of Wnt proteins from human,
mouse and drosophila (FF 4). There is a working example where addition of
the Wnt protein enhances/maintains proliferation of hematopoietic stem cells
in an in vitro culture system (FF 2).
While we agree with the Examiner that Thomson (FF 5-7), Lim (FF
8-9), and Prowse (FF 10-12) indicate that Wnt alone is not sufficient for
stem cell growth, and that feeder cells may be required, both claims 6 and 19
are open “comprising” claims which encompass the use of any other
necessary components for embryonic stem cell growth, including feeder
cells. Appellants expressly acknowledge this, stating that “one of skill in the
art may usefully combine wnt with other agents, feeder layer cells and the
like in cell culture methods” (App. Br. 7).
There is no requirement that the claimed component must alone be
sufficient to obtain the desired effect, only that undue experimentation is not
required to obtain the desired effect, here enhancement of cell growth, using
the teachings of the Specification and the knowledge of the art, here
including other known anti-differentiation factors and feeder cells.
By contrast, Nusse supports the inventive idea that Wnt is important
in enhancing and maintaining stem cells (FF 14-15), while Dravid teaches
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that Wnt stimulates embryonic stem cell proliferation (FF 16-17), teaching
that in “the presence of supportive feeder cells or CM (containing the
antidifferentiation factor), endogenous or feeder-derived Wnt works together
with other survival/proliferation factors, resulting in self-renewing
proliferation of undifferentiated hESCs” (Dravid 1500, col. 1; FF 18).
We recognize that Lange teaches that Wnt is context dependent (FF
19), that Brittan teaches Wnt is present in many different mammalian
species (FF 20) but that the role of Wnt in intestinal stem cells is still not
entirely determined (FF 21-22), and that Yin found that Wnt-3A inhibited
proliferation (FF 23).
We agree with Appellants finding that the skill level is minimally that
of a postdoctoral fellow with some years of experience, as evidenced by the
authors of the literature cited (FF 27). We also agree with Appellants that the
amount of experimentation to determine if any particular stem cell type is
responsive to Wnt would have been relatively routine in light of the
teachings of the Specification regarding expression, lipid modification, and
purification of Wnt, and simply require addition of wnt protein to the known
system for growing or maintaining that particular stem cells to determine the
effect of wnt on stem cell growth (FF 25).
As we balance the factors relating to enablement, including the broad
claims, the presence of a working example, the detailed teachings in the
Specification for purification and lipid modification of Wnt, and the prior art
teaching that Wnt enhances stem cell growth, whether alone or in
combination with other factors or feeder cells, the skill in the art, and the
quantity of experimentation required, we conclude that the balance of the
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Wands factors does not support the Examiner’s finding that the claimed
invention fails to comply with the enablement requirement.
The Examiner also finds that “the art of record establishes that Wnt3a
is insufficient to allow self-renewal and proliferation of embryonic stem
cells” (Ans. 15).
We are not persuaded. In this case, the claims do not require that Wnt
must be sufficient alone to enhance or maintain the proliferation of
embryonic stem cells (see Claims 6 and 19), only that Wnt functions in this
manner in a culture system for embryonic stem cells. “Enablement does not
require the inventor to foresee every means of implementing an invention at
pains of losing his patent franchise.” Invitrogen Corp. v. Clontech
Laboratories, Inc., 429 F.3d 1052, 1071 (Fed. Cir. 2005).
Conclusion of Law
The evidence of record does not support the Examiner’s conclusion
that undue experimentation would have been required to enable the full
scope of claims 1 and 19.
SUMMARY
In summary, we reverse the rejection of claims 6 and 13-18 under 35
U.S.C. § 112, first paragraph as failing to comply with the enablement
requirement.

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We reverse the rejection of claims 19-23 under 35 U.S.C. § 112, first
paragraph as enabling for hematopoietic stem cells but failing to enable the
invention commensurate in scope with the claims.

REVERSED

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