U.S. patent application number 10/338083 was filed with the patent office on 2003-09-04 for dominant negative proteins and methods thereof.
Invention is credited to Dahiyat, Bassil I., Desjarlais, John R., Tansey, Malu Lourdes G..
Application Number | 20030166559 10/338083 |
Document ID | / |
Family ID | 26994550 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166559 |
Kind Code |
A1 |
Desjarlais, John R. ; et
al. |
September 4, 2003 |
Dominant negative proteins and methods thereof
Abstract
The invention relates to novel proteins with TNFSF antagonist
activity and nucleic acids encoding these proteins. The invention
further relates to the use of the novel proteins in the treatment
of TNFSF-related disorders.
Inventors: |
Desjarlais, John R.;
(Pasadena, CA) ; Tansey, Malu Lourdes G.;
(Coppell, TX) ; Dahiyat, Bassil I.; (Altadena,
CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
INTELLECTUAL PROPERTY DEPARTMENT
4 EMBARCADERO CENTER
SUITE 3400
SAN FRANCISCO
CA
94111
US
|
Family ID: |
26994550 |
Appl. No.: |
10/338083 |
Filed: |
January 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60345805 |
Jan 4, 2002 |
|
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60373453 |
Apr 17, 2002 |
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Current U.S.
Class: |
435/69.1 ;
514/1.4; 514/16.6; 514/8.3 |
Current CPC
Class: |
A61P 7/00 20180101; A61P
17/06 20180101; A61P 31/04 20180101; A61P 37/04 20180101; A61P
19/10 20180101; A61P 1/04 20180101; A61P 29/00 20180101; A61K 38/00
20130101; A61P 1/16 20180101; A61P 9/04 20180101; A61P 35/00
20180101; C07K 14/525 20130101; A61P 17/00 20180101; A61P 17/10
20180101; A61P 1/02 20180101; A61P 37/06 20180101; A61P 3/06
20180101; A61P 3/14 20180101; A61P 19/00 20180101; A61P 5/38
20180101; A61P 25/28 20180101; C07K 14/70575 20130101; A61P 3/10
20180101; A61P 31/18 20180101; A61P 19/02 20180101; A61P 25/02
20180101; A61P 35/04 20180101; A61P 19/08 20180101; A61P 37/02
20180101; A61P 9/14 20180101; A61P 17/02 20180101; C07K 2319/00
20130101; A61P 35/02 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/17 |
Claims
We claim:
1. A method of antagonizing a naturally occurring TNFSF protein
comprising contacting a naturally occurring TNFSF monomer protein
with a variant TNFSF monomer protein comprising at least a variant
extracellular domain of a TNFSF protein, to form a mixed TNFSF
oligomer.
2. A method according to claim 1 wherein said mixed oligomers have
reduced receptor signaling as compared to wild-type oligomers.
3. A method according to claim 2 wherein said mixed oligomers are
substantially incapable of activating receptor signaling.
4. A method according to claim 1 wherein said mixed oligomer
interacts with a receptor interface in at least one receptor
binding site to render said receptor substantially incapable of
activating receptor signaling.
5. A method according to claim 1 wherein said variant TNFSF monomer
protein comprises at least one receptor contact domain that has
reduced affinity for a desired receptor as compared to its
corresponding wild-type TNFSF protein and retains the ability to
interact with other TNFSF monomers.
6. A method according to claim 1, wherein said variant TNFSF
monomer protein physically interacts with a naturally occurring
TNFSF monomer protein to reduce the ability of the naturally
occurring TNFSF to activate at least one receptor.
7. A method according to claim 1, wherein said variant TNFSF
protein is a fusion protein.
8. A method according to claim 1 wherein said variant protein is
chemically modified.
9. A method according to claim 8, wherein said chemical
modification is PEGylation.
10. A method according to claim 8, wherein said mixed oligomer
comprises covalent cross-linkages between monomers.
11. A method according to claim 10, wherein said mixed oligomer
comprises at least one linker peptide between monomers.
12. A method according to claim 11, wherein said linker peptide is
a sequence of at least one and not more than about 30 amino acid
residues.
13. A method according to claim 12, wherein said linker peptide is
a sequence of at least 5 and not more than about 20 amino acid
residues.
14. A method according to claim 13, wherein said linker peptide is
a sequence of at least 10 and not more than about 15 amino acid
residues.
15. A method according to claim 11, wherein the linker peptide
comprises at least one of the following amino acid residues: Gly,
Ser, Ala, or Thr.
16. A method of making a mixed TNFSF oligomer comprising contacting
at least one variant TNFSF protein comprising at least a variant
extracellular domain of a TNFSF monomer protein with a
homo-oligomer comprising naturally occurring TNFSF monomer
proteins, under conditions whereby at least one naturally occurring
TNFSF monomer exchanges with a variant monomer to form a mixed
oligomer..
17. A mixed TNFSF oligomer comprising at least one variant TNFSF
protein comprising at least a variant extracellular domain of a
TNFSF monomer protein and a naturally occurring TNFSF monomer
protein.
18. A variant TNFSF monomer protein comprising at least a variant
extracellular domain of a TNFSF protein.
19. A variant TNFSF protein wherein said variant TNFSF protein will
interact with a receptor interface in at least one receptor binding
site to render said receptor substantially incapable of activating
receptor signaling.
20. A variant TNFSF protein according to claim 18 comprising at
least one receptor contact domain that has reduced affinity for a
desired receptor as compared to its corresponding wild-type TNFSF
protein and retains the ability to interact with other receptor
interaction domains.
21. A mixed TNFSF oligomer comprising at least one variant TNFSF
protein monomer according to claim 18, wherein said mixed oligomer
has reduced ability to activate the corresponding receptor as
compared to a wild-type oligomer.
22. A variant TNFSF protein according to claim 18, wherein said
variant TNFSF protein physically interacts with a naturally
occurring TNFSF protein to form mixed trimers.
23. A mixed TNFSF oligomer comprising at least one variant TNFSF
protein monomer according to claim 18 comprising a modification at
a receptor contact position.
24. A variant TNFSF monomer protein according to claim 18, wherein
said variant TNFSF protein comprises a modification at a trimer
interface position.
25. A variant TNFSF protein according to claim 18, wherein said
variant TNFSF protein physically interacts with its corresponding
naturally occurring TNFSF protein.
26. A variant TNFSF protein according to claim 18, wherein said
variant TNFSF protein physically interacts with a non-corresponding
naturally occurring TNFSF protein.
27. A recombinant nucleic acid encoding the variant TNFSF monomer
protein of claims 18-26.
28. An expression vector comprising the recombinant nucleic acid of
claim 27.
29. A host cell comprising the recombinant nucleic acid of claim
27.
30. A host cell comprising the expression vector of claim 28.
31. A method of producing a non-naturally occurring TNFSF protein
comprising culturing the host cell of claim 29 or 30 under
conditions suitable for expression of said nucleic acid.
32. A method according to claim 31, further comprising recovering
said TNFSF protein.
33. A pharmaceutical composition comprising a variant TNFSF protein
according to claims 18-26 and a pharmaceutically acceptable
carrier.
34. A method for treating a TNFSF related disorder comprising
administering a variant TNFSF protein of claim 18-26 to a patient
in need of said treatment.
35. A method according to claim 34, wherein said TNFSF related
disorder is an autoimmune disease.
36. A variant TNFSF protein according to claims 18, wherein at
least one modification is non-conservative.
37. A variant TNFSF protein according to claims 18, wherein at
least one modification is a surface modification.
38. A variant TNFSF protein according to claims 36, wherein said
modification is located within a domain selected from the group
consisting of: Large Domain, Small Domain, DE Loop, Trimer
Interface and combinations thereof.
39. A variant TNFSF protein according to claim 38, wherein at least
one of said Large Domain positions is selected from the group
consisting of TNFA corresponding positions 28, 29, 30, 31, 32, 33,
34, 63, 64, 65, 66, 77, 68, 69, 112, 113, 114, 115, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146 and 147.
40. A variant TNFSF protein according to claim 38, wherein at least
one of said Small Domain positions is selected from the group
consisting of TNFA corresponding positions 72, 73, 74, 75, 76, 78,
79, 95, 96, 97 and 98.
41. A variant TNFSF protein according to claim 38, wherein at least
one of said DE Loop positions is selected from the group consisting
of TNFA corresponding positions 84, 85, 86, 87, 88 and 89.
42. A variant TNFSF protein according to claim 38, wherein at least
one of said Trimer interface positions is selected from the group
consisting of TNFA corresponding positions 11, 13, 15, 34, 36, 53,
54, 55, 57, 59, 61, 63, 72, 73, 75, 77, 119, 87, 91, 92, 93, 94,
95, 96, 97, 98, 99, 102, 103, 104, 109, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 147, 148, 149, 151,
155, 156 and 157.
43. A variant TNFSF protein according to claim 42, wherein at least
one of said Trimer Interface positions is selected from the group
consisting of: 57, 34, and 91.
44. A variant TNFSF protein according to claim 18, wherein said
variant TNFSF protein antagonizes soluble naturally occurring TNFSF
proteins.
Description
[0001] This application claims the benefit of the filing date of
U.S. Ser. No. 60/345,805, filed Jan. 4, 2002 and U.S. Ser. No.
60/373,453, filed Apr. 17, 2002, all of which are expressly
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to novel proteins of the Tumor
Necrosis Factor Super Family (TNFSF) with antagonist activity and
nucleic acids encoding these proteins. The invention further
relates to the use of the novel proteins in the treatment of TNFSF
related disorders, such as autoimmune conditions, including but not
limited to rheumatoid arthritis, sepsis and Crohn's disease, as
well as peripheral nerve injury and demyelinating disorders.
BACKGROUND OF THE INVENTION
[0003] Multicellular organisms consists of an intricate and ordered
society of individual cells that must communicate to maintain and
regulate their functions. This is achieved through a complex and
highly regulated network of hormones, chemica mediators, chemokines
and other cytokines, acting as ligand for intra- or extracellular
receptors. (Bodmer, J-L., et al., (2002) TIBS, 27, 19-26. Such
ligands are often activated by ligand-induced oligomerization or
conformational changes (Heldin, C-H., (1995) Cell, 80, 213-223).
Designing proteins that interfere with intracellular signaling
processes is of considerable interest, as many of these proteins
would be useful in the treatment of anemia, cancer, diabetes,
inflammation, neurological and growth disorders and other disease
states.
[0004] The TNFSF proteins constitute an important class of
cytokines that participate in a variety of cellular and
intracellular signaling processes. The prototype of the family,
Tumor Necrosis Factor Alpha (TNFA), originally discovered for its
in vivo effect causing tumors to regress, is a key mediator of
inflammation. The TNFSF currently constitutes at least 18 unique
cytokines that exist in secreted and membrane-bound forms. These
proteins are important regulators of innate and adaptive immune
responses and developmental events. They are synthesized as type 2
membrane proteins and fold into conserved .beta.-pleated sheet
structures that trimerize. While most TNFSF members form
homotrimers, a few exceptions exist. While lymphotoxin a, but not
.beta., can form homotrimers, the two can also form active
heterotrimers with each other. Similarly, APRIL and BLyS also form
both homotrimers and heterotrimers together. Many of the TNF family
members remain membrane-bound and serve as cell contact mediated
regulators, while others are cleaved from the membrane to release
the extracellular domain as a regulator.
[0005] The receptors for TNF family members also represent a family
of structurally related molecules, including at least 26 receptors
and/or receptor decoy molecules. The extracellular domains of
members of this family are composed of multiple repeats of a
cysteine-rich domain (CRD), a small protein domain containing six
conserved cysteines that form three disulfide bonds. The
intracellular domains of these receptors are more diverse, although
many members of the family contain a death domain that mediates
apoptosis and other receptor signaling events. These members are
all capable of inducing apoptosis via interaction with one or more
intracellular adaptor molecules that also contain death domains.
Other signaling receptors of this family signal via interactions
with a family of adaptor molecules called TRAFs (TNF receptor
associated factors). Signaling through TNFSF receptors is triggered
by binding of a oligomeric (and for the most part, trimeric) TNFSF
ligand.
[0006] The three-dimensional structures of TNFSF members are very
similar, made up of a sandwich of two anti-parallel beta-sheets
with the "jelly roll" or Greek key topology. In addition, all
characterized members of the family assemble into trimeric
complexes. The cognate receptors of the TNF family ligands make up
a related superfamily of receptors. Furthermore, there appears to
be significant conservation of the mode of receptor binding. In
general, each receptor monomer binds within the cleft formed
between two of the ligand monomers. The overall similarity in
tertiary and quaternary structures of both the ligands and their
complexes with receptors suggests that well-proven strategies for
inhibition of one ligand-receptor system may be transferable to the
other proteins in the family. However, a mechanism for extension to
other members of the family has not previously been defined. Thus
the present invention provides methods for the creation of variants
of each member of the TNFSF that are antagonistic.
[0007] A need still exists for proteins that can interfere with
intracellular signaling processes. Thus, it is an object of the
present invention to provide proteins comprising multiple TNF
superfamily receptor-interaction domains that are modified such
that each domain has significantly reduced affinity and/or
signaling capacity for the cognate receptor(s). Such linked domains
preferably retain association with individual monomer domains, but
exhibit a dominant-negative phenotype, antagonizing the action of
related naturally occurring domains via their sequestration into
inactive oligomeric complexes.
SUMMARY OF THE INVENTION
[0008] In accordance with the objects outlined above, the present
invention provides variants of extracellular domains of TNFSF
proteins that antagonize naturally occurring TNFSF proteins.
[0009] The present invention provides variant TNFSF proteins
comprising an amino acid sequence that have at least one
modification as compared to the a naturally occurring TNFSF protein
sequence, wherein said variant TNFSF proteins will physically
interact with a naturally occurring TNFSF proteins to form mixed
oligomers substantially incapable of activating receptor
signaling.
[0010] In another embodiment, variant TNFSF proteins of the
invention are in at least monomeric form and comprise amino acid
sequences that have at least one modification as compared to a
naturally occurring TNFSF protein sequence, wherein said variant
TNFSF proteins will interact with a receptor interface at at least
one receptor binding site to render said receptor substantially
incapable of activating receptor signaling.
[0011] Preferably, a variant TNFSF protein of the present invention
has at least one receptor contact domain that has reduced affinity
for a desired receptor as compared to its corresponding wild-type
TNFSF protein and retains the ability to interact with other
receptor interaction domains.
[0012] More specifically, variant TNFSF proteins of the present
invention physically interact with a naturally occurring TNFSF
protein to reduce the ability of the naturally occurring TNFSF to
activate at least one receptor. Variant TNFSF proteins of the
present invention may interact with its corresponding naturally
occurring TNFSF protein or a non-corresponding naturally occurring
TNFSF protein.
[0013] More specifically, variant TNFSF proteins comprising at
least one modified receptor-contact domain that has reduced
affinity and/or signaling capacity for a desired receptor wherein
said protein cannot substantially activate the desired receptor,
but retains the ability to interact with other TNFSF proteins.
[0014] In an additional aspect, the invention provides methods of
antagonizing a naturally occurring TNFSF protein comprising
contacting a naturally occurring TNFSF monomer protein with a
variant TNFSF monomer protein comprising at least a variant
extracellular domain of a TNFSF protein, to form a mixed TNFSF
oligomer. In some cases, the mixed oligomers have reduced receptor
signaling as compared to wild-type oligomers, for example when the
mixed oligomer interacts with a receptor interface in at least one
receptor binding site to render the receptor substantially
incapable of activating receptor signaling. Alternatively or
additionally, the mixed oligomers are substantially incapable of
activating receptor signaling.
[0015] The present invention relates to the use of variants of TNF
superfamily protein ligands for the inhibition or treatment of a
variety of diseases. The variants are specifically engineered to
remove or reduce their biological signaling capacity, while
retaining their ability to function as oligomeric species, either
in complex with themselves or with naturally occurring members of
the superfamily.
[0016] In a preferred embodiment, variant TNFSF proteins are
engineered to yield significantly reduced signaling through TNFSF
receptors compared to wild-type TNFSF proteins while maintaining
affinity for other TNFSF proteins to allow formation of mixed
olimers, most preferably trimers. Such variant TNFSF proteins are
referred to as "dominant negative TNFSF variants" or "DN-TNFSF".
The dominant negative TNFSF variants act by sequestering one or
more naturally occurring TNFSF proteins in mixed heterotrimers that
are incapable of appreciably activating biological signaling.
Consequently, DN-TNFSF proteins act to antagonize the action of
naturally occurring TNFSF.
[0017] The present invention provides non-naturally occurring
variant TNFSF proteins (e.g. proteins not found in nature)
comprising amino acid sequences with at least one modification
compared to the wild-type TNFSF proteins. Examples of suitable
proteins include but are not limited to TNF-.alpha.,
lymphotoxin-.alpha., lymphotoxin-.beta., Fas ligand (FasL), TRAIL,
CD40 ligand (CD40L), CD30 ligand, CD27 ligand, Ox40 ligand, APRIL,
BLyS, 4-IBBL, TRANCE and RANKL (OPGL), and any other protein that
is recognized to be a member of the TNFSF.
[0018] Preferred embodiments utilize variant TNFSF proteins that
interact with one or more wild-type TNFSF members to form mixed
trimers incapable of substantially activating receptor signaling.
Preferably, variant TNFSF proteins with at least one amino acid
change are used as compared to a wild-type TNFSF protein.
[0019] In another preferred embodiment, modifications may be made
either individually or in combination, with any combination being
possible. Preferred embodiments utilize at least one, and
preferably more, positions in each variant TNFSF protein. For
example, amino acid substitutions may combined to form double
variants or triple point variants.
[0020] In a further embodiment, a TNFSF molecule may be chemically
modified, for example by PEGylation or glycosylation.
[0021] In another aspect, portions of the N- or C-termini may
deleted. In a further embodiment, a TNFSF molecule may be
circularly permuted.
[0022] In an additional aspect, the two or more receptor
interaction domains of the variant proteins are covalently linked
by a linker peptide or by other means. Preferably, the linker
peptide is a sequence of at least one and not more than about 30
amino acid residues and comprises one or more of the following
amino acid residues: Gly, Ser, Ala, or Thr.
[0023] In a further aspect, the invention provides recombinant
nucleic acids encoding the non-naturally occurring variant TNFSF
proteins, expression vectors, and host cells.
[0024] In an additional aspect, the invention provides methods of
producing a non-naturally occurring variant TNFSF protein
comprising culturing the host cell of the invention under
conditions suitable for expression of the nucleic acid.
[0025] In a further aspect, the invention provides pharmaceutical
compositions comprising a variant TNFSF protein of the invention
and a pharmaceutical carrier.
[0026] In a further aspect, the invention provides methods for
treating a TNFSF related disorder comprising administering a
variant TNFSF protein of the invention to a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 illustrates a general mechanism by which a dominant
negative TNFSF protein may antagonize the action of a naturally
occurring TNFSF protein. Ovals represent a TNFSF protein monomer
and triangles represent receptor molecules. Naturally occurring
variant TNFSF typically signal by organizing receptors into an
active complex. Variant TNFSF proteins are modified (bumps and
sticks) within their receptor contact domains such that receptor
binding and/or signaling is reduced. When variant TNFSF trimers are
incubated with naturally occurring TNFSF protein trimers, they
equilibrate to form four different trimer species, three of which
are inactive. At sufficient concentrations of dominant-negative
variant TNFSF, essentially all of the naturally occurring TNFSF
will be sequestered into inactive heterotrimeric complexes. Through
this and related mechanisms of inactivating TNFSF proteins, the
dominant-negative TNFSF variants will exert their therapeutic
effects.
[0028] FIG. 2 depicts the structural superposition of TNFSF Ligand
Monomers. Experimentally determined structures of CD40L (1ALY),
RANKL (1JTZ), TNFB (1TNR), and TRAIL (1DG6) are shown superimposed
onto the structure of TNFA (1TNF).
[0029] FIG. 3 shows a Multiple Sequence Alignment (MSA) of human
TNFSF members. FIG. 3 also shows position numberings of each
individual sequence. For TNF-.alpha. (TNFA) and TNFB (LT-.alpha.),
the numbering is based on current convention. For all other
sequences, the numbering is based on the full-length precursor
sequence of the protein. For sequences in which a structure of the
ligand-receptor complex has been determined experimentally,
residues that lie at the ligand-receptor interface are highlighted
in gray. These interfaces, highlighted in black, are used to define
7 general receptor contact regions of the TNF superfamily ligands.
A generic numbering system, beginning with position number 1, is
also included above the MSA for reference.
[0030] FIG. 4A is a chart showing that TNF-alpha variants are
pre-exchanged with wild-type TNF-alpha to reduce TNF-alpha induced
activation of NFkB in 293T cells. FIG. 4B are photographs of the
immuno-localization of NFkB in HeLa cells showing that the exchange
of wild-type TNFalpha with the A145/Y87H TNFSF variant inhibits
TNFSF-induced nuclear translocation of NFkB in HeLa cells. FIG. 4C
depicts the TNFSF variant A145R/Y87H reduces wild-type
TNFSF-induced activation of the NFkB-driven luciferase
reporter.
[0031] FIG. 5 is a chart showing antagonist activity of TNF-alpha
variants.
[0032] FIGS. 6A-C are dose response curves of caspase activation by
various TNF-alpha variants.
[0033] FIG. 7A shows a model for the dominant-negative inhibitory
RANKL variant strategy. The RANKL variant homotrimers and
heterotrimers with wild-type RANKL do not activate the RANK
receptor. The RANKL variants act in a dominant-negative manner by
rendering the wild-type RANKL protein inactive through the
formation of these heterotrimer species. The bumps and sticks on
the engineered RANKL protein symbolize the rationally designed
mutations in the small and large binding domains of RANKL that
interfere with receptor binding. Through this mechanism of
inactivating wild-type RANKL, the dominant-negative inhibitory
RANKL variants will exert their therapeutic effects.
[0034] FIG. 7B shows the list of computationally designed
inhibitory RANKL variants. These inhibitory variants can be
combined with RANKL solubility variants to produce soluble human
RANKL variants.
[0035] FIG. 7C shows RANKL solubility variant C22S/I247E mediated
osteoclastogenesis is antagonized by OPG and RANK-Fc. The plots
show that commercial RANKL from BioSource is antagonized to the
same extent as Xencor solubility variant. The BSA and Enbrel
controls show that the antagonism is specific to RANKL interacting
with OPG and RANK-Fc. The RANK-Fc and OPG controls show that they
do not mediate osteoclastogenesis.
[0036] FIG. 7D shows aRANKL variant antagonism screen. The ability
of RANKL variants to antagonize osteoclastogenesis was evaluated by
monitoring TRAP levels when RANKL-C221S/I247E is mixed with RANKL
variants, incubated and added to RAW264.7 cells. TRAP is released
from RAW264.7 cells as they undergo RANK-mediated
osteoclastogenesis. This experiment identified RANKL variants that
antagonized this process. FIG. 7E. RANKL variant antagonism screen
using a fixed ratio of RANKL-C221S/I247E to ten fold excess
variants. The ability of RANKL variants to antagonize
osteoclastogenesis was evaluated by monitoring TRAP levels when
RANKL-C221S/I247E is mixed with RANKL variants, incubated, diluted
over 2 logs, and added to RAW264.7 cells. TRAP is released from
RAW264.7 cells as they undergo RANK-mediated osteoclastogenesis.
This experiment identified RANKL variants that antagonized this
process. FIG. 7F shows a RANKL variant antagonism screen. The
ability of RANKL variants to antagonize osteoclastogenesis was
evaluated by monitoring TRAP levels when RANKL-C221S/I247E and
RANKL variants are added to RAW264.7 cells without pre-incubation.
TRAP is released from RAW264.7 cells as they undergo RANK-mediated
osteoclastogenesis. This experiment identified RANKL variants that
antagonized this process. FIG. 7G shows a summary of RANKL
antagonizing variants. These variants were shown to antagonize
RANKL mediated osteoclastogenesis.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention is directed to novel proteins that
exhibit a dominant negative phenotype or mechanism of action. The
dominant-negative therapeutic strategy is based on the design of
novel TNFSF variants that have reduced receptor binding and/or
activation properties as compared to naturally occurring TNFSF
proteins, and the ability to oligomerize with naturally occurring
TNFSF proteins (FIG. 1). In other words, TNFSF variants that result
in reduced activation (preferably do not substantially activate)
TNFSF receptors (as compared to a naturally occurring TNFSF
protein) will exchange with at least one naturally occurring TNFSF
protein and sequester it into reduced or inactive hetero-oligomers,
inhibiting the oligomer's biological activity, e.g. the ability to
bind and/or activate the receptor.
[0038] The dominant-negative TNFSF variants of the present
invention may be designed by modifying TNFSF proteins at key
receptor contact points in order to disrupt the ability of the
ligand to either bind or activate the receptor. The exchange and
physical interaction of these oligomeric TNFSF variants with
naturally occurring TNFSF proteins results in reduced activity or
deactivation of the naturally occurring TNFSF proteins. To help
accomplish this goal more effectively, the TNFSF variants can also
be designed to preferentially hetero-oligomerize with naturally
occurring TNFSF proteins. Alternatively, the variants may be
designed to bind each other and "swamp" out the effect of any
naturally occurring TNFSF proteins due to the amount of variant
oligomers present; e.g. equilibria favors the binding of the
variant mixed oligomers.
[0039] Accordingly, the present invention provides methods and
compositions utilizing variants of an extracellular domain of a
TNFSF protein that antagonize a naturally occurring TNFSF
protein.
[0040] By "extracellular domain" or "ECD" as used herein is meant
the segment of protein existing predominantly outside the cell,
generally soluble when cleaved or isolated away from the rest of
the protein. For transmembrane proteins, this segment can be
tethered to the cell through a transmembrane domain or released
from the cell through proteolytic digestion. Alternatively, the
extracellular domain could comprise the whole protein or amino acid
segments thereof when secreted from the cell. In general, TNFSF
members are expressed as type II transmembrane proteins
(extracellular C terminus). The unprocessed protein generally
contains an atypical signal anchor/intracellular domain of about 10
to 80 amino acids. The extracellular region may be about 140-215
amino acids in length. Soluble forms of TNFSF proteins may result
from proteolytic cleavage of the signal propeptide by matrix
metalloproteinases termed TNF-alpha converting enzymes (TACE) or
directly by recombinant methods. FIG. 3 depicts a number of
extracellular domains from a number of different TNFSF proteins. As
will be appreciated by those in the art, these domains may be
shorter or longer than those depicted in FIG. 3.
[0041] In a preferred embodiment, the extracellular domain can be
defined functionally, as a TNFSF protein or variant protein that is
soluble and will form oligomers, preferably with wild-type
monomers.
[0042] Unless otherwise disclosed, the variant TNFSF proteins of
the present invention are composed of the extracellular domain or
functional equivalents thereof. That is, the variants of the
present invention do not comprise transmembrane domains unless
specifically noted. In certain embodiments of the present
invention, the variant TNFSF proteins antagonize the membrane bound
naturally occurring form of a TNFSF protein and in other
embodiments, the variant TNFSF proteins antagonize the soluble form
of a naturally occurring TNFSF protein, or both.
[0043] The TNFSF proteins of the present invention are variant
proteins. The variant TNFSF proteins and nucleic acids of the
invention are distinguishable from naturally occurring or wild-type
TNFSF. By "naturally occurring", "wild-type", "native", or
grammatical equivalents, herein is meant an amino acid sequence or
a nucleotide sequence that is found in nature and includes allelic
variations; that is, an amino acid sequence or a nucleotide
sequence that usually has not been intentionally modified.
Accordingly, by "non-naturally occurring" or "synthetic" or
"recombinant" or grammatical equivalents thereof, herein is meant
an amino acid sequence or a nucleotide sequence that is not found
in nature; that is, an amino acid sequence or a nucleotide sequence
that usually has been intentionally modified. By the term
"recombinant nucleic acid" herein is meant nucleic acid, originally
formed in vitro, in general, by the manipulation of nucleic acid by
endonucleases, in a form not normally found in nature. Thus an
isolated variant TNFSF nucleic acid, in a linear form, or an
expression vector formed in vitro by ligating DNA molecules that
are not normally joined, are both considered recombinant for the
purposes of this invention. It is understood that once a
recombinant nucleic acid is made and reintroduced into a host cell
or organism, it will replicate non-recombinantly, i.e. using the in
vivo cellular machinery of the host cell rather than in vitro
manipulations; however, such nucleic acids, once produced
recombinantly, although subsequently replicated non-recombinantly,
are still considered recombinant for the purposes of the
invention.
[0044] Similarly, a "recombinant protein" is a protein made using
recombinant techniques, i.e. through the expression of a
recombinant nucleic acid as depicted above. A recombinant protein
is distinguished from naturally occurring protein by at least one
or more characteristics. For example, the protein may be isolated
or purified away from some or all of the proteins and compounds
with which it is normally associated in its wild-type host, and
thus may be substantially pure. For example, an isolated protein is
unaccompanied by at least some of the material with which it is
normally associated in its natural state, preferably constituting
at least about 0.5%, more preferably at least about 5% by weight of
the total protein in a given sample. A substantially pure protein
comprises at least about 75% by weight of the total protein, with
at least about 80% being preferred, and at least about 90% being
particularly preferred. The definition includes the production of a
variant TNFSF protein from one organism in a different organism or
host cell. Alternatively, the protein may be made at a
significantly higher concentration than is normally seen, through
the use of an inducible promoter or high expression promoter, such
that the protein is made at increased concentration levels.
Furthermore, all of the variant TNFSF proteins outlined herein are
in a form not normally found in nature, as they contain amino acid
substitutions, insertions and deletions as compared to the
corresponding wild-type (endogeneous) sequence, with substitutions
being preferred.
[0045] Representative amino acid sequences of naurally occurring
human TNFSF are shown in FIG. 3. It should be noted, that unless
otherwise stated, all positional numbering of variant TNFSF
proteins and variant TNFSF nucleic acids is based on these
sequences. That is, as will be appreciated by those in the art, an
alignment of TNFSF proteins and variant TNFSF proteins may be done
using standard programs, as is outlined below, with the
identification of "equivalent" positions between the two proteins.
Thus, the variant TNFSF proteins and nucleic acids of the invention
are non-naturally occurring; that is, they do not exist in
nature.
[0046] In a preferred embodiment, the variant TNFSF protein
comprises non-conservative modifications (e.g. substitutions). By
"nonconservative" modification herein is meant a modification in
which the wild type residue and the mutant residue differ
significantly in one or more physical properties, including
hydrophobicity, charge, size, and shape. For example, modifications
from a polar residue to a nonpolar residue or vice-versa,
modifications from positively charged residues to negatively
charged residues or vice versa, and modifications from large
residues to small residues or vice versa are nonconservative
modifications. For example, substitutions may be made which more
significantly affect: the structure of the polypeptide backbone in
the area of the alteration, for example the alpha-helical or
beta-sheet structure; the charge or hydrophobicity of the molecule
at the target site; or the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the polypeptide's properties are those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)
an electronegative residue, e.g. glutamyl or aspartyl; or (d) a
residue having a bulky side chain, e.g. phenylalanine, is
substituted for (or by) one not having a side chain, e.g. glycine.
In a preferred embodiment, the variant TNFSF proteins of the
present invention have at least one nonconservative
modification.
[0047] Conservative modifications are generally those shown below,
however, as is known in the art, other substitutions may be
considered conservative:
1 Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly
Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met
Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val
Ile, Leu
[0048] Modifications of the proteins are preferably substitutions
and may include those to surface, boundary and core areas of a
TNFSF member. See, for example, U.S. Pat. Nos. 6,188,965 and
6,269,312, hereby incorporated by reference. In another preferred
embodiment, modifications may be made to surface residues,
particularly when alterations to binding properties are desired
(either to other monomers or to the receptor).
[0049] The variant proteins may be generated, for example, by using
a PDA.TM. system previously described in U.S. Pat. Nos. 6,188,965;
6,296,312; 6,403,312; U.S. Ser. Nos. 09/419,351, 09/782,004,
09/927,790, 09/877,695, and 09/877,695; alanine scanning (see U.S.
Pat. No. 5,506,107), gene shuffling ((WO 01/25277), site saturation
mutagenesis, mean field, sequence homology, or other methods known
to those skill in the art that guide the selection of point
mutation sites and types.
[0050] In a preferred embodiment, sequence and/or structural
alignments may be used to generate the variant TNFSF proteins of
the invention. As is known in the art, there are a number of
sequence-based alignment programs; including for example,
Smith-Waterman searches, Needleman-Wunsch, Double Affine
Smith-Waterman, frame search, Gribskov/GCG profile search,
Gribskov/GCG profile scan, profile frame search, Bucher generalized
profiles, Hidden Markov models, Hframe, Double Frame, Blast,
Psi-Blast, Clustal, and GeneWise. There are also a wide variety of
structural alignment programs known. See for example VAST from the
NCBI (http://www.ncbi.nlm.nih.gov:80/Structure/VAST/vast.shtml);
SSAP (Orengo and Taylor, Methods Enzymol 266(617-635 (1996)) SARF2
(Alexandrov, Protein Eng 9(9):727-732. (1996)) CE (Shinydyalov and
Bourne, Protein Eng 11(9):739-747. (1998)); (Orengo et al.,
Structure 5(8):1093-108 (1997); Dali (Holm et al., Nucleic Acid
Res. 26(1):316-9 (1998), all of which are incorporated by
reference).
[0051] The methods of the present invention can be applied to any
recognized member of the TNFSF or related (e.g. the c1q family of
proteins) system in which individual domains oligomerize to form an
active complex. These domains can be modified in a number of ways
to remove or reduce receptor binding and/or activation. In
addition, each modified domain can be covalently coupled to at
least one additional modified domain to generate dominant negative
proteins with enhanced antagonistic activity.
[0052] In a preferred embodiment, the proteins belong to the TNFSF
(See Oren, D. A., et al., (2002) Nature Structural Biology, 9,
288-292; Bodmer, J-L., et al., (2002) TIBS, 27, 19-26; Locksley, R.
M., et al., (2001) Cell, 104, 487-501; WO 01/25277; all of which
are expressly incorporated herein by reference). Thus, the
definition of "TNFSF" proteins include, but are not limited to,
members of the TNFSF of interest include the ligands for TNF;
osteoprotegerin (OPG) also known as RANKL (U.S. Pat. No. 5,843,678,
incorporated herein by reference), CD40 ligand, BLyS, etc. and
others shown in FIG. 3. However, in some embodiments, the TNFSF
specifically excludes TNF-.alpha. protein.
[0053] The compositions and methods of the present invention may
also be applied to structural homologues of the TNFSF, including
for example, the C1q-related family of proteins, examples of which
include the adipocyte complement-related protein of 30 kDa (ACRP30)
and its human ortholog APM-1. ACRP30 belongs to a C1q-related
family of proteins comprised of at least 23 members either with or
without collagen domains. Those with a collagenous domain include
ACRP30, C1qA, B, and C chains, hibernation-related proteins in
chipmunks (HP-20, 25 and 27), CORS26 and Elastin microfibril
interface-located protein (EMILIN). Those lacking the collagen
domain include multimerin and the precursors of cerebellins 1 and
3. Many of these proteins--which also form trimers or multimers of
trimers--have been implicated in development, and immunological and
physiological homeostasis.
[0054] As outlined herein, there are a variety of mechanisms that
may allow the variant TNFSF proteins to serve as antagonists for
the wild-type proteins. In preferred embodiments, the variant
proteins form hetero-oligomers with endogeneous wild-type TNFSF
proteins and then bind, but do not activate, the corresponding
receptors. That is, as illustrated in FIG. 1, a variant TNFSF
protein is preferably modified such that interactions with a
receptor molecule are disrupted. Preferably, these modifications
would not substantially affect the ability of the variant domain to
interact with and sequester the naturally occurring TNFSF protein.
In a preferred embodiment, these modifications may be combined with
additional modifications that enhance the ability of variant TNFSF
proteins to hetero-oligomerize with one or more naturally occurring
TNFSF proteins. Most preferably, modifications that affect receptor
activation and oligomerization are also combined with chemical
modifications (e.g., glycosylation, PEGylation, fusions, etc.) that
improve pharmacokinetic properties, as further outlined below.
[0055] More preferably, the present invention is also directed to
novel proteins and nucleic acids possessing TNFSF antagonist
activity. Thus, variant proteins possessing "antagonist" activity
ultimately result in a lack of receptor activation as further
defined below. This may be due to variant monomers interacting with
wild-type monomers to "antagonize" the wild-type monomers, e.g.
such that wild-type monomers are incapable of binding and/or
activating the receptors. Generally speaking, the mixed oligomers
comprising at least one variant monomer and at least one wild-type
(e.g. endogeneous) monomer inhibit receptor activation.
[0056] In a preferred embodiment, the variant TNFSF proteins are
dominant negative proteins. By "dominant negative" phenotype or
"mechanism of action" herein is meant a protein comprising at least
one variant TNFSF monomer that has reduced affinity and/or altered
signaling for a desired receptor such that the protein retains the
ability to oligomerize with other receptor interaction domains but
the oligomer cannot substantially interact and/or signal with the
desired receptor (see FIG. 1). Depending on the composition of the
oligomeric ligand complex, i.e., 2 variants:1 native or 1 variant:2
native for heterotrimers, the degree to which ligand mediated
receptor activation is inhibited will vary (see FIG. 1). In other
words, receptor activation may be completely inhibited in a complex
comprising 2variant:1native, whereas activation may be reduced in
complexes comprising other ratios of variant:native. Alternatively,
receptor activation may not be completely inhibited but decreased,
again to varying degrees based on the composition of the
heterodimer and its corresponding mode of action. See also Menart,
V., et al., (2000) Eur J Physiol., 439, R113-R115; U.S. Patent Pub.
Nos. 2002/0039588, 2002/0040132, 2002/0037286, 2002/0037280; all of
which are incorporated herein by reference. Monte carlo simulations
of heterotrimer assembly, as a function of the relative
concentration of variant TNFSF to naturally occurring TNFSF, show
that, in general, greater than 99% of the naturally occurring TNFSF
monomers will be sequestered when a 10-fold excess of variant TNFSF
monomer is added.
[0057] As will be appreciated by those of skill in the art, two
general approaches for creating the dominant negative variants of
the present invention include: (1) modifying individual receptor
interaction domains to reduce or eliminate receptor binding and/or
signaling; and, (2) covalently coupling modified receptor
interaction domains to enhance inhibition of receptor
activation.
[0058] In a preferred embodiment, individual TNFSF proteins are
modified within their receptor contact domains to reduce or
eliminate receptor binding and/or signaling. For example, amino
acid substitutions can be generated as modifications in the
receptor contact domains that reduce or eliminate receptor binding.
In a preferrred embodiment, at least one non-conservative variant
in receptor contact domains may be made to disrupt receptor
interactions. See FIGS. 4A and 4B, and U.S. Pat. No. 5,506,107;
U.S. Ser. Nos. 09/798,789; 09/981,289; 10/262,630; 60/374,035; and
an application filed on even date entitled "NOVEL VARIANTS OF RANKL
PROTEIN", all of which are hereby incorporated by reference.
[0059] Preferable modifications (e.g. substitutions, insertions,
deletions, etc.) that affect receptor binding or signaling may be
identified using a variety of techniques, including structural
alignment methods, sequence alignment methods, etc., as described
above. In many cases, the amino acids in the TNFSF ligand that
interact with the receptor can be identified directly from a
three-dimensional structure of the TNFSF ligand-receptor complex.
Alternatively, equivalent information can be derived by analysis of
the ligand-receptor complex of a related protein. For example,
within the TNFSF, members of the TNF family may be structurally
aligned with a TNFSF protein whose structure has been determined
experimentally (see for example, Oren, D. A., et al., (2002) Nature
Structural Biology, 9, 288-292). Alternatively, if it is not
possible to structurally align residues, sequence alignment may be
used.
[0060] As is known in the art, there are a number of sequence
alignment methodologies that may be used. For example, sequence
homology based alignment methods may be used to create sequence
alignments of TNFSF members (Altschul et al., J. Mol. Biol. 215(3):
403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-3402
(1997), both incorporated by reference).
[0061] In a preferred embodiment, as highlighted in FIG. 3, the
amino acid sequences of members of the TNF superfamily may be
aligned into a multiple sequence alignment (MSA). The alignment
shown in FIG. 3 was derived originally from the Pfam database, and
then further manipulated according to structural alignment (using
CE) of the crystal structures of TNFA, TNFB, CD40L, TRAIL, and
BlyS. The MSA may also be used to extend the known structural
information for additional recognized TNFSFmembers and other
structural homologues and families. Due to the high extent of
structural homology between different TNFSF members, the MSA may be
used as a reliable predictor of the effects of modifications at
various positions within the alignment. For this, the TNFA sequence
and numbering shown in FIG. 3 can be used as an MSA reference point
for any other TNFSF protein sequence. As used herein, referral to
"TNFSF protein positions corresponding to TNFA amino acid X",
represents referral to a collection of equivalent positions in
other recognized TNFSF members and structural homologues and
families. For example, TNFSF protein positions corresponding to
TNFA amino acid L75 corresponds to the following amino acid
positions in the following TNFSF proteins: TNFA:L75, TNFB:Y96,
FASL:P206, LIGHT:T161, VEGI:S99, Lymphotoxin beta:T158, APRIL:T177,
BLyS:A207, CD40L:P188, RANKL:Q237, TRAIL:Q205, CD27L:S121,
4-1BBL:S162, TWEAK:Y176, CD30L:D157, OX40L:N114, AITRL:N106, and
equivalent positions in other proteins recognized as TNFSF
members.
[0062] For example, analysis of a structure of the complex of TNFB
with the p55 (R1) receptor indicates that the amino acid Y108 in
TNFB directly contacts the receptor. The analogous residue Y216
from TRAIL also directly contacts the DR5 receptor. The MSA thus
predicts that the analogous residue 197 from TNFA also contacts a
receptor. Consistent with this prediction, mutation of TNFA-I97 to
R or T results in a significant loss of receptor-binding affinity
and biological signaling activity. The analysis for this contact
position can be extended to all members of the family, predicting
that the following positions are important for receptor
interactions: FASL:Y218, LIGHT:Y173, VEGI:Y111, TNFC:Y170,
APRIL:R189, BlyS:V219, CD40L:R200, RANKL:I249, CD27L:C133,
4-1BBL:A174, TWEAK:A188, CD30L:K169, OX40L:L126, and AITRL:Y118.
This kind of analysis can be performed for all receptor contact
regions of the ligands.
[0063] FIG. 3 highlights 7 canonical receptor contact regions based
on analysis of known structures and mutational data. In preferred
embodiments of the invention, each of the 7 regions highlighted in
FIG. 3 as a receptor-contact region is used to define modification
sites for the creation of variants of each TNFSF member. In
additional preferred embodiments, such modifications reduce
receptor affinity and/or signaling capacity. In additional
preferred embodiments, these modifications also preserve the
ability of each protein to oligomerize with naturally occurring
TNFSF proteins, including, but not necessarily limited to, the
corresponding wild-type sequence of each family member.
[0064] Using the alignment system depicted in FIG. 3 or other
alignment programs discussed above, one can use as a reference
point, the numbering system of any alignment program and may
correlate the relevant positions of the TNFA protein with
equivalent positions in other recognized members of the TNFSF or
structural homologues and families.
[0065] For purposes of the present invention, the areas of TNFSF
proteins to be modified are preferably but not required to be
selected from the group consisting of the Large Domain, Small
Domain, the DE loop, and the trimer interface. The Large Domain,
the Small Domain and the DE loop are three separate receptor
contact domains, each made up of several non-contiguous linear
segments of the protein (i.e. the 7 canonical receptor contact
regions described above). These domains are identified in the TNFSF
proteins--and the MSA--by comparison to the receptor interaction
domains of Lymphotoxin-alpha and TRAIL, two TNFSF proteins whose
structures (PDB entries 1TNR and 1D0G, respectively) have been
defined in complex with their cognate receptors using
crystallographic methods. The trimer interface mediates
interactions between individual TNFSF protein monomers.
Trimerization positions can be identified either directly from the
crystal structure of the appropriate TNFSF protein (e.g. for TNFA,
TNFB, BLyS, TRAIL, CD40L, or RANKL), or by analogy to another TNFSF
protein. In a preferred embodiment, positions from one TNFSF
protein monomer containing atoms that are within 5 angstroms
distance from a neighboring monomer are designated as trimer
interface positions. Modifications may be made solely in one of
these areas or in any combination of these and other areas.
[0066] The Large Domain preferred positions to be modified in TNFSF
proteins include but are not limited to TNFA corresponding
positions 28-34, 63-69, 112-115, and 137-147. For the Small Domain,
the preferred positions to be modified include but are not limited
to TNFA corresponding positions 72-79 and 95-98. For the DE Loop,
the preferred positions to be modified include but are not limited
to TNFA corresponding positions 84-89. The Trimer Interface
positions to be modified include but are not limited toTNFA
corresponding positions 11, 13, 15, 34, 36, 53-55, 57, 59, 61, 63,
72, 73, 75, 77, 119, 87, 91-99, 102-104, 109, 112-125, 147-149,
151, and 155-157. Especially preferred trimer interface positions
to be modified are TNFA corresponding positions 57, 34, and 91. For
example, amino acids X and Y at TNFA corresponding positions 34 and
91 can be replaced simultaneously by similarly charged amino acids
(e.g. X34E+Y91E, X34K+Y91R, etc.) to generate electrostatic
repulsion at the variant monomer-monomer interfaces while not
perturbing the stability of variant-native interfaces.
[0067] In a preferred embodiment, the choice of modification site
and type is made by referring to other sequences in the alignment.
Thus, in a preferred embodiment, the original amino acid X from
sequence A is mutated to amino acid Y from sequence B, such that Y
is a nonconservative substitution relative to amino acid X. For
example, the amino acid Y87 from TNFA aligns with the
non-conservative R189 from APRIL. Indeed, as previous studies have
shown, the Y87R substitution in TNFA leads to a significant
decrease in receptor binding and signaling by TNFA. In additional
embodiments, more conservative mutations can also be utilized. In
additional embodiments, the wild-type residue is mutated to
alanine.
[0068] In a preferred embodiment, useful modifications at receptor
contact and/or trimerization interfaces are selected using protein
design or modeling algorithms such as PDA.TM. technology (see, U.S.
Pat. Nos. 6,188,965; 6,269,312; 6,403,312; U.S. Ser. Nos.
09/714,357; 09/812,034; 09/827,960; 09/837,886; 09/782,004 and
10/218,102, all hereby incorporated by reference). As is known in
the art, algorithms in this class generally use atomic-level or
amino acid level scoring functions to evaluate the compatibility of
amino acid sequences with the overall tertiary and quaternary
structure of a protein. Thus, algorithms of this class can be used
to select receptor-binding disruptions that do not substantially
perturb the ability of variant TNFSF proteins to properly fold and
oligomerize with themselves or their naturally occurring targets.
These technologies typically use high-resolution structural
information of the target protein as input. In a preferred
embodiment, an experimentally determined strucure of the
appropriate TNFSF protein is used as input. In alternative
embodiments, the MSA can be used to guide the construction of
atomic-level homology models for TNFSF members based on the subset
of the family whose three-dimensional structures have been
determined using crystallographic or related methods. For the
TNFSF, high-resolution structures have been determined for TNFA,
TNFB (LT-alpha), TRAIL, CD40L, and BLyS. The homology models can in
turn be used as structural scaffolds to guide the design of variant
TNF superfamily ligands that possess reduced receptor binding
and/or signaling and/or dominant-negative activity.
[0069] In alternative embodiments, protein design algorithms may be
used to generate mutations in individual receptor interaction
domains that create steric repulsion between the receptor
interaction domain and the receptor. Other mutations that may be
generated include, but are not limited to, mutations that create
electrostatic repulsion, and mutations that create unfavorable
desolvation of amino acids.
[0070] In a preferred embodiment, substitutions, insertions,
deletions or other modifications at multiple receptor interaction
and/or trimerization domains may be combined. Such combinations are
frequently advantageous in that they have additive or synergistic
effects on dominant-negative activity. Examples include, but are
not limited to, simultaneous substitution of amino acids at the
large and small domains (e.g. MSA positions 94 and 112), large
domain and DE loop (e.g. MSA positions 95 and 124), large domain
and trimerization domain (e.g. MSA positions 94 and 83), or
multiple substitutions within a single domain. Additional examples
include any and all combinations of substitutions.
[0071] In preferred embodiments, the defined receptor contact
regions constitute sites for insertion, deletion, or substitution
of amino acid residues, or sites for the introduction of chemical
modification sites. In a preferred embodiment, deletions or
insertions are made in accordance with the MSA. For example,
inspection of the MSA reveals that BLyS amino acids 220-223
constitute a 4-residue insertion relative to many of the additional
family members. This region lies in the DE-loop region of the
protein, well known to interact directly with receptors in many of
the TNF ligand-receptor systems. Thus, deletion of these four
residues, while predicted to maintain the structural integrity of
the BLyS protein, is expected to reduce affinity of BLyS for its
receptor.
[0072] In additional embodiments, the variants described above can
be combined with other modifications to the TNF superfamily ligand.
These include, but are not limited to, additional amino acid
substitutions, insertions, or deletions, and/or chemical (e.g.
PEGylation) or posttranslational modifications such as
glycosylation (see WO 99/45026; WO 01/49830; WO 01/49830; WO
02/02597; WO 01/58493; WO 01/51510, U.S. Pat. Nos. 4,002,531;
5,183,550; 5,089,261; 6,153,265; 5,264,209; 5,383,657; 5,766,897;
5,986,068; 4,280,953; 5,089,261; 5,990,237; 6,461,802; 6,495,659;
6,448,369; 6,437,025; 5,900,461; 6,413,507; 5,446,090; 5,672,662;
5,919,455; 6,113,906; 5,985,236; 6,214,966; 6,258,351; 5,932,462;
EP 0786 257; EP 0 902 085; EP 1 064 951; EP 0 544 826; EP 0 424
405; EP 0 400 472; EP 0 311 589; Veronese, F. M. (2001)
Biomaterials, 22: 405-471; all of which are incorporated herein by
reference). In some embodiments, for example in the creation of
animal models of disease, fusion proteins comprising the variant
TNFSF proteins with other sequences may be done, for example using
fusion partners comprising labels (e.g. autofluorescent proteins,
survival and/or selection proteins), stability and/or purification
sequences, toxins, variant proteins from other members of the
superfamily (e.g. analogous to the creation of "bi-specific
antibodies") or any other protein sequences of use. Additional
fusion partners are described below. In some instances, the fusion
partner is not a protein.
[0073] In a preferred embodiment, additional amino acid
substitutions are made to optimize hetero-oligomer interactions
between variant ligand and its endogenous counterpart and/or to
destabilize the oligomeric state of the variant alone. For example,
an L57F mutation in TNFA has been designed in order to promote the
formation of variant:native heterotrimers while disfavoring the
formation of variant homotrimers. Such modifications are useful for
promoting the exchange of variant monomers with native monomers in
order to promote a dominant-negative mechanism of action.
[0074] As will be understood by those in the art, variant TNF
superfamily ligands which have reduced signaling capacity can be
discovered by a large variety of methods, including, but not
limited to, directed evolution (e.g. error prone PCR, DNA
shuffling, etc.), single-site saturation mutagenesis, and
alanine-scanning mutagenesis. Furthermore, it is possible that use
of these or other methods will allow the discovery of
substitutions, insertions, or deletions--which reduce receptor
binding and/or signaling activity--that lie outside of the 7
canonical contact regions described herein.
[0075] In another embodiment, coiled-coil motifs are used to assist
dimer assembly (see Dahiyat et al., Protein Science 6:1333-7 (1997)
and U.S. Ser. No. 09/502,984; both of which are incorporated herein
by reference in their entirety). Coiled coil motifs comprise, but
is not limited to one of the following sequences:
RMEKLEQKVKELLRKNERLEEEVERLKQLVGER, based on the structure of GCN4;
AALESEVSALESEVASLESEVAAL, and LAAVKSKLSAVKSKLASVKSKLAA, coiled-coil
leucine zipper regions defined previously (see Martin et al., EMBO
J. 13(22): 5303-5309 (1994), incorporated by reference). Other
coiled coil sequences from e.g. leucine zipper containing proteins
are known in the art and are used in this invention. See, for
example, Myszka et al., Biochem. 33:2362-2373 (1994), hereby
incorporated by reference).
[0076] Similarly, molecular dynamics calculations can be used to
computationally screen sequences by individually calculating mutant
sequence scores and compiling a list.
[0077] In a preferred embodiment, residue pair potentials can be
used to score sequences (Miyazawa et al., Macromolecules 18(3):
534-552 (1985), expressly incorporated by reference) during
computational screening.
[0078] As will be appreciated by those in the art, additional TNFSF
proteins may be identified and added to the MSA highlighted in FIG.
X. The source of the sequences may vary widely, and include taking
sequences from one or more of the known databases, including, but
not limited to, GenBank (http://www.ncbi.nlm.nih.gov/).
[0079] In addition, sequences from these databases may be subjected
to contiguous analysis or gene prediction; see Wheeler, et al.,
Nucleic Acids Res 28(1):10-14. (2000) and Burge and Karlin, J Mol
Biol 268(1):78-94. (1997).
[0080] As is known in the art, there are a number of sequence
alignment methodologies that may be used. For example, sequence
homology based alignment methods (Altschul et al., J. Mol. Biol.
215(3): 403-410 (1990); Altschul et al., Nucleic Acids Res.
25:3389-3402 (1997), both incorporated by reference) may be used to
create sequence alignments of TNFSF members. These sequence
alignments are then examined to determine the observed sequence
variations. These sequence variations are tabulated to define a set
of variant TNFSF proteins.
[0081] Sequence based alignments may be used in a variety of ways.
For example, a number of related proteins may be aligned, as is
known in the art, and the "variable" and "conserved" residues
defined; that is, the residues that vary or remain identical
between the family members can be defined. These results may be
used to guide the design of variant protein libraries whose
properties can be probed experimentally. For example, the positions
of high variability between family members (i.e. low conservation)
may be randomized, either using all or a subset of amino acids.
Alternatively, the sequence variations may be tabulated and
appropriate substitutions defined from them. Alternatively, the
allowed sequence variations may be used to define the amino acids
considered at each position during a computational modeling and/or
screening process. Another variation is to bias the score for amino
acids that occur in the sequence alignment, thereby increasing the
likelihood that they are found during computational screening but
still allowing consideration of other amino acids. This bias would
result in a focused library of variant TNFSF proteins but would not
eliminate from consideration amino acids not found in the
alignment.
[0082] As used herein variant TNFSF or TNFSF proteins include TNFSF
monomers, dimers or trimers, with the former two preferred when
combined into trimers comprising wild-type protein.
[0083] The TNFSF proteins may be from any number of organisms, with
TNFSF proteins from mammals being particularly preferred. Suitable
mammals include, but are not limited to, rodents (rats, mice,
hamsters, guinea pigs, etc.), primates, farm animals (including
sheep, goats, pigs, cows, horses, etc); and in the most preferred
embodiment, from humans (the sequence of which is depicted in FIG.
6B). As will be appreciated by those in the art, TNFSF proteins
based on TNFSF proteins from mammals other than humans may find use
in animal models of human disease.
[0084] The variant TNFSF proteins of the invention are antagonists
of naturally occurring TNFSF proteins. By "antagonists of naturally
occurring TNFSF" herein is meant that the variant TNFSF protein
inhibits or significantly decreases the activation of receptor
signaling as compared to activation by a naturally occurring member
of the TNFSF in the absence of the antagonist.
[0085] In a preferred embodiment, the variant TNFSF protein
physically interacts with its corresponding wild-type TNFSF protein
(i.e. the endogeneous naturally occurring protein from which the
variant was derived) such that the complex comprising the variant
TNFSF and wild-type TNFSF is incapable of activating TNFSF
receptors. Preferably, the variant TNFSF protein preferentially
interacts with a wild-type TNFSF to form mixed trimers with the
wild-type protein such that receptor binding does not occur and/or
TNFSF signaling is not initiated (FIG. 1).
[0086] In an alternative embodiment, the variant TNFSF protein
physically interacts with a non-corresponding wild-type protein
(i.e. a naturally occurring protein different from the protein from
which the variant was derived). For example, because APRIL is known
to form homotrimers with itself and heterotrimers with BLyS,
dominant-negative variant APRIL protein can be used to inhibit
naturally occurring APRIL or BLyS proteins. Similarly, since
lymphotoxin .alpha. and .beta. can form active heterotrimers with
each other, dominant-negative variant LT-.alpha. proteins of the
invention can be used to inactivate LT-.beta..
[0087] By mixed trimers herein is meant that monomers of native and
variant TNFSF proteins interact to form trimeric TNFSF (FIG. 1).
Mixed trimers may comprise 1 variant TNFSF protein:2 native TNFSF
proteins, 2 variant TNFSF proteins:1 native TNFSF protein. In some
embodiments, trimers may be formed comprising only variant TNFSF
proteins (FIG. 1B).
[0088] In a preferred embodiment, the variant TNFSF antagonist
proteins of the invention are highly specific antagonists for the
corresponding wild-type TNFSF protein. However, in alternative
embodiments, the variant TNFSF antagonistic proteins of the
invention are d specific for more than one wild-type TNFSF protein.
For example, variant APRIL proteins may be specific antagonist of
wild-type APRIL only, wild-type APRIL and BLyS, or wild-type BLyS
only. Additional characteristics of the variant TNFSF antagonist
proteins include improved stability, pharmacokinetics, and high
affinity for native TNFSF. Variants with higher affinity toward
native TNFSF may be generated from variants exhibiting TNFSF
antagonism as outlined above.
[0089] In a preferred embodiment, variant TNFSF proteins exhibit
decreased biological activity as compared to native TNFSF,
including but not limited to, decreased binding to the receptor,
decreased activation and/or ultimately a loss of cytotoxic activity
or otherwise undesired activities that can lead to deleterious side
effects. By "cytotoxic activity" herein refers to the ability of a
TNFSF variant to selectively kill or inhibit cells. Variant TNFSF
proteins that exhibit less than 75-50% biological activity as
compared to native are preferred. More preferred are variant TNFSF
proteins that exhibit less than 25%, even more preferred are
variant proteins that exhibit less than 15%, and most preferred are
variant TNFSF proteins that exhibit less than 10% of a biological
activity of a naturally occurring TNFSF. Suitable assays include,
but are not limited to, TNFSF cytotoxicity assays, DNA binding
assays; transcription assays (using reporter constructs; see
Stavridi, supra); size exclusion chromatography assays and
radiolabeling/immuno-pre- cipitation; see Corcoran et al., supra);
and stability assays (including the use of circular dichroism (CD)
assays and equilibrium studies; see Mateu, supra); all of which are
expressly incorporated by reference. These assays may utilized
labeled variant proteins, with suitable labels including
radioisotopes, chromophores (particularly fluorophores), enzymes,
particles e.g. magnetic particles, etc.
[0090] In one embodiment, at least one property critical for
binding affinity of the variant TNFSF proteins is altered when
compared to the same property of native TNFSF and in particular,
variant TNFSF proteins with altered receptor affinity are
preferred. Also preferred are variant TNFSF with altered affinity
toward oligomerization to native TNFSF.
[0091] Thus, the invention provides variant TNFSF proteins (e.g.
mixed oligomers containing variant monomers) with altered binding
affinities such that the variant TNFSF monomer proteins will
preferentially oligomerize with wild-type (e.g. endogeneous) TNFSF
monomers, but do not substantially agonize TNFSF receptors.
"Preferentially" in this case means that given equal amounts of
variant TNFSF monomers and wild-type TNFSF monomers, at least 10%
and more preferably at least 25% of the resulting trimers are mixed
trimers of variant and wild-type TNFSF, with at least about 50%
being preferred, and at least about 80-90% being particularly
preferred. In other words, it is preferable that the variant TNFSF
proteins of the invention have greater affinity for wild-type TNFSF
protein as compared to wild-type TNFSF proteins. By "do not
substantially interact with TNF receptors" herein is meant that the
variant TNFSF proteins will not be able to associate with TNFSF
receptors to substantially activate the receptors and/or initiate
the TNFSF signaling pathway(s). In a preferred embodiment, at least
a 10% decrease in receptor activation is seen, with greater than
20%, 50%, 76%, 80-90% being preferred. By "agonize TNFSF receptors"
herein is meant that the variant TNFSF protein enhances the
activation of receptor signaling. Generally, variant TNFSF proteins
that function as agonists of wild-type TNFSF are not preferred.
[0092] Variant TNFSF proteins may be experimentally tested and
validated using in vivo and in vitro assays. Suitable assays
include, but are not limited to, activity assays and binding
assays. Screens that may be utilized in identifying TNFSF variants
that are antagonists of TNFSF proteins include, but are not limited
to, caspase activation, NF-kB nuclear translocation (Wei et al.,
Endocrinology 142, 1290-1295, (2001)) or c-Jun (Srivastava et al.,
JBC 276, 8836-8840 (2001)) transcription factor activation assays,
B-cell proliferation assays and IgE secretion assays.
[0093] In a preferred embodiment, binding affinities for the
following interactions are determined and compared: 1) variant
TNFSF oligomer formation, 2) wild-type TNFSF oligomer formation, 3)
variant TNFSF binding to a cognate receptor, 4) wild-type TNFSF
binding to cognate receptor, 5) variant TNFSF binding to decoy
receptor, and 6) wild-type TNFSF binding to decoy receptor.
Suitable assays include, but are not limited to, quantitative
comparisons comparing kinetic and equilibrium binding constants.
The kinetic association rate (Kon) and dissociation rate (Koff),
and the equilibrium binding constants (Kd) may be determined using
surface plasmon resonance on a BIAcore instrument following the
standard procedure in the literature [Pearce et al., Biochemistry
38:81-89 (1999)]. Several alternative methods can also be used to
determine binding affinity and kinetics, including but not limited
to proximity assays such as AlphaScreen.TM. (Packard
BioScience.RTM.).
[0094] TNFSF variants can also be tested to determine whether they
are capable of forming mixed oligomers including but not limited to
mixed trimers. In a preferred embodiment, this is accomplished by
labeling native TNFSF and variant TNFSF with distinguishable tags,
combining native and variant TNFSF, and screening for oligomers
that contain both tag types. For example, FLAG-tagged native TNFSF
and His-tagged variant TNFSF can be combined, and sandwich ELISAs
can be performed to identify trimers that contain both FLAG and His
tag. Another alternative is to run native gels to separate the
mixture into separate species and detect using coomassie staining
or Western blots using both anti-FLAG and anti-His tag antibodies.
This method relies on the fact that FLAG and His tags significantly
perturb protein migration in native gels. As will be appreciated by
those in the art, many alternate protocols could also be used to
measure the formation of mixed trimers.
[0095] As outlined above, the invention provides variant TNFSF
nucleic acids encoding variant TNFSF polypeptides. The variant
TNFSF polypeptide preferably has at least one altered property as
compared to the same property of the corresponding naturally
occurring TNFSF polypeptide. The property of the variant TNFSF
polypeptide is the result of the present invention.
[0096] The term "altered property" or grammatical equivalents
thereof in the context of a polypeptide, as used herein, further
refers to any characteristic or attribute of a polypeptide that can
be selected or detected and compared to the corresponding property
of a naturally occurring protein. These properties include, but are
not limited to cytotoxic activity; oxidative stability, substrate
specificity, substrate binding or catalytic activity, thermal
stability, alkaline stability, pH activity profile, resistance to
proteolytic degradation, kinetic association (Kon) and dissociation
(Koff) rate, protein folding, inducing an immune response, ability
to bind to a ligand, ability to bind to a receptor, ability to be
secreted, ability to be displayed on the surface of a cell, ability
to oligomerize, ability to signal, ability to stimulate cell
proliferation, ability to inhibit cell proliferation, ability to
induce apoptosis, ability to be modified by phosphorylation or
glycosylation, and the ability to treat disease.
[0097] Unless otherwise specified, a substantial change in any of
the above-listed properties, when comparing the property of a
variant TNFSF polypeptide to the property of a naturally occurring
TNFSF protein is preferably at least a 20%, more preferably, 50%,
more preferably at least a 2-fold increase or decrease.
[0098] A change in cytotoxic activity is evidenced by at least a
75% or greater decrease in cell death initiated by a variant TNFSF
protein as compared to wild-type protein.
[0099] A change in binding affinity is evidenced by at least a 5%
or greater increase or decrease in binding affinity to wild-type
TNF receptor proteins or to wild-type TNFSF.
[0100] In a preferred embodiment, the antigenic profile in the host
animal of the variant TNFSF protein is similar, and preferably
identical, to the antigenic profile of the host TNFSF; that is, the
variant TNFSF protein does not significantly stimulate the host
organism (e.g. the patient) to an immune response; that is, any
immune response is not clinically relevant and there is no allergic
response or neutralization of the protein by an antibody. That is,
in a preferred embodiment, the variant TNFSF protein does not
contain additional or different epitopes from the wild-type or
naturally occurring TNFSF. By "epitope" or "determinant" herein is
meant a portion of a protein which will generate and/or bind an
antibody. Thus, in most instances, no significant amounts of
antibodies are generated to a variant TNFSF protein in its native
host. In general, this is accomplished by not significantly
altering surface residues, as outlined below nor by adding any
amino acid residues on the surface which can become glycosylated,
as novel glycosylation can result in an immune response, nor by the
introduction of new MHC binding epitopes.
[0101] The variant TNFSF proteins of the present invention may be
shorter or longer than the amino acid sequences shown in FIG. 3. As
used in this invention, "wild-type TNFSF" is a native mammalian
protein (preferably human). TNFSF may be polymorphic. Thus, in a
preferred embodiment, included within the definition of variant
TNFSF proteins are portions or fragments of the sequences depicted
herein. Fragments of variant TNFSF proteins are considered variant
TNFSF proteins if a) they share at least one antigenic epitope; b)
have at least the indicated homology; c) and preferably have
variant TNFSF biological activity as defined herein.
[0102] In a preferred embodiment, as is more fully outlined below,
the variant TNFSF proteins include further amino acid variations,
as compared to a wild-type TNFSF, than those outlined herein.
Examples include, but are not limited to, amino acid substitutions
introduced to enable soluble expression in E. coli, amino acid
substitutions introduced to optimize solution behavior, and amino
acid substitutions introduced to modulate immunogenicity. In
addition, as outlined herein, any of the variations depicted herein
may be combined in any way to form additional novel variant TNFSF
proteins.
[0103] In addition, variant TNFSF proteins may be made that are
longer than those depicted in the figures, for example, by the
addition of epitope or purification tags, as outlined herein, the
addition of other fusion sequences, etc. For example, the variant
TNFSF proteins of the invention may be fused to other therapeutic
proteins or to other proteins such as Fc or serum albumin for
pharmacokinetic purposes. See for example U.S. Pat. Nos. 5,766,883
and 5,876,969, both of which are expressly incorporated by
reference.
[0104] Variant TNFSF proteins may also be identified as being
encoded by variant TNFSF nucleic acids. In the case of the nucleic
acid, the overall homology of the nucleic acid sequence is
commensurate with amino acid homology but takes into account the
degeneracy in the genetic code and codon bias of different
organisms. Accordingly, the nucleic acid sequence homology may be
either lower or higher than that of the protein sequence, with
lower homology being preferred.
[0105] In a preferred embodiment, a variant TNFSF nucleic acid
encodes a variant TNFSF protein. As will be appreciated by those in
the art, due to the degeneracy of the genetic code, an extremely
large number of nucleic acids may be made, all of which encode the
variant TNFSF proteins of the present invention. Thus, having
identified a particular amino acid sequence, those skilled in the
art could make any number of different nucleic acids, by simply
modifying the sequence of one or more codons in a way which does
not change the amino acid sequence of the variant TNFSF.
[0106] The variant TNFSF proteins and nucleic acids of the present
invention are preferably recombinant (unless made synthetically).
As used herein, "nucleic acid" may refer to either DNA or RNA, or
molecules which contain both deoxy- and ribonucleotides. The
nucleic acids include genomic DNA, cDNA, mRNA and oligonucleotides
including sense and anti-sense nucleic acids. Such nucleic acids
may also contain modifications in the ribose-phosphate backbone to
increase stability and half-life of such molecules in physiological
environments.
[0107] The nucleic acid may be double stranded, single stranded, or
contain portions of both double stranded or single stranded
sequence. As will be appreciated by those in the art, the depiction
of a single strand ("Watson") also defines the sequence of the
other strand ("Crick"); thus the sequence depicted in FIG. 6 also
includes the complement of the sequence.
[0108] Also included within the definition of variant TNFSF
proteins of the present invention are amino acid sequence variants
of the variant TNFSF sequences outlined herein and shown in the
Figures. That is, the variant TNFSF proteins may contain additional
variable positions as compared to human TNFSF other than those used
to generate dominant negative proteins. As for "dominant negative
variable positions", these variants fall into one or more of three
classes: substitutional, insertional or deletional variants. All
variants ordinarily are prepared by site-specific mutagenesis of
nucleotides in the DNA encoding a variant TNFSF protein, using
cassette or PCR mutagenesis or other techniques well known in the
art, to produce DNA encoding the variant, and thereafter expressing
the DNA in recombinant cell culture as outlined above. However,
variant TNFSF protein fragments having up to about 100-150 residues
may be prepared by in vitro synthesis using established techniques.
Amino acid sequence variants are characterized by the predetermined
nature of the variation, a feature that sets them apart from
naturally occurring allelic or interspecies variation of the
variant TNFSF protein amino acid sequence. The variants typically
exhibit the same qualitative biological activity as the naturally
occurring analogue; although variants can also be selected which
have modified characteristics.
[0109] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed variant TNFSF
proteins screened for the optimal combination of desired activity.
Techniques for making substitution mutations at predetermined sites
in DNA having a known sequence are well known, for example, M13
primer mutagenesis and PCR mutagenesis. Screening of the mutants is
done using assays of variant TNFSF protein activities, or
additional properties as outlined here (e.g. stability) for optimum
characteristics.
[0110] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
[0111] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule (e.g. conservative modifications).
However, larger changes may be tolerated in certain circumstances
(e.g. non-conservative modifications).
[0112] The variants typically exhibit the same qualitative
biological activity and will elicit the same immune response as the
original variant TNFSF protein, although variants also are selected
to modify the characteristics of the variant TNFSF proteins as
needed. Alternatively, the variant may be designed such that the
biological activity of the variant TNFSF protein is altered. For
example, glycosylation sites may be altered or removed. Similarly,
the biological function may be altered; for example, in some
instances it may be desirable to have more or less potent TNFSF
activity.
[0113] The variant TNFSF proteins and nucleic acids of the
invention can be made in a number of ways. Individual nucleic acids
and proteins can be made as known in the art and outlined below.
Alternatively, libraries of variant TNFSF proteins can be made for
testing.
[0114] In a preferred embodiment, sets or libraries of variant
TNFSF proteins may be generated in many ways known to those skilled
in the art.
[0115] In a preferred embodiment, the different protein members of
the variant TNFSF library may be chemically synthesized. This is
particularly useful when the designed proteins are short,
preferably less than 150 amino acids in length, with less than 100
amino acids being preferred, and less than 50 amino acids being
particularly preferred, although as is known in the art, longer
proteins may be made chemically or enzymatically. See for example
Wilken et al, Curr. Opin. Biotechnol. 9:412-26 (1998), hereby
expressly incorporated by reference.
[0116] In a preferred embodiment, particularly for longer proteins
or proteins for which large samples are desired, the library
sequences are used to create nucleic acids such as DNA which encode
the member sequences and which may then be cloned into host cells,
expressed and assayed, if desired. Thus, nucleic acids, and
particularly DNA, may be made which encodes each member protein
sequence. This is done using well-known procedures. The choice of
codons, suitable expression vectors and suitable host cells will
vary depending on a number of factors, and may be easily optimized
as needed.
[0117] In a preferred embodiment, for example when libraries of
variants are made for testing for antagonist activity and/or other
desired activities as outlined herein, multiple PCR reactions with
pooled oligonucleotides are done. In this embodiment, overlapping
oligonucleotides are synthesized which correspond to the
full-length gene. Again, these oligonucleotides may represent all
of the different amino acids at each variant position or subsets.
In a preferred embodiment, these oligonucleotides are pooled in
equal proportions and multiple PCR reactions are performed to
create full-length sequences containing the combinations of
mutations defined by the library. In addition, this may be done
using error-prone PCR methods.
[0118] In a preferred embodiment, the different oligonucleotides
are added in relative amounts corresponding to a probability
distribution table as described in U.S. Ser. No. 10/218,102. The
multiple PCR reactions thus result in full-length sequences with
the desired combinations of mutations in the desired
proportions.
[0119] In a preferred embodiment, each overlapping oligonucleotide
comprises only one position to be varied; in alternate embodiments,
the variant positions are too close together to allow this and
multiple variants per oligonucleotide are used to allow complete
recombination of all the possibilities. That is, each oligo may
contain the codon for a single position being mutated, or for more
than one position being mutated. The multiple positions being
mutated must be close in sequence to prevent the oligo length from
being impractical. For multiple mutating positions on an
oligonucleotide, particular combinations of mutations may be
included or excluded in the library by including or excluding the
oligonucleotide encoding that combination. For example, as
discussed herein, there may be correlations between variable
regions; that is, when position X is a certain residue, position Y
must (or must not) be a particular residue. These sets of variable
positions are sometimes referred to herein as a "cluster". When the
clusters are comprised of residues close together, and thus can
reside on one oligonucleotide primer, the clusters can be set to
the "good" correlations, and eliminate the bad combinations that
may decrease the effectiveness of the library. However, if the
residues of the cluster are far apart in sequence, and thus will
reside on different oligonucleotides for synthesis, it may be
desirable to either set the residues to the "good" correlation, or
eliminate them as variable residues entirely. In an alternative
embodiment, the library may be generated in several steps, so that
the cluster mutations only appear together. This procedure, i.e.
the procedure of identifying mutation clusters and either placing
them on the same oligonucleotides or eliminating them from the
library or library generation in several steps preserving clusters,
can considerably enrich the experimental library with properly
folded protein. Identification of clusters may be carried out by a
number of ways, e.g. by using known pattern recognition methods,
comparisons of frequencies of occurrence of mutations or by using
energy analysis of the sequences to be experimentally generated
(for example, if the energy of interaction is high, the positions
are correlated). These correlations may be positional correlations
(e.g. variable positions 1 and 2 always change together or never
change together) or sequence correlations (e.g. if there is residue
A at position 1, there is always residue B at position 2). See:
Pattern discovery in Biomolecular Data: Tools, Techniques, and
Applications; edited by Jason T. L. Wang, Bruce A. Shapiro, Dennis
Shasha. New York: Oxford University, 1999; Andrews, Harry C.
Introduction to mathematical techniques in pattern recognition; New
York, Wiley-lnterscience [1972]; Applications of Pattern
Recognition; Editor, K. S. Fu. Boca Raton, Fla. CRC Press, 1982;
Genetic Algorithms for Pattern Recognition; edited by Sankar K.
Pal, Paul P. Wang. Boca Raton: CRC Press, c1996; Pandya, Abhijit
S., Pattern recognition with neural networks in C++/Abhijit S.
Pandya, Robert B. Macy. Boca Raton, Fla.: CRC Press, 1996; Handbook
of pattern recognition & computer vision/edited by C. H. Chen,
L. F. Pau, P. S. P. Wang. 2nd ed. Singapore; River Edge, N.J.:
World Scientific, c1999; Friedman, Introduction to Pattern
Recognition: Statistical, Structural, Neural, and Fuzzy Logic
Approaches; River Edge, N.J.: World Scientific, c1999, Series
title: Series in machine perception and artificial intelligence;
vol. 32; all of which are expressly incorporated by reference. In
addition, programs used to search for consensus motifs can be used
as well.
[0120] Oligonucleotides with insertions or deletions of codons may
be used to create a library expressing different length proteins.
In particular computational sequence screening for insertions or
deletions may result in secondary libraries defining different
length proteins, which can be expressed by a library of pooled
oligonucleotide of different lengths.
[0121] In another preferred embodiment, variant TNFSF proteins of
the invention are created by shuffling the family (e.g. a set of
variants); that is, some set of the top sequences (if a
rank-ordered list is used) can be shuffled, either with or without
error-prone PCR. "Shuffling" in this context means a recombination
of related sequences, generally in a random way. It can include
"shuffling" as defined and exemplified in U.S. Pat. Nos. 5,830,721;
5,811,238; 5,605,793; 5,837,458 and PCT US/19256, all of which are
expressly incorporated by reference in their entirety. This set of
sequences may also be an artificial set; for example, from a
probability table (for example generated using SCMF) or a Monte
Carlo set. Similarly, the "family" can be the top 10 and the bottom
10 sequences, the top 100 sequence, etc. This may also be done
using error-prone PCR.
[0122] Thus, in a preferred embodiment, in silico shuffling is done
using the computational methods described herein. That is, starting
with two libraries or two sequences, random recombinations of the
sequences may be generated and evaluated.
[0123] In a preferred embodiment variant TNFSF proteins are
chimeras formed from two or more naturally occurring TNFSF
proteins. In a particularly preferred embodiment, the chimeras are
formed by joining one or more receptor contact region from one or
more naturally occurring TNFSF proteins with the amino acid
sequence of another naturally occurring TNFSF protein.
[0124] In a preferred embodiment, error-prone PCR is done to
generate a library of variant TNFSF proteins. See U.S. Pat. Nos.
5,605,793, 5,811,238, and 5,830,721, all of which are hereby
incorporated by reference. This may be done on the optimal sequence
or on top members of the library, or some other artificial set or
family. In this embodiment, the gene for the optimal sequence found
in the computational screen of the primary library may be
synthesized. Error-prone PCR is then performed on the optimal
sequence gene in the presence of oligonucleotides that code for the
mutations at the variant positions of the library (bias
oligonucleotides). The addition of the oligonucleotides will create
a bias favoring the incorporation of the mutations in the library.
Alternatively, only oligonucleotides for certain mutations may be
used to bias the library.
[0125] In a preferred embodiment, gene shuffling with error-prone
PCR can be performed on the gene for the optimal sequence, in the
presence of bias oligonucleotides, to create a DNA sequence library
that reflects the proportion of the mutations found in the variant
TNFSF library. The choice of the bias oligonucleotides can be done
in a variety of ways; they can chosen on the basis of their
frequency, i.e. oligonucleotides encoding high mutational frequency
positions can be used; alternatively, oligonucleotides containing
the most variable positions can be used, such that the diversity is
increased; if the secondary library is ranked, some number of top
scoring positions may be used to generate bias oligonucleotides;
random positions may be chosen; a few top scoring and a few low
scoring ones may be chosen; etc. What is important is to generate
new sequences based on preferred variable positions and
sequences.
[0126] In a preferred embodiment, PCR using a wild-type gene or
other gene may be used, as is schematically depicted in the
Figures. In this embodiment, a starting gene is used; generally,
although this is not required, the gene is usually the wild-type
gene. In some cases it may be the gene encoding the global
optimized sequence, or any other sequence of the list, or a
consensus sequence obtained e.g. from aligning homologous sequences
from different organisms. In this embodiment, oligonucleotides are
used that correspond to the variant positions and contain the
different amino acids of the library. PCR is done using PCR primers
at the termini, as is known in the art. This provides two benefits.
First, this generally requires fewer oligonucleotides and may
result in fewer errors. Second, it has experimental advantages in
that if the wild-type gene is used, it need not be synthesized.
[0127] Using the nucleic acids of the present invention which
encode a variant TNFSF protein, a variety of expression vectors are
made. The expression vectors may be either self-replicating
extrachromosomal vectors or vectors which integrate into a host
genome. Generally, these expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleic acid encoding the variant TNFSF protein. The term "control
sequences" refers to DNA sequences necessary for the expression of
an operably linked coding sequence in a particular host organism.
The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, polyadenylation signals, and enhancers.
[0128] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation.
[0129] In a preferred embodiment, when the endogenous secretory
sequence leads to a low level of secretion of the naturally
occurring protein or of the variant TNFSF protein, a replacement of
the naturally occurring secretory leader sequence is desired. In
this embodiment, an unrelated secretory leader sequence is operably
linked to a variant TNFSF encoding nucleic acid leading to
increased protein secretion. Thus, any secretory leader sequence
resulting in enhanced secretion of the variant TNFSF protein, when
compared to the secretion of TNFSF and its secretory sequence, is
desired. Suitable secretory leader sequences that lead to the
secretion of a protein are known in the art.
[0130] In another preferred embodiment, a secretory leader sequence
of a naturally occurring protein or a protein is removed by
techniques known in the art and subsequent expression results in
intracellular accumulation of the recombinant protein.
[0131] Generally, "operably linked" means that the DNA sequences
being linked are contiguous, and, in the case of a secretory
leader, contiguous and in reading frame. However, enhancers do not
have to be contiguous. Linking is accomplished by ligation at
convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in
accordance with conventional practice. The transcriptional and
translational regulatory nucleic acid will generally be appropriate
to the host cell used to express the fusion protein; for example,
transcriptional and translational regulatory nucleic acid sequences
from Bacillus are preferably used to express the fusion protein in
Bacillus. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of
host cells.
[0132] In general, the transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred embodiment, the regulatory sequences
include a promoter and transcriptional start and stop
sequences.
[0133] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention. In a preferred embodiment, the
promoters are strong promoters, allowing high expression in cells,
particularly mammalian cells, such as the CMV promoter,
particularly in combination with a Tet regulatory element.
[0134] In addition, the expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a prokaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector may be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art.
[0135] In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0136] A preferred expression vector system is a retroviral vector
system such as is generally described in PCT/US97/01019 and
PCT/US97/01048, both of which are hereby expressly incorporated by
reference.
[0137] In a preferred embodiment, the expression vector comprises
the components described above and a gene encoding a variant TNFSF
protein. As will be appreciated by those in the art, all
combinations are possible and accordingly, as used herein, the
combination of components, comprised by one or more vectors, which
may be retroviral or not, is referred to herein as a "vector
composition".
[0138] The variant TNFSF nucleic acids are introduced into the
cells either alone or in combination with an expression vector. By
"introduced into" or grammatical equivalents herein is meant that
the nucleic acids enter the cells in a manner suitable for
subsequent expression of the nucleic acid. The method of
introduction is largely dictated by the targeted cell type,
discussed below. Exemplary methods include CaPO.sub.4
precipitation, liposome fusion, lipofectin.RTM., electroporation,
viral infection, etc. The variant TNFSF nucleic acids may stably
integrate into the genome of the host cell (for example, with
retroviral introduction, outlined below), or may exist either
transiently or stably in the cytoplasm (i.e. through the use of
traditional plasmids, utilizing standard regulatory sequences,
selection markers, etc.).
[0139] The variant TNFSF proteins of the present invention are
produced by culturing a host cell transformed with an expression
vector containing nucleic acid encoding a variant TNFSF protein,
under the appropriate conditions to induce or cause expression of
the variant TNFSF protein. The conditions appropriate for variant
TNFSF protein expression will vary with the choice of the
expression vector and the host cell, and will be easily ascertained
by one skilled in the art through routine experimentation. For
example, the use of constitutive promoters in the expression vector
will require optimizing the growth and proliferation of the host
cell, while the use of an inducible promoter requires the
appropriate growth conditions for induction. In addition, in some
embodiments, the timing of the harvest is important. For example,
the baculoviral systems used in insect cell expression are lytic
viruses, and thus harvest time selection can be crucial for product
yield.
[0140] Appropriate host cells include yeast, bacteria,
archaebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Drosophila melangaster
cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus
subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO,
COS, Pichia pastoris, etc.
[0141] In a preferred embodiment, the variant TNFSF proteins are
expressed in mammalian cells. Mammalian expression systems are also
known in the art, and include retroviral systems. A mammalian
promoter is any DNA sequence capable of binding mammalian RNA
polymerase and initiating the downstream (3') transcription of a
coding sequence for the fusion protein into mRNA. A promoter will
have a transcription initiating region, which is usually placed
proximal to the 5' end of the coding sequence, and a TATA box,
using a located 25-30 base pairs upstream of the transcription
initiation site. The TATA box is thought to direct RNA polymerase
II to begin RNA synthesis at the correct site. A mammalian promoter
will also contain an upstream promoter element (enhancer element),
typically located within 100 to 200 base pairs upstream of the TATA
box. An upstream promoter element determines the rate at which
transcription is initiated and can act in either orientation. Of
particular use as mammalian promoters are the promoters from
mammalian viral genes, since the viral genes are often highly
expressed and have a broad host range. Examples include the SV40
early promoter, mouse mammary tumor virus LTR promoter, adenovirus
major late promoter, herpes simplex virus promoter, and the CMV
promoter.
[0142] Typically, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-translational
cleavage and polyadenylation. Examples of transcription terminator
and polyadenylation signals include those derived from SV40.
[0143] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei. As outlined herein, a particularly preferred
method utilizes retroviral infection, as outlined in PCT
US97/01019, incorporated by reference.
[0144] As will be appreciated by those in the art, the type of
mammalian cells used in the present invention can vary widely.
Basically, any mammalian cells may be used, with mouse, rat,
primate and human cells being particularly preferred, although as
will be appreciated by those in the art, modifications of the
system by pseudotyping allows all eukaryotic cells to be used,
preferably higher eukaryotes. As is more fully described below, a
screen will be set up such that the cells exhibit a selectable
phenotype in the presence of a bioactive peptide. As is more fully
described below, cell types implicated in a wide variety of disease
conditions are particularly useful, so long as a suitable screen
may be designed to allow the selection of cells that exhibit an
altered phenotype as a consequence of the presence of a peptide
within the cell.
[0145] Accordingly, suitable cell types include, but are not
limited to, tumor cells of all types (particularly melanoma,
myeloid leukemia, carcinomas of the lung, breast, ovaries, colon,
kidney, prostate, pancreas and testes), cardiomyocytes, endothelial
cells, epithelial cells, lymphocytes (T-cell and B cell), mast
cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes
including mononuclear leukocytes, stem cells such as haemopoietic,
neural, skin, lung, kidney, liver and myocyte stem cells (for use
in screening for differentiation and de-differentiation factors),
osteoclasts, chondrocytes and other connective tissue cells,
keratinocytes, melanocytes, liver cells, kidney cells, and
adipocytes. Suitable cells also include known research cells,
including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO,
Cos, etc. See the ATCC cell line catalog, hereby expressly
incorporated by reference.
[0146] In one embodiment, the cells may be additionally genetically
engineered, that is, contain exogenous nucleic acid other than the
variant TNFSF nucleic acid.
[0147] In a preferred embodiment, the variant TNFSF proteins are
expressed in bacterial systems. Bacterial expression systems are
well known in the art.
[0148] A suitable bacterial promoter is any nucleic acid sequence
capable of binding bacterial RNA polymerase and initiating the
downstream (3') transcription of the coding sequence of the variant
TNFSF protein into mRNA. A bacterial promoter has a transcription
initiation region which is usually placed proximal to the 5' end of
the coding sequence. This transcription initiation region typically
includes an RNA polymerase binding site and a transcription
initiation site. Sequences encoding metabolic pathway enzymes
provide particularly useful promoter sequences. Examples include
promoter sequences derived from sugar metabolizing enzymes, such as
galactose, lactose and maltose, and sequences derived from
biosynthetic enzymes such as tryptophan. Promoters from
bacteriophage may also be used and are known in the art. In
addition, synthetic promoters and hybrid promoters are also useful;
for example, the tac promoter is a hybrid of the trp and lac
promoter sequences. Furthermore, a bacterial promoter may include
naturally occurring promoters of non-bacterial origin that have the
ability to bind bacterial RNA polymerase and initiate
transcription.
[0149] In addition to a functioning promoter sequence, an efficient
ribosome binding site is desirable. In E. coli, the ribosome
binding site is called the Shine-Delgarno (SD) sequence and
includes an initiation codon and a sequence 3-9 nucleotides in
length located 3-11 nucleotides upstream of the initiation
codon.
[0150] The expression vector may also include a signal peptide
sequence that provides for secretion of the variant TNFSF protein
in bacteria. The signal sequence typically encodes a signal peptide
comprised of hydrophobic amino acids which direct the secretion of
the protein from the cell, as is well known in the art. The protein
is either secreted into the growth media (gram-positive bacteria)
or into the periplasmic space, located between the inner and outer
membrane of the cell (gram-negative bacteria). For expression in
bacteria, usually bacterial secretory leader sequences, operably
linked to a variant TNFSF encoding nucleic acid, are preferred.
[0151] The bacterial expression vector may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed. Suitable selection genes
include genes which render the bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways.
[0152] These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others.
[0153] The bacterial expression vectors are transformed into
bacterial host cells using techniques well known in the art, such
as calcium chloride treatment, electroporation, and others.
[0154] In one embodiment, variant TNFSF proteins are produced in
insect cells. Expression vectors for the transformation of insect
cells, and in particular, baculovirus-based expression vectors, are
well known in the art.
[0155] In a preferred embodiment, variant TNFSF protein is produced
in yeast cells. Yeast expression systems are well known in the art,
and include expression vectors for Saccharomyces cerevisiae,
Candida albicans and C. maltosa, Hansenula polymorpha,
Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.
pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
Preferred promoter sequences for expression in yeast include the
inducible GAL1, 10 promoter, the promoters from alcohol
dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,
phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase,
and the acid phosphatase gene. Yeast selectable markers include
ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to
tunicamycin; the neomycin phosphotransferase gene, which confers
resistance to G418; and the CUP1 gene, which allows yeast to grow
in the presence of copper ions.
[0156] In a preferred embodiment, modified TNFSF variants are
covalently coupled to at least one additional TNFSF variant via a
linker to improve the dominant negative action of the modified
domains. A number of strategies may be used to covalently link
modified receptor domains together. These include, but are not
limited to, linkers, such as polypeptide linkages between N- and
C-termini of two domains, linkage via a disulfide bond between
monomers, and linkage via chemical cross-linking reagents.
Alternatively, the N- and C-termini may be covalently joined by
deletion of portions of the N- and/or C-termini and linking the
remaining fragments via a linker or linking the fragments
directly.
[0157] By "linker", "linker sequence", "spacer", "tethering
sequence" or grammatical equivalents thereof, herein is meant a
molecule or group of molecules (such as a monomer or polymer) that
connects two molecules and often serves to place the two molecules
in a preferred configuration. In one aspect of this embodiment, the
linker is a peptide bond. Choosing a suitable linker for a specific
case where two polypeptide chains are to be connected depends on
various parameters, e.g., the nature of the two polypeptide chains
(e.g., whether they naturally oligomerize (e.g., form a dimer or
not), the distance between the N- and the C-termini to be connected
if known from three-dimensional structure determination, and/or the
stability of the linker towards proteolysis and oxidation.
Furthermore, the linker may contain amino acid residues that
provide flexibility. Thus, the linker peptide may predominantly
include the following amino acid residues: Gly, Ser, Ala, or Thr.
These linked TNFSF proteins have constrained hydrodynamic
properties, that is, they form constitutive dimers) and thus
efficiently interact with other naturally occurring TNFSF proteins
to form a dominant negative heterotrimer.
[0158] The linker peptide should have a length that is adequate to
link two TNFSF variant monomers in such a way that they assume the
correct conformation relative to one another so that they retain
the desired activity as antagonists of the native TNFSF protein.
Suitable lengths for this purpose include at least one and not more
than 30 amino acid residues. Preferably, the linker is from about 1
to 30 amino acids in length, with linkers of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19 and 20 amino acids in
length being preferred. See also WO 01/25277, incorporated herein
by reference in its entirety.
[0159] In addition, the amino acid residues selected for inclusion
in the linker peptide should exhibit properties that do not
interfere significantly with the activity of the polypeptide. Thus,
the linker peptide on the whole should not exhibit a charge that
would be inconsistent with the activity of the polypeptide, or
interfere with internal folding, or form bonds or other
interactions with amino acid residues in one or more of the
monomers that would seriously impede the binding of receptor
monomer domains.
[0160] Useful linkers include glycine-serine polymers (including,
for example, (GS)n, (GSGGS)n (GGGGS)n and (GGGS)n, where n is an
integer of at least one), glycine-alanine polymers, alanine-serine
polymers, and other flexible linkers such as the tether for the
shaker potassium channel, and a large variety of other flexible
linkers, as will be appreciated by those in the art. Glycine-serine
polymers are preferred since both of these amino acids are
relatively unstructured, and therefore may be able to serve as a
neutral tether between components. Secondly, serine is hydrophilic
and therefore able to solubilize what could be a globular glycine
chain. Third, similar chains have been shown to be effective in
joining subunits of recombinant proteins such as single chain
antibodies.
[0161] Suitable linkers may also be identified by screening
databases of known three-dimensional structures for naturally
occurring motifs that can bridge the gap between two polypeptide
chains. Another way of obtaining a suitable linker is by optimizing
a simple linker, e.g., (Gly4Ser)n, through random mutagenesis.
Alternatively, once a suitable polypeptide linker is defined,
additional linker polypeptides can be created by application of
PDA.TM. technology to select amino acids that more optimally
interact with the domains being linked. Other types of linkers that
may be used in the present invention include artificial polypeptide
linkers and inteins. In another preferred embodiment, disulfide
bonds are designed to link the two receptor monomers at
inter-monomer contact sites. In one aspect of this embodiment the
two receptors are linked at distances <5 Angstroms. In addition,
the variant TNFSF polypeptides of the invention may be further
fused to other proteins, if desired, for example to increase
expression or stabilize the protein.
[0162] In one embodiment, the variant TNFSF nucleic acids, proteins
and antibodies of the invention are labeled with a label other than
the scaffold. By "labeled" herein is meant that a compound has at
least one element, isotope or chemical compound attached to enable
the detection of the compound. In general, labels fall into three
classes: a) isotopic labels, which may be radioactive or heavy
isotopes; b) immune labels, which may be antibodies or antigens;
and c) colored or fluorescent dyes. The labels may be incorporated
into the compound at any position.
[0163] Once made, the variant TNFSF proteins may be modified.
Covalent and non-covalent modifications of the protein are included
within the scope of the present invention. Such modifications may
be introduced into a variant TNFSF polypeptide by reacting targeted
amino acid residues of the polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or
terminal residues.
[0164] One type of covalent modification includes reacting targeted
amino acid residues of a variant TNFSF polypeptide with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues of a variant TNFSF
polypeptide. Derivatization with bifunctional agents is useful, for
instance, for cross linking a variant TNFSF protein to a
water-insoluble support matrix or surface for use in the method for
purifying anti-variant TNFSF antibodies or screening assays, as is
more fully described below. Commonly used cross linking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidyl-propionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0165] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the "-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0166] Another type of covalent modification of the variant TNFSF
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence variant TNFSF polypeptide, and/or adding
one or more glycosylation sites that are not present in the native
sequence variant TNFSF polypeptide.
[0167] Addition of glycosylation sites to variant TNFSF
polypeptides may be accomplished by altering the amino acid
sequence thereof. The alteration may be made, for example, by the
addition of, or substitution by, one or more serine or threonine
residues to the native sequence or variant TNFSF polypeptide (for
O-linked glycosylation sites). The variant TNFSF amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the variant TNFSF
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0168] Addition of N-linked glycosylation sites to variant TNFSF
polypeptides may be accomplished by altering the amino acid
sequence thereof. The alteration may be made, for example, by the
addition of, or substitution by, one or more asparagine residues to
the native sequence or variant TNFSF polypeptide. The modification
may be made for example by the incorporation of a canonical
N-linked glycosylation site, including but not limited to, N-X-Y,
where X is any amino acid except for proline and Y is preferably
threonine, serine or cysteine. Another means of increasing the
number of carbohydrate moieties on the variant TNFSF polypeptide is
by chemical or enzymatic coupling of glycosides to the polypeptide.
Such methods are described in the art, e.g., in WO 87/05330
published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
[0169] Removal of carbohydrate moieties present on the variant
TNFSF polypeptide may be accomplished chemically or enzymatically
or by mutational substitution of codons encoding for amino acid
residues that serve as targets for glycosylation. Chemical
deglycosylation techniques are known in the art and described, for
instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52
(1987) and by Edge et al., Anal. Biochem., 118:131 (1981).
Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety of endo-and exo-glycosidases as
described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
[0170] Such derivatized moieties may improve the solubility,
absorption, and permeability across the blood brain barrier
biological half-life, and the like. Such moieties or modifications
of variant TNFSF polypeptides may alternatively eliminate or
attenuate any possible undesirable side effect of the protein and
the like. Moieties capable of mediating such effects are disclosed,
for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack
Publishing Co., Easton, Pa. (1980).
[0171] Another type of covalent modification of variant TNFSF
comprises linking the variant TNFSF polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol ("PEG"),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192; 4,179,337; 5,183,550. These nonproteinaceous polymers
may also be used to enhance the variant TNFSF's ability to disrupt
receptor binding, and/or in vivo stability.
[0172] In another preferred embodiment, cysteines are designed into
variant or wild-type TNFSF in order to incorporate (a) labeling
sites for characterization and (b) incorporate PEGylation sites.
For example, labels that may be used are well known in the art and
include but are not limited to biotin, tag and fluorescent labels
(e.g. fluorescein). These labels may be used in various assays as
are also well known in the art to achieve characterization. A
variety of coupling chemistries may be used to achieve PEGylation,
as is well known in the art. Examples include but are not limited
to, the technologies of Shearwater and Enzon, which allow
modification at primary amines, including but not limited to,
lysine groups and the N-terminus. See, Kinstler et al, Advanced
Drug Deliveries Reviews, 54, 477-485 (2002) and M J Roberts et al,
Advanced Drug Delivery Reviews, 54, 459-476 (2002), both hereby
incorporated by reference.
[0173] Other modifications may be made to the variant TNFSF
proteins of the present invention, including modifications to the
protein that enhance stability, dosage administration (e.g.,
amphiphilic polymers, see WO 0141812A2, commercially available from
Nobex Corporation), clearance (e.g., PEG, aliphatic moieties that
effect binding to HSA), and the like.
[0174] Optimal sites for modification can be chosen using a variety
of criteria, including but not limited to, visual inspection,
structural analysis, sequence analysis and molecular simulation.
Individual residues may be analyzed to identify mutational sites
that will not disrupt the monomer structure. Then the distance from
each side chain of a monomer to another subunit may be calculated
to ensure that chemical modification will not disrupt
oligomerization. It is possible that receptor binding disruption
may occur and may be beneficial to the activity of the TNFSF
variants of this invention.
[0175] In another preferred embodiment, portions of either the N-
or C-termini of the wild-type TNFSF monomer are deleted while still
allowing the TNFSF molecule to fold properly. In addition, these
modified TNFSF proteins would substantially lack receptor binding
and/or activation, and could optionally interact with other
wild-type TNFSF molecules or modified TNFSF proteins to form
trimers (or other oligomers) as described above.
[0176] More specifically, removal or deletion of from about 1 to
about 55 amino acids from either the N or C termini, or both, are
preferred. A more preferred embodiment includes deletions of
N-termini beyond residue 10 and more preferably, deletion of the
first 47 N-terminal amino acids. The deletion of C-terminal leucine
is an alternative embodiment.
[0177] In another preferred embodiment, the wild-type TNFSF or
variants generated by the invention may be circularly permuted. All
natural proteins have an amino acid sequence beginning with an
N-terminus and ending with a C-terminus. The N- and C-termini may
be joined to create a cyclized or circularly permutated TNFSF
proteins while retaining or improving biological properties (e.g.,
such as enhanced stability and activity) as compared to the
wild-type protein. In the case of a TNFSF protein, a novel set of
N- and C-termini are created at amino acid positions normally
internal to the protein's primary structure, and the original N-
and C-termini are joined via a peptide linker consisting of from 0
to 30 amino acids in length (in some cases, some of the amino acids
located near the original termini are removed to accommodate the
linker design). In a preferred embodiment, the novel N- and
C-termini are located in a non-regular secondary structural
element, such as a loop or turn, such that the stability and
activity of the novel protein are similar to those of the original
protein. The circularly permuted TNFSF protein may be further
PEGylated or glycosylated. In a further preferred embodiment
PDA.TM. technology may be used to further optimize the TNFSF
variant, particularly in the regions created by circular
permutation. These include the novel N- and C-termini, as well as
the original termini and linker peptide.
[0178] Various techniques may be used to permutate proteins. See
U.S. Pat. No. 5,981,200; Maki K, Iwakura M., Seikagaku. January
2001; 73(1): 42-6; Pan T., Methods Enzymol. 2000; 317:313-30;
Heinemann U, Hahn M., Prog Biophys Mol Biol. 1995; 64(2-3): 121-43;
Harris M E, Pace N R, Mol Biol Rep. 1995-96; 22(2-3):115-23; Pan T,
Uhlenbeck O C., Mar. 30, 1993; 125(2): 111-4; Nardulli A M, Shapiro
D J. 1993 Winter; 3(4):247-55, EP 1098257 A2; WO 02/22149; WO
01/51629; WO 99/51632; Hennecke, et al., 1999, J. Mol. Biol., 286,
1197-1215; Goldenberg et al J. Mol. Biol 165, 407-413 (1983); Luger
et al, Science, 243, 206-210 (1989); and Zhang et al., Protein Sci
5, 1290-1300 (1996); all hereby incorporated by reference.
[0179] In addition, a completely cyclic TNFSF may be generated,
wherein the protein contains no termini. This is accomplished
utilizing intein technology. Thus, peptides can be cyclized and in
particular inteins may be utilized to accomplish the
cyclization.
[0180] Cyclization and circular permutation may be used to generate
the dominant-negative activity of the TNFSF proteins of the present
invention.
[0181] Variant TNFSF polypeptides of the present invention may also
be modified in a way to form chimeric molecules comprising a
variant TNFSF polypeptide fused to another, heterologous
polypeptide or amino acid sequence. In one embodiment, such a
chimeric molecule comprises a fusion of a variant TNFSF polypeptide
with a tag polypeptide that provides an epitope to which an
anti-tag antibody can selectively bind. The epitope tag is
generally placed at the amino-or carboxyl-terminus of the variant
TNFSF polypeptide. The presence of such epitope-tagged forms of a
variant TNFSF polypeptide can be detected using an antibody against
the tag polypeptide. Also, provision of the epitope tag enables the
variant TNFSF polypeptide to be readily purified by affinity
purification using an anti-tag antibody or another type of affinity
matrix that binds to the epitope tag. In an alternative embodiment,
the chimeric molecule may comprise a fusion of a variant TNFSF
polypeptide with an immunoglobulin or a particular region of an
immunoglobulin. For a bivalent form of the chimeric molecule, such
a fusion could be to the Fc or Fab region of an IgG molecule. Other
fusion entities include human serum albumin (HSA), hydrophilic
peptides, fatty acid molecules, labels, etc.
[0182] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7 6E10, G4, B7
and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering, 3(6): 547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science 255:192-194
(1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.
266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag
[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397
(1 990)].
[0183] In a preferred embodiment, the variant TNFSF protein is
purified or isolated after expression. Variant TNFSF proteins may
be isolated or purified in a variety of ways known to those skilled
in the art depending on what other components are present in the
sample. Standard purification methods include electrophoretic,
molecular, immunological and chromatographic techniques, including
ion exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the variant
TNFSF protein may be purified using a standard anti-library
antibody column. Ultrafiltration and diafiltration techniques, in
conjunction with protein concentration, are also useful. For
general guidance in suitable purification techniques, see Scopes,
R., Protein Purification, Springer-Verlag, NY (1982). The degree of
purification necessary will vary depending on the use of the
variant TNFSF protein. In some instances no purification will be
necessary.
[0184] In a preferred embodiment, the variant TNFSF proteins of the
present invention are produced and purified separated from the
naturally occurring TNFSF proteins that are antagonized. That is,
variant monomers are made and introduced to the wild-type proteins,
either in monomeric or oligomeric form. For example, a preferred
method involves the administration of variant monomers to a
patient, whereby the variant monomers "exchange" into the
homo-oligomers (generally trimers), to produce mixed oligomers that
result in reduced receptor activation. In alternative embodiments,
variant oligomers may be administered that then exchange. However,
in some embodiments, such as in gene therapy applications, the
variant TNFSF proteins may be produced substantially
simulataneously with the naturally occurring TNFSF targets.
[0185] Once made, the variant TNFSF proteins and nucleic acids of
the invention find use in a number of applications. In a preferred
embodiment, the variant TNFSF proteins are administered to a
patient to treat a TNFSF related disorder.
[0186] By "TNFSF related disorder" or "TNFSF responsive disorder"
or "condition" herein is meant a disorder that may be ameliorated
by the administration of a pharmaceutical composition comprising a
variant TNFSF protein, including, but not limited to, autoimmune,
inflammatory and immunological disorders. The variant TNFSF
proteins are major effectors in the pathogenesis of
immune-regulated diseases.
[0187] In a preferred embodiment, the variant TNFSF protein is used
to treat for example, congestive heart failure (CHF), vasculitis,
rosecea, acne, excema, myocarditis and other conditions of the
myocardium, systemic lupus erythematosus, diabetes,
spondylopathies, synovial fibroblasts, and bone marrow stroma; bone
loss; Paget's disease, osteoclastoma; multiple myeloma; breast
cancer; disuse osteopenia; malnutrition, periodontal disease,
Gaucher's disease, Langerhans' cell histiocytosis, spinal cord
injury, acute septic arthritis, osteomalacia, Cushing's syndrome,
monoostotic fibrous dysplasia, polyostotic fibrous dysplasia,
periodontal reconstruction, and bone fractures; sarcoidosis;
multiple myeloma; osteolytic bone cancers, breast cancer, lung
cancer, kidney cancer and rectal cancer; bone metastasis, bone pain
management, and humoral malignant hypercalcemia, ankylosing
spondylitisa and other spondyloarthropathies; transplantation
rejection, viral infections, hematologic neoplasisas and
neoplastic-like conditions for example, Hodgkin's lymphoma;
non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic
lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle
cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma,
marginal zone lymphoma, hairy cell leukemia and lymphoplasmacytic
leukemia), tumors of lymphocyte precursor cells, including B-cell
acute lymphoblastic leukemia/lymphoma, and T-cell acute
lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T
and NK cells, including peripheral T-cell leukemias, adult T-cell
leukemia/T-cell lymphomas and large granular lymphocytic leukemia,
Langerhans cell histocytosis, myeloid neoplasias such as acute
myelogenous leukemias, including AML with maturation, AML without
differentiation, acute promyelocytic leukemia, acute myelomonocytic
leukemia, and acute monocytic leukemias, myelodysplastic syndromes,
and chronic myeloproliferative disorders, including chronic
myelogenous leukemia, tumors of the central nervous system, e.g.,
brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma,
ependymoma, and retinoblastoma), solid tumors (nasopharyngeal
cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile
duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or
cervical cancers, ovarian cancer, primary liver cancer or
endometrial cancer, and tumors of the vascular system (angiosarcoma
and hemagiopericytoma), osteoporosis, hepatitis, HIV, AIDS,
spondyloarthritis, rheumatoid arthritis, inflammatory bowel
diseases (IBD), sepsis and septic shock, Crohn's Disease,
psoriasis, schleraderma, graft versus host disease (GVHD),
allogenic islet graft rejection, hematologic malignancies, such as
multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute
myelogenous leukemia (AML), cancer and the inflammation associated
with tumors, peripheral nerve injury or demyelinating diseases.
[0188] By "therapeutically effective dose" herein is meant a dose
that produces the effects for which it is administered. The exact
dose will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques. In
a preferred embodiment, dosages of about 0.01 to about 50 .mu.g/kg
are used, administered either intravenously or subcutaneously. As
is known in the art, adjustments for variant TNFSF protein
degradation, systemic versus localized delivery, and rate of new
protease synthesis, as well as the age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0189] A "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals, and
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In the preferred embodiment the
patient is a mammal, and in the most preferred embodiment the
patient is human.
[0190] The term "treatment" in the instant invention is meant to
include therapeutic treatment, as well as prophylactic, or
suppressive measures for the disease or disorder. Thus, for
example, successful administration of a variant TNFSF protein prior
to onset of the disease results in "treatment" of the disease. As
another example, successful administration of a variant TNFSF
protein after clinical manifestation of the disease to combat the
symptoms of the disease comprises "treatment" of the disease.
"Treatment" also encompasses administration of a variant TNFSF
protein after the appearance of the disease in order to eradicate
the disease. Successful administration of an agent after onset and
after clinical symptoms have developed, with possible abatement of
clinical symptoms and perhaps amelioration of the disease,
comprises "treatment" of the disease.
[0191] Those "in need of treatment" include mammals already having
the disease or disorder, as well as those prone to having the
disease or disorder, including those in which the disease or
disorder is to be prevented.
[0192] In another embodiment, a therapeutically effective dose of a
variant TNFSF protein, a variant TNFSF gene, or a variant TNFSF
antibody is administered to a patient having a disease involving
inappropriate expression of a TNFSF protein. A "disease involving
inappropriate expression of a TNFSF protein" within the scope of
the present invention is meant to include diseases or disorders
characterized by aberrant TNFSF proteins, either by alterations in
the amount of TNFSF protein present or due to the presence of
mutant TNFSF protein. An overabundance may be due to any cause,
including, but not limited to, overexpression at the molecular
level, prolonged or accumulated appearance at the site of action,
or increased activity of TNFSF protein relative to normal. Included
within this definition are diseases or disorders characterized by a
reduction of TNFSF protein. This reduction may be due to any cause,
including, but not limited to, reduced expression at the molecular
level, shortened or reduced appearance at the site of action,
mutant forms of TNFSF protein, or decreased activity of TNFSF
protein relative to normal. Such an overabundance or reduction of
TNFSF protein can be measured relative to normal expression,
appearance, or activity of TNFSF protein according to, but not
limited to, the assays described and referenced herein.
[0193] The administration of the variant TNFSF proteins of the
present invention, preferably in the form of a sterile aqueous
solution, may be done in a variety of ways, including, but not
limited to, orally, subcutaneously, intravenously, intranasally,
intraotically, transdermally, topically (e.g., gels, salves,
lotions, creams, etc.), intraperitoneally, intramuscularly,
intrapulmonary (e.g., AERx.RTM. inhalable technology commercially
available from Aradigm or Inhance.TM. pulmonary delivery system
commercially available from Inhale Therapeutics), vaginally,
rectally, or intraocularly. In some instances, for example, in the
treatment of wounds, inflammation, etc., the variant TNFSF protein
may be directly applied as a solution or spray. Depending upon the
manner of introduction, the pharmaceutical composition may be
formulated in a variety of ways.
[0194] Also, sustained release or controlled release formulations
may be used for the compositions of the present invention. For
example, ProLease.RTM. (commercially available from Alkermes) a
microsphere-based delivery system composed of the desired bioactive
molecule incorporated into a matrix of poly-DL-lactide-co-glycolide
(PLG) and other pharmaceutically compatible polymeric matrices may
be used to create sustained release formulations.
[0195] The concentration of the therapeutically active variant
TNFSF protein in the formulation may vary from about 0.1 to 100
weight %. In another preferred embodiment, the concentration of the
variant TNFSF protein is in the range of 0.003 to 1.0 molar, with
dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram
of body weight being preferred.
[0196] The pharmaceutical compositions of the present invention
comprise a variant TNFSF protein in a form suitable for
administration to a patient. In the preferred embodiment, the
pharmaceutical compositions are in a water-soluble form, such as
being present as pharmaceutically acceptable salts, which is meant
to include both acid and base addition salts. "Pharmaceutically
acceptable acid addition salt" refers to those salts that retain
the biological effectiveness of the free bases and that are not
biologically or otherwise undesirable, formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid and the like, and organic acids such as
acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic
acid, maleic acid, malonic acid, succinic acid, fumaric acid,
tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic
acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic
acid, salicylic acid and the like. "Pharmaceutically acceptable
base addition salts" include those derived from inorganic bases
such as sodium, potassium, lithium, ammonium, calcium, magnesium,
iron, zinc, copper, manganese, aluminum salts and the like.
Particularly preferred are the ammonium, potassium, sodium,
calcium, and magnesium salts. Salts derived from pharmaceutically
acceptable organic non-toxic bases include salts of primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines and basic ion
exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, and ethanolamine.
[0197] The pharmaceutical compositions may also include one or more
of the following: carrier proteins such as serum albumin; buffers
such as NaOAc; fillers such as microcrystalline cellulose, lactose,
corn and other starches; binding agents; sweeteners and other
flavoring agents; coloring agents; and polyethylene glycol.
Additives are well known in the art, and are used in a variety of
formulations.
[0198] In a further embodiment, the variant TNFSF proteins are
added in a micellular formulation; see U.S. Pat. No. 5,833,948,
hereby expressly incorporated by reference in its entirety.
[0199] Combinations of pharmaceutical compositions may be
administered. Moreover, the compositions may be administered in
combination with other therapeutics.
[0200] In one embodiment provided herein, antibodies, including but
not limited to monoclonal and polyclonal antibodies, are raised
against variant TNFSF proteins using methods known in the art. In a
preferred embodiment, these anti-variant TNFSF antibodies are used
for immunotherapy. Thus, methods of immunotherapy are provided. By
"immunotherapy" is meant treatment of a TNFSF related disorders
with an antibody raised against a variant TNFSF protein. As used
herein, immunotherapy can be passive or active. Passive
immunotherapy, as defined herein, is the passive transfer of
antibody to a recipient (patient). Active immunization is the
induction of antibody and/or T-cell responses in a recipient
(patient). Induction of an immune response can be the consequence
of providing the recipient with a variant TNFSF protein antigen to
which antibodies are raised. As appreciated by one of ordinary
skill in the art, the variant TNFSF protein antigen may be provided
by injecting a variant TNFSF polypeptide against which antibodies
are desired to be raised into a recipient, or contacting the
recipient with a variant TNFSF protein encoding nucleic acid,
capable of expressing the variant TNFSF protein antigen, under
conditions for expression of the variant TNFSF protein antigen.
[0201] In another preferred embodiment, a therapeutic compound is
conjugated to an antibody, preferably an anti-variant TNFSF protein
antibody. The therapeutic compound may be a cytotoxic agent. In
this method, targeting the cytotoxic agent to tumor tissue or
cells, results in a reduction in the number of afflicted cells,
thereby reducing symptoms associated with cancer, and variant TNFSF
protein related disorders. Cytotoxic agents are numerous and varied
and include, but are not limited to, cytotoxic drugs or toxins or
active fragments of such toxins. Suitable toxins and their
corresponding fragments include diphtheria A chain, exotoxin A
chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin,
enomycin and the like. Cytotoxic agents also include radiochemicals
made by conjugating radioisotopes to antibodies raised against cell
cycle proteins, or binding of a radionuclide to a chelating agent
that has been covalently attached to the antibody.
[0202] In a preferred embodiment, variant TNFSF proteins are
administered as therapeutic agents, and can be formulated as
outlined above. Similarly, variant TNFSF genes (including both the
full-length sequence, partial sequences, or regulatory sequences of
the variant TNFSF coding regions) may be administered in gene
therapy applications, as is known in the art. These variant TNFSF
genes can include antisense applications, either as gene therapy
(i.e. for incorporation into the genome) or as antisense
compositions, as will be appreciated by those in the art.
[0203] In a preferred embodiment, the nucleic acid encoding the
variant TNFSF proteins may also be used in gene therapy. In gene
therapy applications, genes are introduced into cells in order to
achieve in vivo synthesis of a therapeutically effective genetic
product, for example for replacement of a defective gene. "Gene
therapy" includes both conventional gene therapy, where a lasting
effect is achieved by a single treatment, and the administration of
gene therapeutic agents, which involves the one time or repeated
administration of a therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for
blocking the expression of certain genes in vivo. It has already
been shown that short antisense oligonucleotides can be imported
into cells where they act as inhibitors, despite their low
intracellular concentrations caused by their restricted uptake by
the cell membrane. [Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A.
83:4143-4146 (1986)]. The oligonucleotides can be modified to
enhance their uptake, e.g. by substituting their negatively charged
phosphodiester groups by uncharged groups.
[0204] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection [Dzau et al., Trends in Biotechnology 11:205-210
(1993)]. In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262:4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. U.S.A.
87:3410-3414 (1990). For review of gene marking and gene therapy
protocols see Anderson et al., Science 256:808-813 (1992).
[0205] In another embodiment, variant TNFSF genes are administered
as DNA vaccines, either single genes or combinations of variant
TNFSF genes. Naked DNA vaccines are generally known in the art.
Brower, Nature Biotechnology, 16:1304-1305 (1998). Methods for the
use of genes as DNA vaccines are well known to one of ordinary
skill in the art, and include placing a variant TNFSF gene or
portion of a variant TNFSF gene under the control of a promoter for
expression in a patient in need of treatment.
[0206] The variant TNFSF gene used for DNA vaccines can encode
full-length variant TNFSF proteins, but more preferably encodes
portions of the variant TNFSF proteins including peptides derived
from the variant TNFSF protein. In a preferred embodiment a patient
is immunized with a DNA vaccine comprising a plurality of
nucleotide sequences derived from a variant TNFSF gene. Similarly,
it is possible to immunize a patient with a plurality of variant
TNFSF genes or portions thereof as defined herein. Without being
bound by theory, expression of the polypeptide encoded by the DNA
vaccine, cytotoxic T-cells, helper T-cells and antibodies are
induced which recognize and destroy or eliminate cells expressing
TNFSF proteins.
[0207] In a preferred embodiment, the DNA vaccines include a gene
encoding an adjuvant molecule with the DNA vaccine. Such adjuvant
molecules include cytokines that increase the immunogenic response
to the variant TNFSF polypeptide encoded by the DNA vaccine.
Additional or alternative adjuvants are known to those of ordinary
skill in the art and find use in the invention.
[0208] All references cited herein, including patents, patent
applications (provisional, utility and PCT), and publications are
incorporated by reference in their entirety.
[0209] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes.
EXAMPLES
[0210] TNF-Alpha Library Expression, Purification, and Activity
Assays for TNF-Alpha Variants
[0211] Methods:
[0212] 1) Overnight Culture Preparation:
[0213] Competent Tuner(DE3)pLysS cells in 96 well-PCR plates were
transformed with 1 ul of TNF-alpha library DNAs and spread on LB
agar plates with 34 mg/ml chloramphenicol and 100 mg/ml ampicillin.
After an overnight growth at 37 degrees C., a colony was picked
from each plate in 1.5 ml of CG media with 34 mg/ml chloramphenicol
and 100 mg/ml ampicillin kept in 96 deep well block. The block was
shaken at 250 rpm at 37 degrees C. overnight.
[0214] 2) Expression:
[0215] Colonies were picked from the plate into 5 ml CG media (34
mg/ml chloramphenicol and 100 mg/ml ampicillin) in 24-well block
and grown at 37 degrees C. at 250 rpm until OD600 0.6 were reached,
at which time IPTG was added to each well to 1 mM concentration.
The culture was grown 4 extra hours.
[0216] 3) Lysis:
[0217] The 24-well block was centrifuged at 3000 rpm for 10
minutes. The pellets were resuspended in 700 ul of lysis buffer (50
mM NaH2PO4, 300 mM NaCl, 10 mM imidazole). After freezing at -80
degrees C. for 20 minutes and thawing at 37 degrees C. twice, MgCl2
was added to 10 mM, and DNase I to 75 mg/ml. The mixture was
incubated at 37 degrees C. for 30 minutes.
[0218] 4) Ni NTA Column Purification:
[0219] Purification was carried out following Qiagen Ni NTA spin
column purification protocol for native condition. The purified
protein was dialyzed against 1.times. PBS for 1 hour at 4 degrees
C. four times. Dialyzed protein was filter sterilized, using
Millipore multiscreenGV filter plate to allow the addition of
protein to the sterile mammalian cell culture assay later on.
[0220] 5) Quantification:
[0221] Purified protein was quantified by SDS PAGE, followed by
Coomassie stain, and by Kodak.RTM. digital image densitometry.
[0222] TNF-Alpha Antagonist Activity
[0223] A) Materials and Methods: Plated cells for the assay: WEHI
plated at 2.5.times.105 cells/ml (50 .mu.l/well) Prepared Assay
Media as follows: 1.times. Assay Medium, 40 ml complete RPMI
medium, 80 .mu.l Actinomycin D (2 .mu.g/ml final concentration).
Prepared assay medium+wild-type TNF-alpha (Wild-type TNF-alpha is
1.1 mg/ml-1 .mu.g/ml: 1:1000; 1 .mu.l of the stock in 1 ml of RPMI
-20 ng/ml: 150 of the 1 .mu.g/ml; 800 .mu.l in 40 ml of assay
medium Dilution of TNF-alpha variants was done as shown below:
2 Assay medium (.mu.l) with 20 ng/ml of Concentration in Final
conc.of Stock (.mu.l) wt TNF-a dilution plate TNF-a in cells K112D:
59 .mu.l 941 100,000 ng/ml 50,000 ng/ml Y115T: 77 .mu.l 923 D143K:
32 .mu.l 968 D143R: 34 .mu.l 966 Y115I: 63 .mu.l 937 D143E: 40
.mu.l 960 A145R: 50 .mu.l 950 A145K: 50 .mu.l 950 A145E: 26 .mu.l
974 E146K: 40 .mu.l 960 E146R: 56 .mu.l 944 500 .mu.l of 500 50,000
ng/ml 25,000 ng/ml 100,000 ng/ml 500 .mu.l of 500 25,000 ng/ml
12,500 ng/ml 50,000 ng/ml 400 .mu.l of 600 10,000 ng/ml 5000 ng/ml
25,000 ng/ml 500 .mu.l of 500 5,000 ng/ml 2,500 ng/ml 10,000 ng/ml
200 .mu.l of 800 1000 ng/ml 500 ng/mL 5000 ng/ml 500 .mu.l of 500
500 ng/ml 50 ng/mL 1000 ng/ml 500 .mu.l of 500 250 ng/ml 125 ng/mL
the 500 ng/ml 400 .mu.l of 600 100 ng/ml 50 ng/mL 250 ng/ml 100
.mu.l of 900 10 ng/ml 5 ng/mL 100 ng/ml 100 .mu.l of 900 1 ng/ml
0.5 ng/mL 10 ng/ml 0 0 0 0
[0224] 5) Dilutions for Inhibition Assay:
[0225] Stocks to dilute TNF Receptor (TNF R) in 1.times. assay
medium: Stock is 100 .mu.g/ml For 20 .mu.g/ml: 1:5 dilution: 60
.mu.l of 100 .mu.g/ml of Stock+240 .mu.l of 1.times. assay medium
with wild-type TNF-alpha
[0226] Diluted TNF R assay medium containing 20 ng/ml of wild-type
TNF-alpha (final on the cell 10 ng/ml) as shown below:
3 Assay medium (.mu.l) Final with TNF- Concentration Concentration
Stock (.mu.l) alpha in dilution plate in cells 300 .mu.l of 20
.mu.g 300 10,000 ng/ml 5000 ng/ml 200 .mu.l of 10,000 ng 300 4000
ng/ml 2000 ng/ml 250 .mu.l of 4000 ng 250 2000 ng/ml 1000 ng/ml 250
.mu.l of 2000 ng 250 1000 ng/ml 500 ng/ml 50 .mu.l of 10,000
.mu.g/ml 950 500 ng/ml 250 ng/ml 200 .mu.l of 500 ng/ml 300 200
ng/ml 100 ng/ml 100 .mu.l of 500 ng/ml 400 100 ng/ml 50 ng/ml 100
.mu.l of 500 ng/ml 900 50 ng/ml 25 ng/ml 200 .mu.l of 50 ng/ml 300
20 ng/ml 10 ng/ml 100 .mu.l 50 ng/ml 400 10 ng/ml 5 ng/ml 50 .mu.l
50 ng/ml 450 5 ng/ml 2.5 ng/ml 0 250 0 0
[0227] All of the above dilutions were done 16 hours prior to
adding to the cells. Then 120 .mu.l of each diluted sample was
incubated at 4.degree. C., and 120 .mu.l of each sample was
incubated at 37.degree. C. The next morning, 50 .mu.l of each
sample was added to the cells. The cells were incubated at
37.degree. C. for 4 hours. After 4 hours of incubation, 100 .mu.l
of the caspase substrate was added to each well, followed by a 2
hour incubation at 37.degree. C. Read fluorescence. Results are
shown in FIG. 6.
[0228] TNF-Alpha Antagonist Activity of Combinatorial TNF-Alpha
Variants
[0229] Materials and Methods:
[0230] Plated cells for the assay: WEHI164-13Var cells plated at
7.5.times.105 cells/ml (50 .mu.l/well), incubate at 37 degrees C.
overnight and prepared Assay Media: (10.times., final concentration
on cells will be 10 ng/mL) (7 ml full RPMI, 5 ul to 310 ug/mL
wild-type his-TNF [Lot#263-56], 140 uL 1 mg/mL ActinomycinD).
Dilution of TN-alpha variants was done as shown below by mixing
these samples three days prior to start of experiment:
4 Conc. Final Before Conc. Conc. on Stock (uL) RPMI 10X After 10X
cells 1 E146K/N34V/V91E (lot 388-3) 1800 ug/mL: 38.6 961.4 69,520
63,200 ng/mL 31,600 ng/mL Y115Q/I97T (380-32) 2000 ug/mL: 34.7
965.3 Y115Q/I97R (380-32) 1400 ug/mL: 49.8 950.2 Y115Q/Y87R
(380-32) 1100 ug/mL: 63.3 936.7 Y115Q/L57Y (380-32) 1100 ug/mL 63.3
936.7 Y115Q/L57F (380-32) 1200 ug/mL 57.8 942.2 A145R/L57F (388-3)
2000 ug/mL 34.7 965.3 A145R/Y87H (378-96) 880 ug/mL 78.7 921.3
Enbrel 25000 ug/mL 997.3 Buffer (PBS pH 8) 100 uL 900 TNF R (500
ug/mL) 70 uL 430 2 316 (158 for TNF R) ul of 63,200 ng/mL 684(342)
22,000 20,000 ng/mL 10,000 ng/mL 3 316 (158 for TNF R) ul of 20000
ng/mL 684(342) 6,952 6,320 ng/mL 3,160 ng/mL 4 316 (158 for TNF R)
ul of 6,320 ng/mL 684(342) 2200 2,000 ng/mL 1000 ng/mL 5 316 (158
for TNF R) ul of 2000 ng/mL 684(342) 695.2 362 ng/mL 316 ng/mL 6
316 (158 for TNF R) ul of 362 ng/mL 684(342) 220 200 ng/mL 100
ng/mL 7 316 (158 for TNF R) ul of 200 ng/mL 684(342) 69.52 63.2
ng/mL 31.6 ng/mL 8 316 (158 for TNF R) ul of 63.2 ng/mL 684(342) 22
20 ng/mL 10 ng/mL 9 316 (158 for TNF R) ul of 20 ng/mL 684(342)
6.95 6.32 ng/mL 3.16 ng/mL 10 316 (158 for TNF R) ul of 6.32 ng/mL
684(342) 2.2 2 ng/mL 1 ng/mL 11 316 (158 for TNF R) ul of 2 ng/mL
684(342) 0.6952 0.632 ng/mL 0.316 ng/mL 12 0 684 0 0 ng/mL 0
[0231] After all dilutions were done at 68.4 (34.2 for TNF R) uL of
10.times. assay media containing WT his TNFa to each dilution well.
Then the 96 well was placed in the incubator for 3 days. 50 ul of
each sample were added to WEHI164-13Var cells for 4 hours. Upon
completion of the incubation, add 100 ul of caspase substrate.
Incubate for 1.5 hours. A R110 curve was also prepared by diluting
the R110 standard 1:100 in RPMI followed by an 8-point half
dilution. Then 100 ul of each dilution were added to a plate
without cells, these dilutions are done right before adding the
substrate to the cells. 100 ul of substrate was also added to R110
curve dilutions. Upon the completion of 1.5-hour incubation at 37
degrees C., all samples were read using the Wallac fluoremeter at
484/535 nm wavelengths. Results are shown in FIG. 6.
[0232] Fixed Equilibrium Screening of TNF-Alpha Variants
[0233] Prepared 1:10 fixed equilibrium ratios of TNF-alpha variants
by mixing together 0.01 mg/mL wild-type his-TNF [lot#263-56] with
0.1 mg/mL variant TNF-alpha in 50 uL reactions in
phosphate-buffered saline (PBS).
5 0.33 mg/ Conc. Volume mL wt Protein Name Lot# (mg/mL) Prot. (uL)
TNF (uL) PBS Y115Q/L57W 380-32 1.3 3.85 1.5 44.65 Y115M/D143N
380-32 0.36 13.8 1.5 34.7 Y115Q/Y87H 380-32 1.1 4.55 1.5 44
Y115Q/A145R 380-32 0.53 9.4 1.5 39.1 Y115Q/A145F 380-32 2.0 2.5 1.5
46 Y115Q/L57Y 380-32 1.1 4.55 1.5 44 Y115M/A145R 380-32 0.74 6.8
1.5 41.7 Y115M/E146K 380-32 0.27 18.5 1.5 30 Y115M/D143Q 380-32
0.37 13.5 1.5 35 Y115Q/L57F 380-32 1.2 4.17 1.5 44.3 A145R/I97R
380-32 0.56 9 1.5 39.5 A145R/Y87H 380-32 1.6 3.13 1.5 45.4
A145R/L75Q 380-32 0.86 5.8 1.5 42.7 A145R/L75K 380-32 0.99 4.9 1.5
43.6 Y115M/A145R 380-32 0.23 21.7 1.5 27 A145R/S86Q 380-32 1.2 4.2
1.5 44.3 E146K/V91E/N34E 380-32 1.2 2.8 1.5 45.7 A145R/S86R 378-95
0.27 18.5 1.5 30 A145R/I97T 378-97 0.47 10.6 1.5 37.9 A145R/L75E
378-94 1.73 2.9 1.5 45.6 Y115Q/S86R 380-32 0.94 4.9 1.5 43.6
Y115Q/Y87R 380-32 1.1 4.6 1.5 43.9 Y115Q/L75K 380-32 0.75 6.7 1.5
41.8 Y115Q/S86Q 380-32 1.0 4.9 1.5 43.6 Y115Q/E146K 380-32 0.38
13.1 1.5 35.4 Y115Q/L75Q 380-32 0.58 8.6 1.5 39.9 Y115Q/I97T 380-32
2.0 2.5 1.5 46 Y115Q/D143N 380-32 0.3 16.7 1.5 31.8 Y115Q/L75E
380-32 0.62 8.1 1.5 40.4 Y115Q/I97R 380-32 1.4 3.6 1.5 44.9
A145R/L57F 388-3 2 2.5 1.5 46
[0234] Prepared this mixture and incubated at 37 degrees C. for 3-4
days. Plated cells for the assay: Human U937 cells plated at
1.times.106 cells/ml (50 .mu.l/well) and incubated at 37 degrees C.
overnight.
[0235] Caspase Assay
[0236] Warmed full RPMI medium and supplement with 2 ug/mL
Actinomycin D. Mixed each entire 50 uL reaction with 450 uL
Actinomycin D supplemented RPMI medium. This mixture was diluted
1:1 eleven times to generate a dose curve for the fixed
equilibrium. 50 uL of the dilution mixture was applied to the cells
in quadruplicate. Cells were incubated in the TNF-alpha/TNF-alpha
variant fixed equilibrium for 1.5 hours. Upon completion of the
incubation, 100 ul of caspase substrate was added and then
incubated for 1.5 hours. A R110 curve was also prepared by diluting
the R110 standard 1:100 in RPMI followed by an 8-point half
dilution. Then 100 ul of each dilution was added to a plate without
cells, these dilutions are done right before adding the substrate
to the cells. 100 ul of substrate was also added to R110 curve
dilutions. Upon the completion of 1.5-hour incubation at 37 degrees
C., all samples were read using the Wallac fluoremeter at 484/535
nm wavelengths. Results are shown in FIGS. 6A-C.
[0237] Binding Assay
[0238] Biotinylation of TNFa was performed by adding 20 molar
excess Sulfo-NHS-LC-biotin to the protein sample and incubating the
sample on ice for 2 hours. Excess biotin was removed from the
sample by dialysis. Coupling ratios ranged between 1 to 4. The
protein concentration of biotinylated TNFa was determined by BCA
protein assay (Pierce). Wells of a microtiter plate were coated
with anti-FLAG antibody at a concentration of 2.5 mg/ml and blocked
with 3% BSA overnight at 4.degree. C. The FLAG-tagged protein TNFR1
receptor was added at a concentration of 10 ng/ml in PBS+1% BSA to
wells of the anti-FLAG-coated microtiter plate, and the plate was
incubated for 2 hours at room temperature. Biotinylated TNFa
proteins ranging in concentrations from 0-1 mg/mL were added in
quadruplicate to anti-FLAG-TNFR1-coated wells to represent total
binding. Non-specific binding was measured by adding biotinylated
TNF-alpha proteins ranging in concentrations from 0-1 .mu.g/ml in
quadruplicate to wells coated only with anti-FLAG antibody. Binding
was allowed to occur overnight at +4.degree. C. to ensure
equilibrium. Alkaline phosphatase conjugated neutravidin (Pierce)
was added to the wells at 1:10,000 dilution in PBS+1% BSA and
incubated for 30 min at room temperature. Luminescence was detected
upon the addition of the CSPD star substrate (Applied Biosystems,
Foster City, Calif.) and was measured (Wallac VICTOR, Perkin Elmer
Life Sciences, Boston, Mass.). The specific binding of TNFa was
calculated by subtracting non-specific binding from total binding.
Data was fit to the binding equation y=(BLmax*x)/(Kd+x).
[0239] The results of the binding assays are shown in FIG. 6. All
variants show a decrease in receptor binding.
Example 7
[0240] TNF-Alpha Variants Exchange with Wild-Type TNF-Alpha to
Reduce Activation of NFk B
[0241] TNF-alpha variants tested were A145R, double variant
A145R/Y87H, and triple variant E146/V91E/N34E. His-tagged TNF-alpha
was pre-incubated with 10-fold excess (1:10) of different variants
for 3-days at 37 degrees C. Wild-type TNF-alpha alone and
pre-exchanged heterotrimers of TNF-alpha variants were then tested
for their ability to activate an NFkB-driven luciferase reporter
(pNFkB-luc, Clontech) in 293T cells. 293T cells were seeded at
1.2.times.104 cells/well in 96-well plates. Cells were then
transfected with pNFkB-luc (NF-kB dependent luciferase reporter) or
pTal (Control: basal promoter driving the luciferase gene, but
without NFkB binding elements) using Fugene transfection reagent
according to the manufacturer's protocol (Roche). 12 hrs after
transfection, cells were treated with a final concentration of 10
ng/ml wild-type TNF-alpha or a pre-exchanged mixtures of 10 ng/ml:
TNF/100 ng/ml variant. 12 hrs after treatment, the cells in 96-well
plates were processed for the luciferase assay using the Steady-Glo
Luciferase Assay System (Promega) according to the manufacturer's
protocol. Luminescence from each well was measured using the
Packard TopCount NXT (Packard Bioscience) luminescence counter.
Treated samples were tested in quadruplicates, and mean values of
luminescence were plotted as bar values including the standard
deviation for each treatment. The results are shown in FIG. 20A.
The graph shows that the TNF-alpha variants of the present
invention were effective in decreasing wild-type TNF-alpha induced
NFkB activation. The TNF-alpha variant A145R/Y87H was most
effective in decreasing TNF-alpha induced NFkB activation.
[0242] Immuno-Localization of NFkB in HeLa Cells
[0243] HeLa cells were seeded onto 12 mm sterile coverslips
(Fisherbrand) at a density of 1.5.times.105 cells/well in 6-well
plates and cultured at 37 degrees C. at 5% CO2 atmosphere. The
following day, the cells were treated with various concentrations
of his-tagged wild-type TNF-alpha, A145R/Y87H variant alone, or the
combination of the his-tagged TNF-alpha and 10-fold excess of the
A145/Y87H variant (pre-exchanged for three days at 37 C.) at
37.degree. C., 5% CO2. After 30 minutes of incubation, the cells
attached to coverslips in 6-well plates were briefly washed with
PBS and fixed in 4% formaldehyde/PBS for 10 minutes. Cells were
then washed an additional five times with PBS or maintained in the
last PBS wash overnight before processing cells for
immunocytochemistry. Fixed cells on coverslips were then treated
with 0.1% Triton X-100/PBS. The buffer was aspirated and cells on
coverslips were blocked in a humidified chamber for 15 minutes with
50 ul of 0.1%BSA/0.1% TX-100/PBS per coverslip at 37.degree. C. The
blocking reagent was then removed and replaced with primary
antibody against p65 subunit of NF-kB (pAb C-20, Santa Cruz
Bioscience). After one hour of incubation at 37 degrees C., the
antibody was removed and coverslips were washed 5 times with PBS.
50 ul of FITC-conjugated secondary antibody (Jackson Immuno
laboratories) diluted in blocking buffer (1:100) was added to each
coverslip (Jackson Immunolaboratories) and coverslips were
incubated in a light-safe humidified chamber for an additional hour
before removing the secondary antibody with 5 washes of PBS.
Coverslips were briefly rinsed with d-water, air-dried in a
light-safe chamber and mounted onto slides using Anti-fade
(Molecular Probes). Digital images of antibody-reacted cells were
captured using a FITC filter and 40.times. objective on a Nikon
Eclipse TS100 microscope coupled to a Cool SNAP-Pro CCD camera
(Media Cybernetics) and operated using Image Pro Plus software
(Media Cybernetics).
[0244] FIG. 4B shows photographs of the immuno-localization of NFkB
in HeLa cells showing that the exchange of wild-type TNF-alpha with
the A145/Y87H TNF-alpha variant inhibits TNF-alpha-induced nuclear
translocation of NFkB in HeLa cells. The TNF-alpha variant
A145R/Y87H alone does not induce NFkB nuclear translocation, unlike
the wild-type TNF-alpha. Moreover, the wild-type TNF-alpha
exchanged (3-days, 37 degrees C.) to form heterotrimers with excess
variant (10 fold excess of TNF-alpha variant A145R/Y87H) loses its
ability to induce NFkB nuclear translocation. This data is
consistent with the effects of this variant in the luciferase
reporter assay.
[0245] TNFA Variant A145R/Y87H Reduced TNF-Alpha Induced Activation
of the NFkB-Driven Luciferase Reporter
[0246] His-tagged wild-type TNF-alpha, TNF-alpha variant A145/Y87H
and the exchanged wild-type TNF-alpha:A145R/Y87H heterotrimer
(1-day exchange with 10-fold excess TNF-alpha variant A145R/Y87H at
37 degrees C.) were tested in the NFkB luciferase reporter assay as
in Example 7A above. The experiment was carried out as in Example
7A, with the exception that a wider range of final TNF-alpha
concentrations and increasing doses were used (0.78, 1.56, 3.13,
6.25,12.5, 25 ng/ml) with 10-fold excess of TNF-alpha variant
(A145R/Y87H) at each TNF-alpha concentration.
[0247] The wild-type TNF-alpha:A145R/Y87H heterotrimer has a
significantly reduced activation level, indicating the TNF-alpha
A145R/Y87H variant's inhibitory effect on wild-type TNF-alpha.
Unlike wild-type TNF-alpha, the TNF-alpha variant A145/Y87H alone
has no significant agonizing effect on NFkB activation as shown by
the lower dotted line in FIG. 4C. Wild-type TNF-alpha induced
activation is dependent on the NFkB activation as the reporter and
without NFkB binding elements is unresponsive to the TNF-alpha as
shown in the solid gray line in FIG. 4C.
[0248] Antagonistic RANKL Variant Library Design
[0249] Antagonistic RANKL variants were generated using the
dominant-negative strategy which includes the single chain variety,
and by the selection of competitive inhibitor antagonists.
[0250] Dominant-Negative RANKL Library Variants
[0251] PDA.TM. was utilized to generate a human structural model of
RANKL in addition to selecting amino acids that disrupt RANK
binding. The lack of a crystal structure for human OPGL
necessitated the creation of a homology model where PDATM was used
for side chain placement based on the human sequence and mouse
RANKL structure (PDB# 1JTZ). This modeling was facilitated by the
87% sequence identity between the mouse and human RANKL sequences.
The dominant-negative RANKL therapeutic strategy is based on the
design of novel RANKL variants that have reduced receptor binding
and/or activation properties and the ability to heterotrimerize
with wild-type RANKL (FIG. 21). In other words, RANKL variants that
do not activate RANK will exchange with wild-type RANKL protein and
sequester it into inactive heterotrimers, inhibiting its activity.
The number and activity of osteoclasts will be lowered as a result
of this treatment leading to a decrease in bone resorption and an
overall increase in bone mineral density.
[0252] The dominant-negative RANKL variants were designed by
substituting the amino acids at key RANKL-RANK contact points with
amino acids that disrupt the ability of the ligand to activate
receptor. PDATM technology was used to select appropriate
substitutions for optimal protein folding. The exchange of these
trimeric RANKL variants with trimeric wild-type RANKL will result
in the deactivation of the wild-type RANKL and reduced osteoclast
formation. To help accomplish this goal more effectively, the RANKL
variants can also be designed to preferentially heterotrimerize
with wild-type RANKL.
[0253] Single Chain Dominant-Negative Polypeptides
[0254] Multiple strategies for covalent linkage of monomers exist.
These included, but are not limited to: polypeptide linkages
between N and C-termini of two domains, made up of zero or more
amino acids (resulting in single chain polypeptides comprising
multiple domains); linkage via a disulfide bond between monomers;
linkage via chemical crosslinking agents.
[0255] Multiple strategies exist for modification of individual
domains such that receptor binding is removed (or reduced). These
include, but are not limited to: amino acid modifications that
create steric repulsion between ligand domain and receptor;
modifications that create electrostatic repulsion; modifications
that create unfavorable desolvation of amino acids; and chemical
modification of amino acids at the ligand/receptor interface (e.g.
PEGylation or glycosylation).
[0256] Linkage of RANKL Monomers into a Trimer
[0257] To improve the dominant negative behavior of these mutants,
single-chain dimers between two modified receptor interaction
domains are being created.
[0258] RANKL Variant Library Construction
[0259] The full-length RANKL cDNA was cloned using RT-PCR on lymph
node cDNAs. Two lengths of RANKL, amino acids 147 to 317 and 159 to
317, were subcloned into pET33b after PCR-mediated addition of TEV
and HIS tags.
[0260] Muatagenesis reactions were performed to introduce the
nucleotide changes that result in the desired amino acid
substitutions. The mutagenesis reactions utilize pairs of divergent
and overlapping oligonucleotides with nucleotide variations that
introduce the specific amino acid changes. These oligonucleotides
are extended using Pfu polymerase through multiple rounds of
thermal cycling and these extension products are transformed into
TOP10 bacterial cells after digesting the products with DpnI.
Clones were sequenced to confirm the designed changes and to insure
no modifications were introduced into the cDNA during the thermal
cycling.
[0261] RANKL Variant Screening for Non-Agonists, Superagonists, and
Antagonists
[0262] A library of 89 human RANKL variants was constructed with
all members of the library comprising the amino acid sequence in
FIG. 1b and the two solubility-imparting modifications, C221S/I247E
in addition to the 89 computationally designed inhibitory
modifications. These 89 human RANKL variant proteins were expressed
and purified from E. coli and 52 were screened for non-agonists,
superagonists, and antagonists in an assay that monitors
osteoclastogenesis in RAW264.7 cells. Treating RAW264.7 cells with
the purified human RANKL variant protein and measuring the amount
of TRAP released identified non-agonist and superagonist variants.
Of the 52 RANKL variants screened, 21 RANKL variants were
identified to be non-agonists, while C221S/I247E/A172R was found to
be a superagonist. In addition, the antagonism screens described
above identified the six antagonizing variants. These antagonizing
variants have the ability to lower the osteoclastogenesis activity
of human RANKL.
[0263] Tartrate-Resistant Acid Phosphatase (TRAP) Activity
Bioassay
[0264] The TRAP activity bioassay was used for assaying
non-agonism, super-agonism, and antagonism. TRAP activity was
measured after adding the RANKL variant protein to cell lines such
as RAW264.7 to identify RANKL variants that are non-agonists or
super-agonists (FIGS. 23-25, 29). An additional screen was
performed to identify antagonist RANKL variants by mixing RANKL
variant protein with one of the active RANKL solubility variants,
C221S/I247E, and adding the protein mixture to the RAW264.7 and
measuring TRAP activity. Antagonist RANKL variants were identified
that lowered the activity of the RANKL solubility variant
C221S/I247E.
[0265] RAW 264.7 cells (ATCC#TIB-71) were cultured and maintained
in 75 cm2 flasks in 5% CO2 humidified incubator at 37.degree. C. in
growth media containing a-MEM w/2mM L-Glutamine
(Gibco-BRL#12571-063), 10% Heat Inactivated Fetal Bovine Serum
(FBS) (Hyclone#SH30071.03) and Penicillin/Streptomycin
(Gibco-BRL#15140-122) 100 units/ml and 100 ug/ml respectively.
[0266] Recombinant mouse RANKL (R&D#462-TR), recombinant human
soluble RANKL (Biosource#PHP0034), and recombinant human soluble
RANKL variants were diluted to their working concentration in assay
media consisting of a-MEM w/I-glutamine (Gibco-BRL#12571-063)
supplemented with 10% Heat Inactivated Fetal Bovine Serum (FBS)
(Hyclone#SH30071.03) and Penicillin/Streptomycin
(Gibco-BRL#15140-122) 100 units/ml and 100 ug/ml respectively. 250
ml of RANKL standards and samples were added to 96-well assay plate
in triplicate or quadruplicate, and serially diluted 1:2 by
transferring 125 ml into subsequent wells containing 125 ml of
assay media.
[0267] Aspirating the exhausted media, adding 10 ml of pre-warmed
media to the flask, and scraping cells from the flask using a cell
scraper harvested the cells. The cells were transferred to a 15 ml
conical tube, spun at 1000 rpm for 5 minutes and resuspended in 10
ml of assay media. The cells were counted and seeded at density of
1.5.times.10.sup.3/125 ml /well in 96-well assay plate. Assay
plates were incubated at 37.degree. C. in 5% CO2 humidified
incubator for 72 hours.
[0268] The amount of osteoclast-like cells induced from the RAW
264.7 cells was determined by measuring the Tartrate-Resistant Acid
Phosphatase (TRAP) activity. TRAP substrate was prepared by adding
20 mg of p-nitrophenol phosphate (pNPP) (ICN#100878) to 10 ml TRAP
buffer pH5.0 (100 mM Sodium Acetate, 11.5 mg/ml Sodium Tartrate
(ICN#195503)). The media was aspirated off the assay plate and
discarded. Cells were fixed for 1 minute with 1:1 ethanol and
acetone solution and washed once with 100-150 ml/well of PBS
(Hyclone#SH30256.02). 100 ml of TRAP solution was added to each
well, and the plate was incubated at 37.degree. C. for 2 hours. The
reaction was stopped by adding 50 ml/well of a 0.2N NaOH solution
and the absorbance was read at 405 nm. If the absorbance reached
saturation, the solutions were diluted 1:5 and read again.
[0269] Tartrate-Resistant Acid Phosphatase (TRAP) Cytological
Staining
[0270] In parallel experiments, osteoclast formation from the RAW
264.7 cells was measured by the presence of Tartrate-Resistant Acid
Phosphatase (TRAP) multinucleated positive cells using cytological
staining. 0.1M Acetate buffer was prepared prior to staining by
combining 35.2 ml of a 0.2M Sodium Acetate solution, 14.8 ml of a
0.2M Acetic Acid solution and 50 ml of water. 10 ml TRAP buffer
pH5.0 (50 mM Acetate Buffer, 30 mM Sodium Tartrate (Sigma#S-8640),
0.1 mg/ml Napthol AS-MX Phosphate Disodium Salt (Sigma#N-5000),
0.1% Triton X-100) was prepared fresh for each assay. The TRAP
buffer was warmed in a 37.degree. C. water bath and 0.3 mg/ml of
Fast Red Violet LB Stain (Sigma#F-3381) was added and the stain was
returned to the 37.degree. C. water bath. The media was aspirated
off the assay plate and discarded. The cells were washed once with
150 ml/well of PBS (Hyclone#SH30256.02) and subsequently fixed with
100 ml/well of a 10% Glutaraldehyde solution for 15 minutes at
37.degree. C. The cells were washed twice with 150 ml/well of
pre-warmed PBS. 100 ml of the pre-warmed TRAP stain was added to
each well and the plate was incubated at 37.degree. C. for 5-10
minutes. The TRAP stain was removed and 100 ml of PBS was added to
each well to prevent cells from drying out. TRAP positive
multinucleated cells (more than three nuclei) were counted. FIG. 7
shows the results.
[0271] Other TNFSF Variants
[0272] BlyS, CD40L, APRIL, OX-40 are generated according to the
protocol disclosed above. They have exchange with their
corresponding wild-type TNFSF member. APRIL and BlyS have exchange
between them and the corresponding APRIL or BlyS. Binding assays,
and dose response curves are done for each TNFSF member.
[0273] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
* * * * *
References