U.S. patent application number 11/433276 was filed with the patent office on 2007-03-08 for compositions and methods for modulating immune responses.
Invention is credited to Janine Bilsborough, Cameron S. Brandt, Erick M. Chadwick, Zeren Gao, Edward D. Howard, Steven D. Levin, Frederick J. Ramsdell, James W. West.
Application Number | 20070054360 11/433276 |
Document ID | / |
Family ID | 37179003 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070054360 |
Kind Code |
A1 |
Gao; Zeren ; et al. |
March 8, 2007 |
Compositions and methods for modulating immune responses
Abstract
The present invention provides a newly identified B7 receptor,
zB7R1 that functions as lymphocyte inhibitory receptor, which is a
PD-1-like molecule and is expressed on T cells. The present
invention also provides the discovery of zB7R1's ability to bind to
CD155. Methods and compositions for modulating zB7R1-mediated
negative signaling and interfering with the interaction of its
counter-receptor for therapeutic, diagnostic and research purposes
are also provided.
Inventors: |
Gao; Zeren; (Redmond,
WA) ; Levin; Steven D.; (Seattle, WA) ;
Bilsborough; Janine; (Seattle, WA) ; West; James
W.; (Seattle, WA) ; Brandt; Cameron S.;
(Seattle, WA) ; Ramsdell; Frederick J.;
(Bainbridge Island, WA) ; Howard; Edward D.;
(Seattle, WA) ; Chadwick; Erick M.; (Bothell,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Family ID: |
37179003 |
Appl. No.: |
11/433276 |
Filed: |
May 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60680374 |
May 12, 2005 |
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60791626 |
Apr 13, 2006 |
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60795005 |
Apr 26, 2006 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/13.2; 514/19.3; 530/350; 530/388.22;
536/23.5 |
Current CPC
Class: |
A61K 39/3955 20130101;
C07K 14/70532 20130101; C07K 16/2818 20130101; G01N 33/505
20130101; C07K 2317/34 20130101; A61P 1/12 20180101; C07K 16/2803
20130101; A61P 43/00 20180101; C07K 2319/00 20130101; C07K 2317/75
20130101; A61K 38/00 20130101; C07K 2317/73 20130101; A61P 35/00
20180101; A61P 37/06 20180101; C07K 14/47 20130101; G01N 33/56972
20130101; G01N 2333/70532 20130101; A61P 29/00 20180101; A61K
2039/507 20130101; C07K 16/2827 20130101; C12Q 1/66 20130101; C07K
2319/30 20130101; C07K 2317/76 20130101; A61P 37/00 20180101; A61P
1/04 20180101; A61P 37/02 20180101 |
Class at
Publication: |
435/069.1 ;
514/012; 530/350; 435/320.1; 435/325; 536/023.5; 530/388.22 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C07K 14/705 20070101
C07K014/705; C07K 16/30 20070101 C07K016/30 |
Claims
1. An isolated soluble zB7R1 polypeptide comprising a sequence of
amino acid residues having at least 95% sequence identity with
amino acid residues 16-140 of SEQ ID NO:2 or amino acid residues
27-208 of SEQ ID NO:6, and wherein the polypeptide mediates T cell
activation.
2. An isolated soluble zB7R1 polypeptide comprising a sequence of
amino acid residues having at least 95% sequence identity with
amino acid residues 16-140 of SEQ ID NO:2 or amino acid residues
27-208 of SEQ ID NO:6, and wherein the polypeptide increases or
upregulates T cell activation.
3. An isolated soluble zB7R1 polypeptide comprising a sequence of
amino acid residues having at least 95% sequence identity with
amino acid residues 16-140 of SEQ ID NO:2 or amino acid residues
27-208 of SEQ ID NO:6, and wherein the polypeptide decreases or
inhibits T cell activation.
4. An isolated soluble zB7R1 polypeptide comprising a sequence of
amino acids selected from the group consisting of: SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID
NO:10.
5. An isolated soluble zB7R1 polypeptide comprising an amino acid
sequence selected from a group consisting of: SEQ ID NO: 25 and SEQ
ID NO:68.
6. An isolated polynucleotide comprising nucleotides selected from
the group consisting of: SEQ ID NO:1, SEQ ID NO:5, and SEQ ID
NO:8.
7. An isolated polynucleotide that hybridizes to a polynucleotide
of claim 6 under stringent conditions of hybridization in buffer
containing 5.times.SSC, 5.times. Denhardt's, 0.5% SDS, 1 mg salmon
sperm/25 mls of hybridization solution incubated at 65.degree. C.
overnight, followed by high stringency washing with
0.2.times.SSC/0.1% SDS at 65.degree. C., wherein the isolated
polynucleotide encodes a soluble polypeptide that inhibits the
costimulation T cells.
8. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
polypeptide of claim 1; and a transcription terminator.
9. A cultured cell into which has been introduced an expression
vector of claim 8, wherein the cell expresses the polypeptide
encoded by the DNA segment.
10. A method of producing a polypeptide comprising: culturing a
cell into which has been introduced an expression vector of claim
8, wherein the cell expresses the polypeptide encoded by the DNA
segment; and recovering the expressed polypeptide.
11. An antibody or antibody fragment that specifically binds to a
polypeptide of claim 1.
12. The antibody or antibody fragment of claim 11, wherein said
antibody blocks or inhibits the interaction of zB7R1 with
CD155.
13. An antibody or antibody fragment that specifically binds to
CD155, wherein said antibody blocks or inhibits the interaction of
CD155 with zB7R1.
14. The antibody or antibody fragment of claim 11, wherein said
antibody replaces or augments the interaction of zB7R1 with its
counter-receptor.
15. The antibody of claim 14, wherein the counter-receptor is
CD155.
16. The antibody of claim 11, wherein the antibody is selected from
the group consisting of a polyclonal antibody, a murine monoclonal
antibody, a humanized antibody derived from a murine monoclonal
antibody, an antibody fragment, neutralizing antibody, and a human
monoclonal antibody.
17. The antibody fragment of claim 11, wherein the antibody
fragment is selected from the group consisting of F(ab'), F(ab),
F(ab').sub.2, Fab', Fab, Fv, scFv, and minimal recognition
unit.
18. An anti-idiotype antibody comprising an anti-idiotype antibody
that specifically binds to the antibody of claim 11.
19. The antibody of claim 13, wherein the antibody is selected from
the group consisting of a polyclonal antibody, a murine monoclonal
antibody, a humanized antibody derived from a murine monoclonal
antibody, an antibody fragment, neutralizing antibody, and a human
monoclonal antibody.
20. The antibody fragment of claim 13, wherein the antibody
fragment is selected from the group consisting of F(ab'), F(ab),
F(ab').sub.2, Fab', Fab, Fv, scFv, and minimal recognition
unit.
21. An anti-idiotype antibody comprising an anti-idiotype antibody
that specifically binds to the antibody of claim 13.
22. A fusion protein comprising a polypeptide comprising a sequence
of amino acid residues having at least 95% sequence identity with
amino acid residues 16-140 of SEQ ID NO:2 or 27-208 of SEQ ID NO:5;
and a polyalkyl oxide moiety, wherein the fusion protein mediates T
cell activation.
23. The fusion protein of claim 22 wherein the polyalkyl oxide
moiety is polyethylene glycol.
24. The fusion protein of claim 23 wherein the polyethylene glycol
is N-terminally or C-terminally attached to the polypeptide.
25. The fusion protein of claim 23 wherein the polyethylene glycol
is mPEG propionaldehyde.
26. The fusion protein of claim 23 wherein the polyethylene glycol
is branched or linear.
27. The fusion protein of claim 23 wherein the polyethylene glycol
has a molecular weight of about 5 kD, 12 kD, 20 kD, 30 kD, 40 kD or
50 kD.
28. A fusion protein comprising a polypeptide comprising a sequence
of amino acid residues having at least 95% sequence identity with
SEQ ID NO:68 or SEQ ID NO:69.
29. The fusion protein of claim 28 wherein the immunoglobulin heavy
chain constant region is an Fc fragment.
30. The fusion protein of claim 28 wherein the immunoglobulin heavy
chain constant region is an isotype selected from the group
consisting of an IgG, IgM, IgE, IgA and IgD.
31. The fusion protein of claim 30 wherein the IgG isotype is IgG1,
IgG2, IgG3, or IgG4.
32. A fusion protein comprising a VASP domain and zB7R1.
33. The fusion protein of claim 32, wherein zB7R1 comprises SEQ ID
NO:3.
34. A fusion protein comprising SEQ ID NO:25.
35. A formulation comprising: an isolated soluble polypeptide
comprising a sequence of amino acid residues having at least 95%
sequence identity with amino acid residues 16-140 of SEQ ID NO:2 or
amino acid residues 27-208 of SEQ ID NO:6; and a pharmaceutically
acceptable vehicle.
36. A kit comprising the formulation of claim 35.
37. A formulation comprising: an antibody or antibody fragment
according to claim 11; and a pharmaceutically acceptable
vehicle.
38. A formulation comprising: an antibody or antibody fragment
according to claim 13; and a pharmaceutically acceptable
vehicle.
39. A kit comprising the formulation of claim 37.
40. A kit comprising the formulation of claim 38.
41. A method for modulating lymphocyte activity, comprising
contacting a zB7R1-positive lymphocyte with a bioactive agent
capable of modulating zB7R1-mediated signaling in an amount
effective to modulate at least one lymphocyte activity.
42. The method according to claim 41, wherein said agent comprises
an antagonist of zB7R1-mediated signaling, and wherein said
contacting inhibits the attenuation of lymphocyte activity mediated
by zB7R1 signaling.
43. The method according to claim 42, wherein said contacting
increases lymphocyte activity.
44. The method according to claim 42, wherein said antagonist
comprises a blocking agent capable of interfering with the
functional interaction of zB7R1 and its counter-receptor.
45. The method of claim 44, wherein said counter-receptor is
CD155.
46. The method according to claim 44, wherein said blocking agent
comprises an anti-zB7R1 antibody capable of specifically binding to
the extracellular domain of zB7R1 (SEQ ID NO:3), and wherein said
binding interferes with the interaction of zB7R1 and its
counter-receptor.
47. The method according to claim 44, wherein said blocking agent
comprises a soluble zB7R1 protein.
48. The method according to claim 47, wherein the soluble zB7R1
protein comprises a sequence of amino acid residues sequence
selected
49. The method according to claim 44, wherein said blocking agent
comprises a soluble zB7R1 fusion protein.
50. The method according to claim 44, wherein the blocking agent is
selected from the group consisting of anti-zB7R1 antibodies, zB7R1
polypeptides, and zB7R1 fusion proteins.
51. The method according to claim 41, wherein said agent comprises
an agonist of zB7R1-mediated signaling, and said contacting
decreases lymphocyte activity.
52. The method according to claim 51, wherein said agonist
comprises a mimicking agent capable of mimicking the functional
interaction of zB7R1 and its counter-receptor.
53. The method according to claim 52, wherein said agonist
comprises a mimicking agent capable of augmenting the functional
interaction of zB7R1 and its counter-receptor.
54. The method according claim 41, wherein said lymphocyte is a T
lymphocyte and said lymphocyte activity is selected from the group
consisting of activation, differentiation, proliferation, survival,
cytolytic activity and cytokine production.
55. The method according claim 41, wherein said lymphocyte is a B
lymphocyte and said lymphocyte activity is selected from the group
consisting of activation, differentiation, proliferation, survival,
and antibody production.
56. The method according to claim 1, wherein said lymphocyte
activity comprises a host immune response to a target antigen, said
target antigen selected from the group consisting of a pathogen
antigen, a vaccine antigen, and a tumor-associated antigen other
than Its counter-receptor.
57. A method for treating cancer in a patient having zB7R1-positive
tumor cells comprising administering to the patient an antagonist
of zB7R1-mediated signaling, wherein said administration is
effective to increase the host immune response against said
zB7R1-positive tumor cell.
58. The method according to claim 57, wherein said antagonist
comprises a blocking agent capable of interfering with the
functional interaction of zB7R1 and its counter-receptor.
59. The method according to claim 51, wherein said blocking agent
comprises an anti-zB7R1 antibody capable of specifically binding to
the extracellular domain of zB7R1 (SEQ ID NO:3), wherein said
binding interferes with the interaction of zB7R1 and its
counter-receptor.
60. A method for treating a patient having an autoimmune disease
characterized by the presence of autoreactive zB7R1-positive
lymphocytes, comprising administering to the patient an agonist of
zB7R1-mediated signaling, wherein said administration is effective
to inhibit an autoreactive immune response against non-lymphoid
non-tumor host cells expressing zB7R1.
61. The method according to claim 60, wherein said agonist
comprises a mimicking agent capable of mimicking the functional
interaction of zB7R1 and its counter-receptor.
62. The method according to claim 40, wherein the counter-structure
is CD155.
63. The method according to claim 41, wherein the counter structure
is CD155.
64. The method according to claim 61, wherein the counter structure
is CD155.
65. A method of inhibiting T cell activation, the method comprising
contacting the T cell with a zB7R1 agonist.
66. A method of upregulating T cell activity, the method comprising
contacting the T cell with a zB7R1 antagonist.
67. The method of claim 66, wherein B cell activity is also
upregulated.
68. A method of inhibiting the co-stimulation a T cell, the method
comprising contacting the T cell with a soluble zB7R1 polypeptide,
the sequence of which comprises a sequence having at least 95%
identify with amino acid residues 16-140 of SEQ ID NO:2 or amino
acid residues 27-208 of SEQ ID NO:6.
69. The method of claim 68, wherein the contacting comprises
culturing the polypeptide with the T cell in vitro.
70. The method of claim 68, wherein the T cell is in a patient.
71. The method of claim 68 wherein the contacting comprises
administering the polypeptide to the patient.
72. The method of claim 70 wherein the contacting comprises
administering a nucleic acid encoding the polypeptide to the
patient.
73. The method of claim 70 wherein comprising (a) providing a
recombinant cell which is the progeny of a cell obtained from the
patient and has been transfected or transformed ex vivo with a
nucleic acid molecule encoding the polypeptide so that the cell
expresses the polypeptide; and (b) administering the cell to the
patient.
74. The method of claim 70 wherein the patient is suffering from an
inflammatory disease selected from the group consisting of Crohn's
disease, ulcerative colitis, graft versus host disease, celiac
disease, and irritable bowel syndrome.
75. A method of treating, preventing, inhibiting the progression
of, delaying the onset of and/or reducing at least one of the
symptoms or conditions associated with a disease selected from the
group consisting of Crohn's disease, ulcerative colitis, celiac
disease, Graft-versus-host disease, and irritable bowel syndrome
comprising administering to the patient an effective amount of the
formulation of claim 35.
76. A method of treating, preventing, inhibiting the progression
of, delaying the onset of and/or reducing at least one of the
symptoms or conditions associated with a disease selected from the
group consisting of Crohn's disease, ulcerative colitis, celiac
disease, Graft-versus-host disease, and irritable bowel syndrome
comprising administering to the patient an effective amount of the
formulation of claim 37.
77. A method of treating, preventing, inhibiting the progression
of, delaying the onset of and/or reducing at least one of the
symptoms or conditions associated with a disease selected from the
group consisting of Crohn's disease, ulcerative colitis, celiac
disease, Graft-versus-host disease, and irritable bowel syndrome
comprising administering to the patient an effective amount of the
formulation of claim 38.
Description
REFERENCE TO RELATED INVENTIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/680,374, filed May 12, 2005, U.S.
Provisional Application Ser. No. 60/791,626, filed Apr. 13, 2006,
and U.S. Provisional Application Ser. No. 60/795,005, filed Apr.
26, 2006 all of which are incorporated in their entirety herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Positive and negative costimulatory signals play critical
roles in the modulation of T cell activity, and the molecules that
mediate these signals have proven to be effective targets for
immunomodulatory agents. Positive costimulation, in addition to T
cell receptor (TCR) engagement, is required for optimal activation
of naive T cells, whereas negative costimulation is believed to be
required for the acquisition of immunologic tolerance to self, as
well as the termination of effector T cell functions. Upon
interaction with B7-1 or B7-2 on the surface of antigen-presenting
cells (APC), CD28, the prototypic T cell costimulatory molecule,
emits signals that promote T cell proliferation and differentiation
in response to TCR engagement, while the CD28 homologue cytotoxic T
lymphocyte antigen-4 (CTLA-4) mediates inhibition of T cell
proliferation and effector functions (Chambers et al., Ann. Rev.
Immunol., 19:565-594, 2001; Egen et al., Nature Immunol.,
3:611-618, 2002).
[0003] Several new molecules with homology to the B7 family have
been discovered (Abbas et al., Nat. Med., 5:1345-6,1999; Coyle et
al., Nat. Immunol., 2: 203-9, 2001; Carreno et al., Annu. Rev.
Immunol., 20: 29-53, 2002; Liang et al., Curr. Opin. Immunol., 14:
384-90, 2002), and their role in T cell activation is just
beginning to be elucidated. These new costimulatory
counter-receptors include B7h2, PD-L1, PD-L2, B7-H3 and B7-H4.
[0004] B7h2 (Swallow et al., Immunity, 11: 423-32, 1999), also
known as B7RP-1 (Yoshinaga et al., Nature, 402: 827-32, 1999), GL50
(Ling, et al., J. Immunol., 164:1653-7, 2000), B7H2 (Wang et al.,
Blood, 96: 2808-13, 2000), and LICOS (Brodie et al., Curr. Biol.,
10: 333-6, 2000), binds to inducible costimulator (ICOS) on
activated T cells, and costimulates T cell proliferation and
production of cytokines such as interleukin 4 (IL-4) and IL-10.
[0005] PD-L1 (Freeman et al., J. Exp. Med., 192: 1027-34, 2000),
also known as B7-R1 in humans (Dong et al., Nat. Med., 5, 1365-9,
1999), and PD-L2 (Latchman et al., Nat. immunol., 2: 261-8, 2001),
also known as B7-DC (Tseng et al., J. Exp. Med., 193, 839-46, 2001)
bind to programmed death 1 (PD-1) receptor on T and B cells,
although at present the function of these interactions is
controversial. Some reports have demonstrated that PD-L1 and PD-L2
have inhibitory effects on T cell responses (Freeman et al., J.
Exp. Med., 192: 1027-34, 2000; Latchman et al., Nat. Immunol., 2:
261-8, 2001), while others have shown that both counter-receptors
(B7-R1 and B7-DC) positively regulate T cell proliferation and
specifically enhance IL-10 or interferon gamma (IFN-.gamma.)
production (Dong et al., Nat. Med., 5, 1365-9, 1999; Tseng et al.,
J. Exp. Med., 193, 839-46, 2001).
[0006] Finally, B7-H3 and B7-H4, both newly identified B7
homologues, bind an as yet currently unknown counter-receptor(s) on
activated T cells, and are reported to enhance proliferation of
CD4+ T helper (Th) cells and CD8+ cytotoxic T lymphocytes (CTLs or
Tcs) and selectively enhance IFN-.gamma. expression (Chapoval et
al., Nat. Immunol., 2, 269-74, 2001; Sun et al., J. Immunol., 168,
6294-7, 2002).
[0007] With the exception of PD-1 counter-receptors, which show
some expression on non-lymphoid tissues, the expression of known B7
family members is largely restricted to lymphoid cells.
Collectively, these studies have revealed that B7 family members
are counter-receptors on lymphoid cells that interact with cognate
receptors on lymphocytes to provide positive or negative
costimulatory signals that play critical roles in the regulation of
cell-mediated immune responses.
[0008] In particular, many autoimmune disorders are known to
involve autoreactive T cells and autoantibodies. Agents that are
capable of inhibiting or eliminating autoreactive lymphocytes
without compromising the immune system's ability to defend against
pathogens are highly desirable. Conversely, many cancer
immunotherapies, such as adoptive immunotherapy, expand
tumor-specific T cell populations and direct them to attack and
kill tumor cells (Dudley et al., Science 298:850-854, 2002;
Pardoll, Nature Biotech., 20:1207-1208, 2002; Egen et al., Nature
Immunol., 3:611-618, 2002). Agents capable of augmenting tumor
attack are highly desirable. In addition, immune responses to many
different antigens (e.g., microbial antigens or tumor antigens),
while detectable, are frequently of insufficient magnitude to
afford protection against a disease process mediated by agents
(e.g., infectious microorganisms or tumor cells) expressing those
antigens. It is often desirable to administer to the subject, in
conjunction with the antigen, an adjuvant that serves to enhance
the immune response to the antigen in the subject. It is also
desirable to inhibit normal immune responses to antigen under
certain circumstances. For example, the suppression of normal
immune responses in a patient receiving a transplant is desirable,
and agents that exhibit such immunosuppressive activity are highly
desirable.
[0009] Costimulatory signals, particularly positive costimulatory
signals, also play a role in the modulation of B cell activity. For
example, B cell activation and the survival of germinal center B
cells require T cell-derived signals in addition to stimulation by
antigen. CD40 counter-receptor present on the surface of helper T
cells interacts with CD40 on the surface of B cells, and mediates
many such T-cell dependent effects in B cells. Interestingly,
negative costimulatory receptors analogous to CTLA-4 have not been
identified on B cells. This suggests fundamental differences may
exist in the way T cells and B cells are induced to respond to
antigen, which has implications for mechanisms of self-tolerance as
well as the inhibition of B cell effector functions, such as
antibody production. Were a functional CTLA-like molecule to be
found on B cells, the finding would dramatically shift our
understanding of the mechanisms of B cell stimulation. Further, the
identification of such receptors could provide for the development
of novel therapeutic agents capable of modulating B cell activation
and antibody production, and useful in the modulation of
immunologic responses.
[0010] Accordingly, there is a need in the art for the
identification of additional B7 family members, their
counter-receptors and molecules derived therefrom, that have either
or both a T cell costimulatory activity and/or a B cell
costimulatory activity . This need is based largely on their
fundamental biological importance and the therapeutic potential of
agents capable of affecting their activity. Such agents capable of
modulating costimulatory signals would find significant use in the
modulation of immune responses, and are highly desirable.
[0011] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
DETAILED DESCRIPTION OF THE INVENTION
1. OVERVIEW
[0012] The present invention is directed to the identification and
characterization of zB7R1, a novel inhibitory lymphocytic receptor,
and the discovery of its ability to bind to CD155 (PVR). Thus, the
present invention provides a newly identified B7 receptor that is a
PD-1-like molecule and is expressed in T lymphocytes. The novel
receptor of the present invention is denominated "zB7R1" and is
distinct from CD28, CTLA-4, ICOS, PD-1 and zB7R1. Methods and
compositions for modulating zB7R1-mediated lymphocyte signaling
such as, e.g., modulating the natural interaction of zB7R1 and its
counter-receptor are also provided, having multiple therapeutic
applications for immunological tolerance, autoimmunity,
immunosuppression, and immunotherapy including cancer
immunotherapy.
[0013] As disclosed for the first time herein, zB7R1 acts a
negative regulator of T lymphocyte activity, wherein signaling
mediated by zB7R1 results in the inhibition of zB7R1-positive
lymphocyte activity. In zB7R1-positive T cells zB7R1 signaling
could, for instance, inhibit TCR-induced T cell responses, such as
cell cycle progression, proliferation, differentiation, survival,
cytokine production and cytolytic activation. Further, in
zB7R1-positive B cells, zB7R1 signaling could an inhibit B cell
antigen receptor-induced B cell responses, such as cell cycle
progression, proliferation, differentiation, survival, antigen
presentation and antibody production. These findings enable the use
of therapeutic agents capable of interfering with the interaction
of zB7R1 and its counter-receptor to modulate lymphocyte activity
for the purpose of treating, among other conditions, cancer and
autoimmune diseases.
[0014] CD155 (PVR) was identified as the counterstructure for
ZB7r1. CD155 has been reported to be the counterstructure for at
least 2 other receptors including CD226 (DNAM-1) and CD96
(Tactile). CD226 and CD96 have been shown to be activating
receptors expressed on T cells and NK cells and CD155 can trigger
activation through these molecules. CD155 has been reported to be
widely expressed in non-hematopoietic tissues and may be
overexpressed in a large number of tumors and transformed cell
types. The role of CD155 on T cell responses to these tumors is
mostly CD155's engagement of zB7R1 which suppresses T and NK cell
responses to the tumor. Thus, a reagent that blocks zB7R1-CD155
interaction, including blocking antibodies to either molecule, or
soluble forms of either protein, will facilitate T and NK cell
responses to the tumor by eliminating or minimizing the inhibitory
signal through ZB7r1. Because of the demonstrated inhibitory effect
of engaging zB7R1 on T cells with agonistic antibodies as shown
herein, agonistic anti-ZB7r1 antibodies or soluble receptors are
suitable candidates to suppress T cell responses in T cell mediated
inflammatory and autoimmune diseases.
[0015] Accordingly, the present invention provides novel uses for
zB7R1 modulators, such as zB7R1 agonists or antagonists. These
modulators could be a soluble receptor or antibodies to zB7R1 or
its counter-receptor, i.e. CD155. The present invention also
provides soluble zB7R1 polypeptide fragments and fusion proteins,
for use in human inflammatory and autoimmune diseases. The zB7R1
antibodies, and soluble zB7R1 receptors of the present invention,
can be used to modulate, agonize, block, increase, inhibit, reduce,
antagonize or neutralize the activity of either zB7R1 or its
counter-receptor(s) (i.e. CD155) in the treatment of specific human
diseases such as cancer, rheumatoid arthritis, psoriasis, psoriatic
arthritis, arthritis, endotoxemia, inflammatory bowel disease
(IBD), colitis, and other inflammatory conditions disclosed
herein.
[0016] An illustrative nucleotide sequence that encodes human zB7R1
(also interchangeably known as zB7R1x1 is provided by SEQ ID NO:1;
the encoded polypeptide is shown in SEQ ID NO:2. zB7R1 is a B7
receptor that binds to yet another B7 family member, or
counter-receptor. Analysis of a human cDNA clone encoding zB7R1(SEQ
ID NO:1) revealed an open reading frame encoding 244 amino acids
(SEQ ID NO:2) comprising an extracellular domain of approximately
125 amino acid residues (residues 16-140 of SEQ ID NO:2; SEQ ID
NO:3), a transmembrane domain of approximately 23 amino acid
residues (residues 141-163 of SEQ ID NO:2), and an intracellular
domain of approximately 81 amino acid residues (residues 164 to 244
of SEQ ID NO:2). zB7R1 also has an IgV domain of approximately 96
amino acid residues (residues 32-127 of SEQ ID NO:2).
[0017] Within zB7R1, there are two ITIM domains, YFNV (amino acid
residues 225-228 of SEQ ID NO:2) and YRSL (amino acid residues
231-234). The presence of an ITIM domain is an indication that
zB7R1 can have an inhibitory effect. Within zB7R1, there are also
two SH-3-kinase binding domains, PSAP (amino acid residues 191-194
of SEQ ID NO:2) and PSPP (amino acid residues 194-197).
[0018] zB7R1 also has a polymorphism at polynucleotide 289 of SEQ
ID NO:1, indicated as n, where n can be either C or T. zB7R1 also
has at least a second polymorphism at polynucleotide 359 of SEQ ID
NO:1, indicated as n, where n can be either A or G, and where the
conversion of A to G leads to a change in the amino acid residue
117 of SEQ ID NO:2 (indicated as Xaa) from Thr to Ala.
[0019] An another illustrative nucleotide sequence that encodes a
variant human zB7R1 (also interchangeably known as zB7R1x2) is
provided by SEQ ID NO:5; the encoded polypeptide is shown in SEQ ID
NO:6. zB7R1x2 is a B7 receptor that binds to yet another B7 family
member, or counter-receptor. Analysis of a human cDNA clone
encoding zB7R1x2 (SEQ ID NO:5) revealed an open reading frame
encoding 311 amino acids (SEQ ID NO:6) comprising an extracellular
domain of approximately 182 amino acid residues (residues 27-208 of
SEQ ID NO:6; SEQ ID NO:7), a transmembrane domain of approximately
22 amino acid residues (residues 209-230 of SEQ ID NO:6), and an
intracellular domain of approximately 81 amino acid residues
(residues 231 to 311 of SEQ ID NO:6).
[0020] An illustrative nucleotide sequence that encodes a murine
zB7R1 is provided by SEQ ID NO:8; the encoded polypeptide is shown
in SEQ ID NO:9. The extracellular domain is shown in SEQ ID
NO:10.
[0021] An illustrative nucleotide sequence that encodes human CD155
(also interchangeably known as PVR) is provided by SEQ ID NO:17;
the encoded polypeptide is shown in SEQ ID NO:18. CD155 has been
shown to bind to zB7R1 and thus is a counter-receptor for this B7
family member. Analysis of a human cDNA clone encoding zB7R1 (SEQ
ID NO:17) revealed an open reading frame encoding 417 amino acids
(SEQ ID NO:18) comprising an extracellular domain of approximately
316 amino acid residues (residues 28-343 of SEQ ID NO:18; SEQ ID
NO:19), a transmembrane domain of approximately 24 amino acid
residues (residues 344-367 of SEQ ID NO:18), and an intracellular
domain of approximately 50 amino acid residues (residues 368-417 of
SEQ ID NO:18).
[0022] An illustrative nucleotide sequence that encodes a murine
CD155 is provided by SEQ ID NO:20; the encoded polypeptide is shown
in SEQ ID NO:21. The extracellular domain is shown in SEQ ID NO:22.
Analysis of a cDNA clone encoding murine CD155 revealed an open
reading frame encoding 408 amino acids (SEQ ID NO:21) comprising an
extracellular domain of approximately 319 amino acid residues
(residues 29-347 of SEQ ID NO:21; SEQ ID NO:22), a transmembrane
domain of approximately 20 amino acid residues (residues 348-367 of
SEQ ID NO:21), and an intracellular domain of approximately 40
amino acid residues (residues 368-408of SEQ ID NO:21)
[0023] Accordingly, in one aspect of the present invention, the
present invention provides nucleic acid sequences encoding zB7R1
polypeptides, which are useful in the modulation of T lymphocyte
activity and in the treatment of immune disorders, including
autoimmune diseases, inflammation, psoriasis, IBD, ulcerative
colitis and SE.
[0024] The present invention also provides isolated polypeptides
and epitopes comprising at least 15 contiguous amino acid residues
of an amino acid sequence of SEQ ID NO:2 or 3. Illustrative
polypeptides include polypeptides that either comprise, or consist
of SEQ ID NO:3, an antigenic epitope thereof, or a functional zB7R1
binding fragment thereof. Moreover, the present invention also
provides isolated polypeptides as disclosed above that agonize,
bind to, block, inhibit, reduce, increase, antagonize or neutralize
the activity of zB7R1.
[0025] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include agonist antibodies, neutralizing
antibodies, polyclonal antibodies, murine monoclonal antibodies,
humanized antibodies derived from murine monoclonal antibodies, and
human monoclonal antibodies. Illustrative antibody fragments
include F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv, scFv, and minimal
recognition units. Neutralizing antibodies preferably bind zB7R1
such that its interaction with its counter-receptor or
counter-receptors is blocked, inhibited, reduced, antagonized or
neutralized; anti-zB7R1 neutralizing antibodies such that its
interaction with its counter-receptor or counter-receptors is
blocked, inhibited, reduced, antagonized or neutralized are also
encompassed by the present invention. The present invention further
includes compositions comprising a carrier and a peptide,
polypeptide, or antibody described herein.
[0026] Thus, in one embodiment, antagonists of zB7R1 signaling are
provided for increasing T cell activation, and possibly B cell
activation. In a preferred embodiment, such antagonists comprise
blocking agents capable of interfering with the natural interaction
of zB7R1 with its counter-receptor or counter-receptors, thereby
inhibiting zB7R1-mediated negative signaling and resulting in an
increase in lymphocyte activation and proliferation and effector
function.
[0027] In an alternative embodiment, agonists of zB7R1 signaling
are provided for inhibiting T cell activation, and possibly B cell
activation. In a preferred embodiment, such bioactive agents
comprise mimicking agents capable of binding to zB7R1 and mimicking
and/or augmenting the natural interaction of zB7R1 with its
counter-receptor or counter-receptors, thereby resulting in
inhibition of T cell activation (and possibly B cell) and
proliferation and effector function.
[0028] In one embodiment, bioactive agents and methods for
increasing and/or up-regulating B and T cell activity are provided.
In a preferred embodiment, such bioactive agents comprise
antagonists of zB7R1-mediated signaling. In a particularly
preferred embodiment, such bioactive agents comprise blocking
agents as described herein, and in a specific embodiment, such
blocking agents are capable of interfering with the interaction of
zB7R1 and Its counter-receptor. In a further embodiment, adjuvant
compositions are provided utilizing zB7R1 and/or Its
counter-receptor blocking agents and other antagonists of
zB7R1-mediated signaling.
[0029] In an alternative embodiment, bioactive agents and methods
for inhibiting and/or down-regulating B and T cell activity are
provided. In a preferred embodiment, such bioactive agents comprise
agonists of zB7R1-mediated signaling. In a particularly preferred
embodiment, such bioactive agents comprise mimicking agents as
described herein, and in a specific embodiment, such mimicking
agents are capable of replacing and/or augmenting the interaction
of zB7R1 and Its counter-receptor. In a further embodiment,
immunsuppressive compositions are provided utilizing zB7R1 and/or
Its counter-receptor mimicking agents and other agonists of
zB7R1-mediated signaling.
[0030] In a further embodiment, methods and compositions for
modulating immunoglobulin production by B cells is provided.
[0031] The methods and compositions described herein will find
advantageous use in immunotherapy, including, e.g., autoimmunity,
immune suppression, cancer immunotherapy and immune adjuvants.
[0032] In addition, the present invention also provides
pharmaceutical compositions comprising a pharmaceutically
acceptable carrier and at least one of such an expression vector or
recombinant virus comprising such expression vectors. The present
invention further includes pharmaceutical compositions, comprising
a pharmaceutically acceptable carrier and a polypeptide or antibody
described herein.
[0033] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 6
or a fragment thereof. An exemplary anti-idiotype antibody binds
with an antibody that specifically binds a polypeptide consisting
of SEQ ID NO:3 or 7.
[0034] The present invention also provides fusion proteins,
comprising a zB7R1 polypeptide and an immunoglobulin moiety. In
such fusion proteins, the immunoglobulin moiety may be an
immunoglobulin heavy chain constant region, such as a human F.sub.c
fragment. The present invention further includes isolated nucleic
acid molecules that encode such fusion proteins.
[0035] The present invention relates to a multmeric zB7R1 protein,
as well as a method of preparing such a multimeric protein,
preferably a tetrameric protein, comprising culturing a host cell
transformed or transfected with an expression vector encoding a
fusion protein comprising a vasodialator-stimulated phosphoprotein
(VASP) domain and a heterologous protein, such as zB7R1 or CD155.
Specifically, the portion of zB7R1 or CD155 that is included in the
fusion protein is the extracellular domain of that protein (i.e.
SEQ ID NO:3 or 7 for zB7R1, or SEQ ID NO:22 for CD155), and the
resulting fusion protein is soluble. In a further embodiment, the
fusion protein comprises a linker sequence. In still another
embodiment of the present invention, the VASP domain can be used to
identify sequences having similar protein structure patterns and
those similar domains are used to make a fusion protein that
multimerizes a heterologous protein or protein domain.
[0036] A further embodiment of the present invention is a method of
preparing a soluble, homo- or hetero-tetrameric zB7R1 or CD155
protein by culturing a host cell transformed or transfected with at
least one, but up to four different expression vectors encoding a
fusion protein comprising a VASP domain and a heterologous protein
such as zB7R1 or CD155 or protein domain thereof. In this
embodiment, the four VASP domains preferentially form a homo- or
hetero-tetramer. This culturing can occur in the same or different
host cells. The VASP domains can be the same or different and the
fusion protein can further comprise a linker sequence. The present
invention also encompasses DNA sequences, expression vectors, and
transformed host cells utilized in the present method and fusion
proteins produced by the present method.
[0037] The present invention also provides polyclonal and
monoclonal antibodies that bind to polypeptides comprising a zB7R1
extracellular domain such as monomeric, homodimeric, heterodimeric
and multimeric receptors, including soluble receptors.
[0038] In another aspect, methods for modulating lymphocyte
activity are provided comprising contacting a B and/or T lymphocyte
with a bioactive agent capable of modulating zB7R1 activity. In one
embodiment, the bioactive agent comprises an antagonist of zB7R1
activity such as, e.g., a zB7R1 or a zB7R1 counter-receptor
blocking agent (i.e. CD155) resulting in an upregulation or
increase in lymphocyte activity by preventing negative
zB7R1-mediated signaling. In an alternative embodiment, the
bioactive agent comprises an agonist of zB7R1 activity such as,
e.g., a zB7R1 or a zB7R1 counter-receptor mimicking agent,
resulting in down-regulation of lymphocyte activity by replacing or
augmenting zB7R1-mediated negative signaling.
[0039] In a further aspect, methods for modulating lymphocyte
activity are provided comprising contacting a B and/or T lymphocyte
with a bioactive agent capable of modulating the interaction of
zB7R1 with a zB7R1 counter-receptor. In one embodiment, a bioactive
agent capable of interfering with the natural interaction of zB7R1
and a zB7R1 counter-receptor (i.e. CD155) is employed to increase
lymphocyte activity and proliferation such as, e.g., a zB7R1
antagonist such as a soluble zB7R1 counter-receptor or a zB7R1
blocking agent. In an alternative embodiment, a bioactive agent
capable augmenting or replacing the natural interaction of zB7R1
and a zB7R1 counter-receptor (i.e. CD155) is employed to inhibit
lymphocyte activity and proliferation.
[0040] Suitable zB7R1 blocking agents may be selected from the
group comprising or consisting of soluble zB7R1 polypeptides and
fusion proteins, anti-zB7R1 antibodies capable of binding to at
least a portion of the extracellular domain of zB7R1 and
interfering with zB7R1-mediated signaling, small molecule
inhibitors of zB7R1 receptor interaction with its ligands, and the
like. Alternative zB7R1 antagonists further include antisense
oligonucleotides directed to the zB7R1 nucleic acid sequence,
inhibitory RNA sequences, small molecule inhibitors of zB7R1
expression and/or intracellular signaling, and the like.
[0041] Similarly, suitable zB7R1 counter-receptor blocking agents
or antagonists may be selected from the group comprising or
consisting of anti-zB7R1 counter-receptor antibodies capable of
binding to at least a portion of the extracellular domain of a
zB7R1 counter-receptor (i.e. CD155; SEQ ID NO:22) and interfering
with the interaction of a zB7R1 counter-receptor and zB7R1, small
molecule inhibitors of the interaction between a zB7R1
counter-receptor and zB7R1, soluble a zB7R1 counter-receptor
polypeptides and fusion proteins having modified a zB7R1
counter-receptor amino acid sequences so as to interfere with the
interaction of a zB7R1 counter-receptor and zB7R1 and incapable of
activating zB7R1-mediated signaling, and the like. Alternative a
zB7R1 counter-receptor antagonists include antisense
olignucleotides directed to the zB7R1 counter-receptor nucleic acid
sequence (i.e. CD155; SEQ ID NO:20), inhibitory RNA molecules,
small molecule inhibitors of a zB7R1 counter-receptor expression,
and the like.
[0042] Suitable zB7R1 mimicking agents or agonists may be selected
from the group comprising or consisting of function-activating
anti-zB7R1 antibodies ("agonistic antibodies") capable of binding
to at least a portion of the extracellular domain of zB7R1 (SEQ ID
NO:3 or 7) and stimulating zB7R1-mediated signaling, gene therapy
vectors capable of recombinantly producing functional zB7R1
molecules intracellularly, small molecule enhancers of zB7R1
expression and/or zB7R1-mediated signaling, and the like.
Similarly, suitable a zB7R1 counter-receptor mimicking agents may
be selected from the group comprising or consisting of soluble a
zB7R1 counter-receptor polypeptides, such as CD155, and fusion
proteins capable of activating zB7R1-mediated signaling, small
molecule enhancers of the interaction between a zB7R1
counter-receptor and zB7R1 as well as enhancers of a zB7R1
counter-receptor expression, gene therapy vectors capable of
recombinantly producing functional a zB7R1 counter-receptor
molecules intracellularly, and the like.
[0043] Thus, in a more specific embodiment methods for stimulating,
augmenting and/or increasing lymphocyte activity are provided
comprising contacting a B or T lymphocyte with an antagonist of
zB7R1-mediated signaling, said antagonist comprising at least one
bioactive agent selected from the group consisting of soluble zB7R1
polypeptides, soluble zB7R1 fusion proteins, anti-zB7R1 antibodies
capable of binding to at least a portion of the extracellular
domain of zB7R1 and interfering with zB7R1-mediated signaling,
small molecule inhibitors of zB7R1 expression and/or zB7R1-mediated
signaling, anti-zB7R1 counter-receptor antibodies capable of
binding to at least a portion of the extracellular domain of a
zB7R1 counter-receptor and interfering with the interaction of a
zB7R1 counter-receptor and zB7R1, small molecule inhibitors of the
interaction between a zB7R1 counter-receptor and zB7R1, soluble a
zB7R1 counter-receptor polypeptides and a zB7R1 counter-receptor
fusion proteins incapable of activating zB7R1-mediated signaling,
and interfering RNA sequences.
[0044] In a particularly preferred embodiment, methods for
increasing a host immune response to antigenic stimulation are
provided, comprising the administration to the host of at least one
of the aforementioned antagonists of zB7R1-mediated signaling.
Desirably, the antigenic stimulation may be from pathogen antigens,
vaccine antigens and/or tumor antigens.
[0045] In a specific embodiment, methods for stimulating a cellular
immune response against tumor antigens other than a zB7R1
counter-receptor are provided, comprising administering to a cancer
patient at least one of the subject antagonists or blocking agents
to inhibit zB7R1-mediated negative signaling and thereby increase
the T cell response directed against tumor antigens other than a
zB7R1 counter-receptor present in the cancerous tissue.
[0046] In a further specific embodiment methods for inhibiting,
attenuating and/or decreasing lymphocyte activity are provided
comprising contacting a B or T lymphocyte with an agonist of
zB7R1-mediated signaling, said agonist selected from the group
consisting of soluble a zB7R1 counter-receptor polypeptides and a
zB7R1 counter-receptor fusion proteins capable of activating
zB7R1-mediated signaling, function-activating anti-zB7R1 antibodies
capable of binding to at least a portion of the extracellular
domain of zB7R1 and stimulating zB7R1-mediated signaling, gene
therapy vectors capable of recombinantly producing functional zB7R1
molecules intracellularly, small molecule enhancers of zB7R1
expression and/or zB7R1-mediated signaling, small molecule
enhancers of the interaction between a zB7R1 counter-receptor and
zB7R1, small molecule enhancers of a zB7R1 counter-receptor
expression, and gene therapy vectors capable of recombinantly
producing functional a zB7R1 counter-receptor molecules
intracellularly.
[0047] In a particularly preferred embodiment, methods for
suppressing a host immune response to antigenic stimulation are
provided, comprising the administration to the host of at least one
of the aforementioned agonists of zB7R1-mediated signaling.
Desirably, the antigenic stimulation may be from self antigens in
the context of autoimmune disease, or from donor antigens present
in transplanted organs and tissues.
[0048] In an alternative aspect, the present invention provides
bioactive agents and methods for modulating the interaction of a a
zB7R1 counter-receptor-expressing cell and a zB7R1-expressing
lymphocyte. In a preferred embodiment, bioactive agents and methods
for interfering with the interaction of a zB7R1 counter-receptor
-positive tumor cells with T cells are provided, resulting in
inhibition of negative zB7R1-mediated signaling. In an especially
preferred embodiment, the T cell is a CD4+ cell or a CD8+ cell. In
a further embodiment, the CD4+ T cell is a Th1 cell.
[0049] In another preferred embodiment, bioactive agents and
methods for mimicking or enhancing the interaction of a zB7R1
counter-recepotr/CD155-positive non-tumor non-lymphoid cells with
zB7R1-positive T cells are provided, thereby decreasing T cell
activity. In an especially preferred embodiment, the T cell is a
CD4+ T cell or a CD8+ T cell. In a further embodiment, the CD4+ T
cell is a Th1 cell.
[0050] In a further aspect, methods for treating cancers
characterized by the presence of a zB7R1
counter-receptor-expressing tumor cells are provided. In one
embodiment, these methods comprise administering to a mammalian
subject at least one of the antagonists of zB7R1-mediated signaling
disclosed herein, either alone or in conjunction with alternative
cancer immunotherapy, chemotherapy and/or radiotherapy protocols.
In a preferred embodiment, at least one zB7R1 antagonist or CD155
antagonist is administered to a subject having a zB7R1
counter-receptor -positive tumor cells, wherein said blocking agent
is capable of interfering with the interaction of zB7R1 and a zB7R1
counter-receptor and inhibiting zB7R1-mediated signaling.
Preferably, administration of said blocking agents is effective to
increase T cell activity directed against tumor antigens other than
a zB7R1 counter-receptor on the tumor cells, and in particular, to
increase cytotoxic T cell activity. Still more preferably,
administration of the subject antagonists is effective to inhibit
the growth of the a zB7R1 counter-receptor -expressing tumor
cells.
[0051] It is also contemplated that the subject zB7R1 and/or a
zB7R1 counter-receptor/CD155 blockade provided herein may find
synergistic combination with CTLA-4 blockade as described in U.S.
Pat. Nos. 5,855,887; 5,811,097;and 6,051,227, and International
Publication WO 00/32231, the disclosures of which are expressly
incorporated herein by reference.
[0052] In a further aspect, methods for treating autoimmune
disorders characterized by the absent or aberrant expression of a
zB7R1 counter-receptor in non-tumor non-lymphoid host cells
subjected to autoimmune attack are provided. In one embodiment,
these methods comprise administering to a mammalian subject at
least one of the agonists of zB7R1-mediated signaling disclosed
herein, either alone or in conjunction with alternative
immunotherapy and/or immunosuppressive protocols. In a preferred
embodiment, at least one zB7R1 or CD15 agonist is administered to a
subject having autoreactive zB7R1-positive lymphocytes, wherein
said agonist is capable of replacing and/or augmenting the
interaction of zB7R1 and CD155 and replacing or increasing
zB7R1-mediated signaling. Preferably, administration of said
agonists is effective in decreasing autoreactive lymphocyte
activity directed against non-tumor non-lymphoid host cells, and
particularly autoreactive CD8+ CTL and CD4+ Th1 activity, and B
cell activity.
[0053] In a still further aspect, methods for improving the outcome
of organ and tissue transplantation and prolonging graft survival
are provided. In one embodiment, these methods comprise
administering to a transplant recipient at least one agent of the
agonists or antagonists of zB7R1-mediated signaling disclosed
herein, either alone or in conjunction with alternative
immunotherapy and/or immunosuppressive protocols. In a preferred
embodiment, at least one zB7R1 mimicking agent (for instance a
soluble receptor that blocks binding a cell-surface zB7R1 to its
counter-receptor, or an angonist antibody that binds to zB7R1 and
induces signaling) is administered to the transplant recipient,
wherein said mimicking agent is capable of replacing and/or
augmenting the interaction of zB7R1 and a zB7R1 counter-receptor
and replacing or increasing zB7R1-mediated signaling. Preferably,
administration of said mimicking agents is effective to decrease
the recipient immune response against donor antigens present in the
graft, particularly the cytolytic CTL response and the B cell
response. Still more preferably, administration of the subject
mimicking agents is effective to bias to T helper cell response
from an unfavorable Th-1 type response to a more favorable Th-2
type response, as described in more detail herein.
Treatment of Autoimune Disease
[0054] The present invention also provides compositions and methods
for inhibiting autoimmune responses. In a preferred embodiment,
compositions and methods for inhibiting the activity of
autoreactive T and B cells that specifically recognize autoantigens
are provided. Desirably, these compositions and methods may be used
to inhibit killing of non-tumor cells mediated by one or more
autoantigens.
[0055] Preferred compositions for use in the treatment of
autoimmune disease comprise agents that mediate zB7R1 signaling
described herein including, e.g., the above-described mimicking
agents, agonists or antagonists. Especially preferred agents
include zB7R1 protein fragments comprising the zB7R1 extracellular
domain (SEQ ID NO:3 or 7), or a portion thereof; zB7R1-Ig fusion
proteins comprising the zB7R1 extracellular domain (SEQ ID NO:3),
or a portion thereof; function-activating anti-zB7R1 or CD155
antibodies; peptides that mimic zB7R1 or its counter-receptor,
CD155 (mimetics); and small molecule chemical compositions that
mimic the natural interaction of zB7R1 with its counter-receptor.
Also preferred are compositions capable of binding to zB7R1, either
in a cross-linking fashion or as polyclonal mixtures.
[0056] Also contemplated in the present invention are genetic
approaches to autoimmune disease. Particularly, gene therapy may be
used to increase the level of zB7R1 expression on T cells, and/or
increase the level of expression of its counter-receptor on
non-lymphoid cells that are subject to attack by autoreactive
lymphocytes. The use of isoforms or variants of zB7R1 that exhibit
elevated specific activity is also contemplated, the object of each
method being to potentiate signaling that is suppressive to T cell
activation.
[0057] The present invention also provides compositions and methods
for treating cancer, and in particular, for increasing the activity
of zB7R1-positive lymphocytes against B7-positive tumor cells.
Desirably, these compositions and methods may be used to inhibit
the growth of tumor cells capable of expressing a B7 family
member.
[0058] Preferred compositions for use in the treatment of cancer
are the antagonists of zB7R1-mediated signaling described herein
including, e.g., zB7R1 blocking agents. Especially preferred agents
include anti-zB7R1 antibodies; protein fragments comprising the
zB7R1 extracellular domain, or a portion thereof; zB7R1-Ig fusion
proteins comprising the BTLA extracellular domain, or a portion
thereof; function-blocking anti-zB7R1 antibody; peptides that mimic
zB7R1 (mimetics); and small molecule chemical compositions that
interfere with the natural interaction of zB7R1 and its
counter-receptor.
[0059] Also contemplated in the present invention are genetic
approaches to the treatment of cancer. Particularly, gene therapy
may be used to decrease the level of zB7R1 expression on T cells,
and/or decrease the level of expression of zB7R1 or its
counter-receptor (i.e. CD155) on tumor cells. The use of isoforms
of zB7R1 that exhibit dominant negative activity is also
contemplated, the object of each method being to inhibit signaling
that is normally suppressive to T cell activation. Genetic
approaches may involve the use of tissue and cell specific
promoters to target expression of zB7R1 dominant negative variants,
antisense nucleic acids, or small inhibitory RNAs to T cells and
tumor cells, respectively. The methods may additionally involve the
use of tumor-targeted viruses, or other delivery vehicles that
specifically recognize tumor cells. The methods may additionally
involve the use of T cell-targeted viruses, or other delivery
vehicles that specifically recognize T cells.
[0060] Particularly preferred are agents that may be selectively
targeted to tumor cells, and effect a decrease in zB7R1 expression
in tumor cells without reducing the level of zB7R1 expression in
non-tumor cells to deleterious levels. Highly preferred are agents
that have a precursor form. These "prodrugs" are converted to their
active form in the vicinity of tumor tissue typically by an
enzymatic activity that is restricted in its distribution to the
vicinity of the tumor.
[0061] Also highly preferred are agents that can be combined with
targeting moieties that selectively deliver the agent to a tumor.
These targeting moieties provide a high local concentration of the
agent in the vicinity of the tumor tissue, and reduce the amount of
agent that must be administered to effect the desired response.
[0062] Also contemplated in the present invention is the use of
combination therapy to treat cancer, as described above.
[0063] In a preferred embodiment, immunization is done to promote a
tumor-specific T cell immune response. In this embodiment, a
bioactive agent that inhibits zB7R1 activation is administered in
combination with a tumor-associated antigen. The combination of a
tumor-associated antigen and a zB7R1-inhibitory/counter-receptor
functional-mimetic promotes a tumor specific T cell response, in
which T cells encounter a lower level of inhibition than exerted by
the tumor tissue in the absence of the bioactive agent.
[0064] In one aspect, the present invention provides a medicament
for the treatment of cancer.
[0065] The present invention also provides compositions and methods
for modulating normal but undesired immune responses involving T
and B cell activity. In a preferred embodiment, compositions and
methods for inhibiting the host lymphocyte response to transplanted
tissue and organs are provided. Desirably, these compositions and
methods may be used to prolong the survival of grafted tissue.
Preferred compositions for use in the prevention of acute and/or
chronic graft rejection comprise the agonists of zB7R1-mediated
signaling described herein including, e.g., the above-described
mimicking agents. Especially preferred agents include zB7R1
polypeptides comprising the zB7R1 extracellular domain (SEQ ID NO:3
or 7), or a portion thereof; zB7R1-Ig fusion proteins comprising
the zB7R1 extracellular domain (SEQ ID NO:3 or 7), or a portion
thereof; function-activating anti-BTLA antibodies; peptides that
mimic its counter-receptor (i.e. CD155) (mimetics); and small
molecule chemical compositions that mimic the natural interaction
of zB7R1 and its counter-receptor. In addition to their utility in
general immunosuppressive strategies, the subject agonists of
zB7R1-mediated signaling described herein may also have important
implications for tolerance induction in tissue and organ
transplantation, by biasing the recipient T helper cell immune
response away from an unfavorable Th-1-type response and towards a
more favorable Th-2 type response.
[0066] In one aspect, the present invention provides a medicament
for use in transplantation and immune suppression.
[0067] Also provided are adjuvant compositions comprising at least
one of the above-described zB7R1 and/or CD155 or other zB7R1
counter-receptor blocking agents as well as other antagonists of
zB7R1-mediated signaling. Also provided are immunosuppressant
compositions comprising at least one of the above-described zB7R1
and/or a zB7R1 counter-receptor mimicking agents as well as other
agonists of zB7R1-mediated signaling.
[0068] It is further contemplated that the subject compositions and
methods may be synergistically combined with immunotherapies based
on modulation of other T cell costimulatory pathways, and with
ICOS, PD-1, CTLA-4 and/or BTLA modulation in particular.
[0069] In an alternative aspect, the present invention provides
methods of screening for bioactive agents that are useful for
modulating T cell activation. Bioactive agents identified by the
screening methods provided herein may be used to react with a zB7R1
counter-receptor-expressing cells or zB7R1-expressing cells in
order to interfere with the interaction between zB7R1-expressing B
and/or T cells and a zB7R1 counter-receptor-expressing non-lymphoid
cells, and thereby antagonize the function of the zB7R1/a zB7R1
counter-receptor interaction. Alternatively, bioactive agents may
be used to react with a zB7R1 counter-receptor-expressing cells or
zB7R1-expressing cells in order to mimic the a zB7R1
counter-receptor/zB7R1 interaction, effecting T cell inhibition in
the absence of a zB7R1/zB7R1 counter-receptor interaction.
Alternatively, bioactive agents may be used to modify the natural
zB7R1/CD155 (or zB7R1 with another zB7R1 counter-receptor)
interaction in some way, for example, to increase the association
and augment the inhibitory signal.
[0070] In an alternative aspect, the invention provides expression
vectors comprising the isolated zB7R1 and/or a zB7R1
counter-receptor nucleic acid sequences disclosed herein (i.e.
CD155; SEQ ID NO:20), recombinant host cells comprising the
recombinant nucleic acid molecules disclosed herein, and methods
for producing zB7R1 and/or zB7R1 counter-receptor polypeptides
comprising culturing the host cells and optionally isolating the
polypeptide produced thereby.
[0071] In a further aspect, transgenic non-human mammals are
provided comprising a nucleic acid encoding a zB7R1, a CD155 and/or
another zB7R1 counter-receptor protein as disclosed herein. The
zB7R1, CD155 or other zB7R1 counter-receptor nucleotides are
introduced into the animal in a manner that allows for increased
expression of levels of a zB7R1 or a zB7R1 counter-receptor
polypeptide, which may include increased circulating levels.
Alternatively, the zB7R1, Cd155 or a zB7R1 counter-receptor nucleic
acid fragments may be used to target endogenous zB7R1, CD155 or a
zB7R1 counter-receptor alleles in order to prevent expression of
endogenous zB7R1 or a zB7R1 counter-receptor nucleic acids (i.e.
generates a transgenic animal possessing a zB7R1 or a zB7R1
counter-receptor protein gene knockout). The transgenic animal is
preferably a mammal, and more preferably a rodent, such as a rat or
a mouse.
[0072] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
2. DEFINITIONS
[0073] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0074] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0075] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence.
[0076] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid molecule that encodes a
polypeptide. Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0077] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0078] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0079] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0080] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0081] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0082] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0083] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0084] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0085] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0086] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0087] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0088] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0089] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0090] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0091] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0092] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces zB7R1 from an expression vector. In contrast,
zB7R1 can be produced by a cell that is a "natural source" of zB7R1
, and that lacks an expression vector.
[0093] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0094] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a fusion protein can comprise at least part
of a zB7R1 polypeptide fused with a polypeptide that binds an
affinity matrix. Such a fusion protein provides a means to isolate
large quantities of zB7R1 using affinity chromatography.
[0095] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "counter-receptor." This
interaction mediates the effect of the counter-receptor on the
cell. Receptors can be membrane bound, cytosolic or nuclear;
monomeric (e.g., thyroid stimulating hormone receptor,
beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,
growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF
receptor, erythropoietin receptor and IL-6 receptor).
Membrane-bound receptors are characterized by a multi-domain
structure comprising an extracellular counter-receptor-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular counter-receptor-binding domain and
the intracellular effector domain are located in separate
polypeptides that comprise the complete functional receptor.
[0096] In general, the binding of counter-receptor to receptor
results in a conformational change in the receptor that causes an
interaction between the effector domain and other molecule(s) in
the cell, which in turn leads to an alteration in the metabolism of
the cell. Metabolic events that are often linked to
receptor-counter-receptor interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids.
[0097] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
counter-receptor-binding polypeptides that lack transmembrane and
cytoplasmic domains, and other linkage to the cell membrane such as
via glycophosphoinositol (gpi). Soluble receptors can comprise
additional amino acid residues, such as affinity tags that provide
for purification of the polypeptide or provide sites for attachment
of the polypeptide to a substrate, or immunoglobulin constant
region sequences. Many cell-surface receptors have naturally
occurring, soluble counterparts that are produced by proteolysis or
translated from alternatively spliced mRNAs. Soluble receptors can
be monomeric, homodimeric, heterodimeric, or multimeric, with
multimeric receptors generally not comprising more than 9 subunits,
preferably not comprising more than 6 subunits, and most preferably
not comprising more than 3 subunits. Receptor polypeptides are said
to be substantially free of transmembrane and intracellular
polypeptide segments when they lack sufficient portions of these
segments to provide membrane anchoring or signal transduction,
respectively. For example, representative soluble receptors for
zB7R1 include, for instance the soluble receptor as shown in SEQ ID
NO:3 or 7. It is well within the level of one of skill in the art
to delineate what sequences of a known B7 family member comprise
the extracellular domain free of a transmsmbrane domain and
intracellular domain. Moreover, one of skill in the art using the
genetic code can readily determine polynucleotides that encode such
soluble receptor polyptides.
[0098] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0099] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, such as 96%, 97%, or
98% or more pure, or greater than 99% pure. One way to show that a
particular protein preparation contains an isolated polypeptide is
by the appearance of a single band following sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis of the protein preparation
and Coomassie Brilliant Blue staining of the gel. However, the term
"isolated" does not exclude the presence of the same polypeptide in
alternative physical forms, such as dimers or alternatively
glycosylated or derivatized forms.
[0100] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0101] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0102] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0103] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and the like, and synthetic
analogs of these molecules.
[0104] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/counter-receptor
pairs, antibody/antigen (or hapten or epitope) pairs,
sense/antisense polynucleotide pairs, and the like. Where
subsequent dissociation of the complement/anti-complement pair is
desirable, the complement/anti-complement pair preferably has a
binding affinity of less than 10.sup.9 M.sup.-1.
[0105] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-zB7R1 antibody, and thus, an anti-idiotype antibody
mimics an epitope of zB7R1.
[0106] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-zB7R1
monoclonal antibody fragment binds with an epitope of zB7R1.
[0107] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0108] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0109] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain. Construction of
humanized antibodies for therapeutic use in humans that are derived
from murine antibodies, such as those that bind to or neutralize a
human protein, is within the skill of one in the art.
[0110] As used herein, a "therapeutic agent" is a molecule or atom
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0111] A "detectable label" is a molecule or atom which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0112] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith
and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P,
FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95 (1991). DNA molecules encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0113] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0114] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0115] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0116] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
zB7R1 polypeptide component. Examples of an antibody fusion protein
include a protein that comprises a zB7R1 extracellular domain, and
either an Fc domain or an antigen-binding region.
[0117] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0118] An "antigenic peptide" is a peptide which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0119] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0120] An "anti-sense oligonucleotide specific for zB7R1" or a
"zB7R1 anti-sense oligonucleotide" is an oligonucleotide having a
sequence (a) capable of forming a stable triplex with a portion of
the zB7R1 gene, or (b) capable of forming a stable duplex with a
portion of an mRNA transcript of the zB7R1 gene.
[0121] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0122] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0123] The term "variant zB7R1 gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:2 (i.e. SEQ ID NO:6). Such
variants include naturally-occurring polymorphisms of zB7R1 genes,
as well as synthetic genes that contain conservative amino acid
substitutions of the amino acid sequence of SEQ ID NO:2. Additional
variant forms of zB7R1 genes are nucleic acid molecules that
contain insertions or deletions of the nucleotide sequences
described herein. A variant zB7R1 gene can be identified, for
example, by determining whether the gene hybridizes with a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:1, or its
complement, under stringent conditions.
[0124] Alternatively, variant zB7R1 genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wisc.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology: Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0125] Regardless of the particular method used to identify a
variant zB7R1 gene or variant zB7R1 polypeptide, a variant gene or
polypeptide encoded by a variant gene may be functionally
characterized the ability to bind specifically to an anti-zB7R1
antibody. A variant zB7R1 gene or variant zB7R1 polypeptide may
also be functionally characterized the ability to bind to its
counter-receptor or counter-receptors, using a biological or
biochemical assay described herein.
[0126] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0127] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0128] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0129] As used herein, the term "immune response" includes both T
and/or B cell responses, i.e., cellular and/or humoral immune
responses. In one embodiment, the compositions and methods
disclosed herein can be used to reduce or enhance helper T cell
(Th) responses, and more preferably, Th1 cell responses. In another
embodiment, the compositions and methods disclosed herein can be
used to reduce or, enhance cytotoxic T cell (Tc) responses. The
claimed methods can be used to reduce or enhance both primary and
secondary immune responses and effector function (e.g., cytolytic
activity, cytokine and antibody production, and antigen
presentation). The immune response of a subject can be readily
determined by the skilled artisan using methods well known in the
art, for example, by assaying for antibody production, immune cell
proliferation, the release of cytokines, the expression of cell
surface markers, cytotoxicity, etc.
[0130] By "zB7R1 signaling", "zB7R1-mediated signaling",
"zB7R1-mediated negative signaling" and variations thereof is meant
intracellular signaling in lymphocytes caused by the binding and/or
activation of the zB7R1 receptor by its corresponding ligand(s)
resulting in attenuation and/or down-regulation of lymphocyte
activity. In one aspect, zB7R1-mediated signaling comprises
activation of SHP-1 and/or SHP-2.
[0131] "Lymphocyte activity" as used herein refers to the
immunological processes of B and T cell activation, proliferation,
differentiation and survival, as well as associated effector immune
functions in lymphocytic cells including cytolytic activity (Tc
cells), cytokine production (Th cells), antibody production (B
cells), and antigen presentation (B cells). As noted above, there
are numerous assays well known to the skilled artisan for detecting
and/or monitoring such processes, including but not limited to the
assays described in the examples provided herein.
[0132] As used herein, the phrase "interaction of zB7R1 and its
counter-receptor" or "interaction of zB7R1 and CD155) refers to
direct physical interaction (e.g. binding) and/or other indirect
interaction of a functional zB7R1 counter-receptor (i.e. CD155)
molecule with a functional zB7R1 receptor on a lymphocyte,
resulting in stimulation of the zB7R1 receptor and associated
intracellular zB7R1 signaling. Similarly, the phrase "natural
interaction of zB7R1 and its counter-receptor" refers to direct
physical interaction (e.g. binding) and/or other indirect
interaction of a functional and endogenously expressed
counter-receptor such as CD155, with a functional and endogenously
expressed zB7R1 receptor on a lymphocyte, resulting in stimulation
of the zB7R1 receptor and associated intracellular zB7R1
signaling.
[0133] As used herein, the term "blocking agent" includes those
agents that interfere with the interaction of zB7R1 and its
counter-receptor, and/or that interfere with the ability of the
counter-receptor to inhibit lymphocyte activity, e.g., as measured
by cytokine production and/or proliferation. The term "blocking
agent" further includes agents that inhibit the ability of zB7R1 to
bind a natural ligand, and/or that interfere with the ability of
zB7R1 to inhibit T cell activity. Exemplary agents include
function-blocking antibodies, as well as peptides that block the
binding zB7R1 with its counter-receptor but which fail to stimulate
zB7R1-mediated signaling in a lymphocyte (e.g., zB7R1 fusion
proteins), peptidomimetics, small molecules, and the like.
Preferred blocking agents include agents capable of inhibiting the
inducible association of zB7R1 with SHP-1 and/or SHP-2, or the
signal transduction that derives from the interaction of SHP-1
and/or SHP-2 with zB7R1.
[0134] As used herein, the term "mimicking agent" includes those
agents that mimick the interaction of zB7R1 and its
counter-receptor, and/or that augment, enhance or increase the
ability of zB7R1 and/or its counter-receptor to inhibit lymphocyte
activity. Exemplary agents include function-activating antibodies,
as well as peptides that augment or enhance the ability of zB7R1 to
bind with its counter-receptor or substitute for the
counter-receptor's role in stimulating zB7R1-mediated signaling
(e.g., Its counter-receptor fusion proteins), peptidomimetics,
small molecules, and the like.
[0135] The present invention includes functional fragments of zB7R1
genes. Within the context of this invention, a "functional
fragment" of a zB7R1 gene refers to a nucleic acid molecule that
encodes a portion of a zB7R1 polypeptide which is a domain
described herein or at least specifically binds with an anti-zB7R1
antibody.
[0136] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
3. PRODUCTION OF zB7R1 POLYNUCLEOTIDES OR GENES
[0137] Nucleic acid molecules encoding a human zB7R1 gene can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon SEQ ID NO:1 or 5. These techniques
are standard and well-established, and may be accomplished using
cloning kits available by commercial suppliers. See, for example,
Ausubel et al. (eds.), Short Protocols in Molecular Biology,
3.sup.rd Edition, John Wiley & Sons 1995; Wu et al., Methods in
Gene Biotechnology, CRC Press, Inc. 1997; Aviv and Leder, Proc.
Nat'l Acad. Sci. USA 69:1408 (1972); Huynh et al., "Constructing
and Screening cDNA Libraries in .lamda.gt10 and .lamda.gt11," in
DNA Cloning: A Practical Approach Vol. 1, Glover (ed.), page 49
(IRL Press, 1985); Wu (1997) at pages 47-52.
[0138] Nucleic acid molecules that encode a human zB7R1 gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the zB7R1 gene or cDNA. General
methods for screening libraries with PCR are provided by, for
example, Yu et al., "Use of the Polymerase Chain Reaction to Screen
Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR
Protocols: Current Methods and Applications, White (ed.), Humana
Press, Inc., 1993. Moreover, techniques for using PCR to isolate
related genes are described by, for example, Preston, "Use of
Degenerate Oligonucleotide Primers and the Polymerase Chain
Reaction to Clone Gene Family Members," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), Humana Press, Inc. 1993. As an alternative, a zB7R1
gene can be obtained by synthesizing nucleic acid molecules using
mutually priming long oligonucleotides and the nucleotide sequences
described herein (see, for example, Ausubel (1995)). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)). For reviews on polynucleotide synthesis, see, for
example, Glick and Pasternak, Molecular Biotechnology, Principles
and Applications of Recombinant DNA (ASM Press 1994), Itakura et
al., Annu. Rev. Biochem. 53:323 (1984), and Climie et al., Proc.
Nat'l Acad. Sci. USA 87:633 (1990).
4. PRODUCTION OF zB7R1 AND CD155 POLYNUCLEOTIDES AND GENE
VARIANTS
[0139] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, that encode the zB7R1
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. Moreover, the present invention also
provides isolated soluble monomeric, homodimeric, heterodimeric and
multimeric receptor polypeptides that comprise at least one zB7R1
receptor subunit that is substantially homologous to the receptor
polypeptide of SEQ ID NO:2 or 5. Thus, the present invention
contemplates zB7R1 polypeptide-encoding nucleic acid molecules
comprising degenerate nucleotides of SEQ ID NO:1, and their RNA
equivalents.
[0140] Table 1 sets forth the one-letter codes to denote degenerate
nucleotide positions. "Resolutions" are the nucleotides denoted by
a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either
C or T, and its complement R denotes A or G, A being complementary
to T, and G being complementary to C. TABLE-US-00001 TABLE 1
Nucleotide Resolution Complement Resolution A A T T C C G G G G C C
T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G
W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T
H A|C|T N A|C|G|T N A|C|G|T
[0141] The degenerate codons, encompassing all possible codons for
a given amino acid, are set forth in Table 2. TABLE-US-00002 TABLE
2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT
TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro
P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG
GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q
CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys
K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG
CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y
TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY
Glu|Gln Z SAR Any X NNN
[0142] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding an amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0143] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). As used herein, the term "preferential codon usage"
or "preferential codons" is a term of art referring to protein
translation codons that are most frequently used in cells of a
certain species, thus favoring one or a few representatives of the
possible codons encoding each amino acid (See Table 2). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequences
disclosed herein serve as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferential
codons can be tested and optimized for expression in various
species, and tested for functionality as disclosed herein.
[0144] A zB7R1-encoding cDNA can be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction with primers designed from the representative human
zB7R1 sequences disclosed herein. In addition, a cDNA library can
be used to transform or transfect host cells, and expression of the
cDNA of interest can be detected with an antibody to zB7R1
polypeptide.
[0145] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human zB7R1,
and that allelic variation and alternative splicing are expected to
occur (i.e. SEQ ID NO:5). Allelic variants of this sequence can be
cloned by probing cDNA or genomic libraries from different
individuals according to standard procedures. Allelic variants of
the nucleotide sequences disclosed herein, including those
containing silent mutations and those in which mutations result in
amino acid sequence changes, are within the scope of the present
invention, as are proteins which are allelic variants of the amino
acid sequences disclosed herein. cDNA molecules generated from
alternatively spliced mRNAs, which retain the properties of the
zB7R1 polypeptide are included within the scope of the present
invention, as are polypeptides encoded by such cDNAs and mRNAs.
Allelic variants and splice variants of these sequences can be
cloned by probing cDNA or genomic libraries from different
individuals or tissues according to standard procedures known in
the art.
[0146] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that comprise a
soluble zB7R1 receptor that is substantially homologous to SEQ ID
NO:2 or 5, or that encodes amino acids of SEQ ID NO:3, 4 or 6, or
allelic variants thereof and retain the counter-receptor-binding
properties of the wild-type zB7R1 receptor. Such polypeptides may
also include additional polypeptide segments as generally disclosed
herein.
[0147] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or to nucleic acid molecules
comprising a nucleotide sequence complementary to SEQ ID NO:1, or
fragments thereof.
[0148] In general, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (T.sub.m) for the
specific sequence at a defined ionic strength and pH. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe.
Following hybridization, the nucleic acid molecules can be washed
to remove non-hybridized nucleic acid molecules under stringent
conditions, or under highly stringent conditions. See, for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition (Cold Spring Harbor Press 1989); Ausubel et al., (eds.),
Current Protocols in Molecular Biology (John Wiley and Sons, Inc.
1987); Berger and Kimmel (eds.), Guide to Molecular Cloning
Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.
Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such
as OLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0
(Premier Biosoft International; Palo Alto, Calif.), as well as
sites on the Internet, are available tools for analyzing a given
sequence and calculating T.sub.m based on user-defined criteria. It
is well within the abilities of one skilled in the art to
adapthybridization and wash conditions for use with a particular
polynucleotide hybrid.
[0149] The present invention also provides isolated zB7R1
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, 3, 6 or 7, or their orthologs. The
term "substantially similar sequence identity" is used herein to
denote polypeptides having at least 70%, at least 80%, at least
90%, at least 95%, such as 96%, 97%, 98%, or greater than 95%
sequence identity to the sequences shown in SEQ ID NO:3, or their
orthologs. For example, variant and orthologous zB7R1 receptors can
be used to generate an immune response and raise cross-reactive
antibodies to human zB7R1. Such antibodies can be humanized, and
modified as described herein, and used therauputically to treat
psoriasis, psoriatic arthritis, IBD, colitis, endotoxemia as well
as in other therapeutic applications described herein.
[0150] The present invention also contemplates zB7R1 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and a hybridization
assay. Such zB7R1 variants include nucleic acid molecules (1) that
remain hybridized with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., and (2) that encode a polypeptide having at least 70%, at least
80%, at least 90%, at least 95%, or greater than 95% such as 96%,
97%, 98%, or 99%, sequence identity to the amino acid sequence of
SEQ ID NO:3. Alternatively, zB7R1 variants can be characterized as
nucleic acid molecules (1) that remain hybridized with a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement) under highly stringent washing conditions, in which the
wash stringency is equivalent to 0.1.times.-0.2.times.SSC with 0.1%
SDS at 50-65.degree. C., and (2) that encode a polypeptide having
at least 70%, at least 80%, at least 90%, at least 95% or greater
than 95%, such as 96%, 97%, 98%, or 99% or greater, sequence
identity to the amino acid sequence of SEQ ID NO:2.
[0151] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0152] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative zB7R1 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID NO:2
or SEQ ID NO:3) and a test sequence that have either the highest
density of identities (if the ktup variable is 1) or pairs of
identities (if ktup=2), without considering conservative amino acid
substitutions, insertions, or deletions. The ten regions with the
highest density of identities are then rescored by comparing the
similarity of all paired amino acids using an amino acid
substitution matrix, and the ends of the regions are "trimmed" to
include only those residues that contribute to the highest score.
If there are several regions with scores greater than the "cutoff"
value (calculated by a predetermined formula based upon the length
of the sequence and the ktup value), then the trimmed initial
regions are examined to determine whether the regions can be joined
to form an approximate alignment with gaps. Finally, the highest
scoring regions of the two amino acid sequences are aligned using a
modification of the Needleman-Wunsch-Sellers algorithm (Needleman
and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl.
Math. 26:787 (1974)), which allows for amino acid insertions and
deletions. Illustrative parameters for FASTA analysis are: ktup=1,
gap opening penalty=10, gap extension penalty=1, and substitution
matrix=BLOSUM62. These parameters can be introduced into a FASTA
program by modifying the scoring matrix file ("SMATRIX"), as
explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63
(1990).
[0153] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as described above.
[0154] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with an amino acid sequence disclosed herein. For example,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:2, in which an alkyl amino acid is
substituted for an alkyl amino acid in a zB7R1 amino acid sequence,
an aromatic amino acid is substituted for an aromatic amino acid in
a zB7R1 amino acid sequence, a sulfur-containing amino acid is
substituted for a sulfur-containing amino acid in a zB7R1 amino
acid sequence, a hydroxy-containing amino acid is substituted for a
hydroxy-containing amino acid in a zB7R1 amino acid sequence, an
acidic amino acid is substituted for an acidic amino acid in a
zB7R1 amino acid sequence, a basic amino acid is substituted for a
basic amino acid in a zB7R1 amino acid sequence, or a dibasic
monocarboxylic amino acid is substituted for a dibasic
monocarboxylic amino acid in a zB7R1 amino acid sequence. Among the
common amino acids, for example, a "conservative amino acid
substitution" is illustrated by a substitution among amino acids
within each of the following groups: (1) glycine, alanine, valine,
leucine, and isoleucine, (2) phenylalanine, tyrosine, and
tryptophan, (3) serine and threonine, (4) aspartate and glutamate,
(5) glutamine and asparagine, and (6) lysine, arginine and
histidine. The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff and Henikoff, Proc.
Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62
substitution frequencies can be used to define conservative amino
acid substitutions that may be introduced into the amino acid
sequences of the present invention. Although it is possible to
design amino acid substitutions based solely upon chemical
properties (as discussed above), the language "conservative amino
acid substitution" preferably refers to a substitution represented
by a BLOSUM62 value of greater than -1. For example, an amino acid
substitution is conservative if the substitution is characterized
by a BLOSUM62 value of 0, 1, 2, or 3. According to this system,
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3). Particular
variants of zB7R1 are characterized by having at least 70%, at
least 80%, at least 90%, at least 95% or greater than 95% such as
96%, 97%, 98%, or 99% or greater sequence identity to the
corresponding amino acid sequence (e.g., SEQ ID NO:2, 3, 6 or 7),
wherein the variation in amino acid sequence is due to one or more
conservative amino acid substitutions.
[0155] Conservative amino acid changes in a zB7R1 gene can be
introduced, for example, by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1 or 5. Such "conservative amino
acid" variants can be obtained by oligonucleotide-directed
mutagenesis, linker-scanning mutagenesis, mutagenesis using the
polymerase chain reaction, and the like (see Ausubel (1995); and
McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL
Press 1991)). A variant zB7R1 polypeptide can be identified by the
ability to specifically bind anti-zB7R1 antibodies.
[0156] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol. 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0157] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0158] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for zB7R1 amino acid residues.
[0159] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Nat'l Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity to
identify amino acid residues that are critical to the activity of
the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699
(1996).
[0160] Although sequence analysis can be used to further define the
zB7R1 counter-receptor binding region, amino acids that play a role
in zB7R1 binding activity (such as binding of zB7R1 to its
counter-receptor or counter-receptors, or to an anti-zB7R1
antibody) can also be determined by physical analysis of structure,
as determined by such techniques as nuclear magnetic resonance,
crystallography, electron diffraction or photoaffinity labeling, in
conjunction with mutation of putative contact site amino acids.
See, for example, de Vos et al., Science 255:306 (1992), Smith et
al., J. Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett.
309:59 (1992).
[0161] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover,
zB7R1 labeled with biotin or FITC can be used for expression
cloning of zB7R1 counter-receptors.
[0162] Variants of the disclosed zB7R1 nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci.
USA 91:10747 (1994), and international publication No. WO 97/20078.
Briefly, variant DNA molecules are generated by in vitro homologous
recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point
mutations. This technique can be modified by using a family of
parent DNA molecules, such as allelic variants or DNA molecules
from different species, to introduce additional variability into
the process. Selection or screening for the desired activity,
followed by additional iterations of mutagenesis and assay provides
for rapid "evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against detrimental
changes.
[0163] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-zB7R1 antibodies, can be recovered
from the host cells and rapidly sequenced using modern equipment.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide of interest, and
can be applied to polypeptides of unknown structure.
[0164] The present invention also includes "functional fragments"
of zB7R1 polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a zB7R1 polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO:1 or 5 can be digested with Bal31 nuclease to obtain a series
of nested deletions. The fragments are then inserted into
expression vectors in proper reading frame, and the expressed
polypeptides are isolated and tested for the ability to bind
anti-zB7R1 antibodies. One alternative to exonuclease digestion is
to use oligonucleotide-directed mutagenesis to introduce deletions
or stop codons to specify production of a desired fragment.
Alternatively, particular fragments of a zB7R1 gene can be
synthesized using the polymerase chain reaction.
[0165] This general approach is exemplified by studies on the
truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0166] The present invention also contemplates functional fragments
of a zB7R1 gene that have amino acid changes, compared with an
amino acid sequence disclosed herein. A variant zB7R1 gene can be
identified on the basis of structure by determining the level of
identity with disclosed nucleotide and amino acid sequences, as
discussed above. An alternative approach to identifying a variant
gene on the basis of structure is to determine whether a nucleic
acid molecule encoding a potential variant zB7R1 gene can hybridize
to a nucleic acid molecule comprising a nucleotide sequence, such
as SEQ ID NO:1 or 5.
[0167] The present invention also includes using functional
fragments of zB7R1 polypeptides, antigenic epitopes,
epitope-bearing portions of zB7R1 polypeptides, and nucleic acid
molecules that encode such functional fragments, antigenic
epitopes, epitope-bearing portions of zB7R1 polypeptides. Such
fragments are used to generate polypeptides for use in generating
antibodies and binding partners that agonize, bind, block, inhibit,
increase, reduce, antagonize or neutralize activity of a B7
receptor. A "functional" zB7R1 polypeptide or fragment thereof as
defined herein is characterized by its ability to bind a zB7R1
counter-recetpr such as CD155, or block, inhibit, reduce,
antagonize or neutralize zB7R1-mediated signaling or inflammatory,
proliferative or differentiating activity; or by its ability to
induce or inhibit specialized cell functions; or by its ability to
bind specifically to an anti-zB7R1 antibody, cell, or B7
counter-receptor. As previously described herein, zB7R1 is
characterized as a B7 family member by its receptor structure and
domains as described herein. Thus, the present invention further
contemplates using fusion proteins encompassing: (a) polypeptide
molecules comprising one or more of the domains described above;
and (b) functional fragments comprising one or more of these
domains. The other polypeptide portion of the fusion protein may be
contributed by another B7 family receptor, such as CD28, CTLA-4,
ICOS, PD-1, HHLA2, or BTLA, or by a non-native and/or an unrelated
secretory signal peptide that facilitates secretion of the fusion
protein.
[0168] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a zB7R1
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0169] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides, antigenic
peptides, epitopes, and polypeptides of the present invention are
useful to raise antibodies that bind with the polypeptides
described herein, as well as to identify and screen anti-zB7R1
monoclonal antibodies that are neutralizing, and that may agonize,
bind, block, inhibit, reduce, antagonize or neutralize the activity
of its counter-receptor. Such neutralizing monoclonal antibodies of
the present invention can bind to a zB7R1 antigenic epitope.
Hopp/Woods hydrophilicity profiles can be used to determine regions
that have the most antigenic potential within SEQ ID NO:3 (Hopp et
al., Proc. Natl. Acad. Sci.78:3824-3828, 1981; Hopp, J. Immun.
Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering
11:153-169, 1998). The profile is based on a sliding six-residue
window. Buried G, S, and T residues and exposed H, Y, and W
residues were ignored. In zB7R1 these regions can be determined by
one of skill in the art. Moreover, zB7R1 antigenic epitopes within
SEQ ID NO:2 as predicted by a Jameson-Wolf plot, e.g., using
DNASTAR Protean program (DNASTAR, Inc., Madison, Wisc.) serve as
preferred antigenic epitpoes, and can be determined by one of skill
in the art. Such antigenic epitpoes include (1) amino acid residues
80 to 86 of SEQ ID NO:2; (2) amino acid residues 163 to 170 of SEQ
ID NO:2; (3) amino acid residues 163 to 190 of SEQ ID NO:2; (4)
amino acid residues 175 to 190 of SEQ ID NO:2; and (5) amino acid
residues 211 to 221 of SEQ ID NO:2. In preferred embodiments,
antigenic epitopes to which neutralizing antibodies of the present
invention bind would contain residues of SEQ ID NO:2 (and
corresponding residues of SEQ ID NO:3) that are important to
counter-receptor-receptor binding.
[0170] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of an amino acid
sequence disclosed herein. Such epitope-bearing peptides and
polypeptides can be produced by fragmenting a zB7R1 polypeptide, or
by chemical peptide synthesis, as described herein. Moreover,
epitopes can be selected by phage display of random peptide
libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.
5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616
(1996)). Standard methods for identifying epitopes and producing
antibodies from small peptides that comprise an epitope are
described, for example, by Mole, "Epitope Mapping," in Methods in
Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana
Press, Inc. 1992), Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0171] For any zB7R1 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise zB7R1
variants based upon the nucleotide and amino acid sequences
described herein.
5. PRODUCTION OF zB7R1 AND CD155 POLYPEPTIDES
[0172] The polypeptides of the present invention, including
full-length polypeptides; soluble monomeric, homodimeric,
heterodimeric and multimeric receptors; full-length receptors;
receptor fragments (e.g. counter-receptor-binding fragments and
antigenic epitopes), functional fragments, and fusion proteins, can
be produced in recombinant host cells following conventional
techniques. To express a zB7R1 or CD155 gene, a nucleic acid
molecule encoding the polypeptide must be operably linked to
regulatory sequences that control transcriptional expression in an
expression vector and then, introduced into a host cell. In
addition to transcriptional regulatory sequences, such as promoters
and enhancers, expression vectors can include translational
regulatory sequences and a marker gene which is suitable for
selection of cells that carry the expression vector.
[0173] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, a zB7R1
expression vector may comprise a zB7R1 gene and a secretory
sequence derived from any secreted gene.
[0174] zB7R1 or CD155 proteins of the present invention may be
expressed in mammalian cells. Examples of suitable mammalian host
cells include African green monkey kidney cells (Vero; ATCC CRL
1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby
hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL
10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster
ovary cells (CHO--K1; ATCC CCL61; CHO DG44 (Chasin et al., Som.
Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[0175] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from mammalian viral sources, for
example, adenovirus, bovine papilloma virus, simian virus, or the
like, in which the regulatory signals are associated with a
particular gene which has a high level of expression. Suitable
transcriptional and translational regulatory sequences also can be
obtained from mammalian genes, for example, actin, collagen,
myosin, and metallothionein genes.
[0176] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0177] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
zB7R1 gene expression in mammalian cells if the prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol.
Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res.
19:4485 (1991)).
[0178] In certain embodiments, a DNA sequence encoding a zB7R1
soluble receptor polypeptide, a fragment of zB7R1 polypeptide, a
CD155 soluble receptor or a fragment of a CD155 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers. Multiple
components of a soluble receptor complex can be co-transfected on
individual expression vectors or be contained in a single
expression vector. Such techniques of expressing multiple
components of protein complexes are well known in the art.
[0179] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0180] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
selection is carried out in the presence of a neomycin-type drug,
such as G-418 or the like. Selection systems can also be used to
increase the expression level of the gene of interest, a process
referred to as "amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of the
selective agent and then increasing the amount of selective agent
to select for cells that produce high levels of the products of the
introduced genes. A suitable amplfifable selectable marker is
dihydrofolate reductase (DHFR), which confers resistance to
methotrexate. Other drug resistance genes (e.g., hygromycin
resistance, multi-drug resistance, puromycin acetyltransferase) can
also be used. Alternatively, markers that introduce an altered
phenotype, such as green fluorescent protein, or cell surface
proteins such as CD4, CD8, Class I MHC, placental alkaline
phosphatase may be used to sort transfected cells from
untransfected cells by such means as FACS sorting or magnetic bead
separation technology.
[0181] zB7R1 polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose include adenovirus, retroviruses, herpesvirus,
vaccinia virus and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994), and
Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages
of the adenovirus system include the accommodation of relatively
large DNA inserts, the ability to grow to high-titer, the ability
to infect a broad range of mammalian cell types, and flexibility
that allows use with a large number of available vectors containing
different promoters.
[0182] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown
as adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0183] zB7R1 or CD155 can also be expressed in other higher
eukaryotic cells, such as avian, fungal, insect, yeast, or plant
cells. The baculovirus system provides an efficient means to
introduce cloned zB7R1 genes into insect cells. Suitable expression
vectors are based upon the Autographa californica multiple nuclear
polyhedrosis virus (AcMNPV), and contain well-known promoters such
as Drosophila heat shock protein (hsp) 70 promoter, Autographa
californica nuclear polyhedrosis virus immediate-early gene
promoter (ie-1) and the delayed early 39K promoter, baculovirus p10
promoter, and the Drosophila metallothionein promoter. A second
method of making recombinant baculovirus utilizes a
transposon-based system described by Luckow (Luckow, et al., J.
Virol. 67:4566 (1993)). This system, which utilizes transfer
vectors, is sold in the BAC-to-BAC kit (Life Technologies,
Rockville, Md.). This system utilizes a transfer vector, PFASTBAC
(Life Technologies) containing a Tn7 transposon to move the DNA
encoding the zB7R1 polypeptide into a baculovirus genome maintained
in E. coli as a large plasmid called a "bacmid." See, Hill-Perkins
and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et al., J. Gen.
Virol. 75:1551 (1994), and Chazenbalk, and Rapoport, J. Biol. Chem.
270:1543 (1995). In addition, transfer vectors can include an
in-frame fusion with DNA encoding an epitope tag at the C- or
N-terminus of the expressed zB7R1 polypeptide, for example, a
Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l Acad. Sci.
82:7952 (1985)). Using a technique known in the art, a transfer
vector containing a zB7R1 gene is transformed into E. coli, and
screened for bacmids which contain an interrupted lacZ gene
indicative of recombinant baculovirus. The bacmid DNA containing
the recombinant baculovirus genome is then isolated using common
techniques.
[0184] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native zB7R1 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native zB7R1 secretory signal
sequence.
[0185] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf900 II.TM. (Life Technologies) or ESF 921.TM. (Expression
Systems) for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences,
Lenexa, Kans.) or Express FiveO.TM. (Life Technologies) for the T.
ni cells. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0186] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0187] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp
vectors, such as YCp19. Methods for transforming S. cerevisiae
cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No.
4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S.
Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, and
Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are
selected by phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e.g., leucine). A suitable vector system for
use in Saccharomyces cerevisiae is the POT1 vector system disclosed
by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows
transformed cells to be selected by growth in glucose-containing
media. Additional suitable promoters and terminators for use in
yeast include those from glycolytic enzyme genes (see, e.g.,
Kawasaki, U.S. Pat. No. 4,599,311, Kingsman et al., U.S. Pat. No.
4,615,974, and Bitter, U.S. Pat. No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154,
5,139,936, and 4,661,454.
[0188] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillernondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0189] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For polypeptide
production in P. methanolica, the promoter and terminator in the
plasmid can be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A suitable selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), and which allows ade2 host cells to grow in the absence
of adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, host cells can be used
in which both methanol utilization genes (AUG1 and AUG2) are
deleted. For production of secreted proteins, host cells can be
deficient in vacuolar protease genes (PEP4 and PRB1).
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. P. methanolica cells can be transformed by
electroporation using an exponentially decaying, pulsed electric
field having a field strength of from 2.5 to 4.5 kV/cm, preferably
about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
[0190] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0191] Alternatively, zB7R1 genes can be expressed in prokaryotic
host cells. Suitable promoters that can be used to express zB7R1
polypeptides in a prokaryotic host are well-known to those of skill
in the art and include promoters capable of recognizing the T4, T3,
Sp6 and T7 polymerases, the P.sub.R and P.sub.L promoters of
bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,
lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
subtilis, the promoters of the bacteriophages of Bacillus,
Streptomyces promoters, the int promoter of bacteriophage lambda,
the bla promoter of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene. Prokaryotic promoters have
been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et
al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins
1987), and by Ausubel et al. (1995).
[0192] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0193] When expressing a zB7R1 polypeptide in bacteria such as E.
coli, the polypeptide may be retained in the cytoplasm, typically
as insoluble granules, or may be directed to the periplasmic space
by a bacterial secretion sequence. In the former case, the cells
are lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0194] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0195] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0196] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0197] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0198] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NO:2. As an illustration, polypeptides can
comprise at least six, at least nine, or at least 15 contiguous
amino acid residues of of SEQ ID NO:2. Within certain embodiments
of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or
more contiguous residues of these amino acid sequences. Nucleic
acid molecules encoding such peptides and polypeptides are useful
as polymerase chain reaction primers and probes.
[0199] Moreover, zB7R1 or CD155 polypeptides and fragments thereof
can be expressed as monomers, homodimers, heterodimers, tetramers
(discussed below) or multimers within higher eukaryotic cells. Such
cells can be used to produce zB7R1 or CD15 monomeric, homodimeric,
heterodimeric, tetrameric and multimeric receptor polypeptides that
comprise at least one zB7R1 or CD155 polypeptide ("zB7R1-comprising
receptors," "zB7R1 -comprising receptor polypeptides," "CD
155-comprising receptors" or "CD 155-comprising receptor
polypeptides"), or can be used as assay cells in screening systems.
Within one aspect of the present invention, a polypeptide of the
present invention comprising the zB7R1 extracellular domain (SEQ ID
NO:3 or 7) is produced by a cultured cell, and the cell is used to
screen for counter-receptors for the receptor, including a natural
counter-receptor, as well as agonists and antagonists of the
natural counter-receptor. To summarize this approach, a cDNA or
gene encoding the receptor is combined with other genetic elements
required for its expression (e.g., a transcription promoter), and
the resulting expression vector is inserted into a host cell. Cells
that express the DNA and produce functional receptor are selected
and used within a variety of screening systems. Each component of
the monomeric, homodimeric, heterodimeric and multimeric receptor
complex can be expressed in the same cell. Moreover, the components
of the monomeric, homodimeric, heterodimeric and multimeric
receptor complex can also be fused to a transmembrane domain or
other membrane fusion moiety to allow complex assembly and
screening of transfectants as described above.
6. zB7R1 AND CD155 TETRAMERIC POLYNUCLEOTIDES, POLYPEPTIDES AND
METHODS OF MAKING THE SAME
[0200] The present invention also encompasses methods of producing
a multimeric, preferably tetrameric, zB7R1 or CD155 polypeptides.
These proteins are described in more detail in U.S. Provisional
Patent Application No. 60/60/791,626, filed Apr. 13, 2006, and
incorporated herein in its entirety. These fusion proteins comprise
a VASP domain and a herterologous protein domain, such as zB7R1 or
CD155. VASP domains are derived from the VASP gene present in many
species. Sequences are selected for their anticipated ability to
form coiled-coil protein structure, as this structure is important
for the ability to form multimeric protein forms. Particularly
desired for the present invention is the ability of coiled-coil
proteins to produce tetrameric protein structures. A particularly
preferred embodiment utilizes amino acids 343 to 376 of the human
VASP sequence (amino acids 5 to 38 of SEQ ID NO:23). The full
length DNA sequence of this protein is SEQ ID NO: 24 and the full
length polypeptide sequence of this protein is SEQ ID NO:25.
[0201] Work with other types of multimerizing sequences, for
examples, the leucine zipper, has shown that a limited number of
conservative amino acid substitutions (even at the d residue) can
be often be tolerated in zipper sequences without the loss of the
ability of the molecules to multimerize (Landschultz et al.,
(1989), supra; ). Thus, conservative changes from the native
sequence for the VASP domain are contemplated within the scope of
the invention. Table 4 shows the conservative changes that are
anticipated to tolerated by the coiled-coil structure.
TABLE-US-00004 TABLE 4 Conservative amino acid substitutions Basic:
arginine Aromatic: phenylalanine lysine tryptophan histidine
tyrosine Acidic: glutamic acid Small: glycine aspartic acid alanine
Polar: glutamine serine asparagine threonine Hydrophobic: leucine
methionine isoleucine valine methionine
[0202] If more than one fusion protein is being used to produce
hetero-multimeric proteins, for example, heterotetramers, the VASP
domain that is used can be the same domain for both fusion proteins
or different VASP domains, as long as the domains have the ability
to associate with each other and form multimeric proteins.
[0203] The VASP domain can be put at either the N or C terminus of
the heterologous protein of interest, based on considerations of
function (i.e., whether the heterologous protein is a type I or
type II membrane protein) and ease of construction of the
construct. Additionally, the VASP domain can be located in the
middle of the protein, effectively creating a double fusion protein
with one heterologous sequence, a VASP domain, and a second
heterologous sequence. The two heterologous sequences for the
double fusion protein can be the same or different.
[0204] Specifically, zB7R1 or CD155 may be linked directly to
another protein to form a fusion protein; alternatively, the
proteins maybe separated by a distance sufficient to ensure the
proteins form proper secondary and tertiary structure needed for
biological activity. Suitable linker sequences will adopt a
flexible extended confirmation and will not exhibit a propensity
for developing an ordered secondary structure which could interact
with the function domains of the fusions proteins, and will have
minimal hydrophobic or charged character which could also interfere
with the function of fusion domains. Linker sequences should be
constructed with the 15 residue repeat in mind, as it may not be in
the best interest of producing a biologically active protein to
tightly constrict the N or C terminus of the heterologous sequence.
Beyond these considerations, the length of the linker sequence may
vary without significantly affecting the biological activity of the
fusion protein. Linker sequences can be used between any and all
components of the fusion protein (or expression construct)
including affinity tags and signal peptides. An example linker is
the GSGG sequence (SEQ ID NO:26).
[0205] A further component of the fusion protein can be an affinity
tag. Such tags do not alter the biological activity of fusion
proteins, are highly antigenic, and provides an epitope that can be
reversibly bound by a specific binding molecule, such as a
monoclonal antibody, enabling repaid detection and purification of
an expressed fusion protein. Affinity tages can also convey
resistence to intracellular degradation if proteins are produced in
bacteria, like E. coli. An exemplary affinity tag is the FLAG Tag
(SEQ ID NO: 27) or the HIS.sub.6 Tag (SEQ ID NO: 28). Methods of
producing fusion proteins utilizing this affinity tag for
purification are described in U.S. Pat. No. 5,011,912.
[0206] A still further component of the fusion protein can be a
signal sequence or leader sequence. These sequences are generally
utilized to allow for secretion of the fusion protein from the host
cell during expression and are also known as a leader sequence,
prepro sequence or pre sequence. The secretory signal sequence may
be that of the heterologous protein being produced, if it has such
a sequence, or may be derived from another secreted protein (e.g.,
t-PA) or synthesized de novo. The secretory signal sequence is
operably linked to fusion protein DNA sequence, i.e., the two
sequences are joined in the correct reading frame and positioned to
direct the newly sythesized polypeptide into the secretory pathway
of the host cell. Secretory signal sequences are commonly
positioned 5' to the DNA sequence encoding the polypeptide of
interest, although certain signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.
5,143,830).
[0207] Thus, the nucleic acid compositions of the present invention
find use in the preparation of all or a portion of the VASP-zB7R1
or VASP-CD155 fusion proteins, as described above. The subject
polynucleotides (including cDNA or the full-length gene) can be
used to express a partial or complete gene product. Constructs
comprising the subject polynucleotides can be generated
synthetically. Alternatively, single-step assembly of a gene and
entire plasmid from large numbers of oligodeoxyribonucleotides is
described by, e.g., Stemmer et al., Gene (Amsterdam) (1995)
164(1):49-53. In this method, assembly PCR (the synthesis of long
DNA sequences from large numbers of oligodeoxyribonucleotides
(oligos)) is described. The method is derived from DNA shuffling
(Stemmer, Nature (1994) 370:389-391), and does not rely on DNA
ligase, but instead relies on DNA polymerase to build increasingly
longer DNA fragments during the assembly process. Appropriate
polynucleotide constructs are purified using standard recombinant
DNA techniques as described in, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold Spring
Harbor Press, Cold Spring Harbor, N.Y., and under current
regulations described in United States Dept. of HHS, National
Institute of Health (NIH) Guidelines for Recombinant DNA
Research.
[0208] Polynucleotide molecules comprising a polynucleotide
sequence provided herein are propagated by placing the molecule in
a vector. Viral and non-viral vectors are used, including plasmids.
The choice of plasmid will depend on the type of cell in which
propagation is desired and the purpose of propagation. Certain
vectors are useful for amplifying and making large amounts of the
desired DNA sequence. Other vectors are suitable for expression in
cells in culture. Still other vectors are suitable for transfer and
expression in cells in a whole animal or person. The choice of
appropriate vector is well within the skill of the art. Many such
vectors are available commercially. The partial or full-length
polynucleotide is inserted into a vector typically by means of DNA
ligase attachment to a cleaved restriction enzyme site in the
vector. Alternatively, the desired nucleotide sequence can be
inserted by homologous recombination in vivo. Typically this is
accomplished by attaching regions of homology to the vector on the
flanks of the desired nucleotide sequence. Regions of homology are
added by ligation of oligonucleotides, or by polymerase chain
reaction using primers comprising both the region of homology and a
portion of the desired nucleotide sequence, for example.
[0209] For expression, an expression cassette or system may be
employed. The gene product encoded by a polynucleotide of the
invention is expressed in any convenient expression system,
including, for example, bacterial, yeast, insect, amphibian and
mammalian systems. Suitable vectors and host cells are described in
U.S. Pat. No. 5,654,173. In the expression vector, the heterologous
protein encoding polynucleotide (such as the extracellular domain
of zB7R1; i.e. SEQ ID NO:3 or 7) is linked to a regulatory sequence
as appropriate to obtain the desired expression properties. These
can include promoters (attached either at the 5' end of the sense
strand or at the 3' end of the antisense strand), enhancers,
terminators, operators, repressors, and inducers. The promoters can
be regulated or constitutive. In some situations it may be
desirable to use conditionally active promoters, such as
tissue-specific or developmental stage-specific promoters. These
are linked to the desired nucleotide sequence using the techniques
described above for linkage to vectors. Any techniques known in the
art can be used. In other words, the expression vector will provide
a transcriptional and translational initiation region, which may be
inducible or constitutive, where the coding region is operably
linked under the transcriptional control of the transcriptional
initiation region, and a transcriptional and translational
termination region. These control regions may be native to the DNA
encoding the VASP-heterologous fusion protein, or may be derived
from exogenous sources.
[0210] Expression vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences encoding heterologous proteins.
A selectable marker operative in the expression host may be
present. Expression vectors may be used for the production of
fusion proteins, where the exogenous fusion peptide provides
additional functionality, i.e. increased protein synthesis,
stability, reactivity with defined antisera, an enzyme marker, e.g.
.beta.-galactosidase, etc.
[0211] Expression cassettes may be prepared comprising a
transcription initiation region, the gene or fragment thereof, and
a transcriptional termination region. Of particular interest is the
use of sequences that allow for the expression of functional
epitopes or domains, usually at least about 8 amino acids in
length, more usually at least about 15 amino acids in length, to
about 25 amino acids, and up to the complete open reading frame of
the gene. After introduction of the DNA, the cells containing the
construct may be selected by means of a selectable marker, the
cells expanded and then used for expression.
[0212] VASP-Heterologous fusion proteins may be expressed in
prokaryotes or eukaryotes in accordance with conventional ways,
depending upon the purpose for expression. For large scale
production of the protein, a unicellular organism, such as E. coli,
B. subtilis, S. cerevisiae, insect cells in combination with
baculovirus vectors, or cells of a higher organism such as
vertebrates, particularly mammals, e.g. COS 7 cells, HEK 293, CHO,
Xenopus Oocytes, etc., may be used as the expression host cells. In
some situations, it is desirable to express a polymorphic VASP
nucleic acid molecule in eukaryotic cells, where the polymorphic
VASP protein will benefit from native folding and
post-translational modifications. Small peptides can also be
synthesized in the laboratory. Polypeptides that are subsets of the
complete VASP sequence may be used to identify and investigate
parts of the protein important for function.
[0213] Specific expression systems of interest include bacterial,
yeast, insect cell and mammalian cell derived expression systems.
Representative systems from each of these categories is are
provided below: Bacteria. Expression systems in bacteria include
those described in Chang et al., Nature (1978) 275:615; Goeddel et
al., Nature (1979) 281:544; Goeddel et al., Nucleic Acids Res.
(1980) 8:4057; EP 0 036,776; U.S. Pat. No. 4,551,433; DeBoer et
al., Proc. Natl. Acad. Sci. (USA) (1983) 80:21-25; and Siebenlist
et al., Cell (1980) 20:269. Yeast. Expression systems in yeast
include those described in Hinnen et al., Proc. Natl. Acad. Sci.
(USA) (1978) 75:1929; Ito et al., J. Bacteriol. (1983) 153:163;
Kurtz et al., Mol. Cell. Biol. (1986) 6:142; Kunze et al., J. Basic
Microbiol. (1985)25:141; Gleeson et al., J. Gen. Microbiol. (1986)
132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202:302; Das
et al., J. Bacteriol. (1984) 158:1165; De Louvencourt et al., J.
Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology
(1990)8:135; Kunze et al., J. Basic Microbiol. (1985)25:141; Cregg
et al., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148
and 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et
al., Curr. Genet. (1985) 10:380; Gaillardin et al., Curr. Genet.
(1985) 10:49; Ballance et al., Biochem. Biophys. Res. Commun.
(1983) 112:284-289; Tilburn et al., Gene (1983) 26:205-221; Yelton
et al., Proc. Natl. Acad. Sci. (USA) (1984) 81:1470-1474; Kelly and
Hynes, EMBO J. (1985) 4:475479; EP 0 244,234; and WO 91/00357.
Insect Cells. Expression of heterologous genes in insects is
accomplished as described in U.S. Pat. No. 4,745,051; Friesen et
al., "The Regulation of Baculovirus Gene Expression", in: The
Molecular Biology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0
127,839; EP 0 155,476; and Vlak et al., J. Gen. Virol. (1988)
69:765-776; Miller et al., Ann. Rev. Microbiol. (1988) 42:177;
Carbonell et al., Gene (1988) 73:409; Maeda et al., Nature (1985)
315:592-594; Lebacq-Verheyden et al., Mol. Cell. Biol. (1988)
8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82:8844;
Miyajima et al., Gene (1987) 58:273; and Martin et al., DNA (1988)
7:99. Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts are described in Luckow et
al., Bio/Technology (1988) 6:47-55, Miller et al., Generic
Engineering (1986) 8:277-279, and Maeda et al., Nature (1985)
315:592-594. Mammalian Cells. Mammalian expression is accomplished
as described in Dijkema et al., EMBO J. (1985) 4:761, Gorman et
al., Proc. Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al.,
Cell (1985) 41:521 and U.S. Pat. No. 4,399,216. Other features of
mammalian expression are facilitated as described in Ham and
Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem.
(1980) 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,
4,560,655, WO 90/103430, WO 87/00195, and U.S. Pat. No. RE
30,985.
[0214] When any of the above host cells, or other appropriate host
cells or organisms, are used to replicate and/or express the
polynucleotides or nucleic acids of the invention, the resulting
replicated nucleic acid, RNA, expressed protein or polypeptide, is
within the scope of the invention as a product of the host cell or
organism. The product is recovered by any appropriate means known
in the art.
[0215] Once the gene corresponding to a selected polynucleotide is
identified, its expression can be regulated-in the cell to which
the gene is native. For example, an endogenous gene of a cell can
be regulated by an exogenous regulatory sequence inserted into the
genome of the cell at location sufficient to at least enhance
expressed of the gene in the cell. The regulatory sequence may be
designed to integrate into the genome via homologous recombination,
as disclosed in U.S. Pat. Nos. 5,641,670 and 5,733,761, the
disclosures of which are herein incorporated by reference, or may
be designed to integrate into the genome via non-homologous
recombination, as described in WO 99/15650, the disclosure of which
is herein incorporated by reference.
[0216] The invention further provides recombinant vectors and host
cells comprising polynucleotides of the invention. In general,
recombinant vectors and host cells of the invention are isolated;
however, a host cell comprising a polynucleotide of the invention
may be part of a genetically modified animal.
[0217] The present invention further provides recombinant vectors
("constructs") comprising a polynucleotide of the invention.
Recombinant vectors include vectors used for propagation of a
polynucleotide of the invention, and expression vectors. Vectors
useful for introduction of the polynucleotide include plasmids and
viral vectors, e.g. retroviral-based vectors, adenovirus vectors,
etc. that are maintained transiently or stably in mammalian cells.
A wide variety of vectors can be employed for transfection and/or
integration of the gene into the genome of the cells.
Alternatively, micro-injection may be employed, fusion, or the like
for introduction of genes into a suitable host cell.
[0218] Expression vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences encoding heterologous proteins.
A selectable marker operative in the expression host may be
present. Expression vectors may be used for the production of
fusion proteins, where the exogenous fusion peptide provides
additional functionality, i.e. increased protein synthesis,
stability, reactivity with defined antisera, an enzyme marker, e.g.
.beta.-galactosidase, etc.
[0219] Expression cassettes may be prepared comprising a
transcription initiation region, the gene or fragment thereof, and
a transcriptional termination region. Of particular interest is the
use of sequences that allow for the expression of functional
epitopes or domains, usually at least about 8 amino acids in
length, more usually at least about 15 amino acids in length, at
least about 25 amino acids, at least about 45 amino acids, and up
to the complete open reading frame of the gene. After introduction
of the DNA, the cells containing the construct may be selected by
means of a selectable marker, the cells expanded and then used for
expression.
[0220] The expression cassettes may be introduced into a variety of
vectors, e.g. plasmid, BAC, YAC, bacteriophage such as lambda, P1,
M13, etc., animal or plant viruses, and the like, where the vectors
are normally characterized by the ability to provide selection of
cells comprising the expression vectors. The vectors may provide
for extrachromosomal maintenance, particularly as plasmids or
viruses, or for integration into the host chromosome. Where
extrachromosomal maintenance is desired, an origin sequence is
provided for the replication of the plasmid, which may be low- or
high copy-number. A wide variety of markers are available for
selection, particularly those which protect against toxins, more
particularly against antibiotics. The particular marker that is
chosen is selected in accordance with the nature of the host, where
in some cases, complementation may be employed with auxotrophic
hosts. Introduction of the DNA construct may use any convenient
method, e.g. conjugation, bacterial transformation,
calcium-precipitated DNA, electroporation, fusion, transfection,
infection with viral vectors, biolistics, etc.
[0221] The present invention further provides host cells, which may
be isolated host cells, comprising polymorphic VASP nucleic acid
molecules of the invention. Suitable host cells include prokaryotes
such as E. coli, B. subtilis, eukaryotes, including insect cells in
combination with baculovirus vectors, yeast cells, such as
Saccharomyces cerevisiae, or cells of a higher organism such as
vertebrates, including amphibians (e.g., Xenopus laevis oocytes),
and mammals, particularly humans, e.g. COS cells, CHO cells, HEK293
cells, and the like, may be used as the host cells. Host cells can
be used for the purposes of propagating a polymorphic VASP nucleic
acid molecule, for production of a polymorphic VASP polypeptide, or
in cell-based methods for identifying agents which modulate a level
of VASP mRNA and/or protein and/or biological activity in a
cell.
[0222] Primary or cloned cells and cell lines may be modified by
the introduction of vectors comprising a DNA encoding the
VASP-heterologous fusion protein polymorphism(s). The isolated
polymorphic VASP nucleic acid molecule may comprise one or more
variant sequences, e.g., a haplotype of commonly occurring
combinations. In one embodiment of the invention, a panel of two or
more genetically modified cell lines, each cell line comprising a
VASP polymorphism, are provided for substrate and/or expression
assays. The panel may further comprise cells genetically modified
with other genetic sequences, including polymorphisms, particularly
other sequences of interest for pharmacogenetic screening, e.g.
other genes/gene mutations associated with obesity, a number of
which are known in the art.
[0223] The subject nucleic acids can be used to generate
genetically modified non-human animals or site specific gene
modifications in cell lines. The term "transgenic" is intended to
encompass genetically modified animals having the addition of DNA
encoding the VASP-heterologous fusion protein or having an
exogenous DNA encoding the VASP-heterologous fusion protein that is
stably transmitted in the host cells. Transgenic animals may be
made through homologous recombination. Alternatively, a nucleic
acid construct is randomly integrated into the genome. Vectors for
stable integration include plasmids, retroviruses and other animal
viruses, YACs, and the like. Of interest are transgenic mammals,
e.g. cows, pigs, goats, horses, etc., and particularly rodents,
e.g. rats, mice, etc.
[0224] DNA constructs for homologous recombination will comprise at
least a portion of the DNA encoding the VASP-heterologous fusion
protein and will include regions of homology to the target locus.
Conveniently, markers for positive and negative selection are
included. Methods for generating cells having targeted gene
modifications through homologous recombination are known in
the-art. For various techniques for transfecting mammalian cells,
see Known et al. (1990) Methods in Enzymology 185:527-537.
[0225] For embryonic stem (ES) cells, an ES cell line may be
employed, or ES cells may be obtained freshly from a host, e.g.
mouse, rat, guinea pig, etc. Such cells are grown on an appropriate
fibroblast-feeder layer or grown in the presence of leukemia
inhibiting factor (LIF). When ES cells have been transformed, they
may be used to produce transgenic animals. After transformation,
the cells are plated onto a feeder layer in an appropriate medium.
Cells containing the construct may be detected by employing a
selective medium. After sufficient time for colonies to grow, they
are picked and analyzed for the occurrence of homologous
recombination. Those colonies that show homologous recombination
may then be used for embryo manipulation and blastocyst injection.
Blastocysts are obtained from 4 to 6 week old superovulated
females. The ES cells are trypsinized, and the modified cells are
injected into the blastocoel of the blastocyst. After injection,
the blastocysts are returned to each uterine horn of pseudopregnant
females. Females are then allowed to go to term and the resulting
litters screened for mutant cells having the construct. By
providing for a different phenotype of the blastocyst and the ES
cells, chimeric progeny can be readily detected. The chimeric
animals are screened for the presence of the DNA encoding the
VASP-heterologous fusion protein and males and females having the
modification are mated to produce homozygous progeny. The
transgenic animals may be any non-human mammal, such as laboratory
animals, domestic animals, etc. The transgenic animals may be used
to determine the effect of a candidate drug in an in vivo
environment.
[0226] The present invention is a method of preparing a soluble,
homo- or hetero-trimeric protein by culturing a host cell
transformed or transfected with at least one or up to four
different expression vectors encoding a fusion protein comprising a
VASP domain and a heterologous protein. In order to produce a
biologically functioning protein, the four VASP domains
preferentially form a homo- or hetero-tetramers. The culturing can
also occur in the same host cell, if efficient production can be
maintained, and homo- or hetero-tetrameric proteins are then
isolated from the medium. Ideally, the four heterologous proteins
are differentially labeled with various tag sequences (i.e., His
tag, FLAG tag, and Glu-Glu tag) to allow analysis of the
composition or purification of the resulting molecules.
Alternatively, the four components can be produced separately and
combined in deliberate ratios to result in the hetero-tetrameric
molecules desired. The VASP domains utilized in making these
hetero-trimeric molecules can be the same or different and the
fusion protein(s) can further comprise a linker sequence. In one
particular embodiment, the heterologous proteins used to form the
homo-tetrameric protein is the soluble domain of zB7R 1.
[0227] One result of the use of the VASP tetramerization domain of
the present invention is the ability to increase the affinity and
avidity of the heterologous protein for its ligand or binding
partner through the formation of the terameric form. By avidity, it
is meant the strength of binding of multiple molecules to a larger
molecule, a situation exemplified but not limited to the binding of
a complex antigen by an antibody. Such a characteristic would be
improved or formed for many heterologous proteins, for example, by
the formation of multiple binding sites for its ligand or ligands
through the tetramerization of the heterologous receptor using the
VASP domain. By affinity, it is meant the strength of binding of a
simple receptor-ligand system. Such a characteristic would be
improved for a subset of heterologous proteins using the
tetramerization domain of the present invention, for example, by
forming a binding site with better binding characteristics for a
single ligand through the tetramerization of the receptor. Avidity
and affinity can be measured using standard assays well known to
one of ordinary skill, for example, the methods described in the
examples below. An improvement in affinity or avidity occurs when
the affinity or avidity value (for example, affinity constant or
Ka) for the tetramerization domain-heterologous protein fusion and
its ligand is higher than for the heterologous protein alone and
its ligand. An alternative means of measuring these characteristics
is the equilibrium constant (Kd) where a decrease would be observed
with the improvement in affinity or avidity using the VASP
tetermerization domain of the present invention.
[0228] Biological activity of recombinant VASP-heterologous fusion
proteins is mediated by binding of the recombinant fusion protein
to a cognate molecule, such as a receptor or cross-receptor. A
cognate molecule is defined as a molecule which binds the
recombinant fusion protein in a non-covalent interaction based upon
the proper conformation of the recombinant fusion protein and the
cognate molecule. For example, for a recombinant fusion protein
comprising an extracellular region of a receptor, the cognate
molecule comprises a ligand which binds the extracellular region of
the receptor. Conversely, for a recombinant soluble fusion protein
comprising a ligand, the cognate molecule comprises a receptor (or
binding protein) which binds the ligand.
[0229] Binding of a recombinant fusion protein to a cognate
molecule is a marker for biological activity. Such binding activity
may be determined, for example, by competition for binding to the
binding domain of the cognate molecule (i.e. competitive binding
assays). One configuration of a competitive binding assay for a
recombinant fusion protein comprising a ligand uses a radiolabeled,
soluble receptor, and intact cells expressing a native form of the
ligand. Similarly, a competitive assay for a recombinant fusion
protein comprising a receptor uses a radiolabeled, soluble ligand,
and intact cells expressing a native form of the receptor. Such an
assay is described in Example 3. Instead of intact cells expressing
a native form of the cognate molecule, one could substitute
purified cognate molecule bound to a solid phase. Competitive
binding assays can be performed using standard methodology.
Qualitative or semi-quantitative results can be obtained by
competitive autoradiographic plate binding assays, or fluorescence
activated cell sorting, or Scatchard plots may be utilized to
generate quantitative results.
[0230] Biological activity may also be measured using bioassays
that are known in the art, such as a cell proliferation assay. An
exemplary bioassay is described in Example 4. The type of cell
proliferation assay used will depend upon the recombinant soluble
fusion protein. For example, a bioassay for a recombinant soluble
fusion protein that in its native form acts upon T cells will
utilize purified T cells obtained by methods that are known in the
art. Such bioassays include costimulation assays in which the
purified T cells are incubated in the presence of the recombinant
soluble fusion protein and a suboptimal level of a mitogen such as
Con A or PHA. Similarly, purified B cells will be used for a
recombinant soluble fusion protein that in its native form acts
upon B cells. Other types of cells may also be selected based upon
the cell type upon which the native form of the recombinant soluble
fusion protein acts. Proliferation is determined by measuring the
incorporation of a radiolabeled substance, such as .sup.3H
thymidine, according to standard methods.
[0231] Yet another type assay for determining biological activity
is induction of secretion of secondary molecules. For example,
certain proteins induce secretion of cytokines by T cells. T cells
are purified and stimulated with a recombinant soluble fusion
protein under the conditions required to induce cytokine secretion
(for example, in the presence of a comitogen). Induction of
cytokine secretion is determined by bioassay, measuring the
proliferation of a cytokine dependent cell line. Similarly,
induction of immunoglobulin secretion is determined by measuring
the amount of immunoglobulin secreted by purified B cells
stimulated with a recombinant soluble fusion protein that acts on B
cells in its native form, using a quantitative (or
semi-quantitative) assay such as an enzyme immunoassay.
[0232] If the binding partner for a particular heterologous protein
is unknown, the VASP-fusion protein can be used in a binding assay
to seek out that binding partner. One method of doing this, called
a secretion trap assay, is described in Example 5, although other
methods of using a VASP-fusion protein to identify binding partners
are well known to one of ordinary skill.
[0233] To assay the zB7R1 agonist and/or antagonist polyepeptides
and antibodies of the present invention, mammalian cells suitable
for use in expressing zB7R1-comprising receptors and transducing a
receptor-mediated signal include cells that express other receptor
subunits that may form a functional complex with zB7R1 (or
zB7R1RA). Within a preferred embodiment, the cell is dependent upon
an exogenously supplied hematopoietic growth factor for its
proliferation. Preferred cell lines of this type are the human TF-1
cell line (ATCC number CRL-2003) and the AML-193 cell line (ATCC
number CRL-9589), which are GM-CSF-dependent human leukemic cell
lines and BaF3 (Palacios and Steinmetz, Cell 41: 727-734, (1985))
which is an IL-3 dependent murine pre-B cell line. Other cell lines
include BHK, COS-1 and CHO cells. Suitable host cells can be
engineered to produce the necessary receptor subunits or other
cellular component needed for the desired cellular response. This
approach is advantageous because cell lines can be engineered to
express receptor subunits from any species, thereby overcoming
potential limitations arising from species specificity. Species
orthologs of the human receptor cDNA can be cloned and used within
cell lines from the same species, such as a mouse cDNA in the BaF3
cell line.
[0234] Cells expressing functional receptor are used within
screening assays. A variety of suitable assays are known in the
art. These assays are based on the detection of a biological
response in a target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by colorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT), (Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An
alternative assay format uses cells that are further engineered to
express a reporter gene. The reporter gene is linked to a promoter
element that is responsive to the receptor-linked pathway, and the
assay detects activation of transcription of the reporter gene. A
preferred promoter element in this regard is a serum response
element, or SRE. See, e.g., Shaw et al., Cell 56:563-572, (1989). A
preferred such reporter gene is a luciferase gene (de Wet et al.,
Mol. Cell. Biol. 7:725, (1987)). Expression of the luciferase gene
is detected by luminescence using methods known in the art (e.g.,
Baumgartner et al., J. Biol. Chem. 269:29094-29101, (1994);
Schenborn and Goiffin, Promega.sub.--Notes 41:11, 1993). Luciferase
activity assay kits are commercially available from, for example,
Promega Corp., Madison, Wisc. Target cell lines of this type can be
used to screen libraries of chemicals, cell-conditioned culture
media, fungal broths, soil samples, water samples, and the like.
For example, a bank of cell-conditioned media samples can be
assayed on a target cell to identify cells that produce
counter-receptor. Positive cells are then used to produce a cDNA
library in a mammalian expression vector, which is divided into
pools, transfected into host cells, and expressed. Media samples
from the transfected cells are then assayed, with subsequent
division of pools, re-transfection, subculturing, and re-assay of
positive cells to isolate a cloned cDNA encoding the
counter-receptor.
[0235] Several zB7R1 responsive cell lines are known in the art or
can be constructed, for example, the Baf3/DIRS1/cytoR11 cell line
(WIPO Publication No. WO 02/072607). Moreover several IL-22
responsive cell lines are known (Dumontier et al., J. Immunol.
164:1814-1819, 2000; Dumoutier, L. et al., Proc. Nat'l. Acad. Sci.
97:10144-10149, 2000; Xie M H et al., J. Biol. Chem. 275:
31335-31339, 2000; Kotenko S V et al., J. Biol. Chem.
276:2725-2732, 2001), as well as those that express the IL-22
receptor subunit zB7R1. For example, the following cells are
responsive to IL-22: TK-10 (Xie M H et al., supra.) (human renal
carcinoma); SW480 (ATCC No. CCL-228) (human colon adenocarcinoma);
HepG2 (ATCC No. HB-8065) (human hepatoma); PC12 (ATCC No. CRL-1721)
(murine neuronal cell model; rat pheochromocytoma); and MES13 (ATCC
No. CRL-1927) (murine kidney mesangial cell line). In addition,
some cell lines express zB7R1 (IL-22 receptor) are also candidates
for responsive cell lines to IL-22: A549 (ATCC No. CCL-185) (human
lung carcinoma); G-361 (ATCC No. CRL-1424) (human melanoma); and
Caki-1 (ATCC No. HTB-46) (human renal carcinoma). In addition,
IL-22-responsive cell lines can be constructed, for example, the
Baf3/cytoR11/CRF2-4 cell line described herein (WIPO Publication
No. WO 02/12345). These cells can be used in assays to assess the
functionality of zB7R1 as an zB7R1 or IL-22 antagonist or
anti-inflammatory factor.
7. PRODUCTION OF zB7R1 OR CD155 FUSION PROTEINS AND CONJUGATES
[0236] One general class of zB7R1 or CD155 analogs are variants
having an amino acid sequence that is a mutation of the amino acid
sequence disclosed herein. Another general class of zB7R1 or CD155
analogs is provided by anti-idiotype antibodies, and fragments
thereof, as described below. Moreover, recombinant antibodies
comprising anti-idiotype variable domains can be used as analogs
(see, for example, Monfardini et al., Proc. Assoc. Am. Physicians
108:420 (1996)). Since the variable domains of anti-idiotype zB7R1
antibodies mimic zB7R1, these domains can provide zB7R1 binding
activity. Methods of producing anti-idiotypic catalytic antibodies
are known to those of skill in the art (see, for example, Joron et
al., Ann. N Y Acad. Sci. 672:216 (1992), Friboulet et al., Appl.
Biochem. Biotechnol. 47:229(1994), and Avalle et al., Ann. N Y
Acad. Sci. 864:118(1998)).
[0237] Another approach to identifying zB7R1 or Cd155 analogs is
provided by the use of combinatorial libraries. Methods for
constructing and screening phage display and other combinatorial
libraries are provided, for example, by Kay et al., Phage Display
of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Pat.
No.5,783,384, Kay, et. al., U.S. Pat. No.5,747,334, and Kauffman et
al., U.S. Pat. No. 5,723,323.
[0238] zB7R1 and CD155 polypeptides have both in vivo and in vitro
uses. As an illustration, a soluble form of zB7R1 can be added to
cell culture medium to inhibit the effects of the zB7R1
counter-receptor produced by the cultured cells.
[0239] Fusion proteins of zB7R1 can be used to express zB7R1 in a
recombinant host, and to isolate the produced zB7R1. As described
below, particular zB7R1 fusion proteins also have uses in diagnosis
and therapy. One type of fusion protein comprises a peptide that
guides a zB7R1 polypeptide from a recombinant host cell. To direct
a zB7R1 polypeptide into the secretory pathway of a eukaryotic host
cell, a secretory signal sequence (also known as a signal peptide,
a leader sequence, prepro sequence or pre sequence) is provided in
the zB7R1 expression vector. While the secretory signal sequence
may be derived from zB7R1, a suitable signal sequence may also be
derived from another secreted protein or synthesized de novo. The
secretory signal sequence is operably linked to a zB7R1-encoding
sequence such that the two sequences are joined in the correct
reading frame and positioned to direct the newly synthesized
polypeptide into the secretory pathway of the host cell. Secretory
signal sequences are commonly positioned 5' to the nucleotide
sequence encoding the polypeptide of interest, although certain
secretory signal sequences may be positioned elsewhere in the
nucleotide sequence of interest (see, e.g., Welch et al., U.S. Pat.
No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).
[0240] Although the secretory signal sequence of zB7R1 or another
protein produced by mammalian cells (e.g., tissue-type plasminogen
activator signal sequence, as described, for example, in U.S. Pat.
No. 5,641,655) is useful for expression of zB7R1 in recombinant
mammalian hosts, a yeast signal sequence is preferred for
expression in yeast cells. Examples of suitable yeast signal
sequences are those derived from yeast mating phermone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0241] zB7R1 soluble receptor polypeptides can be prepared by
expressing a truncated DNA encoding the extracellular domain, for
example, a polypeptide which contains SEQ ID NO:2 or 5, or the
corresponding region of a non-human receptor. It is preferred that
the extracellular domain polypeptides be prepared in a form
substantially free of transmembrane and intracellular polypeptide
segments. To direct the export of the receptor domain from the host
cell, the receptor DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide. To facilitate
purification of the secreted receptor domain, a C-terminal
extension, such as a poly-histidine tag, substance P, Flag.TM.
peptide (Hopp et al., Biotechnology 6:1204-1210, (1988); available
from Eastman Kodak Co., New Haven, Conn.) or another polypeptide or
protein for which an antibody or other specific binding agent is
available, can be fused to the receptor polypeptide. Moreover,
zB7R1 antigenic epitopes from the extracellular cytokine binding
domains are also prepared as described above.
[0242] In an alternative approach, a receptor extracellular domain
of zB7R1 or other B7 receptor component can be expressed as a
fusion with immunoglobulin heavy chain constant regions, typically
an F.sub.c fragment, which contains two constant region domains and
a hinge region but lacks the variable region (See, Sledziewski, A Z
et al., U.S. Pat. Nos. 6,018,026 and 5,750,375). The soluble zB7R1
polypeptides of the present invention include such fusions. Such
fusions are typically secreted as multimeric molecules wherein the
Fc portions are disulfide bonded to each other and two receptor
polypeptides are arrayed in closed proximity to each other. Fusions
of this type can be used to affinity purify the cognate
counter-receptor from solution, as an in vitro assay tool, to
block, inhibit or reduce signals in vitro by specifically titrating
out counter-receptor, and as antagonists in vivo by administering
them parenterally to bind circulating counter-receptor and clear it
from the circulation. To purify counter-receptor, a zB7R1-Ig
chimera is added to a sample containing the counter-receptor (e.g.,
cell-conditioned culture media or tissue extracts) under conditions
that facilitate receptor-counter-receptor binding (typically
near-physiological temperature, pH, and ionic strength). The
chimera-counter-receptor complex is then separated by the mixture
using protein A, which is immobilized on a solid support (e.g.,
insoluble resin beads). The counter-receptor is then eluted using
conventional chemical techniques, such as with a salt or pH
gradient. In the alternative, the chimera itself can be bound to a
solid support, with binding and elution carried out as above. The
chimeras may be used in vivo to regulate inflammatory responses
including acute phase responses such as serum amyloid A (SAA),
C-reactive protein (CRP), and the like. Chimeras with high binding
affinity are administered parenterally (e.g., by intramuscular,
subcutaneous or intravenous injection). Circulating molecules bind
counter-receptor and are cleared from circulation by normal
physiological processes. For use in assays, the chimeras are bound
to a support via the F.sub.c region and used in an ELISA
format.
[0243] To assist in isolating anti-zB7R1 and binding partners of
the present invention, an assay system that uses a
counter-receptor-binding receptor (or an antibody, one member of a
complement/anti-complement pair) or a binding fragment thereof, and
a commercially available biosensor instrument (BIAcore, Pharmacia
Biosensor, Piscataway, N.J.) may be advantageously employed. Such
receptor, antibody, member of a complement/anti-complement pair or
fragment is immobilized onto the surface of a receptor chip. Use of
this instrument is disclosed by Karlsson, J. Immunol. Methods
145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or fragment is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within the flow cell.
A test sample is passed through the cell. If a counter-receptor,
epitope, or opposite member of the complement/anti-complement pair
is present in the sample, it will bind to the immobilized receptor,
antibody or member, respectively, causing a change in the
refractive index of the medium, which is detected as a change in
surface plasmon resonance of the gold film. This system allows the
determination of on- and off-rates, from which binding affinity can
be calculated, and assessment of stoichiometry of binding.
Alternatively, counter-receptor/receptor binding can be analyzed
using SELDI.TM. technology (Ciphergen, Inc., Palo Alto, Calif.).
Moreover, BIACORE technology, described above, can be used to be
used in competition experiments to determine if different
momnoclonal antibodies bind the same or different epitopes on the
zB7R1 polypeptide, and as such, be used to aid in epitope mapping
of antibodies of the present invention.
[0244] Counter-receptor-binding polypeptides (i.e. CD155) can also
be used within other assay systems known in the art. Such systems
include Scatchard analysis for determination of binding affinity
(see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and
calorimetric assays (Cunningham et al., Science 253:545-48, 1991;
Cunningham et al., Science 245:821-25, 1991).
[0245] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a soluble zB7R1 receptor
can be prepared as a fusion to a dimerizing protein as disclosed in
U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred dimerizing
proteins in this regard include immunoglobulin constant region
domains, e.g., IgG.gamma.1, and the human .kappa. light chain.
Immunoglobulin-soluble zB7R1 fusions can be expressed in
genetically engineered cells to produce a variety of multimeric
zB7R1 receptor analogs. Auxiliary domains can be fused to soluble
zB7R1 receptor to target them to specific cells, tissues, or
macromolecules (e.g., collagen, or cells expressing the zB7R1
counter-receptors). A zB7R1 polypeptide can be fused to two or more
moieties, such as an affinity tag for purification and a targeting
domain. Polypeptide fusions can also comprise one or more cleavage
sites, particularly between domains. See, Tuan et al., Connective
Tissue Research 34:1-9, 1996.
[0246] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, zB7R1 can be expressed as a fusion protein
comprising a glutathione S-transferase polypeptide. Glutathione
S-transferease fusion proteins are typically soluble, and easily
purifiable from E. coli lysates on immobilized glutathione columns.
In similar approaches, a zB7R1 fusion protein comprising a maltose
binding protein polypeptide can be isolated with an amylose resin
column, while a fusion protein comprising the C-terminal end of a
truncated Protein A gene can be purified using IgG-Sepharose.
Established techniques for expressing a heterologous polypeptide as
a fusion protein in a bacterial cell are described, for example, by
Williams et al., "Expression of Foreign Proteins in E. coli Using
Plasmid Vectors and Purification of Specific Polyclonal
Antibodies," in DNA Cloning 2: A Practical Approach, 2.sup.nd
Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University
Press 1995). In addition, commercially available expression systems
are available. For example, the PINPOINT Xa protein purification
system (Promega Corporation; Madison, Wisc.) provides a method for
isolating a fusion protein comprising a polypeptide that becomes
biotinylated during expression with a resin that comprises
avidin.
[0247] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyfistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0248] Another form of fusion protein comprises a zB7R1 polypeptide
and an immunoglobulin heavy chain constant region, typically an
F.sub.c fragment, which contains two or three constant region
domains and a hinge region but lacks the variable region. As an
illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
In this fusion protein, an illustrative Fc moiety is a human
.gamma.4 chain, which is stable in solution and has little or no
complement activating activity. Accordingly, the present invention
contemplates a zB7R1 fusion protein that comprises a zB7R1 moiety
and a human Fc fragment, wherein the C-terminus of the zB7R1 moiety
is attached to the N-terminus of the Fc fragment via a peptide
linker. The zB7R1 moiety can be a zB7R1 molecule or a fragment
thereof. For example, a fusion protein can comprise the amino acid
of SEQ ID NO:3 and an Fc fragment (e.g., a human Fc fragment).
[0249] In another variation, a zB7R1 fusion protein comprises an
IgG sequence, a zB7R1 moiety covalently joined to the aminoterminal
end of the IgG sequence, and a signal peptide that is covalently
joined to the aminoterminal of the zB7R1 moiety, wherein the IgG
sequence consists of the following elements in the following order:
a hinge region, a CH.sub.2 domain, and a CH.sub.3 domain.
Accordingly, the IgG sequence lacks a CH.sub.1 domain. The zB7R1
moiety displays a zB7R1 activity, as described herein, such as the
ability to bind with a zB7R1 counter-receptor. This general
approach to producing fusion proteins that comprise both antibody
and nonantibody portions has been described by LaRochelle et al.,
EP 742830 (WO 95/21258).
[0250] Fusion proteins comprising a zB7R1 moiety and an Fc moiety
can be used, for example, as an in vitro assay tool. For example,
the presence of a zB7R1 counter-receptor in a biological sample can
be detected using a zB7R1-immunoglobulin fusion protein, in which
the zB7R1 moiety is used to bind the counter-receptor, and a
macromolecule, such as Protein A or anti-Fc antibody, is used to
bind the fusion protein to a solid support. Such systems can be
used to identify agonists and antagonists that interfere with the
binding of zB7R1 to its counter-receptor.
[0251] Other examples of antibody fusion proteins include
polypeptides that comprise an antigen-binding domain and a zB7R1
fragment that contains a zB7R1 extracellular domain. Such molecules
can be used to target particular tissues for the benefit of zB7R1
binding activity.
[0252] The present invention further provides a variety of other
polypeptide fusions. For example, part or all of a domain(s)
conferring a biological function can be swapped between zB7R1 of
the present invention with the functionally equivalent domain(s)
from another member of the cytokine receptor family. Polypeptide
fusions can be expressed in recombinant host cells to produce a
variety of zB7R1 fusion analogs. A zB7R1 polypeptide can be fused
to two or more moieties or domains, such as an affinity tag for
purification and a targeting domain. Polypeptide fusions can also
comprise one or more cleavage sites, particularly between domains.
See, for example, Tuan et al., Connective Tissue Research 34:1
(1996).
[0253] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0254] zB7R1 binding domains can be further characterized by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction
or photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids of zB7R1 counter-receptor agonists. See,
for example, de Vos et al., Science 255:306 (1992), Smith et al.,
J. Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett.
309:59 (1992).
[0255] The present invention also contemplates chemically modified
zB7R1 compositions, in which a zB7R1 polypeptide is linked with a
polymer. Illustrative zB7R1 polypeptides are soluble polypeptides
that lack a functional transmembrane domain, such as a polypeptide
consisting of amino acid residues SEQ ID NO:3. Typically, the
polymer is water soluble so that the zB7R1 conjugate does not
precipitate in an aqueous environment, such as a physiological
environment. An example of a suitable polymer is one that has been
modified to have a single reactive group, such as an active ester
for acylation, or an aldehyde for alkylation. In this way, the
degree of polymerization can be controlled. An example of a
reactive aldehyde is polyethylene glycol propionaldehyde, or
mono-(C1-C10)alkoxy, or aryloxy derivatives thereof (see, for
example, Harris, et al., U.S. Pat. No. 5,252,714). The polymer may
be branched or unbranched. Moreover, a mixture of polymers can be
used to produce zB7R1 conjugates.
[0256] zB7R1 conjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties.
Suitable water-soluble polymers include polyethylene glycol (PEG),
monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG,
poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
dextran, cellulose, or other carbohydrate-based polymers. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A zB7R1
conjugate can also comprise a mixture of such water-soluble
polymers.
[0257] One example of a zB7R1 conjugate comprises a zB7R1 moiety
and a polyalkyl oxide moiety attached to the N-terminus of the
zB7R1 moiety. PEG is one suitable polyalkyl oxide. As an
illustration, zB7R1 can be modified with PEG, a process known as
"PEGylation." PEGylation of zB7R1 can be carried out by any of the
PEGylation reactions known in the art (see, for example, EP 0 154
316, Delgado et al., Critical Reviews in Therapeutic Drug Carrier
Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet.
27:290 (1994), and Francis et al., Int J Hematol 68:1 (1998)). For
example, PEGylation can be performed by an acylation reaction or by
an alkylation reaction with a reactive polyethylene glycol
molecule. In an alternative approach, zB7R1 conjugates are formed
by condensing activated PEG, in which a terminal hydroxy or amino
group of PEG has been replaced by an activated linker (see, for
example, Karasiewicz et al., U.S. Pat. No. 5,382,657).
[0258] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with a zB7R1 polypeptide. An example
of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between zB7R1 and a water soluble
polymer: amide, carbamate, urethane, and the like. Methods for
preparing PEGylated zB7R1 by acylation will typically comprise the
steps of (a) reacting a zB7R1 polypeptide with PEG (such as a
reactive ester of an aldehyde derivative of PEG) under conditions
whereby one or more PEG groups attach to zB7R1, and (b) obtaining
the reaction product(s). Generally, the optimal reaction conditions
for acylation reactions will be determined based upon known
parameters and desired results. For example, the larger the ratio
of PEG:zB7R1, the greater the percentage of polyPEGylated zB7R1
product.
[0259] The product of PEGylation by acylation is typically a
polyPEGylated zB7R1 product, wherein the lysine .epsilon.-amino
groups are PEGylated via an acyl linking group. An example of a
connecting linkage is an amide. Typically, the resulting zB7R1 will
be at least 95% mono-, di-, or tri-pegylated, although some species
with higher degrees of PEGylation may be formed depending upon the
reaction conditions. PEGylated species can be separated from
unconjugated zB7R1 polypeptides using standard purification
methods, such as dialysis, ultrafiltration, ion exchange
chromatography, affinity chromatography, and the like.
[0260] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with zB7R1 in the presence of a
reducing agent. PEG groups can be attached to the polypeptide via a
--CH.sub.2--NH group.
[0261] Moreover, anti-zB7R1 antibodies or antibody fragments of the
present invention can be PEGylated using methods in the art and
described herein.
[0262] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
E-amino groups of the lysine residues and the .alpha.-amino group
of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups. The
present invention provides a substantially homogenous preparation
of zB7R1 monopolymer conjugates.
[0263] Reductive alkylation to produce a substantially homogenous
population of monopolymer zB7R1 conjugate molecule can comprise the
steps of: (a) reacting a zB7R1 polypeptide with a reactive PEG
under reductive alkylation conditions at a pH suitable to permit
selective modification of the .alpha.-amino group at the amino
terminus of the zB7R1, and (b) obtaining the reaction product(s).
The reducing agent used for reductive alkylation should be stable
in aqueous solution and able to reduce only the Schiff base formed
in the initial process of reductive alkylation. Illustrative
reducing agents include sodium borohydride, sodium
cyanoborohydride, dimethylamine borane, trimethylamine borane, and
pyridine borane.
[0264] For a substantially homogenous population of monopolymer
zB7R1 conjugates, the reductive alkylation reaction conditions are
those that permit the selective attachment of the water-soluble
polymer moiety to the N-terminus of zB7R1. Such reaction conditions
generally provide for pKa differences between the lysine amino
groups and the .alpha.-amino group at the N-terminus. The pH also
affects the ratio of polymer to protein to be used. In general, if
the pH is lower, a larger excess of polymer to protein will be
desired because the less reactive the N-terminal .alpha.-group, the
more polymer is needed to achieve optimal conditions. If the pH is
higher, the polymer:zB7R1 need not be as large because more
reactive groups are available. Typically, the pH will fall within
the range of 3 to 9, or 3 to 6. This method can be employed for
making zB7R1-comprising homodimeric, heterodimeric or multimeric
soluble receptor conjugates.
[0265] Another factor to consider is the molecular weight of the
water-soluble polymer. Generally, the higher the molecular weight
of the polymer, the fewer number of polymer molecules which may be
attached to the protein. For PEGylation reactions, the typical
molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to
about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of
water-soluble polymer to zB7R1 will generally be in the range of
1:1 to 100:1. Typically, the molar ratio of water-soluble polymer
to zB7R1 will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1 for
monoPEGylation.
[0266] General methods for producing conjugates comprising a
polypeptide and water-soluble polymer moieties are known in the
art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738,846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997)). This method can be employed for making
zB7R1-comprising homodimeric, heterodimeric or multimeric soluble
receptor conjugates.
[0267] The present invention contemplates compositions comprising a
peptide or polypeptide, such as a soluble receptor or antibody
described herein. Such compositions can further comprise a carrier.
The carrier can be a conventional organic or inorganic carrier.
Examples of carriers include water, buffer solution, alcohol,
propylene glycol, macrogol, sesame oil, corn oil, and the like.
8. ISOLATION OF zB7R1 OR CD155 POLYPEPTIDES
[0268] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95%, such as 96%, 97%, 98%,
or greater than 99% purity with respect to contaminating
macromolecules, particularly other proteins and nucleic acids, and
free of infectious and pyrogenic agents. The polypeptides of the
present invention may also be purified to a pharmaceutically pure
state, which is greater than 99.9% pure. In certain preparations,
purified polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin.
[0269] Fractionation and/or conventional purification methods can
be used to obtain preparations of zB7R1 (or CD155) purified from
natural sources (e.g., human tissue sources), synthetic zB7R1
polypeptides, and recombinant zB7R1 polypeptides and fusion zB7R1
polypeptides purified from recombinant host cells. In general,
ammonium sulfate precipitation and acid or chaotrope extraction may
be used for fractionation of samples. Exemplary purification steps
may include hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable chromatographic
media include derivatized dextrans, agarose, cellulose,
polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and
Q derivatives are suitable. Exemplary chromatographic media include
those media derivatized with phenyl, butyl, or octyl groups, such
as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate
moieties.
[0270] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0271] Additional variations in zB7R1 (or CD155) isolation and
purification can be devised by those of skill in the art. For
example, anti-zB7R1 antibodies, obtained as described below, can be
used to isolate large quantities of protein by immunoaffinity
purification.
[0272] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification. Moreover, the counter-receptor-binding properties of
zB7R1 extracellular domain can be exploited for purification, for
example, of zB7R1-comprising soluble receptors; for example, by
using affinity chromatography wherein the appropriate
counter-receptor is bound to a column and the zB7R1-comprising
receptor is bound and subsequently eluted using standard
chromatography methods.
[0273] zB7R1 (or CD155) polypeptides or fragments thereof may also
be prepared through chemical synthesis, as described above. zB7R1
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; PEGylated or non-PEGylated; and may or may not
include an initial methionine amino acid residue.
9. PRODUCTION OF ANTIBODIES TO zB7R1 PROTEINS
[0274] Antibodies to zB7R1 can be obtained, for example, using the
product of a zB7R1 expression vector or zB7R1 isolated from a
natural source as an antigen. Particularly useful anti-zB7R1
antibodies "bind specifically" with zB7R1. Antibodies are
considered to be specifically binding if the antibodies exhibit at
least one of the following two properties: (1) antibodies bind to
zB7R1 with a threshold level of binding activity, and (2)
antibodies do not significantly cross-react with polypeptides
related to zB7R1.
[0275] With regard to the first characteristic, antibodies
specifically bind if they bind to a zB7R1 polypeptide, peptide or
epitope with a binding affinity (K.sub.a) of 10.sup.6 M.sup.-1 or
greater, preferably 10.sup.7 M.sup.-1 or greater, more preferably
10.sup.8 M.sup.-1 or greater, and most preferably 109 M.sup.-1 or
greater. The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)).
With regard to the second characteristic, antibodies do not
significantly cross-react with related polypeptide molecules, for
example, if they detect zB7R1, but not presently known polypeptides
using a standard Western blot analysis. Examples of known related
polypeptides include known cytokine receptors.
[0276] Anti-zB7R1 antibodies can be produced using antigenic zB7R1
epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, or between 15 to about 30
amino acids contained within SEQ ID NO:2 or another amino acid
sequence disclosed herein. However, peptides or polypeptides
comprising a larger portion of an amino acid sequence of the
invention, containing from 30 to 50 amino acids, or any length up
to and including the entire amino acid sequence of a polypeptide of
the invention, also are useful for inducing antibodies that bind
with zB7R1. It is desirable that the amino acid sequence of the
epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues, while hydrophobic residues are
typically avoided). Moreover, amino acid sequences containing
proline residues may be also be desirable for antibody
production.
[0277] As an illustration, potential antigenic sites in zB7R1 were
identified using the Jameson-Wolf method, Jameson and Wolf, CABIOS
4:181, (1988), as implemented by the PROTEAN program (version 3.14)
of LASERGENE (DNASTAR; Madison, Wisc.). Default parameters were
used in this analysis.
[0278] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), conformation table=64 proteins;
.alpha. region threshold=103; .beta. region threshold=105;
Garnier-Robson parameters: .alpha. and .beta. decision
constants=0). In the sixth subroutine, flexibility parameters and
hydropathy/solvent accessibility factors were combined to determine
a surface contour value, designated as the "antigenic index."
Finally, a peak broadening function was applied to the antigenic
index, which broadens major surface peaks by adding 20, 40, 60, or
80% of the respective peak value to account for additional free
energy derived from the mobility of surface regions relative to
interior regions. This calculation was not applied, however, to any
major peak that resides in a helical region, since helical regions
tend to be less flexible.
[0279] The results of this analysis indicated that the following
amino acid sequences of SEQ ID NO:2 would provide suitable
antigenic peptides: Hopp/Woods hydrophilicity profiles can be used
to determine regions that have the most antigenic potential within
SEQ ID NO:3 (Hopp et al., Proc. Natl. Acad. Sci.78:3824-3828, 1981;
Hopp, J. Immun. Meth. 88:1-18, 1986 and Triquier et al., Protein
Engineering 11:153-169, 1998). The profile is based on a sliding
six-residue window. Buried G, S, and T residues and exposed H, Y,
and W residues were ignored. Moreover, zB7R1 antigenic epitopes
within SEQ ID NO:2 as predicted by a Jameson-Wolf plot, e.g., using
DNASTAR Protean program (DNASTAR, Inc., Madison, Wisc.) serve as
preferred antigenic epitopes, and can be determined by one of skill
in the art. Such antigenic epitopes include (1) (1) amino acid
residues 80 to 86 of SEQ ID NO:2; (2) amino acid residues 163 to
170 of SEQ ID NO:2; (3) amino acid residues 163 to 190 of SEQ ID
NO:2; (4) amino acid residues 175 to 190 of SEQ ID NO:2; and (5)
amino acid residues 211 to 221 of SEQ ID NO:2. The present
invention contemplates the use of any one of antigenic peptides 1
to 5 to generate antibodies to zB7R1 or as a tool to screen or
identify neutralizing monoclonal antibodies of the present
invention. The present invention contemplates the use of any
antigenic peptides or epitopes described herein to generate
antibodies to zB7R1, as well as to identify and screen anti-zB7R1
monoclonal antibodies that may bind, agonize, block, inhibit,
reduce, increase, antagonize or neutralize the activity of a zB7R1
counter-receptor.
[0280] Polyclonal antibodies to recombinant zB7R1 protein or to
zB7R1 isolated from natural sources can be prepared using methods
well-known to those of skill in the art. See, for example, Green et
al., "Production of Polyclonal Antisera," in Immunochemical
Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using
plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a zB7R1 polypeptide can be increased through the
use of an adjuvant, such as alum (aluminum hydroxide) or Freund's
complete or incomplete adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of
zB7R1 or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like," such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0281] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-zB7R1 antibody of the present
invention may also be derived from a subhuman primate antibody.
General techniques for raising diagnostically and therapeutically
useful antibodies in baboons may be found, for example, in
Goldenberg et al., international patent publication No. WO
91/11465, and in Losman et al., Int. J. Cancer 46:310 (1990).
[0282] Alternatively, monoclonal anti-zB7R1 antibodies can be
generated. Rodent monoclonal antibodies to specific antigens may be
obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0283] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a zB7R1 gene product, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0284] In addition, an anti-zB7R1 antibody of the present invention
may be derived from a human monoclonal antibody. Human monoclonal
antibodies are obtained from transgenic mice that have been
engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al., Int. Immun. 6:579 (1994).
[0285] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0286] For particular uses, it may be desirable to prepare
fragments of anti-zB7R1 antibodies. Such antibody fragments can be
obtained, for example, by proteolytic hydrolysis of the antibody.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. As an illustration,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0287] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0288] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0289] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0290] As an illustration, a scFV can be obtained by exposing
lymphocytes to zB7R1 polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled zB7R1 protein or peptide).
Genes encoding polypeptides having potential zB7R1 polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides which interact with a
known target which can be a protein or polypeptide, such as a
counter-receptor or receptor, a biological or synthetic
macromolecule, or organic or inorganic substances. Techniques for
creating and screening such random peptide display libraries are
known in the art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et
al., U.S. Pat. No. 4,946,778, Ladner et al., U.S. Pat. No.
5,403,484, Ladner et al., U.S. Pat. No. 5,571,698, and Kay et al.,
Phage Display of Peptides and Proteins (Academic Press, Inc. 1996))
and random peptide display libraries and kits for screening such
libraries are available commercially, for instance from CLONTECH
Laboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the zB7R1 sequences disclosed
herein to identify proteins which bind to zB7R1.
[0291] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0292] Alternatively, an anti-zB7R1 antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0293] Moreover, anti-zB7R1 antibodies or antibody fragments of the
present invention can be PEGylated using methods in the art and
described herein.
[0294] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-zB7R1 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-zB7R1 antibodies or antibody fragments as immunogens with the
techniques, described above. As another alternative, humanized
anti-idiotype antibodies or subhuman primate anti-idiotype
antibodies can be prepared using the above-described techniques.
Methods for producing anti-idiotype antibodies are described, for
example, by Irie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S.
Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.
77:1875 (1996).
[0295] An anti-zB7R1 antibody can be conjugated with a detectable
label to form an anti-zB7R1 immunoconjugate. Suitable detectable
labels include, for example, a radioisotope, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label or
colloidal gold. Methods of making and detecting such
detectably-labeled immunoconjugates are well-known to those of
ordinary skill in the art, and are described in more detail
below.
[0296] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0297] Anti-zB7R1 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0298] Alternatively, anti-zB7R1 immunoconjugates can be detectably
labeled by coupling an antibody component to a chemiluminescent
compound. The presence of the chemiluminescent-tagged
immunoconjugate is determined by detecting the presence of
luminescence that arises during the course of a chemical reaction.
Examples of chemiluminescent labeling compounds include luminol,
isoluminol, an aromatic acridinium ester, an imidazole, an
acridinium salt and an oxalate ester.
[0299] Similarly, a bioluminescent compound can be used to label
anti-zB7R1 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0300] Alternatively, anti-zB7R1 immunoconjugates can be detectably
labeled by linking an anti-zB7R1 antibody component to an enzyme.
When the anti-zB7R1-enzyme conjugate is incubated in the presence
of the appropriate substrate, the enzyme moiety reacts with the
substrate to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorometric or visual means.
Examples of enzymes that can be used to detectably label
polyspecific immunoconjugates include .beta.-galactosidase, glucose
oxidase, peroxidase and alkaline phosphatase.
[0301] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to anti-zB7R1 antibodies can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein et al.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0302] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-zB7R1 antibodies that have
been conjugated with avidin, streptavidin, and biotin (see, for
example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods
In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,
"Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0303] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0304] The present invention also contemplates kits for performing
an immunological diagnostic assay for zB7R1 gene expression. Such
kits comprise at least one container comprising an anti-zB7R1
antibody, or antibody fragment. A kit may also comprise a second
container comprising one or more reagents capable of indicating the
presence of zB7R1 antibody or antibody fragments. Examples of such
indicator reagents include detectable labels such as a radioactive
label, a fluorescent label, a chemiluminescent label, an enzyme
label, a bioluminescent label, colloidal gold, and the like. A kit
may also comprise a means for conveying to the user that zB7R1
antibodies or antibody fragments are used to detect zB7R1 protein.
For example, written instructions may state that the enclosed
antibody or antibody fragment can be used to detect zB7R1. The
written material can be applied directly to a container, or the
written material can be provided in the form of a packaging
insert.
10. USE OF ANTI-zB7R1 ANTIBODIES TO AGONIZE OR ANTAGONIZE zB7R1
BINDING TO ITS COUNTER-RECEPTOR
[0305] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to soluble zB7R1 receptor polypeptides or fragments thereof, such
as antigenic epitopes, and selection of antibody display libraries
in phage or similar vectors (for instance, through use of
immobilized or labeled soluble zB7R1 receptor polypeptides or
fragments thereof, such as antigenic epitopes). Genes encoding
polypeptides having potential binding domains such as soluble zB7R1
receptor polypeptides or fragments thereof, such as antigenic
epitopes can be obtained by screening random peptide libraries
displayed on phage (phage display) or on bacteria, such as E. coli.
Nucleotide sequences encoding the polypeptides can be obtained in a
number of ways, such as through random mutagenesis and random
polynucleotide synthesis. These random peptide display libraries
can be used to screen for peptides that interact with a known
target that can be a protein or polypeptide, such as a
counter-receptor (i.e. CD155) or receptor, a biological or
synthetic macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display
libraries are known in the art (Ladner et al., U.S. Pat. No.
5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al.,
U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698)
and random peptide display libraries and kits for screening such
libraries are available commercially, for instance from Clontech
(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the soluble zB7R1 receptor
polypeptides or fragments thereof, such as antigenic epitope
polypeptide sequences disclosed herein to identify proteins which
bind to zB7R1-comprising receptor polypeptides. These "binding
polypeptides," which interact with soluble zB7R1-comprising
receptor polypeptides, can be used for tagging cells; for isolating
homolog polypeptides by affinity purification; they can be directly
or indirectly conjugated to drugs, toxins, radionuclides and the
like. These binding polypeptides can also be used in analytical
methods such as for screening expression libraries and for
agonizing and/or neutralizing activity, e.g., for binding,
blocking, inhibiting, reducing, antagonizing or neutralizing
interaction between zB7R1 and its counter-receptor. The binding
polypeptides can also be used for diagnostic assays for determining
circulating levels of soluble zB7R1-comprising receptor
polypeptides; for detecting or quantitating soluble or non-soluble
zB7R1-comprising receptors as marker of underlying pathology or
disease. These binding polypeptides can also act as "antagonists"
to block or inhibit soluble or membrane-bound zB7R1 monomeric
receptor or zB7R1 homodimeric, heterodimeric or multimeric
polypeptide binding (e.g. to counter-receptor) and signal
transduction in vitro and in vivo. Again, these binding
polypeptides serve as anti-zB7R1 monomeric receptor or anti-zB7R1
homodimeric, heterodimeric or multimeric polypeptides and are
useful for inhibiting zB7R1 activity, as well as zB7R1
counter-receptor activity or protein-binding. Antibodies raised to
the natural receptor complexes of the present invention, and
zB7R1-epitope-binding antibodies, and anti-zB7R1 neutralizing
monoclonal antibodies may be preferred embodiments, as they may act
more specifically against the zB7R1 and can inhibit its binding to
its counter-receptor. Moreover, the agonistic, antagonistic and
binding activity of the antibodies of the present invention can be
assayed in a zB7R1 proliferation, signal trap, luciferase or
binding assays in the presence of its counter-receptor or any other
B7 family receptor, and zB7R1-comprising soluble receptors, and
other biological or biochemical assays described herein.
[0306] Antibodies to zB7R1 receptor polypeptides (e.g., antibodies
to SEQ ID NO:2 or 5) or fragments thereof, such as antigenic
epitopes may be used for inhibiting the inflammatory effects of
zB7R1 in vivo, for theraputic use against rheumatoid arthritis,
psoriasis, atopic dermatitis, inflammatory skin conditions,
endotoxemia, arthritis, asthma, IBD, colitis, psoriatic arthritis
or other B7-induced inflammatory conditions; tagging cells that
express zB7R1 receptors; for isolating soluble zB7R1-comprising
receptor polypeptides by affinity purification; for diagnostic
assays for determining circulating levels of soluble
zB7R1-comprising receptor polypeptides; for detecting or
quantitating soluble zB7R1-comprising receptors as marker of
underlying pathology or disease; in analytical methods employing
FACS; for screening expression libraries; for generating
anti-idiotypic antibodies that can act as zB7R1 agonists; and as
neutralizing antibodies or as antagonists to bind, block, inhibit,
reduce, or antagonize zB7R1 receptor function, or to bind, block,
inhibit, reduce, antagonize or neutralize zB7R1 activity in vitro
and in vivo. Suitable direct tags or labels include radionuclides,
enzymes, substrates, cofactors, biotin, inhibitors, fluorescent
markers, chemiluminescent markers, magnetic particles and the like;
indirect tags or labels may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to soluble zB7R1-comprising receptor polypeptides, or
fragments thereof may be used in vitro to detect denatured or
non-denatured zB7R1-comprising receptor polypeptides or fragments
thereof in assays, for example, Western Blots or other assays known
in the art.
[0307] Antibodies to soluble zB7R1 receptor or soluble zB7R1
homodimeric, heterodimeric or multimeric receptor polypeptides are
useful for tagging cells that express the corresponding receptors
and assaying their expression levels, for affinity purification,
within diagnostic assays for determining circulating levels of
receptor polypeptides, analytical methods employing
fluorescence-activated cell sorting. Moreover, divalent antibodies,
and anti-idiotypic antibodies may be used as agonists to mimic the
effect of zB7R1.
[0308] Antibodies herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
For instance, antibodies or binding polypeptides which recognize
soluble zB7R1 receptor or soluble zB7R1 homodimeric, heterodimeric
or multimeric receptor polypeptides can be used to identify or
treat tissues or organs that express a corresponding
anti-complementary molecule (i.e., a zB7R1-comprising soluble or
membrane-bound receptor). More specifically, antibodies to soluble
zB7R1-comprising receptor polypeptides, or bioactive fragments or
portions thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues or organs
that express the zB7R1-comprising receptor such as zB7R1-expressing
cancers.
[0309] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind zB7R1-comprising receptor
polypeptides, such as "binding polypeptides," (including binding
peptides disclosed above), antibodies, or bioactive fragments or
portions thereof. Suitable detectable molecules include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles
and the like. Suitable cytotoxic molecules may be directly or
indirectly attached to the polypeptide or antibody, and include
bacterial or plant toxins (for instance, diphtheria toxin,
Pseudomonas exotoxin, ricin, abrin and the like), as well as
therapeutic radionuclides, such as iodine-131, rhenium-188 or
yttrium-90 (either directly attached to the polypeptide or
antibody, or indirectly attached through means of a chelating
moiety, for instance). Binding polypeptides or antibodies may also
be conjugated to cytotoxic drugs, such as adriamycin. For indirect
attachment of a detectable or cytotoxic molecule, the detectable or
cytotoxic molecule can be conjugated with a member of a
complementary/anticomplementary pair, where the other member is
bound to the binding polypeptide or antibody portion. For these
purposes, biotin/streptavidin is an exemplary
complementary/anticomplementary pair.
[0310] In another embodiment, binding polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for targeted
cell or tissue inhibition or ablation (for instance, to treat
cancer cells or tissues). Alternatively, if the binding polypeptide
has multiple functional domains (i.e., an activation domain or a
counter-receptor binding domain, plus a targeting domain), a fusion
protein including only the targeting domain may be suitable for
directing a detectable molecule, a cytotoxic molecule or a
complementary molecule to a cell or tissue type of interest. In
instances where the fusion protein including only a single domain
includes a complementary molecule, the anti-complementary molecule
can be conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell/tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0311] Alternatively, zB7R1 receptor binding polypeptides or
antibody fusion proteins described herein can be used for enhancing
in vivo killing of target tissues by directly stimulating a zB7R1
receptor-modulated apoptotic pathway, resulting in cell death of
hyperproliferative cells expressing zB7R1-comprising receptors.
11. THERAPEUTIC USES OF POLYPEPTIDES HAVING zB7R1 ACTIVITY OR
ANTIBODIES TO zB7R1
[0312] Amino acid sequences having soluble zB7R1 activity can be
used to modulate the immune system by binding zB7R1
counter-receptors such as CD155, and thus, preventing the binding
of zB7R1 counter-receptor with endogenous zB7R1 receptor. zB7R1
antagonists, such as anti-zB7R1 antibodies, can also be used to
modulate the immune system by inhibiting the binding of zB7R1
counter-receptor with the endogenous zB7R1 receptor. Accordingly,
the present invention includes the use of proteins, polypeptides,
and peptides having zB7R1 activity (such as soluble zB7R1
polypeptides, zB7R1 polypeptide fragments, zB7R1 analogs (e.g.,
anti-zB7R1 anti-idiotype antibodies), and zB7R1 fusion proteins) to
a subject which lacks an adequate amount of this polypeptide, or
which produces an excess of zB7R1 counter-receptor. zB7R1
antagonists (e.g., anti-zB7R1 antibodies) can be also used to treat
a subject which produces an excess of either zB7R1 counter-receptor
or zB7R1. Suitable subjects include mammals, such as humans. For
example, such zB7R1 polypeptides and anti-zB7R1 antibodies are
useful in binding, blocking, inhibiting, reducing, antagonizing or
neutralizing zB7R1 and CD155 (either singly or together), in the
treatment of psoriasis, atopic dermatitis, inflammatory skin
conditions, psoriatic arthritis, arthritis, endotoxemia, asthma,
inflammatory bowel disease (IBD), colitis, and other inflammatory
conditions disclosed herein.
[0313] zB7R1 may be involved in the pathology of psoriasis. The
present invention is in particular a method for treating psoriasis
by administering agents that bind, block, inhibit, reduce,
antagonize or neutralize zB7R1. The agonists to zB7R1 can either be
a soluble receptor that binds to zB7R1, or antibodies, single chain
antibodies or fragments of antibodies that bind to either zB7R1 or
the zB7R1 counter-receptor, e.g., anti-zB7R1 antibodies. The
antagonists will thus prevent activation of the zB7R1 receptor.
[0314] Psoriasis is one of the most common dermatologic diseases,
affecting up to 1 to 2 percent of the world's population. It is a
chronic inflammatory skin disorder characterized by erythematous,
sharply demarcated papules and rounded plaques, covered by silvery
micaceous scale. The skin lesions of psoriasis are variably
pruritic. Traumatized areas often develop lesions of psoriasis.
Additionally, other external factors may exacerbate psoriasis
including infections, stress, and medications, e.g. lithium, beta
blockers, and anti-malarials.
[0315] The most common variety of psoriasis is called plaque type.
Patients with plaque-type psoriasis will have stable, slowly
growing plaques, which remain basically unchanged for long periods
of time. The most common areas for plaque psoriasis to occur are
the elbows knees, gluteal cleft, and the scalp. Involvement tends
to be symmetrical. Inverse psoriasis affects the intertriginous
regions including the axilla, groin, submammary region, and navel,
and it also tends to affect the scalp, palms, and soles. The
individual lesions are sharply demarcated plaques but may be moist
due to their location. Plaque-type psoriasis generally develops
slowly and runs an indolent course. It rarely spontaneously
remits.
[0316] Eruptive psoriasis (guttate psoriasis) is most common in
children and young adults. It develops acutely in individuals
without psoriasis or in those with chronic plaque psoriasis.
Patients present with many small erythematous, scaling papules,
frequently after upper respiratory tract infection with
beta-hemolytic streptococci. Patients with psoriasis may also
develop pustular lesions. These may be localized to the palms and
soles or may be generalized and associated with fever, malaise,
diarrhea, and arthralgias.
[0317] About half of all patients with psoriasis have fingernail
involvement, appearing as punctate pitting, nail thickening or
subungual hyperkeratosis. About 5 to 10 percent of patients with
psoriasis have associated joint complaints, and these are most
often found in patients with fingernail involvement. Although some
have the coincident occurrence of classic Although some have the
coincident occurrence of classic rheumatoid arthritis, many have
joint disease that falls into one of five type associated with
psoriasis: (1) disease limited to a single or a few small joints
(70 percent of cases); (2) a seronegative rheumatoid arthritis-like
disease; (3) involvement of the distal interphalangeal joints; (4)
severe destructive arthritis with the development of "arthritis
mutilans"; and (5) disease limited to the spine.
[0318] Psoriasis can be treated by administering agents that act as
zB7R1 agonists. The preferred antagonists are either a soluble
receptor to zB7R1 such as zB7R1 (SEQ ID NO:3) or antibodies,
antibody fragments or single chain antibodies that bind to the
zB7R1 or itys counter-receptor. Such antagonists can be
administered alone or in combination with other established
therapies such as lubricants, keratolytics, topical
corticosteroids, topical vitamin D derivatives, anthralin, systemic
antimetabolites such as methotrexate, psoralen-ultraviolet-light
therapy (PUVA), etretinate, isotretinoin, cyclosporine, and the
topical vitamin D3 derivative calcipotriol. Moreover, such
antagonists can be administered to individual subcutaneously,
intravenously, or transdermally using a cream or transdermal patch
that contains the antagonist. If administered subcutaneously, the
antagonist can be injected into one or more psoriatic plaques. If
administered transdermally, the antagonists can be administered
directly on the plaques using a cream, ointment, salve, or solution
containing the antagonist.
[0319] Agonists to zB7R1 can be administered to a person who has
asthma, bronchitis or cystic fibrosis or other inflammatory lung
disease to treat the disease. The antagonists can be administered
by any suitable method including intravenous, subcutaneous,
bronchial lavage, and the use of inhalant containing the
antagonist.
[0320] Thus, particular embodiments of the present invention are
directed toward use of soluble zB7R1 and anti-zB7R1 antibodies as
agonists in inflammatory and immune diseases or conditions such as
psoriasis, psoriatic arthritis, atopic dermatitis, inflammatory
skin conditions, rheumatoid arthritis, inflammatory bowel disease
(IBD), Crohn's Disease, diverticulosis, asthma, pancreatitis, type
I diabetes (IDDM), pancreatic cancer, pancreatitis, Graves Disease,
colon and intestinal cancer, autoimmune disease, sepsis, organ or
bone marrow transplant; inflammation due to endotoxemia, trauma,
sugery or infection; amyloidosis; splenomegaly; graft versus host
disease; and where inhibition of inflammation, immune suppression,
reduction of proliferation of hematopoietic, immune, inflammatory
or lymphoid cells, macrophages, T-cells (including Th1 and Th2
cells), suppression of immune response to a pathogen or antigen, or
other instances where inhibition of zB7R1 is desired.
[0321] Moreover, antibodies or binding polypeptides that bind zB7R1
polypeptides described herein, and zB7R1 polypeptides themselves
are useful to:
[0322] Block, inhibit, reduce, antagonize or neutralize signaling
via zB7R1 in the treatment of acute inflammation, inflammation as a
result of trauma, tissue injury, surgery, sepsis or infection, and
chronic inflammatory diseases such as asthma, inflammatory bowel
disease (IBD), chronic colitis, splenomegaly, rheumatoid arthritis,
recurrent acute inflammatory episodes (e.g., tuberculosis), and
treatment of amyloidosis, and atherosclerosis, Castleman's Disease,
asthma, and other diseases associated with the induction of
acute-phase response.
[0323] Block, inhibit, reduce, antagonize or neutralize signaling
via zB7R1 in the treatment of autoimmune diseases such as IDDM,
multiple sclerosis (MS), systemic Lupus erythematosus (SLE),
myasthenia gravis, rheumatoid arthritis, and IBD to prevent or
inhibit signaling in immune cells (e.g. lymphocytes, monocytes,
leukocytes) via zB7R1 (Hughes C et al., J. Immunol 153: 3319-3325,
1994). Alternatively antibodies, such as monoclonal antibodies
(MAb) to zB7R1, can also be used as an antagonist to deplete
unwanted immune cells to treat autoimmune disease. Asthma, allergy
and other atopic disease may be treated with an MAb against, for
example, soluble zB7R1 soluble receptors to inhibit the immune
response or to deplete offending cells. Blocking, inhibiting,
reducing, or antagonizing signaling via zB7R1, using the
polypeptides and antibodies of the present invention, may also
benefit diseases of the pancreas, kidney, pituitary and neuronal
cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinoma may
benefit. zB7R1 may serve as a target for MAb therapy of cancer
where an antagonizing MAb inhibits cancer growth and targets
immune-mediated killing. (Holliger P, and Hoogenboom, H: Nature
Biotech. 16: 1015-1016, 1998). Mabs to soluble zB7R1 may also be
useful to treat nephropathies such as glomerulosclerosis,
membranous neuropathy, amyloidosis (which also affects the kidney
among other tissues), renal arteriosclerosis, glomerulonephritis of
various origins, fibroproliferative diseases of the kidney, as well
as kidney dysfunction associated with SLE, IDDM, type II diabetes
(NIDDM), renal tumors and other diseases.
[0324] Agonize, enhance, increase or initiate signaling via zB7R1
in the treatment of autoimmune diseases such as IDDM, MS, SLE,
myasthenia gravis, rheumatoid arthritis, and IBD. Anti-zB7R1
neutralizing and monoclonal antibodies may signal lymphocytes or
other immune cells to differentiate, alter proliferation, or change
production of cytokines or cell surface proteins that ameliorate
autoimmunity. Specifically, modulation of a T-cell response may
deviate an autoimmune response to ameliorate disease (Smith J A et
al., J. Immunol. 160:4841-4849, 1998). Similarly, agonistic
anti-zB7R1 monoclonal antibodies may be used to signal, deplete and
deviate immune cells involved in rheumatoid arthritis, asthma,
allergy and atopoic disease. Signaling via zB7R1 may also benefit
diseases of the pancreas, kidney, pituitary and neuronal cells.
IDDM, NIDDM, pancreatitis, and pancreatic carcinoma may benefit.
zB7R1 may serve as a target for MAb therapy of pancreatic cancer
where a signaling MAb inhibits cancer growth and targets
immune-mediated killing (Tutt, A L et al., J Immunol. 161:
3175-3185, 1998). Similarly renal cell carcinoma may be treated
with monoclonal antibodies to zB7R1-comprising soluble receptors of
the present invention.
[0325] Soluble zB7R1 polypeptides described herein can be used to
bind, block, inhibit, reduce, antagonize or neutralize zB7R1
activity, either singly or together; in the treatment of autoimmune
disease, atopic disease, NIDDM, pancreatitis and kidney dysfunction
as described above. A soluble form of zB7R1 may be used to promote
an antibody response mediated by Th cells and/or to promote the
production of IL-4 or other cytokines by lymphocytes or other
immune cells.
[0326] Moreover, inflammation is a protective response by an
organism to fend off an invading agent. Inflammation is a cascading
event that involves many cellular and humoral mediators. On one
hand, suppression of inflammatory responses can leave a host
immunocompromised; however, if left unchecked, inflammation can
lead to serious complications including chronic inflammatory
diseases (e.g., psoriasis, arthritis, rheumatoid arthritis,
multiple sclerosis, inflammatory bowel disease and the like),
septic shock and multiple organ failure. Importantly, these diverse
disease states share common inflammatory mediators. The collective
diseases that are characterized by inflammation have a large impact
on human morbidity and mortality. Therefore it is clear that
molecules that are inimtaely involved in the costimualtion and/or
inhibition of immune responses, such as zB7R1, its
counter-receptor, and anti-zB7R1 antibodies, could have crucial
therapeutic potential for a vast number of human and animal
diseases, from asthma and allergy to autoimmunity and septic
shock.
[0327] 1. Arthritis
[0328] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury, and the like, are common
inflammatory conditions which would benefit from the therapeutic
use of anti-inflammatory proteins, such as the zB7R1 molecules of
the present invention. For example, rheumatoid arthritis (RA) is a
systemic disease that affects the entire body and is one of the
most common forms of arthritis. It is characterized by the
inflammation of the membrane lining the joint, which causes pain,
stiffness, warmth, redness and swelling. Inflammatory cells release
enzymes that may digest bone and cartilage. As a result of
rheumatoid arthritis, the inflamed joint lining, the synovium, can
invade and damage bone and cartilage leading to joint deterioration
and severe pain amongst other physiologic effects. The involved
joint can lose its shape and alignment, resulting in pain and loss
of movement.
[0329] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).
One of those mediators could be a soluble zB7R1 protein or an
anti-zB7R1 antibody and as such a molecule that binds or mediates
zB7R1, such as soluble B7R1-Fc, B7R1m-VASP CH6 or antibodies or
binding partners as described herein, could serve as a valuable
therapeutic to reduce inflammation in rheumatoid arthritis, and
other arthritic diseases.
[0330] There are several animal models for rheumatoid arthritis
known in the art. For example, in the collagen-induced arthritis
(CIA) model, mice develop chronic inflammatory arthritis that
closely resembles human rheumatoid arthritis. Since CIA shares
similar immunological and pathological features with RA, this makes
it an ideal model for screening potential human anti-inflammatory
compounds. The CIA model is a well-known model in mice that depends
on both an immune response, and an inflammatory response, in order
to occur. The immune response comprises the interaction of B-cells
and CD4+ T-cells in response to collagen, which is given as
antigen, and leads to the production of anti-collagen antibodies.
The inflammatory phase is the result of tissue responses from
mediators of inflammation, as a consequence of some of these
antibodies cross-reacting to the mouse's native collagen and
activating the complement cascade. An advantage in using the CIA
model is that the basic mechanisms of pathogenesis are known. The
relevant T-cell and B-cell epitopes on type II collagen have been
identified, and various immunological (e.g., delayed-type
hypersensitivity and anti-collagen antibody) and inflammatory
(e.g., cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediated arthritis have been
determined, and can thus be used to assess test compound efficacy
in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20, 1999;
Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life
Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959,
1995).
[0331] As shown in Example 21, mRNA levels of murine B7R1 are
higher in the affected paws and draining (popliteal) lymph nodes
from mice with CIA compared to mice without CIA, and the levels are
associated with disease severity. Furthermore, one group has shown
that the delivery of a neutralizing antibody to another B7 family
member, B7 homologous protein (B7h), reduces symptoms in a mouse
CIA-model relative to control mice (Iwai et al, J. Immunol.
169:4332, 2002), thus supporting the idea that soluble B7R1-Fc and
B7R1m-VASP CH6 may be beneficial in treating human disease, such as
arthritis. The administration of a neutralizing anti-B7h antibody
reduced the symptoms of arthritis in the animals when introduced
prophylactically or after symptoms of arthritis were already
present in the model (Iwai et al, J. Immunol. 169:4332, 2002).
Therefore, B7R1-Fc or B7R1m-VASP CH6 can be used to treat of
specific human diseases such as cancer, rheumatoid arthritis,
psoriasis, psoriatic arthritis, arthritis, endotoxemia,
inflammatory bowel disease (IBD), colitis, and other inflammatory
conditions disclosed herein.
[0332] The administration of soluble B7R1 comprising polypeptides,
such as B7R1-Fc or B7R1m-VASP CH6 or other zB7R1 soluble and fusion
proteins to these CIA model mice is used to evaluate the use of
soluble B7R1-Fc to ameliorate symptoms and alter the course of
disease. Furthermore, since inflammation is implicated in the
pathogenesis and progression of rheumatoid arthritis, the systemic
or local administration of soluble B7R1 comprising polypeptides,
such as B7R1-Fc, B7R1m-VASP CH6 or other soluble receptors and
anti-zB7R1 antibodies, and fusion proteins can potentially suppress
the inflammatory response in RA. By way of example and without
limitation, the injection of 10-200 ug B7R1-Fc or B7R1m-VASP CH6
per mouse (one to seven times a week for up to but not limited to 4
weeks via s.c., i.p., or i.m route of administration) can
significantly reduce the disease score (paw score, incident of
inflammation, or disease). Depending on the initiation of B7R1-Fc
administration (e.g. prior to or at the time of collagen
immunization, or at any time point following the second collagen
immunization, including those time points at which the disease has
already progressed), B7R1-Fc or B7R1m-VASP CH6 can be efficacious
in preventing rheumatoid arthritis, as well as preventing its
progression. Other potential therapeutics include CD155
polypeptides or anti-CD155 antibodies.
[0333] 2. Endotoxemia
[0334] Endotoxemia is a severe condition commonly resulting from
infectious agents such as bacteria and other infectious disease
agents, sepsis, toxic shock syndrome, or in immunocompromised
patients subjected to opportunistic infections, and the like.
Therapeutically useful of anti-inflammatory proteins, such as zB7R1
polypeptides and antibodies of the present invention, could aid in
preventing and treating endotoxemia in humans and animals. zB7R1
polypeptides, anti-EL22RA antibodies, or anti IL-22 antibodies or
binding partners, could serve as a valuable therapeutic to reduce
inflammation and pathological effects in endotoxemia.
[0335] Lipopolysaccharide (LPS) induced endotoxemia engages many of
the proinflammatory mediators that produce pathological effects in
the infectious diseases and LPS induced endotoxemia in rodents is a
widely used and acceptable model for studying the pharmacological
effects of potential pro-inflammatory or immunomodulating agents.
LPS, produced in gram-negative bacteria, is a major causative agent
in the pathogenesis of septic shock (Glausner et al., Lancet
338:732, 1991). A shock-like state can indeed be induced
experimentally by a single injection of LPS into animals. Molecules
produced by cells responding to LPS can target pathogens directly
or indirectly. Although these biological responses protect the host
against invading pathogens, they may also cause harm. Thus, massive
stimulation of innate immunity, occurring as a result of severe
Gram-negative bacterial infection, leads to excess production of
cytokines and other molecules, and the development of a fatal
syndrome, septic shock syndrome, which is characterized by fever,
hypotension, disseminated intravascular coagulation, and multiple
organ failure (Dumitru et al. Cell 103:1071-1083, 2000).
[0336] These toxic effects of LPS are mostly related to macrophage
activation leading to the release of multiple inflammatory
mediators. Among these mediators, TNF appears to play a crucial
role, as indicated by the prevention of LPS toxicity by the
administration of neutralizing anti-TNF antibodies (Beutler et al.,
Science 229:869, 1985). It is well established that lug injection
of E. coli LPS into a C57B1/6 mouse will result in significant
increases in circulating IL-6, TNF-alpha, IL-1, and acute phase
proteins (for example, SAA) approximately 2 hours post injection.
The toxicity of LPS appears to be mediated by these cytokines as
passive immunization against these mediators can result in
decreased mortality (Beutler et al., Science 229:869, 1985). The
potential immunointervention strategies for the prevention and/or
treatment of septic shock include anti-TNF mAb, IL-1 receptor
antagonist, LIF, IL-10, and G-CSF.
[0337] The administration of anti-zB7R1 antibodies or other zB7R1
soluble and fusion proteins to these LPS-induced model can be used
to to evaluate the use of zB7R1 to ameliorate symptoms and alter
the course of LPS-induced disease.
[0338] 3 Inflammatory Bowel Disease. IBD
[0339] In the United States approximately 500,000 people suffer
from Inflammatory Bowel Disease (IBD) which can affect either colon
and rectum (Ulcerative colitis) or both, small and large intestine
(Crohn's Disease). The pathogenesis of these diseases is unclear,
but they involve chronic inflammation of the affected tissues.
zB7R1 polypeptides, anti-zB7R1 antibodies, or or binding partners,
could serve as a valuable therapeutic to reduce inflammation and
pathological effects in IBD and related diseases.
[0340] Ulcerative colitis (UC) is an inflammatory disease of the
large intestine, commonly called the colon, characterized by
inflammation and ulceration of the mucosa or innermost lining of
the colon. This inflammation causes the colon to empty frequently,
resulting in diarrhea. Symptoms include loosening of the stool and
associated abdominal cramping, fever and weight loss. Although the
exact cause of UC is unknown, recent research suggests that the
body's natural defenses are operating against proteins in the body
which the body thinks are foreign (an "autoimmune reaction").
Perhaps because they resemble bacterial proteins in the gut, these
proteins may either instigate or stimulate the inflammatory process
that begins to destroy the lining of the colon. As the lining of
the colon is destroyed, ulcers form releasing mucus, pus and blood.
The disease usually begins in the rectal area and may eventually
extend through the entire large bowel. Repeated episodes of
inflammation lead to thickening of the wall of the intestine and
rectum with scar tissue. Death of colon tissue or sepsis may occur
with severe disease. The symptoms of ulcerative colitis vary in
severity and their onset may be gradual or sudden. Attacks may be
provoked by many factors, including respiratory infections or
stress.
[0341] Although there is currently no cure for UC available,
treatments are focused on suppressing the abnormal inflammatory
process in the colon lining. Treatments including corticosteroids
immunosuppressives (eg. azathioprine, mercaptopurine, and
methotrexate) and aminosalicytates are available to treat the
disease. However, the long-term use of immunosuppressives such as
corticosteroids and azathioprine can result in serious side effects
including thinning of bones, cataracts, infection, and liver and
bone marrow effects. In the patients in whom current therapies are
not successful, surgery is an option. The surgery involves the
removal of the entire colon and the rectum.
[0342] There are several animal models that can partially mimic
chronic ulcerative colitis. The most widely used model is the
2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis
model, which induces chronic inflammation and ulceration in the
colon. When TNBS is introduced into the colon of susceptible mice
via intra-rectal instillation, it induces T-cell mediated immune
response in the colonic mucosa, in this case leading to a massive
mucosal inflammation characterized by the dense infiltration of
T-cells and macrophages throughout the entire wall of the large
bowel. Moreover, this histopathologic picture is accompanies by the
clinical picture of progressive weight loss (wasting), bloody
diarrhea, rectal prolapse, and large bowel wall thickening (Neurath
et al. Intern. Rev. Immunol. 19:51-62, 2000).
[0343] Another colitis model uses dextran sulfate sodium (DSS),
which induces an acute colitis manifested by bloody diarrhea,
weight loss, shortening of the colon and mucosal ulceration with
neutrophil infiltration. DSS-induced colitis is characterized
histologically by infiltration of inflammatory cells into the
lamina propria, with lymphoid hyperplasia, focal crypt damage, and
epithelial ulceration. These changes are thought to develop due to
a toxic effect of DSS on the epithelium and by phagocytosis of
lamina propria cells and production of TNF-alpha and IFN-gamma.
Despite its common use, several issues regarding the mechanisms of
DSS about the relevance to the human disease remain unresolved. DSS
is regarded as a T cell-independent model because it is observed in
T cell-deficient animals such as SCID mice.
[0344] The administration of anti-zB7R1 antibodies or other zB7R1
soluble and fusion proteins to these TNBS or DSS models can be used
to evaluate the use of zB7R1 to ameliorate symptoms and alter the
course of gastrointestinal disease. Moreover, the results showing
inhibition of T cell signaling by zB7R1 provide proof of concept
that other zB7R1 antagonists, such as zB7R1 or antibodies thereto,
can also be used to ameliorate symptoms in the colitis/IBD models
and alter the course of disease.
[0345] 4. Psoriasis
[0346] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. zB7R1
polypeptides, anti-zB7R1 antibodies, or anti IL-22 and anti zB7R1
antibodies or binding partners, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in
psoriasis, other inflammatory skin diseases, skin and mucosal
allergies, and related diseases.
[0347] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy.
[0348] Moreover, anti-zB7R1 antibodies and zB7R1 soluble
receptorsof the present invention can be used in the prevention and
therapy against weight loss associated with a number of
inflammatory diseases described herein, as well as for cancer
(e.g., chemotherapy and cachexia), and infectious diseases. For
example, severe weight loss is a key marker associated with models
for septicemia, MS, RA, and tumor models. In addition, weight loss
is a key parameter for many human diseases including cancer,
infectious disease and inflammatory disease. Anti-zB7R1 antibodies
and zB7R1 antagonists such as the soluble zB7R1 receptors and
antibodies thereto of the present invention, can be tested for
their ability to prevent and treat weight loss in mice injected
with zB7R1 andenovires described herein. Methods of determining a
prophylactic or therapeutic regimen for such zB7R1 antagonists is
known in the art and can be determined using the methods described
herein.
[0349] zB7R1 soluble receptor polypeptides and antibodies thereto
may also be used within diagnostic systems for the detection of
circulating levels of zB7R1 or zB7R1 counter-receptor, and in the
detection of zB7R1 associated with acute phase inflammatory
response. Within a related embodiment, antibodies or other agents
that specifically bind to zB7R1 soluble receptors of the present
invention can be used to detect circulating receptor polypeptides;
conversely, zB7R1 soluble receptors themselves can be used to
detect circulating or locally-acting zB7R1 polypeptides. Elevated
or depressed levels of zB7R1 counter-receptor or zB7R1 polypeptides
may be indicative of pathological conditions, including
inflammation or cancer. Moreover, detection of acute phase proteins
or molecules such as zB7R1 can be indicative of a chronic
inflammatory condition in certain disease states (e.g., psoriasis,
rheumatoid arthritis, colitis, IBD). Detection of such conditions
serves to aid in disease diagnosis as well as help a physician in
choosing proper therapy.
[0350] For example, neutralizing antibodies to zB7R1 include
antibodies, such as neutralizing monoclonal antibodies that can
bind zB7R1 antigenic epitopes and neutralize zB7R1 activity.
Accordingly, antigenic epitope-bearing peptides and polypeptides of
zB7R1 are useful to raise antibodies that bind with the zB7R1
polypeptides described herein, as well as to identify and screen
anti-zB7R1 monoclonal antibodies that are neutralizing, and that
may bind, block, inhibit, reduce, antagonize or neutralize the
activity of zB7R1. Such neutralizing monoclonal antibodies of the
present invention can bind to an zB7R1 antigenic epitope.
[0351] In addition to other disease models described herein, the
activity of anti-zB7R1 antibodies on inflammatory tissue derived
from human psoriatic lesions can be measured in vivo using a severe
combined immune deficient (SCID) mouse model. Several mouse models
have been developed in which human cells are implanted into
immunodeficient mice (collectively referred to as xenograft
models); see, for example, Cattan A R, Douglas E, Leuk. Res.
18:513-22, 1994 and Flavell, D J, Hematological Oncology 14:67-82,
1996. As an in vivo xenograft model for psoriasis, human psoriatic
skin tissue is implanted into the SCID mouse model, and challenged
with an appropriate antagonist. Moreover, other psoriasis animal
models in ther art may be used to evaluate zB7R1 antagonists, such
as human psoriatic skin grafts implanted into AGR129 mouse model,
and challenged with an appropriate antagonist (e.g., see, Boyman,
O. et al., J. Exp. Med. Online publication #20031482, 2004,
incorporated hereing by reference). Anti-zB7R1 antibodies that
bind, block, inhibit, reduce, antagonize or neutralize the activity
of zB7R1 are preferred antagonists, however, anti-zB7R1 antibodies
(alone or in combination with other B7 antagonists), soluble zB7R1,
as well as other zB7R1 antagonists can be used in this model.
Similarly, tissues or cells derived from human colitis, IBD,
arthritis, or other inflammatory lestions can be used in the SCID
model to assess the anti-inflammatory properties of the zB7R1
antagonists described herein.
[0352] Therapies designed to abolish, retard, or reduce
inflammation using anti-zB7R1 antibodies or its derivatives,
agonists, conjugates or variants can be tested by administration of
anti-zB7R1 antibodies or soluble zB7R1 compounds to SCID mice
bearing human inflammatory tissue (e.g., psoriatic lesions and the
like), or other models described herein. Efficacy of treatment is
measured and statistically evaluated as increased anti-inflammatory
effect within the treated population over time using methods well
known in the art. Some exemplary methods include, but are not
limited to measuring for example, in a psoriasis model, epidermal
thickness, the number of inflammatory cells in the upper dermis,
and the grades of parakeratosis. Such methods are known in the art
and described herein. For example, see Zeigler, M. et al. Lab
Invest 81:1253, 2001; Zollner, T. M. et al. J. Clin. Invest.
109:671, 2002; Yamanaka, N. et al. Microbio.l Immunol. 45:507,
2001; Raychaudhuri, S. P. et al. Br. J. Dermatol. 144:931, 2001;
Boehncke, W. H et al. Arch. Dermatol. Res. 291:104, 1999; Boehncke,
W. H et al. J. Invest. Dermatol. 116:596, 2001; Nickoloff, B. J. et
al. Am. J. Pathol. 146:580, 1995; Boehncke, W. H et al. J. Cutan.
Pathol. 24:1, 1997; Sugai, J., M. et al. J. Dermatol. Sci. 17:85,
1998; and Villadsen L. S. et al. J. Clin. Invest. 112:1571, 2003.
Inflammation may also be monitored over time using well-known
methods such as flow cytometry (or PCR) to quantitate the number of
inflammatory or lesional cells present in a sample, score (weight
loss, diarrhea, rectal bleeding, colon length) for IBD, paw disease
score and inflammation score for CIA RA model. For example,
therapeutic strategies appropriate for testing in such a model
include direct treatment using anti-zB7R1 antibodies, other zB7R1
antagonists (singly or together with other B7 antagonists), or
related conjugates or antagonists based on the disrupting
interaction of anti-zB7R1 antibodies with zB7R1, or for cell-based
therapies utilizing anti-zB7R1 antibodies or its derivatives,
agonists, conjugates or variants.
[0353] Moreover, psoriasis is a chronic inflammatory skin disease
that is associated with hyperplastic epidermal keratinocytes and
infiltrating mononuclear cells, including CD4+ memory T cells,
neutrophils and macrophages (Christophers, Int. Arch. Allergy
Immunol., 110: 199, 1996). It is currently believed that
environmental antigens play a significant role in initiating and
contributing to the pathology of the disease. However, it is the
loss of tolerance to self-antigens that is thought to mediate the
pathology of psoriasis. Dendritic cells and CD4.sup.+ T cells are
thought to play an important role in antigen presentation and
recognition that mediate the immune response leading to the
pathology. We have recently developed a model of psoriasis based on
the CD4+CD45RB transfer model (Davenport et al., Internat.
Immunopharmacol., 2:653-672). Anti-zB7R1 antibodies of the present
invention, or soluble zB7R1, are administered to the mice.
Inhibition of disease scores (skin lesions, inflammatory cytokines)
indicates the effectiveness of zB7R1 antagonists in psoriasis,
e.g., anti-zB7R1 antibodies or zB7R1 soluble receptors, or other
antagonists such as antibodies against the zB7R1
counter-receptor.
[0354] 5. Atopic Dermatitis.
[0355] AD is a common chronic inflammatory disease that is
characterized by hyperactivated cytokines of the helper T cell
subset 2 (Th2). Although the exact etiology of AD is unknown,
multiple factors have been implicated, including hyperactive Th2
immune responses, autoimmunity, infection, allergens, and genetic
predisposition. Key features of the disease include xerosis
(dryness of the skin), pruritus (itchiness of the skin),
conjunctivitis, inflammatory skin lesions, Staphylococcus aureus
infection, elevated blood eosinophilia, elevation of serum IgE and
IgG1, and chronic dermatitis with T cell, mast cell, macrophage and
eosinophil infiltration. Colonization or infection with S. aureus
has been recognized to exacerbate AD and perpetuate chronicity of
this skin disease.
[0356] AD is often found in patients with asthma and allergic
rhinitis, and is frequently the initial manifestation of allergic
disease. About 20% of the population in Western countries suffer
from these allergic diseases, and the incidence of AD in developed
countries is rising for unknown reasons. AD typically begins in
childhood and can often persist through adolescence into adulthood.
Current treatments for AD include topical corticosteroids, oral
cyclosporin A, non-corticosteroid immunosuppressants such as
tacrolimus (FK506 in ointment form), and interferon-gamma. Despite
the variety of treatments for AD, many patients' symptoms do not
improve, or they have adverse reactions to medications, requiring
the search for other, more effective therapeutic agents. The
soluble zB7R1 polypeptides and anti-zB7R1 antibodies of the present
invention, can be used to neutralize zB7R1 in the treatment of
specific human diseases such as atoptic dermatitis, inflammatory
skin conditions, and other inflammatory conditions disclosed
herein.
[0357] For pharmaceutical use, the soluble zB7R1 or anti-zB7R1
antibodies of the present invention are formulated for parenteral,
particularly intravenous or subcutaneous, delivery according to
conventional methods. Intravenous administration will be by bolus
injection, controlled release, e.g, using mini-pumps or other
appropriate technology, or by infusion over a typical period of one
to several hours. In general, pharmaceutical formulations will
include a hematopoietic protein in combination with a
pharmaceutically acceptable vehicle, such as saline, buffered
saline, 5% dextrose in water or the like. Formulations may further
include one or more excipients, preservatives, solubilizers,
buffering agents, albumin to provent protein loss on vial surfaces,
etc. When utilizing such a combination therapy, the cytokines may
be combined in a single formulation or may be administered in
separate formulations. Methods of formulation are well known in the
art and are disclosed, for example, in Remington's Pharmaceutical
Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa., 1990,
which is incorporated herein by reference. Therapeutic doses will
generally be in the range of 0.1 to 100 mg/kg of patient weight per
day, preferably 0.5-20 mg/kg per day, with the exact dose
determined by the clinician according to accepted standards, taking
into account the nature and severity of the condition to be
treated, patient traits, etc. Determination of dose is within the
level of ordinary skill in the art. The proteins will commonly be
administered over a period of up to 28 days following chemotherapy
or bone-marrow transplant or until a platelet count of
>20,000/mm.sup.3, preferably >50,000/mm.sup.3, is achieved.
More commonly, the proteins will be administered over one week or
less, often over a period of one to three days. In general, a
therapeutically effective amount of soluble zB7R1 or anti-zB7R1
antibodies of the present invention is an amount sufficient to
produce a clinically significant increase in the proliferation
and/or differentiation of lymphoid or myeloid progenitor cells,
which will be manifested as an increase in circulating levels of
mature cells (e.g. platelets or neutrophils). Treatment of platelet
disorders will thus be continued until a platelet count of at least
20,000/mm.sup.3, preferably 50,000/mm.sup.3, is reached. The
soluble zB7R1 or anti-zB7R1 antibodies of the present invention can
also be administered in combination with other cytokines such as
IL-3, -6 and -11; stem cell factor; erythropoietin; G-CSF and
GM-CSF. Within regimens of combination therapy, daily doses of
other cytokines will in general be: EPO, 150 U/kg; GM-CSF, 5-15
lg/kg; IL-3, 1-5 lg/kg; and G-CSF, 1-25 lg/kg. Combination therapy
with EPO, for example, is indicated in anemic patients with low EPO
levels.
[0358] Generally, the dosage of administered soluble zB7R1 (or
zB7R1 analog or fusion protein) or anti-zB7R1 antibodies will vary
depending upon such factors as the patient's age, weight, height,
sex, general medical condition and previous medical history.
Typically, it is desirable to provide the recipient with a dosage
of soluble zB7R1 or anti-zB7R1 antibodies which is in the range of
from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of
patient), although a lower or higher dosage also may be
administered as circumstances dictate.
[0359] Administration of soluble zB7R1 or anti-zB7R1 antibodies to
a subject can be intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, intrapleural, intrathecal, by
perfusion through a regional catheter, or by direct intralesional
injection. When administering therapeutic proteins by injection,
the administration may be by continuous infusion or by single or
multiple boluses.
[0360] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising zB7R1 can be prepared and inhaled with the aid of
dry-powder dispersers, liquid aerosol generators, or nebulizers
(e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al.,
Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated
by the AERX diabetes management system, which is a hand-held
electronic inhaler that delivers aerosolized insulin into the
lungs. Studies have shown that proteins as large as 48,000 kDa have
been delivered across skin at therapeutic concentrations with the
aid of low-frequency ultrasound, which illustrates the feasibility
of trascutaneous administration (Mitragotri et al., Science 269:850
(1995)). Transdermal delivery using electroporation provides
another means to administer a molecule having zB7R1 binding
activity (Potts et al., Pharm. Biotechnol. 10:213 (1997)).
[0361] A pharmaceutical composition comprising a soluble zB7R1 or
anti-zB7R1 antibody can be formulated according to known methods to
prepare pharmaceutically useful compositions, whereby the
therapeutic proteins are combined in a mixture with a
pharmaceutically acceptable carrier. A composition is said to be a
"pharmaceutically acceptable carrier" if its administration can be
tolerated by a recipient patient. Sterile phosphate-buffered saline
is one example of a pharmaceutically acceptable carrier. Other
suitable carriers are well-known to those in the art. See, for
example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th
Edition (Mack Publishing Company 1995).
[0362] For purposes of therapy, soluble zB7R1 or anti-zB7R1
antibody molecules and a pharmaceutically acceptable carrier are
administered to a patient in a therapeutically effective amount. A
combination of a therapeutic molecule of the present invention and
a pharmaceutically acceptable carrier is said to be administered in
a "therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient. For example, an agent used to
treat inflammation is physiologically significant if its presence
alleviates the inflammatory response.
[0363] A pharmaceutical composition comprising zB7R1 (or zB7R1
analog or fusion protein) or anti-zB7R1 antibody can be furnished
in liquid form, in an aerosol, or in solid form. Liquid forms, are
illustrated by injectable solutions and oral suspensions. Exemplary
solid forms include capsules, tablets, and controlled-release
forms. The latter form is illustrated by miniosmotic pumps and
implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade,
"Implants in Drug Delivery," in Drug Delivery Systems, Ranade and
Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,
"Protein Delivery with Infusion Pumps," in Protein Delivery:
Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum
Press 1997); Yewey et al., "Delivery of Proteins from a Controlled
Release Injectable Implant," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[0364] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0365] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0366] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0367] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or counter-receptors into the liposomes. For example,
liposomes, prepared with a high content of a nonionic surfactant,
have been used to target the liver (Hayakawa et al., Japanese
Patent 04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)).
These formulations were prepared by mixing soybean
phospatidylcholine, .alpha.-tocopherol, and ethoxylated
hydrogenated castor oil (HCO-60) in methanol, concentrating the
mixture under vacuum, and then reconstituting the mixture with
water. A liposomal formulation of dipalmitoylphosphatidylcholine
(DPPC) with a soybean-derived sterylglucoside mixture (SG) and
cholesterol (Ch) has also been shown to target the liver (Shimizu
et al., Biol. Pharm. Bull. 20:881 (1997)).
[0368] Alternatively, various targeting counter-receptors can be
bound to the surface of the liposome, such as antibodies, antibody
fragments, carbohydrates, vitamins, and transport proteins. For
example, liposomes can be modified with branched type
galactosyllipid derivatives to target asialoglycoprotein
(galactose) receptors, which are exclusively expressed on the
surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug
Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm. Bull.
20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),
have shown that labeling liposomes with asialofetuin led to a
shortened liposome plasma half-life and greatly enhanced uptake of
asialofetuin-labeled liposome by hepatocytes. On the other hand,
hepatic accumulation of liposomes comprising branched type
galactosyllipid derivatives can be inhibited by preinjection of
asialofetuin (Murahashi et al., Biol. Pharm. Bull. 20:259 (1997)).
Polyaconitylated human serum albumin liposomes provide another
approach for targeting liposomes to liver cells (Kamps et al.,
Proc. Nat'l Acad. Sci. USA 94:11681 (1997)). Moreover, Geho, et al.
U.S. Pat. No. 4,603,044, describe a hepatocyte-directed liposome
vesicle delivery system, which has specificity for hepatobiliary
receptors associated with the specialized metabolic cells of the
liver.
[0369] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a
counter-receptor expressed by the target cell (Harasym et al., Adv.
Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free
antibody, streptavidin-conjugated liposomes are administered. In
another approach, targeting antibodies are directly attached to
liposomes (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0370] Polypeptides and antibodies can be encapsulated within
liposomes using standard techniques of protein microencapsulation
(see, for example, Anderson et al., Infect. Immun. 31:1099 (1981),
Anderson et al., Cancer Res. 50:1853 (1990), and Cohen et al.,
Biochim. Biophys. Acta 1063:95 (1991), Alving et al. "Preparation
and Use of Liposomes in Immunological Studies," in Liposome
Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRC
Press 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). As
noted above, therapeutically useful liposomes may contain a variety
of components. For example, liposomes may comprise lipid
derivatives of poly(ethylene glycol) (Allen et al., Biochim.
Biophys. Acta 1150:9 (1993)).
[0371] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0372] The present invention also contemplates chemically modified
polypeptides having binding zB7R1 activity such as zB7R1 monomeric,
homodimeric, heterodimeric or multimeric soluble receptors, and
zB7R1 antagonists, for example anti-zB7R1 antibodies or binding
polypeptides, or neutralizing anti-zB7R1 antibodies, which a
polypeptide is linked with a polymer, as discussed above.
[0373] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0374] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a zB7R1 extracellular domain, e.g., zB7R1
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, or a zB7R1 antagonist (e.g., an antibody or antibody
fragment that binds a zB7R1 polypeptide, or neutralizing anti-zB7R1
antibody). Therapeutic polypeptides can be provided in the form of
an injectable solution for single or multiple doses, or as a
sterile powder that will be reconstituted before injection.
Alternatively, such a kit can include a dry-powder disperser,
liquid aerosol generator, or nebulizer for administration of a
therapeutic polypeptide. Such a kit may further comprise written
information on indications and usage of the pharmaceutical
composition. Moreover, such information may include a statement
that the zB7R1 composition is contraindicated in patients with
known hypersensitivity to zB7R1.
[0375] A pharmaceutical composition comprising Anti-zB7R1
antibodies or binding partners (or Anti-zB7R1 antibody fragments,
antibody fusions, humanized antibodies and the like), or zB7R1
soluble receptor, can be furnished in liquid form, in an aerosol,
or in solid form. Liquid forms, are illustrated by injectable
solutions, aerosols, droplets, topological solutions and oral
suspensions. Exemplary solid forms include capsules, tablets, and
controlled-release forms. The latter form is illustrated by
miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
10:239 (1997); Ranade, "Implants in Drug Delivery," in Drug
Delivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC
Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps,"
in Protein Delivery: Physical Systems, Sanders and Hendren (eds.),
pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of
Proteins from a Controlled Release Injectable Implant," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages
93-117 (Plenum Press 1997)). Other solid forms include creams,
pastes, other topological applications, and the like.
[0376] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0377] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0378] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0379] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or counter-receptors into the liposomes. For example,
liposomes, prepared with a high content of a nonionic surfactant,
have been used to target the liver (Hayakawa et al., Japanese
Patent 04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)).
These formulations were prepared by mixing soybean
phospatidylcholine, .alpha.-tocopherol, and ethoxylated
hydrogenated castor oil (HCO-60) in methanol, concentrating the
mixture under vacuum, and then reconstituting the mixture with
water. A liposomal formulation of dipalmitoylphosphatidylcholine
(DPPC) with a soybean-derived sterylglucoside mixture (SG) and
cholesterol (Ch) has also been shown to target the liver (Shimizu
et al., Biol. Pharm. Bull. 20:881 (1997)).
[0380] Alternatively, various targeting counter-receptors can be
bound to the surface of the liposome, such as antibodies, antibody
fragments, carbohydrates, vitamins, and transport proteins. For
example, liposomes can be modified with branched type
galactosyllipid derivatives to target asialoglycoprotein
(galactose) receptors, which are exclusively expressed on the
surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug
Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm. Bull.
20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),
have shown that labeling liposomes with asialofetuin led to a
shortened liposome plasma half-life and greatly enhanced uptake of
asialofetuin-labeled liposome by hepatocytes. On the other hand,
hepatic accumulation of liposomes comprising branched type
galactosyllipid derivatives can be inhibited by preinjection of
asialofetuin (Murahashi et al., Biol. Pharm. Bull. 20:259 (1997)).
Polyaconitylated human serum albumin liposomes provide another
approach for targeting liposomes to liver cells (Kamps et al.,
Proc. Nat'l Acad. Sci. USA 94:11681 (1997)). Moreover, Geho, et al.
U.S. Pat. No. 4,603,044, describe a hepatocyte-directed liposome
vesicle delivery system, which has specificity for hepatobiliary
receptors associated with the specialized metabolic cells of the
liver.
[0381] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a
counter-receptor expressed by the target cell (Harasym et al., Adv.
Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free
antibody, streptavidin-conjugated liposomes are administered. In
another approach, targeting antibodies are directly attached to
liposomes (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0382] Anti-zB7R1 neutralizing antibodies and binding partners with
zB7R1 binding activity, or zB7R1 soluble receptor, can be
encapsulated within liposomes using standard techniques of protein
microencapsulation (see, for example, Anderson et al., Infect.
Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990),
and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving et
al. "Preparation and Use of Liposomes in Immunological Studies," in
Liposome Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page
317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124
(1987)). As noted above, therapeutically useful liposomes may
contain a variety of components. For example, liposomes may
comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,
Biochim. Biophys. Acta 1150:9 (1993)).
[0383] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0384] The present invention also contemplates chemically modified
Anti-zB7R1 antibody or binding partner, for example anti-zB7R1
antibodies or zB7R1 soluble receptor, linked with a polymer, as
discussed above.
[0385] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0386] The present invention contemplates compositions of
anti-zB7R1 antibodies, and methods and therapeutic uses comprising
an antibody, peptide or polypeptide described herein. Such
compositions can further comprise a carrier. The carrier can be a
conventional organic or inorganic carrier. Examples of carriers
include water, buffer solution, alcohol, propylene glycol,
macrogol, sesame oil, corn oil, and the like.
[0387] 12. Production of Transgenic Mice
[0388] Nucleic acids which encode zB7R1 or modified forms thereof
can also be used to generate either transgenic animals or "knock
out" animals which, in turn, are useful in the development and
screening of therapeutically useful reagents. A transgenic animal
(e.g., a mouse or rat) is an animal having cells that contain a
transgene, which transgene was introduced into the animal or an
ancestor of the animal at a prenatal, e.g., an embryonic stage. A
transgene is a DNA which is integrated into the genome of a cell
from which a transgenic animal develops. In one embodiment, cDNA
encoding a zB7R1 protein can be used to clone genomic DNA encoding
a zB7R1 protein in accordance with established techniques and the
genomic sequences used to generate transgenic animals that contain
cells which express the desired DNA. Methods for generating
transgenic animals, particularly animals such as mice or rats, have
become conventional in the art and are described, for example, in
U.S. Pat. Nos. 4,736,866 and 4,870,009.
[0389] Alternatively, non-human homologues of zB7R1 can be used to
construct a "knock out" animal which has a defective or altered
gene encoding a zB7R1 protein as a result of homologous
recombination between the endogenous gene and an altered genomic
DNA encoding zB7R1, which is introduced into an embryonic cell of
the animal. For example, cDNA encoding a zB7R1 protein can be used
to clone genomic DNA encoding a zB7R1 protein in accordance with
established techniques. A portion of the genomic DNA encoding a
zB7R1 protein can be deleted or replaced with another gene, such as
a gene encoding a selectable marker which can be used to monitor
integration. Typically, several kilobases of unaltered flanking DNA
(both at the 5' and 3' ends) are included in the vector. See e.g.,
Thomas and Capecchi, Cell, 51:503 (1987). The vector is introduced
into an embryonic stem cell line (e.g., by electroporation) and
cells in which the introduced DNA has homologously recombined with
the endogenous DNA are selected. See e.g., Li et al., Cell, 69:915
(1992). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse or rat) to form aggregation chimeras. See
e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.
113-152. A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term
to create a "knock out" animal. Progeny harboring the homologously
recombined DNA in their germ cells can be identified by standard
techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knockout animals
can be characterized for instance, for their ability to defend
against certain pathological conditions and for their development
of pathological conditions due to absence of the zB7R1 protein. It
is understood that the models described herein can be varied. For
example, "knock-in" models can be formed, or the models can be
cell-based rather than animal models.
[0390] The invention is further illustrated by the following
non-limiting examples.
EXAMPLE 1
Murine zB7R1 Expression Construct
[0391] An expression plasmid containing a polynucleotide encoding
the full-length mouse zB7R1 (SEQ ID NO:8) was constructed via
homologous recombination. A fragment of mouse zB7R1 cDNA was
isolated by PCR using the polynucleotide sequence as identified by
SEQ ID NO:29 with flanking regions at the 5' and 3' ends
corresponding to the vector sequences flanking the mouse zB7R1
insertion point using primers zc51280 (SEQ ID NO:30) and zc51314
(SEQ ID NO:31).
[0392] The PCR reaction mixture was run on a 2% agarose gel and a
band corresponding to the size of the insert is gel-extracted using
a QIAquick.TM. Gel Extraction Kit (Qiagen, Valencia, Calif.).
Plasmid pZMP21 is a mammalian expression vector containing an
expression cassette having the MPSV promoter, multiple restriction
sites for insertion of coding sequences, a stop codon, an E. coli
origin of replication; a mammalian selectable marker expression
unit comprising an SV40 promoter, enhancer and origin of
replication, a DHFR gene, and the SV40 terminator; and URA3 and
CEN-ARS sequences required for selection and replication in S.
cerevisiae. It was constructed from pZP9 (deposited at the American
Type Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, under Accession No. 98668) with the yeast genetic
elements taken from pRS316 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
under Accession No. 77145), an internal ribosome entry site (IRES)
element from poliovirus, and the extracellular domain of CD8
truncated at the C-terminal end of the transmembrane domain.
Plasmid pZMP21 was digested with BglII, and used for recombination
with the PCR insert.
[0393] The recombination was performed using the BD In-Fusion.TM.
Dry-Down PCR Cloning kit (BD Biosciences, Palo Alto, Calif.). The
mixture of the PCR fragment and the digested vector in 10 .mu.l was
added to the lyophilized cloning reagents and incubated at
37.degree. C. for 15 minutes and 50.degree. C. for 15 minutes. The
reaction was ready for transformation. 2 .mu.l of recombination
reaction was transformed into One Shot TOP10 Chemical Competent
Cells (Invitrogen, Carlbad, Calif.); the transformation was
incubated on ice for 10 minutes and heat shocked at 42.degree. C.
for 30 seconds. The reaction was incubated on ice for 2 minutes
(helping transformed cells to recover). After the 2 minutes
incubation, 300 .mu.l of SOC (2% Bacto.TM. Tryptone (Difco,
Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM
KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was added
and the transformation was incubated at 37.degree. C. with shaker
for one hour. The whole transformation was plated on one LB AMP
plates (LB broth (Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L
Ampicillin).
[0394] The colonies were screened by PCR using primers zc51280 (SEQ
ID NO:30) and zc51314 (SEQ ID NO:31), respectively. The positive
colonies were verified by sequencing. The correct construct was
designated as mzB7R1FLpZMP21.
EXAMPLE 2
Mouse zB7R1mFc2pZMP21
[0395] An expression plasmid containing a polynucleotide encoding
the extra-cellular domain of mouse zB7R1 and the mouse Fc2 portion
can be constructed via homologous recombination. A DNA fragment of
the extra-cellular domain of mouse zB7R1 is isolated by PCR using
SEQ ID NO:32 with flanking regions at the 5' and 3' ends
corresponding to the vector sequence and the mouse Fc2 sequence
flanking the mouse zB7R1 insertion point using primers zc50437 (SEQ
ID NO:33) and zc50438 (SEQ ID NO:34).
[0396] The PCR reaction mixture is run on a 2% agarose gel and a
band corresponding to the size of the insert is gel-extracted using
a QIAquick.TM. Gel Extraction Kit (Qiagen, Valencia, Calif.). The
initial plasmid used is pZMP21 that used pZMP21 as a base vector
and has the mouse Fc2 portion built into it. Plasmid pZMP21 is a
mammalian expression vector containing an expression cassette
having the MPSV promoter, multiple restriction sites for insertion
of coding sequences, a stop codon, an E. coli origin of
replication; a mammalian selectable marker expression unit
comprising an SV40 promoter, enhancer and origin of replication, a
DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences
required for selection and replication in S. cerevisiae. It is
constructed from pZP9 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
under Accession No. 98668) with the yeast genetic elements taken
from pRS316 (deposited at the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209, under
Accession No. 77145), an internal ribosome entry site (IRES)
element from poliovirus, and the extracellular domain of CD8
truncated at the C-terminal end of the transmembrane domain.
Plasmid hBTLA mFc2 pZMP21 was digested with EcoR1/BglII to cleave
off human BTLA and used for recombination with the PCR insert.
[0397] The recombination was performed using the BD In-Fusion.TM.
Dry-Down PCR Cloning kit (BD Biosciences, Palo Alto, Calif.). The
mixture of the PCR fragment and the digested vector in 10 .mu.l was
added to the lyophilized cloning reagents and incubated at
37.degree. C. for 15 minutes and 50.degree. C. for 15 minutes. The
reaction was ready for transformation. 2 .mu.l of recombination
reaction was transformed into One Shot TOP10 Chemical Competent
Cells (Invitrogen, Carlbad, Calif.); the transformation was
incubated on ice for 10 minutes and heat shocked at 42.degree. C.
for 30 seconds. The reaction was incubated on ice for 2 minutes
(helping transformed cells to recover). After the 2 minutes
incubation, 300 .mu.l of SOC (2% Bacto.TM. Tryptone (Difco,
Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM
KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was added
and the transformation was incubated at 37.degree. C. with shaker
for one hour. The whole transformation was plated on one LB AMP
plates (LB broth (Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L
Ampicillin).
[0398] The colonies were screened by PCR using primers zc50437 (SEQ
ID NO:33) and zc50438 (SEQ ID NO:34). The positive colonies were
verified by sequencing. The correct construct was designated as
mB7R1mFc2pZMP21 (SEQ ID NO: 69).
EXAMPLE 3
B7/mFc2 Expression Constructs
[0399] An expression vector, pZMP21 hB7R1/mFc2 (SEQ ID NO: 68), was
prepared to express a c-terminally Fc tagged soluble version of
zB7R1. A 734 base pair fragment was generated by PCR containing the
extracellular domain of zB7R1 (SEQ ID NO:3) and the first two amino
acids of mFc (glutamine and proline) with EcoRI and BglII sites
coded on the 5' and 3' ends, respectively.
[0400] This PCR fragment was generated using primers zc48914 (SEQ
ID NO:35) and zc48908 (SEQ ID NO:36) by amplification from a human
placenta cDNA library. The PCR reaction conditions were as follows:
25 cycles of 94.degree. C. for 1 minute, 60.degree. C. for 1
minute, and 72.degree. C. for 2 minutes; 1 cycle at 72.degree. C.
for 10 minutes; followed by a 4.degree. C. soak. A 699 base pair
fragment was generated by PCR containing the constant 2 and
constant 3 domains of effector function minus BALB-C IgG gamma 2a
(mFc2). This PCR fragment was generated using primers zc48911 and
ac48915 by amplification from an expression vector containing mFc2
(mTACI/mFc2 construct #998). The PCR reaction conditions were as
follows: 25 cycles of 94.degree. C. for 1 minute, 60.degree. C. for
1 minute, and 72.degree. C. for 2 minutes; 1 cycle at 72.degree. C.
for 10 minutes; followed by a 4.degree. C. soak. The 734 base pair
zB7R1 fragment and the 699 base pair mFc2 fragment were purified by
1% agarose gel electrophoresis and band purification using a
QiaQuick gel extraction kit (Qiagen: 28704). 1/5.sup.th and
1/25.sup.th of the total of the purified bands each for the zB7R1
and the mFc2 fragments, respectively were recombined into pZMP21
that had been linearized by BglII digestion and purified by band
purification, as described above, using the yeast strain
SF838-9Dalpha. Yeast that were able to grow out of uracil deficient
agar plates were lysed and DNA was extracted by ethanol
precipitation. 2 .mu.l of the ligation mix was electroporated in 37
.mu.l DH10B electrocompetent E.coli (Gibco 18297-010) according to
the manufacturer's directions. The transformed cells were diluted
in 400 .mu.l of LB media and plated onto LB plates containing 100
.mu.g/ml ampicillin. Clones were analyzed by restriction digests
and positive clones were sent for DNA sequencing to confirm PCR
accuracy.
[0401] The expression vector, pZMP21 hB7R1/mfc2, described above,
was then used to build a series of mFc2 soluble chimeric proteins.
zB7R1/mFc2 was built by PCRing a 438 base pair fragment using
oligos zc 50136 (SEQ ID NO:37) and zc50138 (SEQ ID NO:38) with
clonetrack #101632 as template. The resulting PCR product was band
purfied, as described above, and disgested with EcoRI and BglII.
The resulting product was again band purfied. PZMP21 hB7R1/mFc2 was
also digested with EcoRI and BglII and the 9721 base pair vector
backbone plus mFc2 was isolated. 1/50.sup.th of the pZMP21
hB7R1/mFc2 product was ligated to 3/50.sup.th of the 438 base pair
fragment using T4 DNA ligase. 2 .mu.l of the ligation mix was
electroporated in 37 .mu.l DH10B electrocompetent E.coli (Gibco
18297-010) according to the manufacturer's directions. The
transformed cells were diluted in 400 .mu.l of LB media and plated
onto LB plates containing 100 .mu.g/ml ampicillin. Clones were
analyzed by restriction digests and positive clones were sent for
DNA sequencing to confirm PCR accuracy. Three sets of 200 .mu.g of
the pZMP21 hB7R1/mFc2 construct were then each digested with 200
units of Pvu I at 37.degree. C. for three hours and then were
precipitated with IPA and spun down in a 1.5 mL microfuge tube. The
supernatant was decanted off the pellet, and the pellet was washed
with 1 mL of 70% ethanol and allowed to incubate for 5 minutes at
room temperature. The tube was spun in a microfuge for 10 minutes
at 14,000 RPM and the supernatant was decanted off the pellet. The
pellet was then resuspended in 750 .mu.l of PF--CHO media in a
sterile environment, allowed to incubate at 60.degree. C. for 30
minutes, and was allowed to cool to room temperature. 5E6 APFDXB11
cells were spun down in each of three tubes and were resuspended
using the DNA-media solution. The DNA/cell mixtures were placed in
a 0.4 cm gap cuvette and electroporated using the following
parameters: 950 .mu.F, high capacitance, and 300 V. The contents of
the cuvettes were then removed, pooled, and diluted to 25 mLs with
PF--CHO media and placed in a 125 mL shake flask. The flask was
placed in an incubator on a shaker at 37.degree. C., 6% CO.sub.2,
and shaking at 120 RPM. The cell line was subjected to nutrient
selection followed by step amplification to 200 nM methotrexate
(MTX), and then to 500 nM MTX. Expression was confirmed by western
blot, and the cell line was scaled-up and protein purification
followed.
EXAMPLE 4
Mouse zB7R1Avi-HIS TagpZMP21
[0402] In the effort to create the tetramer molecules an expression
plasmid containing a polynucleotide encoding the extra-cellular
domain of mouse zB7R1, the Avi Tag and HIS Tag was constructed. A
DNA fragment of the extra-cellular domain of mouse zB7R1 is
isolated by PCR using SEQ ID NO:39 with flanking regions at the 5'
and 3' ends corresponding to the vector sequence and part of the
Avi Tag sequence flanking the mouse zB7R1 insertion point using
primers zc51100 (SEQ ID NO:40) and zc51101 (SEQ ID NO:41).
[0403] The PCR reaction mixture is run on a 2% agarose gel and a
band corresponding to the size of the insert is gel-extracted using
a QIAquick.TM. Gel Extraction Kit (Qiagen, Valencia, Calif.).
Plasmid pZMP21 is a mammalian expression vector containing an
expression cassette having the MPSV promoter, multiple restriction
sites for insertion of coding sequences, a stop codon, an E. coli
origin of replication; a mammalian selectable marker expression
unit comprising an SV40 promoter, enhancer and origin of
replication, a DHFR gene, and the SV40 terminator; and URA3 and
CEN-ARS sequences required for selection and replication in S.
cerevisiae. It is constructed from pZP9 (deposited at the American
Type Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, under Accession No. 98668) with the yeast genetic
elements taken from pRS316 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
under Accession No. 77145), an internal ribosome entry site (IRES)
element from poliovirus, and the extracellular domain of CD8
truncated at the C-terminal end of the transmembrane domain.
Plasmid pZMP21AviHIS was digested with EcoR1 and used for
recombination with the PCR insert. 1397] The recombination was
performed using the BD In-Fusion.TM. Dry-Down PCR Cloning kit (BD
Biosciences, Palo Alto, Calif.). The mixture of the PCR fragment
and the digested vector in 10 .mu.l was added to the lyophilized
cloning reagents and incubated at 37.degree. C. for 15 minutes and
50.degree. C. for 15 minutes. The reaction was ready for
transformation. 2 .mu.l of recombination reaction was transformed
into One Shot TOP10 Chemical Competent Cells (Invitrogen, Carlbad,
Calif.); the transformation was incubated on ice for 10 minutes and
heat shocked at 42.degree. C. for 30 seconds. The reaction was
incubated on ice for 2 minutes (helping transformed cells to
recover). After the 2 minutes incubation, 300 .mu.l of SOC (2%
Bacto.TM. Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract
(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM
MgSO.sub.4, 20 mM glucose) was added and the transformation was
incubated at 37.degree. C. with shaker for one hour. The whole
transformation was plated on one LB AMP plates (LB broth (Lennox),
1.8% Bacto.TM. Agar (Difco), 100 mg/L Ampicillin).
[0404] The colonies were screened by PCR using primers zc51100 (SEQ
ID NO:40) and zc51101 (SEQ ID NO:41). The positive colonies were
verified by sequencing. The correct construct was designated as
mB7R1AviHISpZMP21.
EXAMPLE 5
Human zB7R1Avi-HIS TagpZMP21
[0405] In the effort to create the tetramer molecules an expression
plasmid containing a polynucleotide encoding the extra-cellular
domain of human zB7R1 (SEQ ID NO:3), the Avi Tag and HIS Tag was
constructed. A DNA fragment of the extra-cellular domain of human
zB7R1 is isolated by PCR using SEQ ID NO:42 with flanking regions
at the 5' and 3' ends corresponding to the vector sequence and the
Avi Tag (SEQ IO NO:43) and HIS Tag (SEQ ID NO:44) sequences
flanking the human zB7R1 insertion point using primers zc50485 (SEQ
ID NO:45) and zc50729 (SEQ ID NO:46).
[0406] The PCR reaction mixture is run on a 2% agarose gel and a
band corresponding to the size of the insert is gel-extracted using
a QIAquick.TM. Gel Extraction Kit (Qiagen, Valencia, Calif.).
Plasmid pZMP21 is a mammalian expression vector containing an
expression cassette having the MPSV promoter, multiple restriction
sites for insertion of coding sequences, a stop codon, an E. coli
origin of replication; a mammalian selectable marker expression
unit comprising an SV40 promoter, enhancer and origin of
replication, a DHFR gene, and the SV40 terminator; and URA3 and
CEN-ARS sequences required for selection and replication in S.
cerevisiae. It is constructed from pZP9 (deposited at the American
Type Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, under Accession No. 98668) with the yeast genetic
elements taken from pRS316 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
under Accession No. 77145), an internal ribosome entry site (IRES)
element from poliovirus, and the extracellular domain of CD8
truncated at the C-terminal end of the transmembrane domain.
Plasmid pZMP21 was digested with EcoR1/BglII to cleave off the PTA
leader and used for recombination with the PCR insert.
[0407] The recombination was performed using the BD In-Fusion.TM.
Dry-Down PCR Cloning kit (BD Biosciences, Palo Alto, Calif.). The
mixture of the PCR fragment and the digested vector in 10 .mu.l was
added to the lyophilized cloning reagents and incubated at
37.degree. C. for 15 minutes and 50.degree. C. for 15 minutes. The
reaction was ready for transformation. 2 .mu.l of recombination
reaction was transformed into One Shot TOP10 Chemical Competent
Cells (Invitrogen, Carlbad, Calif.); the transformation was
incubated on ice for 10 minutes and heat shocked at 42.degree. C.
for 30 seconds. The reaction was incubated on ice for 2 minutes
(helping transformed cells to recover). After the 2 minutes
incubation, 300 .mu.l of SOC (2% Bacto.TM. Tryptone (Difco,
Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM
KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was added
and the transformation was incubated at 37.degree. C. with shaker
for one hour. The whole transformation was plated on one LB AMP
plates (LB broth (Lennox), 1.8% Bactom Agar (Difco), 100 mg/L
Ampicillin).
[0408] The colonies were screened by PCR using primers zc50485 (SEQ
ID NO:45) and zc50729 (SEQ ID NO:46). The positive colonies were
verified by sequencing. The correct construct was designated as
hB7R1AviHISpZMP21.
EXAMPLE 6
Stimulation Conditions for the Expression of zB7R1 and Other B7
Family Members
A. Introduction
[0409] Stimulation conditions under which known B7 family members
are expressed and/or upregulated on murine bone marrow derived
dendritic cells (BMDCs) would be helpful in the assessment of
zB7R1'a ability to bind cultured DCs. First, the regulation of
known B7 family members was investigated using various stimulation
conditions in FLT3L and GM-CSF/IL4 cultures from BALB/c mice.
Secondly, the binding of the available murine soluble Fc-fusion
proteins to FLT3L, GM-CSF, and GM-CSF/IL4 cultured bone marrow
cells from both BALB/c and C57BL/6 strains of mice were tested.
This is described in more detail below.
B. Methods
1) Isolating Bone Marrow
[0410] Bone marrow from 8-week-old female BALB/c mice or
4-month-old C57B1/6 mice was collected from the femurs. The bone
marrow was filtered through a 100 .mu.M cell strainer, the red
blood cells lysed with ACK Lysis buffer, and the cells resuspended
in RPMI "complete" media (10% FCS, 2 mM L-Glutamine, 1 mM
Na-Pyruvate, 0.1 mM NEAA, 0.05 mM .beta.-ME). Cells were then
plated in 6-well plates at 1.times.10.sup.6 cells per ml with the
appropriate culturing conditions.
[0411] (i) Generation of Flt3L BMDCs Cell Cultures
[0412] Bone marrow cells were cultured in the presence of 100 ng/ml
of recombinant human Flt3 ligand. One half of the media was
replaced on day 5 of culture with fresh Flt3L containing media.
Cells were collected on day 7 of culture.
[0413] (ii) Generation of GM-CSF/IL-4 BMDCs Cell Cultures
[0414] Bone marrow cells were cultured in the presence of 10 ng/ml
each of recombinant murine GM-CSF and recombinant murine IL-4 (both
R&D Systems). One half of the media was replaced on day 3 of
culture with fresh GM-CSF/IL4 containing media. Cells were
collected on day 6 of culture.
[0415] (iii) Generation of GM-CSF BMDCs Cell Cultures
[0416] Bone marrow cells were cultured in the presence of 20 ng/ml
recombinant murine GM-CSF in 4 ml in 6-well plates. On day 3 of
culture 2 ml of fresh GM-CSF containing media was added to each
well, on day 6, one half of the media (3 ml) was replaced with
fresh GM-CSF containing media. Cells were collected on day 7 of
culture.
2) FACS Analysis
[0417] All stains and dilutions were performed in FACS wash buffer
(PBS, 1% BSA, 0.1% NaN.sub.3) Prior to staining, FcBlock (0.25
ug/10.sup.6 cells) was added to the cells and incubated
approximately 5-10 minutes. Cells were co-stained with CD11c and
B220. All data was acquired on BD FACSCalibur.
C. Stimulation of Cell Cultures:
1) Investigation of Regulation of Known B7-Family Molecules
[0418] Cultured cells were plated at 1.times.10.sup.6 cells per ml
in 2 ml in 24-well plates and stimulated with 100 ng/ml LPS, 20
ng/ml IFN.gamma., 1 ug/ml CD40L, or a TLR ligand mix containing 0.1
ug/ml MALP-2, 12.5 ug/ml Poly I:C, 100 ng/ml LPS, 0.1 ug/ml
Flagellin, 1 ug/ml R848, and 125 ng/ml CpG ODN1826. Cells were
assayed by flow cytometry for B7 family expression at t=0, 24 h, 48
h, 72 h, and 96 h.
[0419] Cells were stained with one of the PE-conjugated B7 family
antibodies B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-DC, ICOS, or
PD-1. 7-AAD was used to gate out dead cells at the 72 and 96 h
timepoints.
[0420] Flt3L BMDCs
[0421] (i) B7-1
[0422] At all timepoints, unstimulated cells expressed B7-1, and
expression was further upregulated by LPS and the TLR ligand mix.
The IFN.gamma. and CD40L treatment had no effect on B7-1 expression
relative to unstimulated cells.
[0423] (ii) B7-2
[0424] At all timepoints, unstimulated cells expressed B7-2, and
expression was further upregulated by LPS, IFN.gamma. and the TLR
ligand mix. The CD40L treatment had no effect on B7-2 expression
relative to unstimulated cells.
[0425] (iii) B7-H1
[0426] At 24 h unstimulated cells do not express B7-H1, but LPS,
IFN.gamma. and the TLR ligand mix do induce expression.
Unstimulated cells begin to express low levels of B7-H1 by 48 h,
and LPS, IFN.gamma. and the TLR ligand mix continue to show
upregulation of the B7-H1 relative to the unstimulated control. The
CD40L treatment had no effect on B7-H1 expression relative to
unstimulated cells.
[0427] (iv) B7-H2
[0428] At all timepoints, unstimulated cells expressed B7-H2. The
IFN.gamma. and CD40L treatment had little to no effect on B7-H2
expression relative to unstimulated cells. Treatment with the TLR
ligand cocktail appeared to decrease B7-H2 expression.
[0429] (v) PD-1
[0430] Unstimulated Flt3L dendritic cells were negative for PD-1
expression, however TLR ligand stimulation induced expression at
all timepoints, and LPS (weakly) and IFN.gamma. induced
upregulation at 48 h, 72 h, and 96 h timepoints.
[0431] (vi) B7-H3, B7-H4, B7-DC, and ICOS
[0432] B7-H3, B7-H4, B7-DC, and ICOS were negative for expression,
at all timepoints, with all stimulation conditions, in Flt3L
generated dendritic cells.
GM-CSF/IL-4 BMDCs
[0433] (i) B7-1
[0434] At all timepoints, unstimulated cells expressed B7-1, and
expression was further upregulated by TLR ligand mix at 72 h and 96
h timepoints. The LPS, IFN.gamma. and CD40L treatment had no effect
on B7-1 expression relative to unstimulated cells.
[0435] (ii) B7-2
[0436] At all timepoints, unstimulated cells expressed B7-2, and
expression was further upregulated by IFN.gamma. and the TLR ligand
mix at 72 h and 96 h timepoints. LPS decreased B7-2 expression at
the 72 and 96 h timepoints. The CD40L treatment had no effect on
B7-2 expression relative to unstimulated cells.
[0437] (iii) B7-H1
[0438] Unstimulated GM-CSF/IL-4 BMDCs highly express B7-H1. LPS
upregulated expression at only the 24 h timepoint, and IFN.gamma.
and the TLR ligand mix upregulated expression at all timepoints.
The CD40L treatment had no effect on B7-H1 expression relative to
unstimulated cells.
[0439] (iv) B7-H2
[0440] At all timepoints, unstimulated cells expressed B7-H2.
IFN.gamma. treatment slightly upregulated B7-H2 expression relative
to unstimulated cells at 48, 72, and 96 h. All other stimulation
conditions had no effect on B7-H2 expression relative to
unstimulated cells.
[0441] (v) B7-DC
[0442] Unstimulated GM-CSF/IL4 dendritic cells were positive for
PD-1 expression, and IFN.gamma. stimulation induced increased
expression at 48 h, 72 h, and 96 h timepoints LPS, CD40L, and TLR
ligand cocktail had no effect on B&-DC expression relative to
the unstimulated control.
[0443] (vi) B7-H3, B7-H4, PD-1, and ICOS
[0444] B7-H3, B7-H4, PD-1, and ICOS were negative for expression,
at all timepoints, with all stimulation conditions, in GM-CSF/IL-4
generated dendritic cells.
2) Binding of Soluble Fc-Fusion Proteins
[0445] Cells were cultured as described above, however the
stimulation conditions were modified to (i) LPS 100 ng/ml, (ii)
CD40L (1 ug/ml) and IFN.gamma. (20 ng/ml) and (iii) TLR ligand
cocktail, with the some modifications (we omitted LPS, and used CpG
ODN 2395 instead of CpG ODN 1826 (both murine TLR 9 ligands) and
used Polyuridylic acid instead of R848 (both TLR 7/8 ligands)).
Cells were assayed for binding to the available Fc-fusion proteins
of interest at 48 h by flow cytometry.
[0446] Murine Fc-fusion proteins pBTLA, zB7R1, zB7-H4mL, and
zB7-H4mS as well as human zB7-H3x2 (negative control) and murine
ICOS-Fc purchased from R&D Systems (positive control) were
labeled with PE using Zenon Mouse IgG Labeling Kits (Molecular
Probes) and used to stain the cells. The dye 7-AAD was used to gate
out dead cells.
[0447] There did not appear to be any binding of murine pBTLA-Fc,
zB7-H4mL-Fc or zB7-H4mS-Fc under any of the conditions tested.
[0448] However, murine zB7R1-Fc did appear to bind in
CD40L+IFN.gamma. stimulated Flt3L cultured cells in both BALB/c and
C57B1/6 strains of mice. Binding of zB7R1-Fc appeared negative in
both the GM-CSF and GM-CSF/IL-4 cultured cells under conditions
tested.
D. Conclusion
[0449] Conditions under which B7-1, B7-2, B7-H1, B7-H2, B7-DC, and
PD-1 are expressed and/or upregulated in cell cultures enriched for
dendritic cells were identified. Furthermore, binding of
fluorescently labeled Fc fusion proteins of the orphan receptors
and ligands to cells cultured under these conditions to define
unknown counterparts for the unpaired B7 family members were
tested. Specifically, one stimulation condition (IFNg+CD40L) was
identified, which induced binding of zB7R1.
EXAMPLE 7
Identification of Cells Expressing zB7R1
A. Introduction
[0450] The identification of a cell source expressing the
counter-receptor for zB7R1 would help understand the biology of
zB7R1 and also would assist in cloning the counter-receptor. A
direct fluorochrome conjugate of zB7R1-mFc2a and mouse splenocytes
surface stained with a cocktail of fluorochrome conjugated
antibodies specific for lineage markers to identify activated
CD11c.sup.+ cells as the primary cell type that binds zB7R1 was
used.
B. Procedure
[0451] A DO11.10 mouse spleen transgenic for a TCR specific for the
Ova peptide 323-339 was collected and mashed between frosted glass
slides to obtain a single cell suspension. Red cells in the
suspension were lysed using ACK lysis buffer. The resulting cell
suspension was adjusted to 1.times.10.sup.6 cells per well in media
(RPMI+10% FBS, glutamine, pyruvate, pen-strep and 2-mercaptoethanol
at 5.times.10.sup.-5M) and incubated with 1 uM OVA peptide 323-339
at 37 C. Cells were collected for analysis by flow cytometry at
times 0, 24, 48, and 72 hours.
[0452] zB7R1-mIgGFc2a fusion protein was directly labeled with PE
using the Zenon.TM. R-Phycoerythrin mouse IgG2a labeling kit
(Molecular Probes, Eugene Oreg., cat. #Z25155) following
manufacturer's instructions. Cells collected at each timepoint were
incubated in Facs buffer (PBS+2% BSA+0.02% NaN.sub.3) with 5 ug/ml
of Zenon.TM.-PE labeled zB7R1-mFc2a. In some cases, these binding
experiments were also performed in the presence of 40-fold excess
unlabeled zB7R1-mFc2a (specific blocking) or 40 fold excess
pB7H4L-mFc2a (non-specific blocking). Control wells were incubated
with the Zenon.TM. labeling reagent alone or with an irrelevant
Fc-fusion protein labeled in the same way. Cells were
simultaneously incubated with antibodies to the following antigens:
CD11c-APC, CD11b-PerCP-CY5, CD49b-APC-CY7, CD3-PeCy7 CD19-FITC (BD
Pharmingen), CD8-PE-Texas Red and CD4-A405 (Caltag) at appropriate
dilutions in FACS buffer on ice for 30 minutes. Cells were washed
twice (adding Facs buffer at 4.times. the labeling volume and
centrifuging the cells at 300.times.g for 5 minutes, decanting the
supernatant for each wash), then fixed with 2% paraformaldehyde in
PBS for 20 minutes. Cells were centrifuged at 300.times.g for 5
minutes and resuspended in 200 ul FACs buffer per 2.times.10.sup.5
cells and stored at 4 C for up to 5 days. Samples were analyzed
using a flow cytometer (FACSAria, Becton Dickenson) and FACS Diva
software.
C. Results
[0453] Viable cells were gated on using forward and side scatter
dot plots. Viable cells were then analyzed for CD11b and CD11c
expression, as well as for the other surface markers in the
staining combination. In one experiment, binding of zB7R1-mFc2a was
observed on CD11c cells at all timepoints. In the same experiment,
binding of zB7R1-mFc2a on CD11b CD11c double positive cells was
detectable only at 48 and 72 hours. In another experiment, binding
of zB7R1-mFc2a was observed on CD11c cells and CD11c CD11b double
positive cells at 48 and 72 hours. Cells positive for CD11b but
negative for CD11c did not bind zB7R1 significantly higher than
cells stained with the labeling reagent alone or with an irrelevant
Fc-fusion protein at all timepoints in both experiments. Cells with
a CD11c.sup.+ CD11b.sup..+-. surface phenotype bound zB7R1 with a
mean fluorescence of 1952 channels versus 660 for the control. The
sample specifically blocked with excess unlabeled zB7R1-mFc2a had a
mean channel fluorescence of 781 compared to 1682 for the sample
non-specifically blocked with excess pB7H4L-mFc2a.
D. Conclusion
[0454] The CD11c surface marker is found on most dendritic cells
and is used to identify them in the mixture of activated and
resting spleen cells responses. The binding of zB7R1-mFc2a to the
surface of dendritic cells indicates the presence of the cognate
ligand on the surface of these cells. The binding increases on
activated dendritic cells. The interaction of zB7R1 on dendritic
cells and zB7R1 on T cells and possibly other cell types influences
the progression of an immune response.
EXAMPLE 8
Murine zB7R1 mRNA is Regulated in Select Tissues in Murine Models
of Disease Compared to Non-Diseased Controls
A. Procedure
[0455] Tissues were obtained from the following murine models of
disease: Colitis, Asthma, Experimental Allergic Encephalomyelitis
(EAE), Psoriasis and Collagen Induced Arthritis (CIA). Animal
models were run following standard procedures and included
appropriate non-diseased controls. Colitis was induced by dextran
sodium sulfate (DSS) in the drinking water and the tissues isolated
from the model included distal colon, proximal colon and mesenteric
lymph nodes. Asthma was induced by sensitization and intranasal
challenge to the antigen ovalbumin. The tissues isolated included
lung, spleen and lymph node. EAE was induced by immunizing with
MOG35-55 peptide in RIBI adjuvant. Tissues isolated included brain,
lymph node, and spinal cord. Psoriasis was induced by adoptive
transfer of naive T cells into minor histocompatibility mismatched
or syngeneic immunocompromised mice. Tissues isolated included
lesional skin and adjacent skin. CIA was induced by collagen
injections and tissues isolated included foot and lymph node. RNA
was isolated from all tissues using standard procedures. In brief,
tissues were collected and immediately frozen in liquid N2 and then
transferred to -80.degree. C. until processing. For processing,
tissues were placed in Qiazol reagent (Qiagen, Valencia, Calif.)
and RNA was isolated using the Qiagen Rneasy kit according to
manufacturer's recommendations. Expression of murine zB7R1 mRNA was
measured with multiplex real-time quantitative RT-PCR method
(TaqMan) and the ABI PRISM 7900 sequence detection system (PE
Applied Biosystems). zB7R1 mRNA levels were normalized to the
expression of the murine hypoxanthine guanine physphoribosyl
transferase mRNA and determined by the comparative threshold cycle
method (User Bulletin 2; PE Applied Biosystems). The primers and
probe for murine zB7R1 included a 5' forward primer (SEQ ID NO:47),
reverse 5' primer (SEQ ID NO:48) and a probe (SEQ ID NO:49).
B. Results
[0456] Murine zB7R1 mRNA expression was detected in all tissues
tested. Highest levels of expression were observed in the lymph
node and spleen tissues. Lower levels of expression were found in
skin, colon, lung, brain, foot, and spinal cord tissues.
[0457] Murine zB7R1 mRNA was increased in tissues from a chronic
model of DSS colitis compared to tissues from non-diseased
controls. Zb7r1 was increased 1.65 fold in the LN, 3.2 fold in the
distal colon and 2.6 fold in the proximal colon compared to
non-diseased controls.
[0458] zB7R1 mRNA was increased in tissues from the murine model of
asthma compared to tissues from non-diseased controls. Zb7r1 was
increased 5.4 fold in lung, 1.4 fold in spleen and 1.7 fold in
lymph node.
[0459] Zb7r1 RNA was increased in tissues from the EAE model
compared to tissues from non-diseased controls. Zb7r1 mRNA was
increased 16.87 fold in the brain of animals from the early onset
of disease and 5.63 fold in animals with severe disease scores.
Zb7r1 mRNA was increased 4.15 fold in the spinal cord of animals
from the early onset of disease and 6.93 fold in animals with
severe disease scores.
[0460] zB7R1 mRNA was increased in skin tissues from the psoriasis
model compared to skin tissues from non-diseased controls. Zb7r1
mRNA was increased 2.24 fold in a skin lesion and 3.07 fold in skin
tissue adjacent to the psoriatic lesion.
[0461] zB7R1 mRNA was increased in whole foot tissue from mice in
the CIA model of arthritis compared to foot tissue from
non-diseased controls. Zb7r1 mRNA was increased 2.31 fold in
animals scored with mild disease and 3.4 fold in animals with
severe disease.
EXAMPLE 9
Cloning and Construction of VASP Expression Vector
[0462] Human vasodialator-activated phosphoprotein (VASP) is
described by Kuhnel, et al., (2004) Proc. Nat'l. Acad. Sci. 101:
17027. VASP nucleotide and amino acid sequences are provided as SEQ
ID NOs: 13 and 14. Two overlapping oligonucleotides, which encoded
both sense and antisense strands of the tetramerization domain of
human VASP protein, were synthesized by solid phased synthesis
using oligonucleotide zc50629 (SEQ ID NO:50) and oligonucleotide ZC
50630 (SEQ ID NO:51). These oligonucleotides were annealed at
55.degree. C., and amplified by PCR with the olignucleotide primers
zc50955 (SEQ ID NO:52) and zc50956 (SEQ ID NO:53).
[0463] The amplified DNA was fractionated on 1.5% agarose gel and
then isolated using a Qiagen gel isolation kit according to
manufacturer's protocol (Qiagen, Valiencia, Calif.). The isolated
DNA was inserted into BglII cleaved pzmp21 vector by yeast
recombination. DNA sequencing confirmed the expected sequence of
the vector, which was designated pzmp21VASP-His.sub.6.
[0464] The extra cellular domain of human zB7R1 was generated by
restriction enzyme digestion of human zB7R1mFc2 (SEQ ID No:61). A
double digest with EcoRI and BglII (Roche Indianapolis, Ind.) was
performed to obtain the extra cellular domain. The fragment was
fractionated on 2% agarose gel (Invitrogen Carlsbad, Calif.)and
then isolated using a Qiagen gel isolation kit according to
manufacturer's protocol (Qiagen Valencia Calif.). The isolated
fragment was inserted into EcoRI/BglII cleaved pZMP21VASP-His.sub.6
vector by ligation (Fast Link Ligase EPICENTRE Madison, Wisc.). The
construct was designated as hzB7R1VASPpZMP21 (SEQ ID No: 62).
[0465] The extra cellular domain of mouse zB7R1 was generated by
restriction enzyme digestion of mouse zB7R1mFc2 SEQ ID No: 63. A
double digest with EcoRI and BglII (Roche Indianapolis, Ind.) was
performed to obtain the extra cellular domain. The fragment was
fractionated on 2% agarose gel (Invitrogen Carlsbad, Calif.) and
then isolated using a Qiagen gel isolation kit according to
manufacturer's protocol (Qiagen Valencia Calif.). The isolated
fragment was inserted into EcoRI/BglII cleaved pZMP21VASP-His.sub.6
vector by ligation (Fast Link Ligase EPICENTRE Madison, Wisc.). The
construct was designated as mzB7R1VASPpZMP21 SEQ ID No: 64.
[0466] These vector includes the coding sequence for the zB7R1
extracellular domain (including the native signal sequence)
comprising amino acids 1 to 140 of the full length gene (amino
acids 1-140 of SEQ ID NO:2), the flexible linker GSGG (SEQ ID NO:
27), the VASP tetramerization domain (amino acids 5 to 38 of SEQ ID
NO: 54), the flexible linker GSGG (SEQ ID NO:27), and the His6 tag
amino acid residues (amino acids 43 to 48 of SEQ ID NO: 54).
EXAMPLE 10
Expression and Purification of B7R1VASP-HIS.sub.6
[0467] The pzmp21B7R1VASP-His.sub.6 vector was transfected into
BHK570 cells using Lipofectamine 2000 according to manufacturer's
protocol (Invitrogen, Carlsbad, Calif.) and the cultures were
selected for transfectants resistance to 10 .mu.M methotrexate.
Resistant colonies were transferred to tissue culture dishes,
expanded and analyzed for secretion of B7R1VASP-His.sub.6 by
western blot analysis with Anti-His (C-terminal) Antibody
(Invitrogen, Carlsbad, Calif.). The resulting cell line,
BHK.B7R1VASP-His.sub.6.2, was expanded.
A. Purification of B7R1VASP-His.sub.6 from BHK Cells
[0468] The purification was performed at 4.degree. C. About 2 L of
conditioned media from BHK:B7R1VASP-His.sub.6.2 was concentrated to
0.2 L using Pellicon-2 5 k filters (Millipore, Bedford, Mass.),
then buffer-exchanged tenfold with 20 mM NaPO.sub.4, 0.5M NaCl, 15
mM Imidazole, pH 7.5. The final 0.2 L sample was passed-through a
0.2 mm filter (Millipore, Bedford, Mass.).
[0469] A Talon (BD Biosciences, San Diego, Calif.) column with a 20
mL bed-volume was packed and equilibrated with 20 mM NaPi, 15 mM
Imidazole, 0.5 M NaCl, pH 7.5. The media was loaded onto the column
at a flow-rate of 0.2-0.4 mL/min then washed with 5-6 CV of the
equilibration buffer. B7R1VASP-His.sub.6 was eluted from the column
with 20 mM NaPO.sub.4, 0.5 M NaCl, 0.5 M Imidazole, pH 7.5 at a
flow-rate of 4 mL/min. 10 mL fractions were collected and analyzed
for the presence of B7R1VASP-His.sub.6 by Coomassie-stained
SDS-PAGE.
[0470] A combined pool of Talon eluates obtained from three
identical runs as described above was concentrated from 60 mL to 3
mL using an Amicon Ultra 5 k centrifugal filter (Millipore,
Bedford, Mass.). A Superdex 200 column with a bed-volume of 318 mL
was equilibrated with 50 mM NaPi, 110 mM NaCl, pH 7.3, and the 3 mL
sample was injected into the column at a flow-rate of 0.5 mL/min.
Two 280 nm absorbance peaks were observed eluting from the column,
one at 0.38 CV and the other at 0.44 CV. The fractions eluting
around 0.44 CV, believed to contain tetrameric B7R1VASP-His.sub.6,
were pooled and concentrated, sterile-filtered through a 0.2 mm
Acrodisc filter (Pall Corporation, East Hills, N.Y.), and stored at
-80.degree. C. Concentration of the final sample was determined by
BCA (Pierce, Rockford, Ill.).
B. SEC-MALS Analysis of B7R1VASP-CH.sub.6
[0471] The purpose of size exclusion chromatography (SEC) is to
separate molecules on the basis of size for estimation of molecular
weight (M.sub.w). If static light scattering detection is added to
a SEC system, absolute measurements of molecular weight can be
made. This is possible because the intensity of light scattered by
the analyte is directly proportional to its mass and concentration,
and is completely independent of SEC elution position, conformation
or interaction with the column matrix. Additionally, by combining
SEC, multi-angle laser light scattering (GALS) and refractive index
detection (RI), the molecular mass, association state, and degree
of glycosylation can be determined. The limit of accuracy of these
measurements for a sample that is monodisperse with respect to
M.sub.w is .+-.2%.
EXAMPLE 11
CD155 Binds Soluble zB7R1
[0472] A soluble form of zB7R1 was produced either as an in-frame
fusion with a mouse Fc-region or with the tetramerization domain
from the Vasp protein (both of which are described herein). These
proteins were labeled with either biotin or conjugated to a
fluorochrome for use as a FACS reagent or for fluorescence
microscopy. These reagents were used to interrogate a variety of
primary cell types from mouse bone marrow and spleen for binding.
Dendritic cells (DC's) from bone marrow grown seven days in Flt-3
ligand (Flt3L) and then activated with CD40 ligand (CD40L) and
interferon-g (IFNg) were found to bind fluorochrome conjugated or
biotinylated forms of both zB7R1 proteins. An expression library
was produced from this activated DC population and this library was
introduced into COS cells by transient transfection. Transfected
pools of cells were then screened for zB7R1 binding using the
biotinylated ZB7r1-Vasp protein and fluorescence microscopy.
Positive pools were broken down systematically until a single
plasmid was recovered that conveyed binding activity. Nucleic acid
sequencing revealed this plasmid encoded the mouse homolog of the
human poliovirus receptor (PVR), CD155 (SEQ ID NOs:17 and 18).
CD155 binds zB7R1 transfected cells and, thus, it is one
counter-receptor now known to bind zB7R1.
EXAMPLE 12
VASP-zB7R1 Expression for the Secretion Trap Assay
[0473] Three sets of 50 .mu.g of the mzB7R1/Vasp fusion protein
construct were each digested with 50 units of Pvu I at 37.degree.
C. for three hours and then were precipitated with EPA and spun
down in a 1.5 mL microfuge tube. The supernatant was decanted off
the pellet, and the pellet was washed with 1 mL of 70% ethanol and
allowed to incubate for 5 minutes at room temperature. The tube was
spun in a microfuge for 10 minutes at 14,000 RPM and the
supernatant was decanted off the pellet. The pellet was then
resuspended in 750 .mu.l of PF--CHO media in a sterile environment,
allowed to incubate at 60.degree. C. for 30 minutes, and was
allowed to cool to room temperature. 5E6 APFDXB11 cells were spun
down in each of three tubes and were resuspended using the
DNA-media solution. The DNA/cell mixtures were placed in a 0.4 cm
gap cuvette and electroporated using the following parameters: 950
.mu.F, high capacitance, and 300 V. The contents of the cuvettes
were then removed, pooled, and diluted to 25 mLs with PF--CHO media
and placed in a 125 mL shake flask. The flask was placed in an
incubator on a shaker at 37.degree. C., 6% CO.sub.2, and shaking at
120 RPM.
[0474] The cell line was subjected to nutrient selection followed
by step amplification to 200 nM methotrexate (MTX), and then to 500
nM MTX. Expression was confirmed by western blot, and the cell line
was scaled-up and protein purification followed.
EXAMPLE 13
Use of VASP-zB7R1 Fusion Protein to Screen for Ligands
[0475] zB7R1VASP fusion protein was made as described in the above
Example 12. This protein was then used to screen for its
corresponding ligand as described below.
A) Screening of the mBMDC Library:
[0476] A secretion trap assay was used to pair mzB7R1 to mCD155
(SEQ ID NO:18). The soluble mzB7R1/Vasp fusion protein that had
been biotinylated was used as a binding reagent in a secretion trap
assay. A pZP-7NX cDNA library from stimulated mouse bone marrow
(mBMDC) was transiently transfected into COS cells in pools of 800
clones. The binding of mzB7R1/Vasp-biotin to transfected COS cells
was carried out using the secretion trap assay described below.
Positive binding was seen in 26 of 72 pools screened. One of these
pools was selected and electroporated into DR10B. 400 single
colonies were picked into 1.2 mls LB+100 ug/ml ampicillin in deep
well 96-well blocks, grown overnight followed by DNA isolation from
each plate. After transfection and secretion trap probe, a single
positive well was identified from this breakdown and submitted to
sequencing and was identified as being mCD155. This purified cDNA
was transfected and probed with mB7R1/Vasp-biotin along with
additional controls to verifiy that mCD155 specifically and
reproducibly bound mB7R1/Vasp-biotin but not other vasp
chimeras.
B) COS Cell Transfections
[0477] The COS cell transfection was performed as follows: Mix 1 ug
pooled DNA in 25 ul of serum free DMEM media (500 mls DMEM with 5
mls non-essential amino acids) and 1 ul Cosfectin.TM. in 25 ul
serum free DMEM media. The diluted DNA and cosfectin are then
combined followed by incubating at room temperature for 30 minutes.
Add this 50 ul mixture onto 8.5.times.10.sup.5 COS cells/well that
had been plated on the previous day in 12-well tissue culture
plates and incubate overnight at 37.degree. C.
C) Secretion Trap Assay
[0478] The secretion trap was performed as follows: Media was
aspirated from the wells and then the cells were fixed for 15
minutes with 1.8% formaldehyde in PBS. Cells were then washed with
TNT (0.1M Tris-HCL, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O),
and permeabilized with 0.1% Triton-X in PBS for 15 minutes, and
again washed with TNT. Cells were blocked for 1 hour with TNB (0.1M
Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance
TSA-Direct Kit) in H.sub.2O), and washed again with TNT. The cells
were incubated for 1 hour with 2 .mu.g/ml mzB7R1/Vasp-biotin
soluble receptor fusion protein. Cells were then washed with TNT.
Cells were fixed a second time for 15 minutes with 1.8%
formaldehyde in PBS. After washing with TNT, cells were incubated
for another hour with 1:1000 diluted streptavidin HRP. Again cells
were washed with TNT.
[0479] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit) and incubated for
5 minutes, and washed with TNT. Cells were preserved with
Vectashield Mounting Media (Vector Labs Burlingame, Calif.) diluted
1:5 in TNT. Cells were visualized using a FITC filter on
fluorescent microscope.
EXAMPLE 14
Biological Activity of the VASP-zB7R1 Fusion Protein
[0480] T-cells are isolated from peripheral blood by negative
selection (Mitenyi Biotec, Auburn, Calif.). T-cells are plated into
each well of a 96 well dish that had been pre-coated with anti-CD3
(BD Bioscience, San Diego, Calif.). Anti-CD28 (BD Bioscience, San
Diego, Calif.), and increasing concentration of zB7R1/VASP are
added to appropriate wells. The cultures are incubated at
37.degree. C. for 4 days and then labeled overnight with 1
.quadrature.Ci [.sup.3H] thymidine per well. Proliferation is
measured as [.sup.3H] thymidine incorporated, and culture cytokine
content is quantitated using Luminex (Austen, Tex.). zB7R1/VASP
does potently inhibit both T-cell proliferation and cytokine
release (Dong et al., Nature Med. 5: 1365-1369, 1999).
EXAMPLE 15
zB7R1 Monoclonal Antibodies
[0481] BALB/c mice were immunized with DNA encoding the human zB7R1
extracellular domain (SEQ ID NO:3) expressed as a membrane protein.
Mice with positive serum titers to cellular expressed human zB7R1
were given a prefusion boost of soluble zB7R1-Fc fusion
protein.
[0482] Splenocytes were harvested from one high-titer mouse and
fused to P3-X63-Ag8/ATCC (mouse) myeloma cells in an optimized
PEG-mediated fusion protocol (Rockland Immunochemicals). Following
9 days growth post-fusion, specific antibody-producing hybridoma
pools were identified by ELISA using 500 ng/ml each of the purified
recombinant fusion protein zB7R1-mFc2 as the specific antibody
target and a pTACI mFc2 fusion protein as a non-specific antibody
target. To check for cross-reactivity, the samples were also
checked against mouse zB7R1. Hybridoma pools positive to the
specific antibody target only were analyzed further for ability to
bind via FACS analysis to p815/zB7R1 cells as antibody target.
[0483] Hybridoma pools yielding a specific positive result in the
ELISA assay and positive results in the FACS assay were cloned at
least two times by limiting dilution.
[0484] The following five clones were harvested and purified for
use in assays: 318.4.1.1, 318.28.2.1, 318.39.1.1, 318.59.3.1,
318.77.1.10
EXAMPLE 16
Bioassays for the Detection of Anti-zB7R1 Signaling Antibodies
[0485] In an effort to develop an assay that could be used to
detect and evaluate signaling antibodies, a Baf3-STAT-luciferase
reporter cell line was constructed expressing a chimera of the
extracellular domain of the molecule of interest (i.e. zB7R1), and
the transmembrane and intracellular domains of mouse GCSFR.
Antibodies against the molecule of interest may mediate
dimerization of their target molecule on the cell surface, leading
in turn to the dimerization of the mGCSFR intracellular domains and
consequent phosphorylation of STAT signaling molecules. These
phosphorylated STATs then migrate to the nucleus where they bind to
STAT responsive elements located on a recombinant,
enhancer/promoter/cDNA construct. This binding results in the
transcription and synthesis of a luciferase protein that can be
measured quantitatively utilizing a simple assay.
[0486] The assay cell line was constructed by placing an expression
vector (pZMP21Z) containing the human zB7R1/mGCSFR chimera, into a
previously utilized BaF3/KZ134 cell line. This expression vector
and subsequent cell line were built using the following steps.
Generation of Human zB7R1 Extracellular Domain and Mouse GCSFr
Transmembrane and Intracellular Domain PCR Products
[0487] A 465 bp, human B7r1 extracellular domain, DNA fragment was
created by PCR using Expand reagents (Roche, Applied Sciences,
Indianapolis, Ind.), and ZC53051 (SEQ ID NO:55) and ZC54199 (SEQ ID
NO:56). These zB7R1 amplification primers added complimentary
regions to mGCSFR and the pZMP21 vector allowing for overlap PCR
and yeast recombination respectively.
[0488] A 1562 bp transmembrane and intracellular domain mouse GCSFr
DNA fragment was created by PCR using Expand reagents (Roche,
Applied Sciences, Indianapolis, Ind.), and ZC54198 (SEQ ID NO:57)
and ZC53248 (SEQ ID NO:58) These mGCSF amplification primers added
complimentary regions to hB7R1 and the pZMP21 vector allowing for
overlap PCR and yeast recombination respectively.
Generation of Human zB7R1-m.GCSFr Overlap PCR Product for Use in
Yeast Recombination
[0489] Plasmids containing the zB7r1 and mouse GCSFr cDNAs were
used as templates. PCR amplification of the zB7r1 and mouse GCSFr
fragments were performed as follows: One cycle of 95.degree. C. for
2 minutes; then thirty cycles at 95.degree. C. for 30 seconds,
56.degree. C. for 30 seconds, 72.degree. C. for 1.5 minutes,
followed by one cycle of 72.degree. C. for 7 minutes and then a
4.degree. C. hold. The reactions were visualized on a 1.2% agarose
gel and the appropriate bands were excised and purified using
QIAquick Gel Extraction kit (Qiagen, Santa Clarita, Calif.)
[0490] The zB7R1 and mouse GCSFr purified PCR products were used as
templates in an overlap PCR reaction to create a chimeric
B7r1-m.GCSFr product of 1995 bp. Expand reagents (Roche, Applied
Sciences, Indianapolis, Ind.), and ZC53051 (SEQ ID NO:59) and
ZC53248 (SEQ ID NO:60) as PCR primers were used.
[0491] PCR amplification of the B7r1-mouse GCSFr fragment was
performed as follows: One cycle of 95.degree. C. for 2 minutes;
then thirty cycles at 95.degree. C. for 30 seconds, 56.degree. C.
for 30 seconds, 72.degree. C. for 1.5 minutes, followed by one
cycle of 72.degree. C. for 7 minutes and a 4.degree. C. hold. The
reaction was visualized on a 1.2% agarose gel and the appropriate
band was excised and purified using QIAquick Gel Extraction kit
(Qiagen, Santa Clarita, Calif.)
Yeast Recombination of Human zB7R1-m.GCSFr Purified PCR Product
into pZMP21Z
[0492] Competent yeast cells strain SF838-9D.quadrature.were thawed
on ice. One .mu.l of pZMP21Z vector digested with BglII by standard
restriction digest methods was mixed with 6 .mu.l h.zB7r1-m.GCSFR
purified PCR product, or 6 .mu.l TE buffer as a negative control.
The DNA mixture was added to 45 .mu.l yeast cells, mixed and
transferred to separate 2 mm disposable electroporation chambers
(VWR, West Chester, Pa.). Cells were electroporated using a Biorad
Genepulser.TM. (Hercules, Calif.) set to 750 V, 25 .mu.FD, infinite
resistance. 600 .mu.l cold 1.2 M sorbitol was immediately added to
each chamber. 150 .mu.l and 300 .mu.l from each chamber was plated
on -URA DS agar plates and incubated for 72 hours at 30.degree. C.
Yeast colonies from each plate were suspended in 1 ml H.sub.2O and
transferred to 1.5 ml eppendorf tubes. Cells were pelleted by
centrifugation and the supernatant was removed. An amount
equivalent to 50 .mu.l packed yeast of each sample was transferred
to another 1.5 ml eppendorf tube and resuspended in 100 .mu.l Yeast
Lysis Buffer (25 Triton X, 1% SDS, 100 mM NaCl, 10 mM Tris HCl,
pH8.0, 1 mM EDTA). To each tube 2 .mu.l (10 U) Zymolase (Zymo
Research, Cat #E1001/E1002) was added followed by a 30 minute
incubation at 37.degree. C. The lysed cells were miniprepped by
adding 150 .mu.l Buffer P1 (Qiagen) and then proceeding with the
QIAprep Spin Miniprep Kit at step 2. Plasmid DNA thus purified from
yeast was electroporated into DH10b Electormax cells (Invitrogen,
Carlsbad, Calif.) following the manufacturers recommendations.
Clones were isolated, sequenced, and large scale plasmid isolations
were preformed using standard methods.
Construction of BaF3/KZ134 Cells Expressing Chimeric Human
zB7R1-Mouse GCSFr
[0493] BaF3, an interleukin-3 (IL-3) dependent prelymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6.:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3)
(R+D, Minneapolis, Minn.), 2 mM L-glutamine (Gibco-BRL), and 1 mM
Sodium Pyruvate (Gibco-BRL).
[0494] The KZ134 plasmid was constructed with complementary
oligonucleotides that contain STAT transcription factor binding
elements from 4 genes, which includes a modified c-fos Sis
inducible element (m67SIE, or hSIE) (Sadowski, h. et al., Science
261: 1739-1744, 1993) the p21 SIE1 from the p21 WAF1 gene (Chin, Y.
et al., Science 272: 719-722, 1996), the mammary gland response
element of the .quadrature.-casein gene (Schmitt-Ney, M. et al.,
Mol. Cell. Biol. 11:3745-3755, 1991), and a STAT inducible element
of the Fc.gamma. RI gene, (Seidel, H. et al., Proc. Natl. Acad.
Sci. 92:3041-3045, 1995). These oligonucleotides contain
Asp718-XhoI compatible ends and were ligated, using standard
methods, into a recipient firefly luciferase reporter vector with a
c-fos promoter (Poulsen, L. K. et al., J. Biol. Chem.
273:6229-6232, 1998) digested with the same enzymes and containing
a neomycin selectable marker. The KZ134 plasmid was used to stably
transfect BaF3 cells, using standard transfection and selection
methods, to make the BaF3/KZ134 cell line.
[0495] BaF3/KZ134 cells were prepared for electroporation by
washing twice in RPMI medium (JRH Bioscience Inc., Lenexa, Kans.)
and then resuspending in RPMI at a cell density of 10.sup.7
cells/ml. One ml of resuspended BaF3 cells was mixed with 30 .mu.g
of the pZPMPZ/h.zB7r1-m.GCSFr plasmid DNA and transferred to
separate disposable electroporation chambers (Gibco-BRL). The cells
were then given 2 serial shocks (800 lFad/300V; 1180 lFad/300V.)
delivered by an electroporation apparatus (CELL-PORATOR.TM.;
Gibco-BRL, Bethesda, Md.). The electroporated cells were
subsequently transferred to 20 mls of complete media containing 2
.mu.g/ml Puromycin (Clontech, PaloAlto, Calif.) and placed in an
incubator for 24 hours (37.degree. C., 5% CO.sub.2). The cells were
then spun down and resuspended in 20 mls of complete media
containing .mu.g/ml Puromycin and 240 .mu.g/ml Zeocin (Invitrogen,
Carlsbad, Calif.) selection in a T75 flask to isolate the Zeocin
resistant pool. The resulting stable cell line was called
BaF3/KZ134/h.zB7r1-m.GCSFr.
HzB7R1 Antibodies Specifically Activate STAT Signaling in
BaF3/KZ134/h.B7r1-m.GCSFr Cells
[0496] The antibodies tested on the BaF3/KZ134/h.B7r1-m.GCSFr cells
were: mouse anti-human zB7r1 318.4.1.1 (E9310), 318.28.2.1 (E9296),
318.39.1.1 (E9311), 318.59.3.1 (E9400). These antibodies were
coupled to Dynabeads M-450 Tosylactivated, (Dynal Biotech ASA,
Oslo, Norway) as follows: 50 .mu.l (2.times.10.sup.7 beads) per
sample was washed once with 1 ml 0.1M sodium phosphate buffer, pH
7.4-8.0 in a 2.0 ml eppendorf tube. The tube was placed in a magnet
for 1 minute and the supernatant was removed. The beads were
resuspended in the original volume using the sodium phosphate
buffer. 10 .mu.g of each antibody was combined with 50 .mu.l washed
beads in 2.0 ml eppendorf tubes. A beads only (no antibody) control
was included. The tubes were placed on a Clay Adams Nutator mixer
(Bectin-Dickinson, Franklin Lakes, N.J.) at room temperature for 48
hours. The tubes were then placed in a magnet for 1 minute and the
supernatant was removed. The coated beads were then washed 4 times
with 1 ml PBS (without Ca2+ and Mg2+), 0.1% BSA (w/v) and 2 mM
EDTA, pH 7.4.
[0497] In setting up the cell assay, the coated beads and a beads
only control were plated in Falcon U-bottomed 96 well plates
(Bectin-Dickinson, Franklin Lakes, N.J.) at concentrations of
480,000, 240,000, 120,000, 60,000, 30,000, 15,000, and 7500 beads
per well in 100 .mu.l. Unbound antibody was also plated at
concentrations of 2, 1, 0.5, 0.25, 0.13, 0.6, and 0.3 .mu.g/ml in
100 .mu.l. Each sample was plated in triplicate. As a positive
control for STAT signaling, mouse IL3 dilutions were included at
concentrations of 2, 1, 0.5, 0.25, 0.13, 0.6, and 0.3 pg/ml in 100
.mu.l.
[0498] The BaF3/KZ134/h.B7r1-m.GCSFr Zeocin resistant cells were
washed three times in RPMI and counted using a hemocytometer. Cells
were resuspended in RPMI and plated at a concentration of 30,000
cells per well in 100 .mu.l into the plate containing the samples
for total well volume of 200 .mu.l.
[0499] The assay was incubated at 37.degree. C., 5% CO.sub.2 for 24
hours at which time the BaF3 cells were pelleted by centrifugation
at 1500 rpm for 10 min., the media was aspirated and 25 .mu.l of
lysis buffer (Promega) was added. After allowing 10 minutes for
cell lysis at room temperature, the plates were measured for
activation of the STAT reporter construct by reading them on a
luminometer (EG&G Berthold, model Microlumat Plus LB 96V) which
added 40 .mu.l of luciferase assay substrate (Promega) and measured
the light generated in the 10 seconds following substrate
addition.
[0500] The results of this assay showed that the B7r1 antibody-bead
complex bound to the B7r1-m.GCSFr in a dose dependent manner and
caused dimerization leading to STAT formation and signal
transduction. Neither unbound antibodies nor undecorated beads
elicited a STAT response.
[0501] In this example, the extracellular domain of a B7 family
type I protein (B7r1) and the transmembrane and intracellular
domain of a type I cytokine receptor superfamily protein (GCSFR)
were expressed as a chimera and induced dimerization and STAT
signaling when exposed to antibody. This method may also be used
with chimeras from other receptor families. Examples of chimeras
utilizing mouse GCSFr for signaling have included the extracellular
ligand binding domains of CD28, zTNFR14, and Fas, among others.
Variations on this method could be used with chimeras from other
receptor families paired with cells line assays sensitive to
appropriate signaling pathways. Examples may include chimeras
signaling through the NFkB pathway. These chimeras may be expressed
in NIH3T3 cells also expressing an NFkB responsive promoter fused
to a luciferase cDNA plasmid such as KZ142. These chimeras may be
built with the transmembrane and intracellular domain of a TNF
family molecule such as pTNFRSF4, known to signal through NFkB, and
could include the extracellular domain of molecules such as B7r1,
CD28, TNFR14, and Fas, among others.
[0502] Additional invitro assays utilizing chimeric receptors will
be useful in examining the signaling properties of the zB7R1
intracellular domain and in identifying antibodies directed against
the extracellular domain that mediate signaling. The type of cell
signal that the zB7R1 intracellular domain generates in response to
ligand binding may be elucidated in the following way. The
extracellular domain of hCD28, a B7 family member, is fused to the
transmembrane and intracellular domains of murine zB7R1. This
chimera is then transfected into the murine T cell hybridoma cell
line, Tea. This murine cell line responds to T cell receptor (TCR)
ligation by secreting IL2. Some B7 family members have been shown
to modify the magnitude of the T cell response; for example,
simultaneous ligation of CD28 alongside CD3 (TCR) yields a
significant increase in IL2 secretion over CD3 ligation alone.
Utilizing this chimera, antibodies directed against the human CD28
extracellular domain may mediate the ligation of mzB7R1
intracellular domain and subsequent signaling. IL2 levels may be
quantitated by ELISA in invitro CD3/CD28 costimulation assays
revealing the nature of the hzB7R1 signaling domain.
[0503] Additionally, human zB7R1 extracellular domains may be fused
with mCD28 intracellular domains in TEa hybridomas. Such chimeras
would allow for the screening of antibodies or other ligands
directed against the zB7R1 extracellular domain. This binding may
result in dimerization and signaling through the mCD28
intracellular domain that would likely increase IL2 secretion. Such
screening for molecules active on the zB7R1 extracellular domain
may thus be initiated prior to a complete understanding of the
zB7R1 signaling mechanism.
EXAMPLE 17
zB7R1 Expression on Human PBMNC
[0504] In order to culture the cells, blood from normal in-house
donors was separated on a ficol gradient, and the PBMNC interface
collected and washed inPBS. The cells were counted and plated in 96
well round bottom plates at 2e.sup.5 cells/well in 200 ul culture
medium with either LPS at 100 ng/ml or with anti-CD3+ anti-CD28
mabs (50 ng/ml and 1 ug/ml respectively). Some cells were reserved
for the time 0 timepoint. Cells were collected for staining at
times 24, 48 and 72 hours.
[0505] At each timepoint, cells in 96 well plates are spun, the
media flicked out, and a combination of fluor-conjugated antibodies
to surface lineage markers added in 50 ul Facs staining buffer
(CD56-A488, CD19-PE, CD45RA-Cychrome, CD45RO-PE-Cy7, CD4-A405,
CD8-A700, and CD14-A750). The combination included either mab
anti-B7R1 (318.4.1) coupled to A647 dye, or a control mab similarly
coupled. In some experiments, the binding of mab anti-B7R1 was
competed with 20 fold (g/g) excess mB7R1 receptor. Each condition
was stained in triplicate wells. Cells were incubated with a stain
combo for 30 minutes on ice, then are washed 1.5.times. with Facs
buffer and fixed with 2% paraformaldehyde, 100 ul/well, for 10
minutes, at room temp. Plates were spun, the paraformaldehyde
flicked out, and cells resuspended in 200 ul Facs buffer and stored
at 4 C. foil-covered until they were read on the LSRII.
[0506] The LSRII data was analyzed using FacsDiva software.
FSC.times.SSC dot plots were used to determine a viable cell
population gate. Viable cells were then analyzed for anti-B7R1
binding using dot plots of anti-B7R1-A647 vs specific lineage
markers.
[0507] For the kinetic analysis of B7R1 expression, the background
fluorescence (either determined with the control mab-A647 or with
blocking using 20.times.g/g soluble receptor) was subtracted from
the anti-B7R1-A647 staining for each lineage over time.
[0508] The results indicated that zB7R1 is expressed on resting
CD8+ and NK cells and that expression is upregulated with
activation on CD4+, CD8+ and NK cells. There is no detectable
binding on CD19+ and there is no competable binding to CD14+ or
CD11c cells. Expression of zB7R1 was higher on memory T cells
relative to naive T cells.
EXAMPLE 18
T-Cell Proliferation is Inhibited by zB7R1 Antibodies
[0509] The proliferation of purified CD4 and CD8 T cells from human
peripheral blood mononuclear cells (PBMC) was inhibited by antibody
to zB7r1 in vitro. An antibody to CD3 (BD Biosciences 555329)
mimiced T cell antigen recognition. Engagement of CD3 and the T
cell receptor by antibody provided a signal to proliferate in
vitro. This signal was enhanced or inhibited by additional signals.
An antibody to zB7r1, covalently coupled to tosylactivated
4.5.quadrature. beads (Dynal 140.13), inhibited the
anti-CD3-induced proliferation of T cells in vitro. The addition of
co-stimulatory anti-CD28 (BD Biosciences 555725) did not overcome
the inhibitory effect of anti-zB7r1. Moreover, anti-zB7r1 inhibited
the expression of the early activation markers CD69 and the IL-2
receptor CD25 as well as the production of IL-2.
[0510] Tosylactivated beads were used as a solid phase platform to
present anti-CD3 and anti-zB7r1 to T cells. Human PBMC from healthy
volunteers were collected by Ficoll-Paque (GE Healthcare) density
gradient. CD4 and CD8 were co-purified from PBMC by magnetic bead
columns (Miltenyi Biotec). T cells were labeled with CFSE
(Invitrogen) to assess proliferation by flow cytometry.
1.times.10E5 CFSE-labeled T cells and 1.times.10E5 beads were
plated per well. Cultures were maintained for 1 day to assess early
activation markers or 3 days to assess proliferation in humidified
incubators at 5% CO2. Proliferation of CD4s and CD8s was measured
on an LSRII (Becton Dickinson).
[0511] Anti-zB7r1 inhibited CD4 memory and naive T cells
equivalently. Specifically, CD4 T cells were purified as before
then sorted into CD45RA high (naive) and CD45RA low (memory)
populations via cell sorting on the FACSAria (BD Biosciences).
Cells were cultured as above then assessed for proliferation at 72
hr. Anti-CD3 was titrated in combination with fixed amount of
zb7r1, control or anti-CTLA4. CD4 memory and naive cells were
inhibited in proliferation to an equivalent extent.
[0512] Anti-zB7r1 inhibited IL-2 production by memory and naive
CD4s. Specifically, IL-2 production of CD3-activated memory and
naive CD4 cells is inhibited by anti-zB7r1. T cells and beads were
cultured as above. IL-2 production at 24 h was assessed in culture
supernatants by Luminex technology (Bio-Rad).
EXAMPLE 19
zB7R1-VASP in Acute Graft Versus Host Disease (GVHD)
[0513] The purpose of this experiment was to determine if
prophylactic treatment of B7R1-VASP soluble protein influences the
development and severity of an acute GVHD response in mice.
[0514] To initiate GVHD, 75 million spleen cells from C57B1/6 mice
are injected by intravenous delivery into DBA2 X C57B1/6 F1 mice
(BDF1) on day 0. Mice are treated with 150 ug of B7R1-VASP protein
intraperitoneally every other day starting the day before cell
transfer and continuing throughout the duration of the experiment.
Body weight is monitored daily and mice are sacrificed on day 12
after spleen transfer. Spleens are collected for FACS analysis and
blood is collected for serum.
[0515] Prophylactic delivery of B7R1-VASP significantly decreases
the severity of body weight loss during acute GVHD.
EXAMPLE 20
Delayed Type Hypersensitivity in zB7R1-Fc-Treated Mice
[0516] Delayed Type Hypersensitivity (DTH) is a measure of T cell
responses to specific antigen. In this response, mice are immunized
with a specific protein in adjuvant (e.g., chicken ovalbumin, OVA)
and then later challenged with the same antigen (without adjuvant)
in the ear. Increase in ear thickness (measured with calipers)
after the challenge is a measure of specific immune response to the
antigen. DTH is a form of cell-mediated immunity that occurs in
three distinct phases 1) the cognitive phase, in which T cells
recognize foreign protein antigens presented on the surface of
antigen presenting cells (APCs), 2) the activation/sensitization
phase, in which T cells secrete cytokines (especially
interferon-gamma; IFN-.gamma.) and proliferate, and 3) the effector
phase, which includes both inflammation (including infiltration of
activated macrophages and neutrophils) and the ultimate resolution
of the infection. This reaction is the primary defense mechanism
against intracellular bacteria, and can be induced by soluble
protein antigens or chemically reactive haptens. A classical DTH
response occurs in individuals challenged with purified protein
derivative (PPD) from Mycobacterium tuberculosis, when those
individuals injected have recovered from primary TB or have been
vaccinated against TB. Induration, the hallmark of DTH, is
detectable by about 18 hours after injection of antigen and is
maximal by 24-72 hours. The lag in the onset of palpable induration
is the reason for naming the response "delayed type." In all
species, DTH reactions are critically dependent on the presence of
antigen-sensitized CD4+ (and, to a lesser extent, CD8+) T cells,
which produce the principal initiating cytokine involved in DTH,
IFN-.gamma..
[0517] In order to test for anti-inflammatory effects of mB7R1-Fc,
a DTH experiment was conducted with six groups of C57B1/6 mice
treated with: I) control plasmid, II) 25 ug mCTLA-4-Fc plasmid, and
III) 25 ug mB7R1-Fc plasmid. All of these plasmids were injected
hydrodynamically through the tail vein. In short, 25 ug of plasmid
was resuspended in 2 mL of sterile injectable saline. Each mouse
received a single intravenous injection of 2 mL saline containing
25 ug plasmid via its tail vein. Injections were accomplished
within 4-8 seconds/mouse, leading to the hydrodynamic pressure that
results in cellular transfection in multiple organs in the mouse.
Treatments were given one day prior to the OVA/RIBI sensitization
(groups 1-3) or one day prior to OVA re-challenge (groups 4-6). The
mice (6 per group) were first immunized in the back with 100 ug
chicken ovalbumin (OVA) emulsified in Ribi in a total volume of 200
ul. Seven days later, the mice were re-challenged intradermally in
the left ear with 10 ul PBS (control) or in the right ear with 10
ug OVA in PBS (no adjuvant) in a volume of 10 ul. Ear thickness of
all mice was measured before injecting mice in the ear (0
measurement). Ear thickness was measured 24, and 48 hours after
challenge. The difference in ear thickness between the 0
measurement and the 24 hour measurement is shown in TABLE 1.
Control mice in the control plasmid treatment group developed a
strong DTH reaction as shown by increase in the ear thickness at 24
and 48 hours post-challenge. In contrast, mice treated with
CTLA-4Fc or B7R1Fc at the challenge phase had a lesser degree of
ear thickness compared to controls. B7R1-Fc injection also
inhibited ear thickness at the sensitization phase but only at the
24 hr time point. These differences were statistically significant,
as determined by Student's t-test (Table 5, p values vs. control
plasmid). TABLE-US-00005 TABLE 5 zB7R1 inhibits the Delayed Type
Hypersensitivity (DTH) reaction when administered either at the
challenge or at the sensitization phase of the response CHANGE IN
EAR TIME/ROUTE THICKNESS (.times.10.sup.-3 inch) p value OF LEFT
EAR RIGHT EAR vs. EXPT # TREATMENT TREATMENT (PBS) (OVA) control 24
hr 24 hr 1 Control plasmid Sensitization (d-1) 0.42 +/- 0.80 6.72
+/- 1.04 -- (n = 6) mCTLA-4-Fc i.v. 0.5 +/- 0.63 7.11 +/- 2.69
0.7484 mB7R1-Fc 1.19 +/- 0.54 4.44 +/- 0.86 0.002 Control plasmid
Challenge (d6) 0.08 +/- 0.66 10 +/- 1.84 -- mCTLA-4-Fc i.v. 0.55
+/- 0.08 5.94 +/- 0.78 0.0006 mB7R1-Fc 0.65 +/- 0.62 7.08 +/- 1.28
0.0099 48 hr 48 hr 1 Control plasmid Sensitization (d-1) 0.2 +/-
0.54 5.91 +/- 1.3 -- (n = 6) mCTLA-4-Fc i.v. 0.66 +/- 0.40 7.69 +/-
2.69 0.1758 mB7R1-Fc 0.94 +/- 0.88 6.33 +/- 1.12 0.5650 Control
plasmid Challenge (d6) 0.55 +/- 0.62 11.38 +/- 2.67 -- mCTLA-4-Fc
i.v. 0.05 +/- 0.08 6.97 +/- 1.26 0.0045 mB7R1-Fc 0.05 +/- 0.13 6.30
+/- 0.96 0.0014
EXAMPLE 21
B7R1 is Regulated in Tissues From Mice With Collagen Induced
Arthritis (CIA) Compared to Non-Disease Tissue
[0518] Experimental Protocol: Tissues were obtained from mice with
verying degrees of disease in the collagen-induced arthritis (CIA)
model. The model was performed following standard procedures of
immunizing male DBA/1J mice with collagen (see Example 22 below)
and included appropriate non-diseased controls. Tissues isolated
included affected paws and popliteal lymph nodes. RNA was isolated
from all tissues using standard procedures. In brief, tissues were
collected and immediately frozen in liquid N2 and then transferred
to -80.degree. C. until processing. For processing, tissues were
placed in Qiazol reagent (Qiagen, Valencia, Calif.) and RNA was
isolated using the Qigen Rneasy kit according to manufacturer's
recommendations. Expression of murine zB7R1 mRNA was measured with
multiplex real-time quantitative RT-PCR methods (TaqMan) and the
ABI PRISM 7900 sequence detection system (PE Applied Biosystems).
Murine zB7R1 mRNA levels were normalized to the expression of
murine hypoxanthine guanine physphoribosyl transferase mRNA and
determined by the comparative threshold cycle method (User Bullein
2: PE Applied Biosystems). The primers and probe for murine B7R1
included forward primer 5' SEQ ID NO:65, reverse primer 5' SEQ ID
NO:66, and probe SEQ ID NO:67.
[0519] Results: Murine B7R1 mRNA expression was detected in the
tissues tested. Higher levels of expression were observed in lymph
nodes compared to the paws. B7R1 mRNA was increased in the
popliteal lymph nodes and the paws from mice in the CIA model of
arthritis compared to tissues obtained from non-diseased controls,
and the levels were associated with disease severity. B7R1 mRNA was
increased in the paws approximately 2.3-fold in mice with mild
disease and approximately 4-fold in mice with severe disease
compared to non-diseased controls. B7R1 mRNA was increased in the
lymph node approximately 1.5-fold in mice with mild disease and
approximately 1.8-fold in mice with severe disease compared to
non-diseased controls.
EXAMPLE 22
B7R1m-mFc and B7R1m-VASP CH6 Decreases Disease Incidence and
Progression in Mouse Collagen Induced Arthritis (CIA) Model
[0520] Mouse Collagen Induced Arthritis (CIA) Model: Ten week old
male DBA/1J mice (Jackson Labs) were divided into 3 groups of 13
mice/group. On day-21, animals were given an intradermal tail
injection of 50-100 .mu.l of 1 mg/ml chick Type II collagen
formulated in Complete Freund's Adjuvant (prepared by Chondrex,
Redmond, Wash.), and three weeks later on Day 0 they were given the
same injection except prepared in Incomplete Freund's Adjuvant.
B7R1m-mFc or B7R1m-VASP CH6 was administered as an intraperitoneal
injection every other day for 1.5 weeks (although dosing may be
extended to as must as four weeks), at different time points
ranging from Day-1 to a day in which the majority of mice exhibit
moderate symptoms of disease. Groups received 150 .mu.g of
B7R1m-mFc or B7R1m-VASP CH6 per animal per dose, and control groups
received the vehicle control, PBS (Life Technologies, Rockville,
Md.). Animals began to show symptoms of arthritis following the
second collagen injection, with most animals developing
inflammation within 1.5-3 weeks. The extent of disease was
evaluated in each paw by using a caliper to measure paw thickness,
and by assigning a clinical score (0-3) to each paw: 0=Normal,
0.5=Toe(s) inflamed, 1=Mild paw inflammation, 2=Moderate paw
inflammation, and 3=Severe paw inflammation as detailed below.
[0521] Monitoring Disease: Animals can begin to show signs of paw
inflammation soon after the second collagen injection, and some
animals may even begin to have signs of toe inflammation prior to
the second collagen injection. Most animals develop arthritis
within 1-3 weeks of the boost injection, but some may require a
longer period of time. Incidence of disease in this model is
typically 95-100%, and 0-2 non-responders (determined after 6 weeks
of observation) are typically seen in a study using 40 animals.
Note that as inflammation begins, a common transient occurrence of
variable low-grade paw or toe inflammation can occur. For this
reason, an animal is not considered to have established disease
until marked, persistent paw swelling has developed.
[0522] All animals were observed daily to assess the status of the
disease in their paws, which is done by assigning a qualitative
clinical score to each of the paws. Every day, each animal had its
4 paws scored according to its state of clinical disease. To
determine the clinical score, the paw can be thought of as having 3
zones, the toes, the paw itself (manus or pes), and the wrist or
ankle joint. The extent and severity of the inflammation relative
to these zones was noted including: observation of each toe for
swelling; torn nails or redness of toes; notation of any evidence
of edema or redness in any of the paws; notation of any loss of
fine anatomic demarcation of tendons or bones; evaluation of the
wrist or ankle for any edema or redness; and notation if the
inflammation extends proximally up the leg. A paw score of 1, 2, or
3 is based first on the overall impression of severity, and second
on how many zones are involved. The scale used for clinical scoring
is shown below.
[0523] Clinical Score: [0524] 0=Normal [0525] 0.5=One or more toes
involved, but only the toes are inflamed [0526] 1=mild inflammation
involving the paw (1 zone), and may include a toe or toes [0527]
2=moderate inflammation in the paw and may include some of the toes
and/or the wrist/ankle (2 zones) [0528] 3=severe inflammation in
the paw, wrist/ankle, and some or all of the toes (3 zones)
[0529] Established disease is defined as a qualitative score of paw
inflammation ranking 2 or more, that persists for two days in a
row. Once established disease is present, the date is recorded and
designated as that animal's first day with "established
disease".
[0530] Blood is collected throughout the experiment to monitor
serum levels of anti-collagen antibodies, as well as serum
immunoglobulin and cytokine levels. Serum anti-collagen antibodies
correlate well with severity of disease. Animals are euthanized on
a determined day, and blood collected for serum. From each animal,
one affected paw may be?? collected in 10% NBF for histology and
one is frozen in liquid nitrogen and stored at -80.degree. C. for
mRNA analysis. Also, 1/2 spleen, 1/2 thymus, 1/2 mesenteric lymph
node, one liver lobe and the left kidney are collected in RNAlater
for RNA analysis, and 1/2 spleen, 1/2 thymus, 1/2 mesenteric lymph
node, the remaining liver, and the right kidney are collected in
10% NBF for histology. Serum is collected and frozen at -80.degree.
C. for immunoglobulin and cytokine assays.
[0531] Groups of mice that received soluble zB7R1-Fc fusion protein
as described herein and zB7R1-VASP CH6 as described herein, at all
time points tested (prophylactic and therapeutic delivery) were
characterized by a delay in the incidence (for prophylactic
administration), onset and/or progression of paw inflammation. On
day 8 of the model, mice that received PBS prophylactically had
100% disease incidence and had significant swelling of the majority
of their paws. However, mice that received zB7R1-Fc fusion protein
prophylactically had significantly reduced paw swelling (2.3-fold
lower arthritis score compared to PBS-treated mice) and 80%
incidence. Moreover, mice treated prophlyactically with zB7R1-VASP
CH6 fusion protein were greatly protected from disease, as only 40%
of these mice developed arthritis symptoms, which was associated
with markedly reduced arthritis scores (3.5-fold lower than
PBS-treated mice). zB7R1-VASP CH6 fusion protein was also able to
reduce arthritis symptoms when administered after disease onset,
such that mice treated therapeutically with zB7R1-VASP CH6 fusion
protein had approximately 2-fold lower arthritis scores than mice
treated therapeutically with PBS. These results indicate that
soluble zB7R1 fusion proteins of the present invention reduce
inflammation, as well as disease incidence and progression.
[0532] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
69 1 902 DNA Homo sapiens CDS (11)...(745) misc_feature
(289)...(289) n = c or t misc_feature (359)...(359) n = a or g 1
gggcagaagc atg cgc tgg tgt ctc ctc ctg atc tgg gcc cag ggg ctg 49
Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu 1 5 10 agg cag
gct ccc ctc gcc tca gga atg atg aca ggc aca ata gaa aca 97 Arg Gln
Ala Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr 15 20 25
acg ggg aac att tct gca gag aaa ggt ggc tct atc atc tta caa tgt 145
Thr Gly Asn Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys 30
35 40 45 cac ctc tcc tcc acc acg gca caa gtg acc cag gtc aac tgg
gag cag 193 His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp
Glu Gln 50 55 60 cag gac cag ctt ctg gcc att tgt aat gct gac ttg
ggg tgg cac atc 241 Gln Asp Gln Leu Leu Ala Ile Cys Asn Ala Asp Leu
Gly Trp His Ile 65 70 75 tcc cca tcc ttc aag gat cga gtg gcc cca
ggt ccc ggc ctg ggc ctn 289 Ser Pro Ser Phe Lys Asp Arg Val Ala Pro
Gly Pro Gly Leu Gly Leu 80 85 90 acc ctc cag tcg ctg acc gtg aac
gat aca ggg gag tac ttc tgc atc 337 Thr Leu Gln Ser Leu Thr Val Asn
Asp Thr Gly Glu Tyr Phe Cys Ile 95 100 105 tat cac acc tac cct gat
ggg ncg tac act ggg aga atc ttc ctg gag 385 Tyr His Thr Tyr Pro Asp
Gly Xaa Tyr Thr Gly Arg Ile Phe Leu Glu 110 115 120 125 gtc cta gaa
agc tca gtg gct gag cac ggt gcc agg ttc cag att cca 433 Val Leu Glu
Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro 130 135 140 ttg
ctt gga gcc atg gcc gcg acg ctg gtg gtc atc tgc aca gca gtc 481 Leu
Leu Gly Ala Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val 145 150
155 atc gtg gtg gtc gcg ttg act aga aag aag aaa gcc ctc aga atc cat
529 Ile Val Val Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His
160 165 170 tct gtg gaa ggt gac ctc agg aga aaa tca gct gga cag gag
gaa tgg 577 Ser Val Glu Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu
Glu Trp 175 180 185 agc ccc agt gct ccc tca ccc cca gga agc tgt gtc
cag gca gaa gct 625 Ser Pro Ser Ala Pro Ser Pro Pro Gly Ser Cys Val
Gln Ala Glu Ala 190 195 200 205 gca cct gct ggg ctc tgt gga gag cag
cgg gga gag gac tgt gcc gag 673 Ala Pro Ala Gly Leu Cys Gly Glu Gln
Arg Gly Glu Asp Cys Ala Glu 210 215 220 ctg cat gac tac ttc aat gtc
ctg agt tac aga agc ctg ggt aac tgc 721 Leu His Asp Tyr Phe Asn Val
Leu Ser Tyr Arg Ser Leu Gly Asn Cys 225 230 235 agc ttc ttc aca gag
act ggt tag caaccagagg catcttctgg aagatacact 775 Ser Phe Phe Thr
Glu Thr Gly * 240 tttgtctttg ctattataga tgaatatata agcagctgta
ctctccatca gtgctgcgtg 835 tgtgtgtgtg tgtgtgtgta tgtgtgtgtg
tgttcagttg agtgaataaa tgtcatcctc 895 ttctcca 902 2 244 PRT Homo
sapiens VARIANT (117)...(117) Xaa = Thr or Ala 2 Met Arg Trp Cys
Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu
Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30
Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35
40 45 Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp
Gln 50 55 60 Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile
Ser Pro Ser 65 70 75 80 Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu
Gly Leu Thr Leu Gln 85 90 95 Ser Leu Thr Val Asn Asp Thr Gly Glu
Tyr Phe Cys Ile Tyr His Thr 100 105 110 Tyr Pro Asp Gly Xaa Tyr Thr
Gly Arg Ile Phe Leu Glu Val Leu Glu 115 120 125 Ser Ser Val Ala Glu
His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly 130 135 140 Ala Met Ala
Ala Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val 145 150 155 160
Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu 165
170 175 Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro
Ser 180 185 190 Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala Glu Ala
Ala Pro Ala 195 200 205 Gly Leu Cys Gly Glu Gln Arg Gly Glu Asp Cys
Ala Glu Leu His Asp 210 215 220 Tyr Phe Asn Val Leu Ser Tyr Arg Ser
Leu Gly Asn Cys Ser Phe Phe 225 230 235 240 Thr Glu Thr Gly 3 141
PRT Homo sapiens 3 Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly
Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly Met Met Thr Gly Thr
Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala Glu Lys Gly Gly Ser
Ile Ile Leu Gln Cys His Leu Ser 35 40 45 Ser Thr Thr Ala Gln Val
Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55 60 Leu Leu Ala Ile
Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser 65 70 75 80 Phe Lys
Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln 85 90 95
Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr 100
105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu
Glu 115 120 125 Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro
130 135 140 4 140 PRT Homo sapiens 4 Met Arg Trp Cys Leu Leu Leu
Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala Ser Gly
Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile Ser Ala
Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35 40 45 Ser
Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln 50 55
60 Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser
65 70 75 80 Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr
Leu Gln 85 90 95 Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys
Ile Tyr His Thr 100 105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile
Phe Leu Glu Val Leu Glu 115 120 125 Ser Ser Val Ala Glu His Gly Ala
Arg Phe Gln Ile 130 135 140 5 1711 DNA Homo sapiens CDS
(755)...(1690) 5 aaactatttg agggtagggg ctgtgattat ttactctcat
atcctcagag cctggtgttg 60 aggttggtgc tttgtaggca cccagggact
ttcaaatgaa tgaagggagg gagggaggaa 120 agaaggatgg gtccatagta
ggacctggtg atgggctggg agctccaggc aaatgtcaac 180 caatccctct
cctgggtcag ctcccagggg ctcacccttc tttgcatttc cagctctcat 240
gaggtcattg tgcacaggaa agctctctcc tctaatctcc tctgatccta ctgcaccaga
300 gaaatcaagc cagaattcaa caaagtctca gtccagataa acaagacaaa
agaaataaga 360 ttcgagtaga agatctcctt caagggaaag ttgctgtgtt
tgtccaagac ctttgtccca 420 tccatgtatc atcccccaag taaacacttc
ttgttcacct gttcattaga tttcaagtgc 480 agtccctggc ctgtaagtcc
ctacaatgat aagtttctct tatcattgca cattcttcat 540 caggaggatg
ccagaggagc tcagccaaca gttcctcatc agtagcagat tcttcagaat 600
cttgggcact acacagatgc ccttgagctc tttgaataaa ggctgatttt tagaaaaaac
660 attaagacag aacttaaaaa caatagattg actataatcc aaagacgagt
gtacctctaa 720 ccacaatttt catttatttt taaatgtttc cttc atg gcc ttt
ctt gtg gct cac 775 Met Ala Phe Leu Val Ala His 1 5 cct atg cag ttt
gtg tat ttg ttg aca act tta tgt gtt ttt aat atg 823 Pro Met Gln Phe
Val Tyr Leu Leu Thr Thr Leu Cys Val Phe Asn Met 10 15 20 gtt ttt
gcc aaa ctt ggt ttt tcc gag acc gtc ttt tct cag agg ctc 871 Val Phe
Ala Lys Leu Gly Phe Ser Glu Thr Val Phe Ser Gln Arg Leu 25 30 35
agt ttt acc gtc cta tct gca gtc ggc tac ttt cag tgg cag aag agg 919
Ser Phe Thr Val Leu Ser Ala Val Gly Tyr Phe Gln Trp Gln Lys Arg 40
45 50 55 cca cat ctg ctt cct gta ggc cct ctg ggc aga agc atg cgc
tgg tgt 967 Pro His Leu Leu Pro Val Gly Pro Leu Gly Arg Ser Met Arg
Trp Cys 60 65 70 ctc ctc ctg atc tgg gcc cag ggg ctg agg cag gct
ccc ctc gcc tca 1015 Leu Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln
Ala Pro Leu Ala Ser 75 80 85 gga atg atg aca ggc aca ata gaa aca
acg ggg aac att tct gca gag 1063 Gly Met Met Thr Gly Thr Ile Glu
Thr Thr Gly Asn Ile Ser Ala Glu 90 95 100 aaa ggt ggc tct atc atc
tta caa tgt cac ctc tcc tcc acc acg gca 1111 Lys Gly Gly Ser Ile
Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala 105 110 115 caa gtg acc
cag gtc aac tgg gag cag cag gac cag ctt ctg gcc att 1159 Gln Val
Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile 120 125 130
135 tgt aat gct gac ttg ggg tgg cac atc tcc cca tcc ttc aag gat cga
1207 Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp
Arg 140 145 150 gtg gcc cca ggt ccc ggc ctg ggc ctc acc ctc cag tcg
ctg acc gtg 1255 Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln
Ser Leu Thr Val 155 160 165 aac gat aca ggg gag tac ttc tgc atc tat
cac acc tac cct gat ggg 1303 Asn Asp Thr Gly Glu Tyr Phe Cys Ile
Tyr His Thr Tyr Pro Asp Gly 170 175 180 acg tac act ggg aga atc ttc
ctg gag gtc cta gaa agc tca gtg gct 1351 Thr Tyr Thr Gly Arg Ile
Phe Leu Glu Val Leu Glu Ser Ser Val Ala 185 190 195 gag cac ggt gcc
agg ttc cag att cca ttg ctt gga gcc atg gcc gcg 1399 Glu His Gly
Ala Arg Phe Gln Ile Pro Leu Leu Gly Ala Met Ala Ala 200 205 210 215
acg ctg gtg gtc atc tgc aca gca gtc atc gtg gtg gtc gcg ttg act
1447 Thr Leu Val Val Ile Cys Thr Ala Val Ile Val Val Val Ala Leu
Thr 220 225 230 aga aag aag aaa gcc ctc aga atc cat tct gtg gaa ggt
gac ctc agg 1495 Arg Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu
Gly Asp Leu Arg 235 240 245 aga aaa tca gct gga cag gag gaa tgg agc
ccc agt gct ccc tca ccc 1543 Arg Lys Ser Ala Gly Gln Glu Glu Trp
Ser Pro Ser Ala Pro Ser Pro 250 255 260 cca gga agc tgt gtc cag gca
gaa gct gca cct gct ggg ctc tgt gga 1591 Pro Gly Ser Cys Val Gln
Ala Glu Ala Ala Pro Ala Gly Leu Cys Gly 265 270 275 gag cag cgg gga
gag gac tgt gcc gag ctg cat gac tac ttc aat gtc 1639 Glu Gln Arg
Gly Glu Asp Cys Ala Glu Leu His Asp Tyr Phe Asn Val 280 285 290 295
ctg agt tac aga agc ctg ggt aac tgc agc ttc ttc aca gag act ggt
1687 Leu Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe Thr Glu Thr
Gly 300 305 310 tag caaccagagg catcttctgg a 1711 * 6 311 PRT Homo
sapiens 6 Met Ala Phe Leu Val Ala His Pro Met Gln Phe Val Tyr Leu
Leu Thr 1 5 10 15 Thr Leu Cys Val Phe Asn Met Val Phe Ala Lys Leu
Gly Phe Ser Glu 20 25 30 Thr Val Phe Ser Gln Arg Leu Ser Phe Thr
Val Leu Ser Ala Val Gly 35 40 45 Tyr Phe Gln Trp Gln Lys Arg Pro
His Leu Leu Pro Val Gly Pro Leu 50 55 60 Gly Arg Ser Met Arg Trp
Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu 65 70 75 80 Arg Gln Ala Pro
Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr 85 90 95 Thr Gly
Asn Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys 100 105 110
His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln 115
120 125 Gln Asp Gln Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His
Ile 130 135 140 Ser Pro Ser Phe Lys Asp Arg Val Ala Pro Gly Pro Gly
Leu Gly Leu 145 150 155 160 Thr Leu Gln Ser Leu Thr Val Asn Asp Thr
Gly Glu Tyr Phe Cys Ile 165 170 175 Tyr His Thr Tyr Pro Asp Gly Thr
Tyr Thr Gly Arg Ile Phe Leu Glu 180 185 190 Val Leu Glu Ser Ser Val
Ala Glu His Gly Ala Arg Phe Gln Ile Pro 195 200 205 Leu Leu Gly Ala
Met Ala Ala Thr Leu Val Val Ile Cys Thr Ala Val 210 215 220 Ile Val
Val Val Ala Leu Thr Arg Lys Lys Lys Ala Leu Arg Ile His 225 230 235
240 Ser Val Glu Gly Asp Leu Arg Arg Lys Ser Ala Gly Gln Glu Glu Trp
245 250 255 Ser Pro Ser Ala Pro Ser Pro Pro Gly Ser Cys Val Gln Ala
Glu Ala 260 265 270 Ala Pro Ala Gly Leu Cys Gly Glu Gln Arg Gly Glu
Asp Cys Ala Glu 275 280 285 Leu His Asp Tyr Phe Asn Val Leu Ser Tyr
Arg Ser Leu Gly Asn Cys 290 295 300 Ser Phe Phe Thr Glu Thr Gly 305
310 7 208 PRT Homo sapiens 7 Met Ala Phe Leu Val Ala His Pro Met
Gln Phe Val Tyr Leu Leu Thr 1 5 10 15 Thr Leu Cys Val Phe Asn Met
Val Phe Ala Lys Leu Gly Phe Ser Glu 20 25 30 Thr Val Phe Ser Gln
Arg Leu Ser Phe Thr Val Leu Ser Ala Val Gly 35 40 45 Tyr Phe Gln
Trp Gln Lys Arg Pro His Leu Leu Pro Val Gly Pro Leu 50 55 60 Gly
Arg Ser Met Arg Trp Cys Leu Leu Leu Ile Trp Ala Gln Gly Leu 65 70
75 80 Arg Gln Ala Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu
Thr 85 90 95 Thr Gly Asn Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile
Leu Gln Cys 100 105 110 His Leu Ser Ser Thr Thr Ala Gln Val Thr Gln
Val Asn Trp Glu Gln 115 120 125 Gln Asp Gln Leu Leu Ala Ile Cys Asn
Ala Asp Leu Gly Trp His Ile 130 135 140 Ser Pro Ser Phe Lys Asp Arg
Val Ala Pro Gly Pro Gly Leu Gly Leu 145 150 155 160 Thr Leu Gln Ser
Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile 165 170 175 Tyr His
Thr Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu 180 185 190
Val Leu Glu Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro 195
200 205 8 726 DNA Mus musculus CDS (1)...(726) 8 atg cat ggc tgg
ctg ctc ctg gtc tgg gtc cag ggg ctg ata cag gct 48 Met His Gly Trp
Leu Leu Leu Val Trp Val Gln Gly Leu Ile Gln Ala 1 5 10 15 gcc ttc
ctc gct aca gga gcc aca gca ggc acg ata gat aca aag agg 96 Ala Phe
Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg 20 25 30
aac atc tct gca gag gaa ggt ggc tct gtc atc tta cag tgt cac ttc 144
Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe 35
40 45 tcc tct gac aca gct gaa gtg acc caa gtc gac tgg aag cag cag
gac 192 Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln
Asp 50 55 60 cag ctt ctg gcc att tat agt gtt gac ctg ggg tgg cat
gtc gct tca 240 Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His
Val Ala Ser 65 70 75 80 gtc ttc agt gat cgg gtg gtc cca ggc ccc agc
cta ggc ctc acc ttc 288 Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser
Leu Gly Leu Thr Phe 85 90 95 cag tct ctg aca atg aat gac acg gga
gag tac ttc tgt acc tat cat 336 Gln Ser Leu Thr Met Asn Asp Thr Gly
Glu Tyr Phe Cys Thr Tyr His 100 105 110 acg tat cct ggt ggg att tac
aag ggg aga ata ttc ctg aag gtc caa 384 Thr Tyr Pro Gly Gly Ile Tyr
Lys Gly Arg Ile Phe Leu Lys Val Gln 115 120 125 gaa agc tca gtg gct
cag ttc cag act gcc ccg ctt gga gga acc atg 432 Glu Ser Ser Val Ala
Gln Phe Gln Thr Ala Pro Leu Gly Gly Thr Met 130 135 140 gct gct gtg
ctg gga ctc att tgc tta atg gtc aca gga gtg act gta 480 Ala Ala Val
Leu Gly Leu Ile Cys Leu Met Val Thr Gly Val Thr Val 145 150 155 160
ctg gct aga aag aag tct att aga atg cat tct ata gaa agt ggc ctt 528
Leu Ala Arg Lys Lys Ser Ile Arg Met His Ser Ile Glu Ser Gly Leu 165
170 175 ggg aga
aca gaa gcg gag cca cag gaa tgg aac ctg agg agt ctc tca 576 Gly Arg
Thr Glu Ala Glu Pro Gln Glu Trp Asn Leu Arg Ser Leu Ser 180 185 190
tcc cct gga agc cct gtc cag aca caa act gcc cct gct ggt ccc tgt 624
Ser Pro Gly Ser Pro Val Gln Thr Gln Thr Ala Pro Ala Gly Pro Cys 195
200 205 gga gag cag gca gaa gat gac tat gct gac cca cag gaa tac ttt
aat 672 Gly Glu Gln Ala Glu Asp Asp Tyr Ala Asp Pro Gln Glu Tyr Phe
Asn 210 215 220 gtc ctg agc tac aga agc cta gag agc ttc att gct gta
tcg aag act 720 Val Leu Ser Tyr Arg Ser Leu Glu Ser Phe Ile Ala Val
Ser Lys Thr 225 230 235 240 ggc taa 726 Gly * 9 241 PRT Mus
musculus 9 Met His Gly Trp Leu Leu Leu Val Trp Val Gln Gly Leu Ile
Gln Ala 1 5 10 15 Ala Phe Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile
Asp Thr Lys Arg 20 25 30 Asn Ile Ser Ala Glu Glu Gly Gly Ser Val
Ile Leu Gln Cys His Phe 35 40 45 Ser Ser Asp Thr Ala Glu Val Thr
Gln Val Asp Trp Lys Gln Gln Asp 50 55 60 Gln Leu Leu Ala Ile Tyr
Ser Val Asp Leu Gly Trp His Val Ala Ser 65 70 75 80 Val Phe Ser Asp
Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe 85 90 95 Gln Ser
Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His 100 105 110
Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln 115
120 125 Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Pro Leu Gly Gly Thr
Met 130 135 140 Ala Ala Val Leu Gly Leu Ile Cys Leu Met Val Thr Gly
Val Thr Val 145 150 155 160 Leu Ala Arg Lys Lys Ser Ile Arg Met His
Ser Ile Glu Ser Gly Leu 165 170 175 Gly Arg Thr Glu Ala Glu Pro Gln
Glu Trp Asn Leu Arg Ser Leu Ser 180 185 190 Ser Pro Gly Ser Pro Val
Gln Thr Gln Thr Ala Pro Ala Gly Pro Cys 195 200 205 Gly Glu Gln Ala
Glu Asp Asp Tyr Ala Asp Pro Gln Glu Tyr Phe Asn 210 215 220 Val Leu
Ser Tyr Arg Ser Leu Glu Ser Phe Ile Ala Val Ser Lys Thr 225 230 235
240 Gly 10 139 PRT Mus musculus 10 Met His Gly Trp Leu Leu Leu Val
Trp Val Gln Gly Leu Ile Gln Ala 1 5 10 15 Ala Phe Leu Ala Thr Gly
Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg 20 25 30 Asn Ile Ser Ala
Glu Glu Gly Gly Ser Val Ile Leu Gln Cys His Phe 35 40 45 Ser Ser
Asp Thr Ala Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp 50 55 60
Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser 65
70 75 80 Val Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu
Thr Phe 85 90 95 Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe
Cys Thr Tyr His 100 105 110 Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg
Ile Phe Leu Lys Val Gln 115 120 125 Glu Ser Ser Val Ala Gln Phe Gln
Thr Ala Pro 130 135 11 140 PRT Homo sapiens 11 Met Arg Trp Cys Leu
Leu Leu Ile Trp Ala Gln Gly Leu Arg Gln Ala 1 5 10 15 Pro Leu Ala
Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn 20 25 30 Ile
Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser 35 40
45 Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln
50 55 60 Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser
Pro Ser 65 70 75 80 Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly
Leu Thr Leu Gln 85 90 95 Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr
Phe Cys Ile Tyr His Thr 100 105 110 Tyr Pro Asp Gly Thr Tyr Thr Gly
Arg Ile Phe Leu Glu Val Leu Glu 115 120 125 Ser Ser Val Ala Glu His
Gly Ala Arg Phe Gln Ile 130 135 140 12 138 PRT Mus musculus 12 Met
His Gly Trp Leu Leu Leu Val Trp Val Gln Gly Leu Ile Gln Ala 1 5 10
15 Ala Phe Leu Ala Thr Gly Ala Thr Ala Gly Thr Ile Asp Thr Lys Arg
20 25 30 Asn Ile Ser Ala Glu Glu Gly Gly Ser Val Ile Leu Gln Cys
His Phe 35 40 45 Ser Ser Asp Thr Ala Glu Val Thr Gln Val Asp Trp
Lys Gln Gln Asp 50 55 60 Gln Leu Leu Ala Ile Tyr Ser Val Asp Leu
Gly Trp His Val Ala Ser 65 70 75 80 Val Phe Ser Asp Arg Val Val Pro
Gly Pro Ser Leu Gly Leu Thr Phe 85 90 95 Gln Ser Leu Thr Met Asn
Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His 100 105 110 Thr Tyr Pro Gly
Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys Val Gln 115 120 125 Glu Ser
Ser Val Ala Gln Phe Gln Thr Ala 130 135 13 147 DNA Artificial
Sequence VASP-His6 Tetramerizing Domain 13 ggctccggtg gctccgacct
acagagggtg aaacaggagc ttctggaaga ggtgaagaag 60 gaattgcaga
aagtgaaaga ggaaatcatt gaagccttcg tccaggagct gaggggttcc 120
ggtggccatc accatcacca tcactga 147 14 48 PRT Artificial Sequence
VASP-His6 tetramerizing domain 14 Gly Ser Gly Gly Ser Asp Leu Gln
Arg Val Lys Gln Glu Leu Leu Glu 1 5 10 15 Glu Val Lys Lys Glu Leu
Gln Lys Val Lys Glu Glu Ile Ile Glu Ala 20 25 30 Phe Val Gln Glu
Leu Arg Gly Ser Gly Gly His His His His His His 35 40 45 15 4 PRT
Artificial Sequence Linker 15 Gly Ser Gly Gly 1 16 6 PRT Artificial
Sequence Linker 16 His His His His His His 1 5 17 3200 DNA
Artificial Sequence CDS (184)...(1425) Human CD155 17 cttgaagaag
tgggtattcc ccttcccacc ccaggcactg gaggagcggc cccccgggga 60
ttccaggacc tgagctccgg gagctggact cgcagcgacc gcggcagagc gagctggcgc
120 cgggaagcga ggagacgccc gcgggaggcc cagctgctcg gagcaactgg
catggcccga 180 gcc atg gcc gcc gcg tgg ccg ctg ctg ctg gtg gcg cta
ctg gtg ctg 228 Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu Leu
Val Leu 1 5 10 15 tcc tgg cca ccc cca gga acc ggg gac gtc gtc gtg
cag gcg ccc acc 276 Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val
Gln Ala Pro Thr 20 25 30 cag gtg ccc ggc ttc ttg ggc gac tcc gtg
acg ctg ccc tgc tac cta 324 Gln Val Pro Gly Phe Leu Gly Asp Ser Val
Thr Leu Pro Cys Tyr Leu 35 40 45 cag gtg ccc aac atg gag gtg acg
cat gtg tca cag ctg act tgg gcg 372 Gln Val Pro Asn Met Glu Val Thr
His Val Ser Gln Leu Thr Trp Ala 50 55 60 cgg cat ggt gaa tct ggc
agc atg gcc gtc ttc cac caa acg cag ggc 420 Arg His Gly Glu Ser Gly
Ser Met Ala Val Phe His Gln Thr Gln Gly 65 70 75 ccc agc tat tcg
gag tcc aaa cgg ctg gaa ttc gtg gca gcc aga ctg 468 Pro Ser Tyr Ser
Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu 80 85 90 95 ggc gcg
gag ctg cgg aat gcc tcg ctg agg atg ttc ggg ttg cgc gta 516 Gly Ala
Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val 100 105 110
gag gat gaa ggc aac tac acc tgc ctg ttc gtc acg ttc ccg cag ggc 564
Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly 115
120 125 agc agg agc gtg gat atc tgg ctc cga gtg ctt gcc aag ccc cag
aac 612 Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln
Asn 130 135 140 aca gct gag gtt cag aag gtc cag ctc act gga gag cca
gtg ccc atg 660 Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro
Val Pro Met 145 150 155 gcc cgc tgc gtc tcc aca ggg ggt cgc ccg cca
gcc caa atc acc tgg 708 Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro
Ala Gln Ile Thr Trp 160 165 170 175 cac tca gac ctg ggc ggg atg ccc
aat acg agc cag gtg cca ggg ttc 756 His Ser Asp Leu Gly Gly Met Pro
Asn Thr Ser Gln Val Pro Gly Phe 180 185 190 ctg tct ggc aca gtc act
gtc acc agc ctc tgg ata ttg gtg ccc tca 804 Leu Ser Gly Thr Val Thr
Val Thr Ser Leu Trp Ile Leu Val Pro Ser 195 200 205 agc cag gtg gac
ggc aag aat gtg acc tgc aag gtg gag cac gag agc 852 Ser Gln Val Asp
Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser 210 215 220 ttt gag
aag cct cag ctg ctg act gtg aac ctc acc gtg tac tac ccc 900 Phe Glu
Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro 225 230 235
cca gag gta tcc atc tct ggc tat gat aac aac tgg tac ctt ggc cag 948
Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln 240
245 250 255 aat gag gcc acc ctg acc tgc gat gct cgc agc aac cca gag
ccc aca 996 Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu
Pro Thr 260 265 270 ggc tat aat tgg agc acg acc atg ggt ccc ctg cca
ccc ttt gct gtg 1044 Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu
Pro Pro Phe Ala Val 275 280 285 gcc cag ggc gcc cag ctc ctg atc cgt
cct gtg gac aaa cca atc aac 1092 Ala Gln Gly Ala Gln Leu Leu Ile
Arg Pro Val Asp Lys Pro Ile Asn 290 295 300 aca act tta atc tgc aac
gtc acc aat gcc cta gga gct cgc cag gca 1140 Thr Thr Leu Ile Cys
Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala 305 310 315 gaa ctg acc
gtc cag gtc aaa gag gga cct ccc agt gag cac tca ggc 1188 Glu Leu
Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly 320 325 330
335 atg tcc cgt aac gcc atc atc ttc ctg gtt ctg gga atc ctg gtt ttt
1236 Met Ser Arg Asn Ala Ile Ile Phe Leu Val Leu Gly Ile Leu Val
Phe 340 345 350 ctg atc ctg ctg ggg atc ggg att tat ttc tat tgg tcc
aaa tgt tcc 1284 Leu Ile Leu Leu Gly Ile Gly Ile Tyr Phe Tyr Trp
Ser Lys Cys Ser 355 360 365 cgt gag gtc ctt tgg cac tgt cat ctg tgt
ccc tcg agt aca gag cat 1332 Arg Glu Val Leu Trp His Cys His Leu
Cys Pro Ser Ser Thr Glu His 370 375 380 gcc agc gcc tca gct aat ggg
cat gtc tcc tat tca gct gtg agc aga 1380 Ala Ser Ala Ser Ala Asn
Gly His Val Ser Tyr Ser Ala Val Ser Arg 385 390 395 gag aac agc tct
tcc cag gat cca cag aca gag ggc aca agg tga 1425 Glu Asn Ser Ser
Ser Gln Asp Pro Gln Thr Glu Gly Thr Arg * 400 405 410 cagcgtcggg
actgagaggg gagagagact ggagctggca aggacgtggg cctccagagt 1485
tggacccgac cccaatggat gaagaccccc tccaaagaga ccagcctccc tccctgtgcc
1545 agacctcaaa acgacggggg caggtgcaag ttcataggtc tccaagacca
ccctcctttc 1605 atttgctaga aggactcact agactcagga aagctgttag
gctcacagtt acagtttatt 1665 acagtaaaag gacagagatt aagatcagca
aagggaggag gtgcacagca cacgttccac 1725 gacagatgag gcgacggctt
ccatctgccc tctcccagtg gagccatata ggcagcacct 1785 gattctcaca
gcaacatgtg acaacatgca agaagtactg ccaatactgc caaccagagc 1845
agctcactcg agatctttgt gtccagagtt ttttgtttgt cttgagacag ggtctggctc
1905 tgttggcaga ctagagtaca gtggtgagat cacagttcat tgcagccttg
acttctcaac 1965 gccaagtcat cctcccacct cagcctcctg agtagctatg
actacaggta tgtgccacca 2025 cgtctggcta atctttttat tatttgtaaa
gtcgaggttt ccctgtgttg cccaggctgg 2085 tcttgaactc ttggctccaa
gtgatacttc tgccttggcc tcccaaagtg ctgaattaag 2145 cagctcacca
tccacacggc tgacctcata catcaagcca ataccgtgtg gcccaagacc 2205
cccaccataa atcacatcat tagcatgaac cacccagagt ggcccaagac tcccagatca
2265 gctaccaggc aggatattcc aagggcttag agatgaatgc ccaggagctg
aggataaagg 2325 gcccgatctt tctttgggca aggttaagcc tttactgcat
agcagaccac acagaagggt 2385 gtgggccacc agagaatttt ggtaaaaatt
tggcctctgg ccttgagctt ctaaatctct 2445 gtatccgtca gatctctgtg
gttacaagaa acagccactg accctggtca ccagaggctg 2505 caattcaggc
cgcaagcagc tgcctagggg gtgtccaagg agcagagaaa actactagat 2565
gtgaacttga agaaggttgt cagctgcagc cactttctgc cagcatctgc agccactttc
2625 tgccagcatc tgcagccagc aagctgggac tggcaggaaa taacccacaa
aagaagcaaa 2685 tgcaatttcc aacacaaggg ggaagggatg cagggggagg
cagcgctgca gttgctcagg 2745 acacgctcct ataggaccaa gatggatgcg
acccaagacc caggaggccc agctgctcag 2805 tgcaactgac aagttaaaaa
ggtctatgat cttgagggca gacagcagaa ttcctcttat 2865 aaagaaaact
gtttgggaaa atacgttgag ggagagaaga ccttgggcca agatgctaaa 2925
tgggaatgca aagcttgagc tgctctgcaa gagaaaataa gcaggacaga ggatttgctc
2985 tggacagaga tggaagagcc gggaacagag aagtgtgggg aagagatagg
aaccagcagg 3045 atggcagggg caaagggctc aagggtgagg aggccagtgg
gaccccacag agttggggag 3105 ataaaggaac attggttgct ttggtggcac
gtaagctcct tgtctgtctc cagcacccag 3165 aatctcatta aagcttattt
attgtacctc caaaa 3200 18 413 PRT Artificial Sequence Human CD155 18
Met Ala Ala Ala Trp Pro Leu Leu Leu Val Ala Leu Leu Val Leu Ser 1 5
10 15 Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr
Gln 20 25 30 Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys
Tyr Leu Gln 35 40 45 Val Pro Asn Met Glu Val Thr His Val Ser Gln
Leu Thr Trp Ala Arg 50 55 60 His Gly Glu Ser Gly Ser Met Ala Val
Phe His Gln Thr Gln Gly Pro 65 70 75 80 Ser Tyr Ser Glu Ser Lys Arg
Leu Glu Phe Val Ala Ala Arg Leu Gly 85 90 95 Ala Glu Leu Arg Asn
Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu 100 105 110 Asp Glu Gly
Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser 115 120 125 Arg
Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr 130 135
140 Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala
145 150 155 160 Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile
Thr Trp His 165 170 175 Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln
Val Pro Gly Phe Leu 180 185 190 Ser Gly Thr Val Thr Val Thr Ser Leu
Trp Ile Leu Val Pro Ser Ser 195 200 205 Gln Val Asp Gly Lys Asn Val
Thr Cys Lys Val Glu His Glu Ser Phe 210 215 220 Glu Lys Pro Gln Leu
Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro 225 230 235 240 Glu Val
Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn 245 250 255
Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly 260
265 270 Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val
Ala 275 280 285 Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro
Ile Asn Thr 290 295 300 Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly
Ala Arg Gln Ala Glu 305 310 315 320 Leu Thr Val Gln Val Lys Glu Gly
Pro Pro Ser Glu His Ser Gly Met 325 330 335 Ser Arg Asn Ala Ile Ile
Phe Leu Val Leu Gly Ile Leu Val Phe Leu 340 345 350 Ile Leu Leu Gly
Ile Gly Ile Tyr Phe Tyr Trp Ser Lys Cys Ser Arg 355 360 365 Glu Val
Leu Trp His Cys His Leu Cys Pro Ser Ser Thr Glu His Ala 370 375 380
Ser Ala Ser Ala Asn Gly His Val Ser Tyr Ser Ala Val Ser Arg Glu 385
390 395 400 Asn Ser Ser Ser Gln Asp Pro Gln Thr Glu Gly Thr Arg 405
410 19 316 PRT Artificial Sequence Human CD155 19 Gln Ala Pro Thr
Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu 1 5 10 15 Pro Cys
Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln 20 25 30
Leu Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His 35
40 45 Gln Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe
Val 50 55 60 Ala Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu
Arg Met Phe 65 70 75 80 Gly Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr
Cys Leu Phe Val Thr 85 90 95 Phe Pro Gln Gly Ser Arg Ser Val Asp
Ile Trp Leu Arg Val Leu Ala 100 105 110 Lys Pro Gln Asn Thr Ala Glu
Val Gln Lys Val Gln Leu Thr Gly Glu 115 120 125 Pro Val Pro
Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala 130 135 140 Gln
Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln 145 150
155 160 Val Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp
Ile 165 170 175 Leu Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr
Cys Lys Val 180 185 190 Glu His Glu Ser Phe Glu Lys Pro Gln Leu Leu
Thr Val Asn Leu Thr 195 200 205 Val Tyr Tyr Pro Pro Glu Val Ser Ile
Ser Gly Tyr Asp Asn Asn Trp 210 215 220 Tyr Leu Gly Gln Asn Glu Ala
Thr Leu Thr Cys Asp Ala Arg Ser Asn 225 230 235 240 Pro Glu Pro Thr
Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro 245 250 255 Pro Phe
Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp 260 265 270
Lys Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly 275
280 285 Ala Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro
Ser 290 295 300 Glu His Ser Gly Met Ser Arg Asn Ala Ile Ile Phe 305
310 315 20 2838 DNA Murine CDS (62)...(1288) 20 gagataaggc
gcttggccgt tactaactgg actacaaaga gctggatcgg accggaacca 60 c atg gct
caa ctc gcc cga gcc acc cgc tcc ccg ctg tca tgg ctg ctg 109 Met Ala
Gln Leu Ala Arg Ala Thr Arg Ser Pro Leu Ser Trp Leu Leu 1 5 10 15
ctg ctg ttc tgc tat gca ctc cgg aaa gcg ggt ggg gat ata cgt gtg 157
Leu Leu Phe Cys Tyr Ala Leu Arg Lys Ala Gly Gly Asp Ile Arg Val 20
25 30 ctg gtg ccc tac aat tcg aca ggc gtc ttg gga ggg tcg acc acc
ttg 205 Leu Val Pro Tyr Asn Ser Thr Gly Val Leu Gly Gly Ser Thr Thr
Leu 35 40 45 cac tgt agt ctg act tct aat gag aat gtg act atc act
caa ata acc 253 His Cys Ser Leu Thr Ser Asn Glu Asn Val Thr Ile Thr
Gln Ile Thr 50 55 60 tgg atg aag aag gat tca ggt gga tcc cac gct
ctt gtg gct gtc ttc 301 Trp Met Lys Lys Asp Ser Gly Gly Ser His Ala
Leu Val Ala Val Phe 65 70 75 80 cac ccc aag aag ggg ccc aac atc aaa
gag cca gag agg gtg aaa ttc 349 His Pro Lys Lys Gly Pro Asn Ile Lys
Glu Pro Glu Arg Val Lys Phe 85 90 95 ttg gct gcc caa cag gat ctg
agg aac gca tct ctg gcc atc tcg aac 397 Leu Ala Ala Gln Gln Asp Leu
Arg Asn Ala Ser Leu Ala Ile Ser Asn 100 105 110 tta agt gta gaa gac
gaa ggc atc tat gaa tgt cag att gcc aca ttc 445 Leu Ser Val Glu Asp
Glu Gly Ile Tyr Glu Cys Gln Ile Ala Thr Phe 115 120 125 ccc aga ggc
agt aga agc acc aat gcc tgg ctg aag gtg caa gcc cga 493 Pro Arg Gly
Ser Arg Ser Thr Asn Ala Trp Leu Lys Val Gln Ala Arg 130 135 140 cct
aag aac act gca gag gcc ctg gag ccc tct ccc acc ttg ata ctg 541 Pro
Lys Asn Thr Ala Glu Ala Leu Glu Pro Ser Pro Thr Leu Ile Leu 145 150
155 160 cag gat gtg gct aaa tgc atc tct gcc aat ggt cac cct cct gga
cga 589 Gln Asp Val Ala Lys Cys Ile Ser Ala Asn Gly His Pro Pro Gly
Arg 165 170 175 atc tct tgg ccc tcg aat gtg aat gga agt cac cgt gaa
atg aag gaa 637 Ile Ser Trp Pro Ser Asn Val Asn Gly Ser His Arg Glu
Met Lys Glu 180 185 190 cca ggg tcc cag ccg ggc acc acc aca gtt acc
agc tac ctc tcc atg 685 Pro Gly Ser Gln Pro Gly Thr Thr Thr Val Thr
Ser Tyr Leu Ser Met 195 200 205 gta cct tct cgc cag gca gac ggc aag
aac atc acc tgc acg gtg gag 733 Val Pro Ser Arg Gln Ala Asp Gly Lys
Asn Ile Thr Cys Thr Val Glu 210 215 220 cat gaa agc tta cag gag ctg
gac cag ctg ctg gtg acc ctt tcc caa 781 His Glu Ser Leu Gln Glu Leu
Asp Gln Leu Leu Val Thr Leu Ser Gln 225 230 235 240 ccc tat cca cct
gaa aac gtg tcc atc tct ggc tat gac ggc aac tgg 829 Pro Tyr Pro Pro
Glu Asn Val Ser Ile Ser Gly Tyr Asp Gly Asn Trp 245 250 255 tat gtt
ggc ctc act aac ttg acc ctg acc tgt gaa gct cac agc aaa 877 Tyr Val
Gly Leu Thr Asn Leu Thr Leu Thr Cys Glu Ala His Ser Lys 260 265 270
cca gcg cct gac atg gct gga tat aac tgg agc acg aac acg ggt gac 925
Pro Ala Pro Asp Met Ala Gly Tyr Asn Trp Ser Thr Asn Thr Gly Asp 275
280 285 ttt ccc aac tct gtt aag cgc cag ggc aat atg ctt cta atc tcc
acc 973 Phe Pro Asn Ser Val Lys Arg Gln Gly Asn Met Leu Leu Ile Ser
Thr 290 295 300 gta gag gat ggt ctc aat aac acg gtc att gtg tgc gaa
gtc acc aat 1021 Val Glu Asp Gly Leu Asn Asn Thr Val Ile Val Cys
Glu Val Thr Asn 305 310 315 320 gcc cta ggg tct ggg cag ggc caa gtg
cac atc att gtt aaa gag aaa 1069 Ala Leu Gly Ser Gly Gln Gly Gln
Val His Ile Ile Val Lys Glu Lys 325 330 335 cct gag aat atg cag caa
aat aca aga tta cac cta ggc tac atc ttt 1117 Pro Glu Asn Met Gln
Gln Asn Thr Arg Leu His Leu Gly Tyr Ile Phe 340 345 350 ctt atc gtc
ttt gtc ctc gct gta gtc atc atc atc gca gca cta tac 1165 Leu Ile
Val Phe Val Leu Ala Val Val Ile Ile Ile Ala Ala Leu Tyr 355 360 365
act ata cga aga tgc agg cat ggt cgt gct ctg cag tcc aat ccc tca
1213 Thr Ile Arg Arg Cys Arg His Gly Arg Ala Leu Gln Ser Asn Pro
Ser 370 375 380 gag agg gag aac gtc cag tat tca tct gtg aac ggc gac
tgt aga ctg 1261 Glu Arg Glu Asn Val Gln Tyr Ser Ser Val Asn Gly
Asp Cys Arg Leu 385 390 395 400 aac atg gag cca aac agc aca agg tga
cggtgctggg tagacagaac 1308 Asn Met Glu Pro Asn Ser Thr Arg * 405
taaggaactt gaaggcatag caactggaac cctactctca taaatgaaga agcctccaga
1368 gagactggct gctcagtgtg atgagcatag caagtttggg gggtctccca
ggatgctgcc 1428 gaattccacg ttgtcaaaag gacccatgga ggccagtgtg
ttggctcact cttgacatct 1488 cagcaagctg gggggggggg ggggagcata
aagcgaggtt gagtctagct tgggctatag 1548 agcaaagccc tgtccataca
caaacaagct aaggggcttt gagacggtca gaaactgaag 1608 tcttgctttg
ggtaaggtaa atcctctacc gcatgtatgt gctagacttg aaagacttcc 1668
acacagacct ctttataagt tgactccatt ggggctatcc cctcctctct ggacaaggtc
1728 tctgtatgta gccaaggcta ggctcaaact cacagagata tgtctgcttc
tacctcccca 1788 gtgctagagt tgaaagtatt tgtgccactg cacttttcta
ggtcttcttt taatgaagta 1848 aagtatatat ttataaaaag ctatttagtt
atatatatat atatttttga gactatttca 1908 tagagcccaa gctaacctca
aacttactat gtagccaaga gtgatggtaa actaatttat 1968 tttaatttat
ttgtcttcaa ttttaaccat cacccaaccc ctgctccctt ccatatcttc 2028
tttcaatcca tttcattgtc tttttcttcc cagacactat tctgacttac gtctccatta
2088 caaacatttt attgaactac ataaaaatgt gtgaaccaca aaaaaaaaat
gtatttgtca 2148 aaattgtagt tgtctttctg aggctgacct gagttctctg
ataccattct ctccagttgt 2208 atccagtttc ctgtaaacaa tgtgactttg
tttttctcag tagctaaaac atcccaatta 2268 tgtgagtgta cactttcttt
actcattcct ctgtgggcca ccagctgggt tggttccata 2328 tctgagctat
tgtgcatgga attgtctctg tggtgggttt agtaaactcc caggaatgcc 2388
tgtacatgtt tgtagaggcc agaagaaggc acaaaatctt gagccaggct tacatgcact
2448 tgtgagtagc cccacatagg tgctaagaac ccagttcagg tcctctgctg
tgggatggtg 2508 ggctgtgcac agaaagcctg gtcccggtct agcaaaggtc
tggaactccg gagccggtgg 2568 gctgtgattt acaccagcat gggatggaag
gagttggacc tcgcctcctg ggcacctggc 2628 tcctgtcaca tagctacagc
ctcccacagc ccccctatag ggaggtatgc agcatcaatc 2688 acatagtagc
tgcactaagc cctcccacat gcaaataagg tttccccaaa ctctcagtcc 2748
aagccaatga aaagtacctg ctgtcaaacc ctaaatcatc cccaaaactc tgtaagtcct
2808 atcagggaat aaaatgtgtg tgaaaactaa 2838 21 408 PRT Murine 21 Met
Ala Gln Leu Ala Arg Ala Thr Arg Ser Pro Leu Ser Trp Leu Leu 1 5 10
15 Leu Leu Phe Cys Tyr Ala Leu Arg Lys Ala Gly Gly Asp Ile Arg Val
20 25 30 Leu Val Pro Tyr Asn Ser Thr Gly Val Leu Gly Gly Ser Thr
Thr Leu 35 40 45 His Cys Ser Leu Thr Ser Asn Glu Asn Val Thr Ile
Thr Gln Ile Thr 50 55 60 Trp Met Lys Lys Asp Ser Gly Gly Ser His
Ala Leu Val Ala Val Phe 65 70 75 80 His Pro Lys Lys Gly Pro Asn Ile
Lys Glu Pro Glu Arg Val Lys Phe 85 90 95 Leu Ala Ala Gln Gln Asp
Leu Arg Asn Ala Ser Leu Ala Ile Ser Asn 100 105 110 Leu Ser Val Glu
Asp Glu Gly Ile Tyr Glu Cys Gln Ile Ala Thr Phe 115 120 125 Pro Arg
Gly Ser Arg Ser Thr Asn Ala Trp Leu Lys Val Gln Ala Arg 130 135 140
Pro Lys Asn Thr Ala Glu Ala Leu Glu Pro Ser Pro Thr Leu Ile Leu 145
150 155 160 Gln Asp Val Ala Lys Cys Ile Ser Ala Asn Gly His Pro Pro
Gly Arg 165 170 175 Ile Ser Trp Pro Ser Asn Val Asn Gly Ser His Arg
Glu Met Lys Glu 180 185 190 Pro Gly Ser Gln Pro Gly Thr Thr Thr Val
Thr Ser Tyr Leu Ser Met 195 200 205 Val Pro Ser Arg Gln Ala Asp Gly
Lys Asn Ile Thr Cys Thr Val Glu 210 215 220 His Glu Ser Leu Gln Glu
Leu Asp Gln Leu Leu Val Thr Leu Ser Gln 225 230 235 240 Pro Tyr Pro
Pro Glu Asn Val Ser Ile Ser Gly Tyr Asp Gly Asn Trp 245 250 255 Tyr
Val Gly Leu Thr Asn Leu Thr Leu Thr Cys Glu Ala His Ser Lys 260 265
270 Pro Ala Pro Asp Met Ala Gly Tyr Asn Trp Ser Thr Asn Thr Gly Asp
275 280 285 Phe Pro Asn Ser Val Lys Arg Gln Gly Asn Met Leu Leu Ile
Ser Thr 290 295 300 Val Glu Asp Gly Leu Asn Asn Thr Val Ile Val Cys
Glu Val Thr Asn 305 310 315 320 Ala Leu Gly Ser Gly Gln Gly Gln Val
His Ile Ile Val Lys Glu Lys 325 330 335 Pro Glu Asn Met Gln Gln Asn
Thr Arg Leu His Leu Gly Tyr Ile Phe 340 345 350 Leu Ile Val Phe Val
Leu Ala Val Val Ile Ile Ile Ala Ala Leu Tyr 355 360 365 Thr Ile Arg
Arg Cys Arg His Gly Arg Ala Leu Gln Ser Asn Pro Ser 370 375 380 Glu
Arg Glu Asn Val Gln Tyr Ser Ser Val Asn Gly Asp Cys Arg Leu 385 390
395 400 Asn Met Glu Pro Asn Ser Thr Arg 405 22 319 PRT Murine 22
Asp Ile Arg Val Leu Val Pro Tyr Asn Ser Thr Gly Val Leu Gly Gly 1 5
10 15 Ser Thr Thr Leu His Cys Ser Leu Thr Ser Asn Glu Asn Val Thr
Ile 20 25 30 Thr Gln Ile Thr Trp Met Lys Lys Asp Ser Gly Gly Ser
His Ala Leu 35 40 45 Val Ala Val Phe His Pro Lys Lys Gly Pro Asn
Ile Lys Glu Pro Glu 50 55 60 Arg Val Lys Phe Leu Ala Ala Gln Gln
Asp Leu Arg Asn Ala Ser Leu 65 70 75 80 Ala Ile Ser Asn Leu Ser Val
Glu Asp Glu Gly Ile Tyr Glu Cys Gln 85 90 95 Ile Ala Thr Phe Pro
Arg Gly Ser Arg Ser Thr Asn Ala Trp Leu Lys 100 105 110 Val Gln Ala
Arg Pro Lys Asn Thr Ala Glu Ala Leu Glu Pro Ser Pro 115 120 125 Thr
Leu Ile Leu Gln Asp Val Ala Lys Cys Ile Ser Ala Asn Gly His 130 135
140 Pro Pro Gly Arg Ile Ser Trp Pro Ser Asn Val Asn Gly Ser His Arg
145 150 155 160 Glu Met Lys Glu Pro Gly Ser Gln Pro Gly Thr Thr Thr
Val Thr Ser 165 170 175 Tyr Leu Ser Met Val Pro Ser Arg Gln Ala Asp
Gly Lys Asn Ile Thr 180 185 190 Cys Thr Val Glu His Glu Ser Leu Gln
Glu Leu Asp Gln Leu Leu Val 195 200 205 Thr Leu Ser Gln Pro Tyr Pro
Pro Glu Asn Val Ser Ile Ser Gly Tyr 210 215 220 Asp Gly Asn Trp Tyr
Val Gly Leu Thr Asn Leu Thr Leu Thr Cys Glu 225 230 235 240 Ala His
Ser Lys Pro Ala Pro Asp Met Ala Gly Tyr Asn Trp Ser Thr 245 250 255
Asn Thr Gly Asp Phe Pro Asn Ser Val Lys Arg Gln Gly Asn Met Leu 260
265 270 Leu Ile Ser Thr Val Glu Asp Gly Leu Asn Asn Thr Val Ile Val
Cys 275 280 285 Glu Val Thr Asn Ala Leu Gly Ser Gly Gln Gly Gln Val
His Ile Ile 290 295 300 Val Lys Glu Lys Pro Glu Asn Met Gln Gln Asn
Thr Arg Leu His 305 310 315 23 48 PRT Artificial Sequence VASP-His6
Tetramerizing Domain 23 Gly Ser Gly Gly Ser Asp Leu Gln Arg Val Lys
Gln Glu Leu Leu Glu 1 5 10 15 Glu Val Lys Lys Glu Leu Gln Lys Val
Lys Glu Glu Ile Ile Glu Ala 20 25 30 Phe Val Gln Glu Leu Arg Gly
Ser Gly Gly His His His His His His 35 40 45 24 2207 DNA Homo
sapiens 24 ccccttcctg tggggttcat tggggcatcc cctttctgct gcaggaacct
ctcatcagac 60 cgcctgaggg aagcggcgcc cggagacccg ccccggcccg
gtccacattc tccccaggaa 120 gccggactct atggggcggg accctggggg
agcctgagcc gagcccggag ccagccccga 180 acccctgaac ctccagccag
gggcgccccg ggagcagcca gcccgtgggc gagccgcccg 240 cccgccgagc
agccatgagc gagacggtca tctgttccag ccgggccact gtgatgcttt 300
atgatgatgg caacaagcga tggctccctg ctggcacggg tccccaggcc ttcagccgcg
360 tccagatcta ccacaacccc acggccaatt cctttcgcgt cgtgggccgg
aagatgcagc 420 ccgaccagca ggtggtcatc aactgtgcca tcgtccgggg
tgtcaagtat aaccaggcca 480 cccccaactt ccatcagtgg cgcgacgctc
gccaggtctg gggcctcaac ttcggcagca 540 aggaggatgc ggcccagttt
gccgccggca tggccagtgc cctagaggcg ttggaaggag 600 gtgggccccc
tccaccccca gcacttccca cctggtcggt cccgaacggc ccctccccgg 660
aggaggtgga gcagcagaaa aggcagcagc ccggcccgtc ggagcacata gagcgccggg
720 tctccaatgc aggaggccca cctgctcccc ccgctggggg tccaccccca
ccaccaggac 780 ctccccctcc tccaggtccc cccccacccc caggtttgcc
cccttcgggg gtcccagctg 840 cagcgcacgg agcaggggga ggaccacccc
ctgcaccccc tctcccggca gcacagggcc 900 ctggtggtgg gggagctggg
gccccaggcc tggccgcagc tattgctgga gccaaactca 960 ggaaagtcag
caagcaggag gaggcctcag gggggcccac agcccccaaa gctgagagtg 1020
gtcgaagcgg aggtggggga ctcatggaag agatgaacgc catgctggcc cggagaagga
1080 aagccacgca agttggggag aaaaccccca aggatgaatc tgccaatcag
gaggagccag 1140 aggccagagt cccggcccag agtgaatctg tgcggagacc
ctgggagaag aacagcacaa 1200 ccttgccaag gatgaagtcg tcttcttcgg
tgaccacttc cgagacccaa ccctgcacgc 1260 ccagctccag tgattactcg
gacctacaga gggtgaaaca ggagcttctg gaagaggtga 1320 agaaggaatt
gcagaaagtg aaagaggaaa tcattgaagc cttcgtccag gagctgagga 1380
agcggggttc tccctgacca cagggaccca gaagacccgc ttctcctttc cgcacacccg
1440 gcctgtcacc ctgctttccc tgcctctact tgacttggaa ttggctgaag
acacaggaat 1500 gcatcgttcc cactccccat cccacttgga aaactccaag
ggggtgtggc ttccctgctc 1560 acacccacac tggctgctga ttggctgggg
aggcccccgc ccttttctcc ctttggtcct 1620 tcccctctgc catccccttg
gggccggtcc ctctgctggg gatgcaccaa tgaaccccac 1680 aggaaggggg
aaggaaggag ggaatttcac attcccttgt tctagattca ctttaacgct 1740
taatgccttc aaagttttgg tttttttaag aaaaaaaaat atatatatat ttgggttttg
1800 ggggaaaagg gaaatttttt tttctctttg gttttgataa aatgggatgt
gggagttttt 1860 aaatgctata gccctgggct tgccccattt ggggcagcta
tttaagggga ggggatgtct 1920 caccgggctg ggggtgagat atccccccac
cccagggact ccccttccct ctggctcctt 1980 ccccttttct atgaggaaat
aagatgctgt aactttttgg aacctcagtt ttttgatttt 2040 ttatttgggt
aggttttggg gtccaggcca ttttttttac cccttggagg aaataagatg 2100
agggagaaag gagaagggga ggaaacttct cccctcccac cttcaccttt agcttcttga
2160 aaatgggccc ctgcagaata aatctgccag tttttataaa aaaaaaa 2207 25
380 PRT homo sapiens 25 Met Ser Glu Thr Val Ile Cys Ser Ser Arg Ala
Thr Val Met Leu Tyr 1 5 10 15 Asp Asp Gly Asn Lys Arg Trp Leu Pro
Ala Gly Thr Gly Pro Gln Ala 20 25 30 Phe Ser Arg Val Gln Ile Tyr
His Asn Pro Thr Ala Asn Ser Phe Arg 35 40 45 Val Val Gly Arg Lys
Met Gln Pro Asp Gln Gln Val Val Ile Asn Cys 50 55 60 Ala Ile Val
Arg Gly Val Lys Tyr Asn Gln Ala Thr Pro Asn Phe His 65 70 75 80 Gln
Trp Arg Asp Ala Arg Gln Val Trp Gly Leu Asn Phe Gly Ser Lys 85 90
95 Glu Asp Ala Ala Gln Phe Ala Ala Gly Met Ala Ser Ala Leu Glu Ala
100 105 110 Leu Glu Gly Gly Gly Pro Pro Pro Pro Pro Ala Leu Pro Thr
Trp Ser 115 120 125 Val Pro Asn Gly Pro Ser Pro Glu Glu Val Glu Gln
Gln Lys Arg Gln 130 135 140 Gln Pro Gly Pro Ser Glu His Ile Glu Arg
Arg Val Ser Asn Ala Gly 145 150 155 160 Gly Pro Pro Ala Pro Pro Ala
Gly Gly Pro Pro Pro Pro Pro Gly Pro 165 170 175 Pro Pro Pro Pro
Gly Pro Pro Pro Pro Pro Gly Leu Pro Pro Ser Gly 180 185 190 Val Pro
Ala Ala Ala His Gly Ala Gly Gly Gly Pro Pro Pro Ala Pro 195 200 205
Pro Leu Pro Ala Ala Gln Gly Pro Gly Gly Gly Gly Ala Gly Ala Pro 210
215 220 Gly Leu Ala Ala Ala Ile Ala Gly Ala Lys Leu Arg Lys Val Ser
Lys 225 230 235 240 Gln Glu Glu Ala Ser Gly Gly Pro Thr Ala Pro Lys
Ala Glu Ser Gly 245 250 255 Arg Ser Gly Gly Gly Gly Leu Met Glu Glu
Met Asn Ala Met Leu Ala 260 265 270 Arg Arg Arg Lys Ala Thr Gln Val
Gly Glu Lys Thr Pro Lys Asp Glu 275 280 285 Ser Ala Asn Gln Glu Glu
Pro Glu Ala Arg Val Pro Ala Gln Ser Glu 290 295 300 Ser Val Arg Arg
Pro Trp Glu Lys Asn Ser Thr Thr Leu Pro Arg Met 305 310 315 320 Lys
Ser Ser Ser Ser Val Thr Thr Ser Glu Thr Gln Pro Cys Thr Pro 325 330
335 Ser Ser Ser Asp Tyr Ser Asp Leu Gln Arg Val Lys Gln Glu Leu Leu
340 345 350 Glu Glu Val Lys Lys Glu Leu Gln Lys Val Lys Glu Glu Ile
Ile Glu 355 360 365 Ala Phe Val Gln Glu Leu Arg Lys Arg Gly Ser Pro
370 375 380 26 4 PRT Artificial Sequence Linker 26 Gly Ser Gly Gly
1 27 8 PRT Artificial Sequence Tag 27 Asp Tyr Lys Asp Asp Asp Asp
Lys 1 5 28 6 PRT Artificial Sequence Tag 28 His His His His His His
1 5 29 726 DNA murine 29 atgcatggct ggctgctcct ggtctgggtc
caggggctga tacaggctgc cttcctcgct 60 acaggagcca cagcaggcac
gatagataca aagaggaaca tctctgcaga ggaaggtggc 120 tctgtcatct
tacagtgtca cttctcctct gacacagctg aagtgaccca agtcgactgg 180
aagcagcagg accagcttct ggccatttat agtgttgacc tggggtggca tgtcgcttca
240 gtcttcagtg atcgggtggt cccaggcccc agcctaggcc tcaccttcca
gtctctgaca 300 atgaatgaca cgggagagta cttctgtacc tatcatacgt
atcctggtgg gatttacaag 360 gggagaatat tcctgaaggt ccaagaaagc
tcagtggctc agttccagac tgccccgctt 420 ggaggaacca tggctgctgt
gctgggactc atttgcttaa tggtcacagg agtgactgta 480 ctggctagaa
agaagtctat tagaatgcat tctatagaaa gtggccttgg gagaacagaa 540
gcggagccac aggaatggaa cctgaggagt ctctcatccc ctggaagccc tgtccagaca
600 caaactgccc ctgctggtcc ctgtggagag caggcagaag atgactatgc
tgacccacag 660 gaatacttta atgtcctgag ctacagaagc ctagagagct
tcattgctgt atcgaagact 720 ggctaa 726 30 43 DNA Artificial Sequence
primer 30 tccacaggtg tccagggaat tcaccatgca tggctggctg ctc 43 31 36
DNA Artificial Sequence primer 31 aggcgcgcct ctagattagc cagtcttcga
tacagc 36 32 414 DNA Artificial Sequence oligonucleotide 32
atgcatggct ggctgctcct ggtctgggtc caggggctga tacaggctgc cttcctcgct
60 acaggagcca cagcaggcac gatagataca aagaggaaca tctctgcaga
ggaaggtggc 120 tctgtcatct tacagtgtca cttctcctct gacacagctg
aagtgaccca agtcgactgg 180 aagcagcagg accagcttct ggccatttat
agtgttgacc tggggtggca tgtcgcttca 240 gtcttcagtg atcgggtggt
cccaggcccc agcctaggcc tcaccttcca gtctctgaca 300 atgaatgaca
cgggagagta cttctgtacc tatcatacgt atcctggtgg gatttacaag 360
gggagaatat tcctgaaggt ccaagaaagc tcagtggctc agttccagac tgcc 414 33
43 DNA Artificial Sequence Primer 33 tccacaggtg tccagggaat
tcaccatgca tggctggctg ctc 43 34 46 DNA Artificial Sequence Primer
34 agggcttgat tgtgggagat ctgggctcgg cagtctggaa ctgagc 46 35 61 DNA
Artificial Sequence Primer 35 ccactttgcc tttctctcca caggtgtcca
gggaattcgc aagatgagga tatttgctgt 60 c 61 36 57 DNA Artificial
Sequence Primer 36 gcatggagga cagggcttga ttgtgggaga tctgggctct
tcatttggag gatgtgc 57 37 33 DNA Artificial Sequence Primer 37
gcatgaattc gcaagatgcg ctggtgtctc ctc 33 38 34 DNA Artificial
Sequence Primer 38 atgcagatct gggctcaatc tggaacctgg cacc 34 39 414
DNA Artificial Sequence Primer 39 atgcatggct ggctgctcct ggtctgggtc
caggggctga tacaggctgc cttcctcgct 60 acaggagcca cagcaggcac
gatagataca aagaggaaca tctctgcaga ggaaggtggc 120 tctgtcatct
tacagtgtca cttctcctct gacacagctg aagtgaccca agtcgactgg 180
aagcagcagg accagcttct ggccatttat agtgttgacc tggggtggca tgtcgcttca
240 gtcttcagtg atcgggtggt cccaggcccc agcctaggcc tcaccttcca
gtctctgaca 300 atgaatgaca cgggagagta cttctgtacc tatcatacgt
atcctggtgg gatttacaag 360 gggagaatat tcctgaaggt ccaagaaagc
tcagtggctc agttccagac tgcc 414 40 44 DNA Artificial Sequence Primer
40 ccacaggtgt ccagggaatt cgcaagatgc atggctggct gctc 44 41 37 DNA
Artificial Sequence Primer 41 ctccaccaga tcccttgcgg gcagtctgga
actgagc 37 42 420 DNA Artificial Sequence Primer 42 atgcgctggt
gtctcctcct gatctgggcc caggggctga ggcaggctcc cctcgcctca 60
ggaatgatga caggcacaat agaaacaacg gggaacattt ctgcagagaa aggtggctct
120 atcatcttac aatgtcacct ctcctccacc acggcacaag tgacccaggt
caactgggag 180 cagcaggacc agcttctggc catttgtaat gctgacttgg
ggtggcacat ctccccatcc 240 ttcaaggatc gagtggcccc aggtcccggc
ctgggcctca ccctccagtc gctgaccgtg 300 aacgatacag gggagtactt
ctgcatctat cacacctacc ctgatgggac gtacactggg 360 agaatcttcc
tggaggtcct agaaagctca gtggctgagc acggtgccag gttccagatt 420 43 45
DNA Artificial Sequence Tag 43 ggtctgaacg acatcttcga agctcagaaa
atcgaatggc acgaa 45 44 18 DNA Artificial Sequence Tag 44 catcaccatc
accatcac 18 45 43 DNA Artificial Sequence Primer 45 cacaggtgtc
cagggaattc gcaagatgcg ctggtgtctc ctc 43 46 123 DNA Artificial
Sequence Primer 46 aggcgcgcct ctagattagt gatggtgatg gtgatgtcca
ccagatcctt cgtgccattc 60 gattttctga gcttcgaaga tgtcgttcag
acctccacca gatccaatct ggaacctggc 120 acc 123 47 27 DNA Artificial
Sequence Primer 47 ggagtgactg tactggctag aaagaag 27 48 23 DNA
Artificial Sequence Primer 48 gagactcctc aggttccatt cct 23 49 23
DNA Artificial Sequence Primer 49 agctcagtgg ctgagcacgg tgc 23 50
103 DNA Artificial Sequence Primer 50 acgcttccgt agatctggtt
ccggaggctc cggtggctcc gacctacaga gggtgaaaca 60 ggagcttctg
gaagaggtga agaaggaatt gcagaagtga aag 103 51 103 DNA Artificial
Sequence Primer 51 aaggcgcgcc tctagatcag tgatggtgat ggtgatggcc
accggaaccc ctcagctcct 60 ggacgaaggc ttcaatgatt tcctctttca
ctttctgcaa ttc 103 52 47 DNA Artificial Sequence Primer 52
ctcagccagg aaatccatgc cgagttgaga cgcttccgta gatctgg 47 53 47 DNA
Artificial Sequence Primer 53 ggggtggggt acaaccccag agctgtttta
aggcgcgcct ctagatc 47 54 290 PRT Artificial Sequence VASP
tetramerization domain 54 Met Arg Ile Phe Ala Val Phe Ile Phe Met
Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro
Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile
Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala
Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80
Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85
90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val
Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg
Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln
Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu
Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile
Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr
Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205
Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210
215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr
His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly
Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met
Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys
Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 55 64 DNA
Artificial Sequence primer 55 caggtgtcca gggaattcat ataggccggc
caccatgcgc tggtgtctcc tcctgatctg 60 ggcc 64 56 33 DNA Artificial
Sequence primer 56 agtctgatgg tgggggcgaa cctggcaccg tgc 33 57 33
DNA Artificial Sequence primer 57 gcacggtgcc aggttcgccc ccaccatcag
act 33 58 63 DNA Artificial Sequence primer 58 aaccccagag
ctgttttaag gcgcgcctct agactagaaa cccccttgtt cttcaactcc 60 atg 63 59
64 DNA Artificial Sequence primer 59 caggtgtcca gggaattcat
ataggccggc caccatgcgc tggtgtctcc tcctgatctg 60 ggcc 64 60 63 DNA
Artificial Sequence primer 60 aaccccagag ctgttttaag gcgcgcctct
agactagaaa cccccttgtt cttcaactcc 60 atg 63 61 1133 DNA Artificial
Sequence human zB7R1mFc2 61 gaattcgcaa gatgcgctgg tgtctcctcc
tgatctgggc ccaggggctg aggcaggctc 60 ccctcgcctc aggaatgatg
acaggcacaa tagaaacaac ggggaacatt tctgcagaga 120 aaggtggctc
tatcatctta caatgtcacc tctcctccac cacggcacaa gtgacccagg 180
tcaactggga gcagcaggac cagcttctgg ccatttgtaa tgctgacttg gggtggcaca
240 tctccccatc cttcaaggat cgagtggccc caggtcccgg cctgggcctc
accctccagt 300 cgctgaccgt gaacgataca ggggagtact tctgcatcta
tcacacctac cctgatggga 360 cgtacactgg gagaatcttc ctggaggtcc
tagaaagctc agtggctgag cacggtgcca 420 ggttccagat tgagcccaga
tctcccacaa tcaagccctg tcctccatgc aaatgcccag 480 cacctaacct
cgagggtgga ccatccgtct tcatcttccc tccaaagatc aaggatgtac 540
tcatgatctc cctgagcccc atagtcacat gtgtggtggt ggatgtgagc gaggatgacc
600 cagatgtcca gatcagctgg tttgtgaaca acgtggaagt acacacagct
cagacacaaa 660 cccatagaga ggattacaac agtactctcc gggtggtcag
tgccctcccc atccagcacc 720 aggactggat gagtggcaaa gctttcgcat
gcgcggtcaa caacaaagac ctcccagcgc 780 ccatcgagag aaccatctca
aaacccaaag ggtcagtaag agctccacag gtatatgtct 840 tgcctccacc
agaagaagag atgactaaga aacaggtcac tctgacctgc atggtcacag 900
acttcatgcc tgaagacatt tacgtggagt ggaccaacaa cgggaaaaca gagctaaact
960 acaagaacac tgaaccagtc ctggactctg atggttctta cttcatgtac
agcaagctga 1020 gagtggaaaa gaagaactgg gtggaaagaa atagctactc
ctgttcagtg gtccacgagg 1080 gtctgcacaa tcaccacacg actaagagct
tctcccggac tccgggtaaa taa 1133 62 605 DNA Artificial Sequence
hzB7R1VASPpZMP21 62 gaattcgcaa gatgcgctgg tgtctcctcc tgatctgggc
ccaggggctg aggcaggctc 60 ccctcgcctc aggaatgatg acaggcacaa
tagaaacaac ggggaacatt tctgcagaga 120 aaggtggctc tatcatctta
caatgtcacc tctcctccac cacggcacaa gtgacccagg 180 tcaactggga
gcagcaggac cagcttctgg ccatttgtaa tgctgacttg gggtggcaca 240
tctccccatc cttcaaggat cgagtggccc caggtcccgg cctgggcctc accctccagt
300 cgctgaccgt gaacgataca ggggagtact tctgcatcta tcacacctac
cctgatggga 360 cgtacactgg gagaatcttc ctggaggtcc tagaaagctc
agtggctgag cacggtgcca 420 ggttccagat tgagcccaga tctggttccg
gaggctccgg tggctccgac ctacagaggg 480 tgaaacagga gcttctggaa
gaggtgaaga aggaattgca gaaagtgaaa gaggaaatca 540 ttgaagcctt
cgtccaggag ctgaggggtt ccggtggcca tcaccatcac catcactgat 600 ctaga
605 63 1116 DNA Artificial Sequence murine zB7R1mFc2 63 atgcatggct
ggctgctcct ggtctgggtc caggggctga tacaggctgc cttcctcgct 60
acaggagcca cagcaggcac gatagataca aagaggaaca tctctgcaga ggaaggtggc
120 tctgtcatct tacagtgtca cttctcctct gacacagctg aagtgaccca
agtcgactgg 180 aagcagcagg accagcttct ggccatttat agtgttgacc
tggggtggca tgtcgcttca 240 gtcttcagtg atcgggtggt cccaggcccc
agcctaggcc tcaccttcca gtctctgaca 300 atgaatgaca cgggagagta
cttctgtacc tatcatacgt atcctggtgg gatttacaag 360 gggagaatat
tcctgaaggt ccaagaaagc tcagtggctc agttccagac tgccgagccc 420
agatctccca caatcaagcc ctgtcctcca tgcaaatgcc cagcacctaa cctcgagggt
480 ggaccatccg tcttcatctt ccctccaaag atcaaggatg tactcatgat
ctccctgagc 540 cccatagtca catgtgtggt ggtggatgtg agcgaggatg
acccagatgt ccagatcagc 600 tggtttgtga acaacgtgga agtacacaca
gctcagacac aaacccatag agaggattac 660 aacagtactc tccgggtggt
cagtgccctc cccatccagc accaggactg gatgagtggc 720 aaagctttcg
catgcgcggt caacaacaaa gacctcccag cgcccatcga gagaaccatc 780
tcaaaaccca aagggtcagt aagagctcca caggtatatg tcttgcctcc accagaagaa
840 gagatgacta agaaacaggt cactctgacc tgcatggtca cagacttcat
gcctgaagac 900 atttacgtgg agtggaccaa caacgggaaa acagagctaa
actacaagaa cactgaacca 960 gtcctggact ctgatggttc ttacttcatg
tacagcaagc tgagagtgga aaagaagaac 1020 tgggtggaaa gaaatagcta
ctcctgttca gtggtccacg agggtctgca caatcaccac 1080 acgactaaga
gcttctcccg gactccgggt aaataa 1116 64 582 DNA Artificial Sequence
murine zB7R1VASPpZMP21 64 atgcatggct ggctgctcct ggtctgggtc
caggggctga tacaggctgc cttcctcgct 60 acaggagcca cagcaggcac
gatagataca aagaggaaca tctctgcaga ggaaggtggc 120 tctgtcatct
tacagtgtca cttctcctct gacacagctg aagtgaccca agtcgactgg 180
aagcagcagg accagcttct ggccatttat agtgttgacc tggggtggca tgtcgcttca
240 gtcttcagtg atcgggtggt cccaggcccc agcctaggcc tcaccttcca
gtctctgaca 300 atgaatgaca cgggagagta cttctgtacc tatcatacgt
atcctggtgg gatttacaag 360 gggagaatat tcctgaaggt ccaagaaagc
tcagtggctc agttccagac tgccgagccc 420 agatctggtt ccggaggctc
cggtggctcc gacctacaga gggtgaaaca ggagcttctg 480 gaagaggtga
agaaggaatt gcagaaagtg aaagaggaaa tcattgaagc cttcgtccag 540
gagctgaggg gttccggtgg ccatcaccat caccatcact ga 582 65 27 DNA
Artificial Sequence primer 65 ggagtgactg tactggctag aaagaag 27 66
23 DNA Artificial Sequence primer 66 gagactcctc aggttccatt cct 23
67 24 DNA Artificial Sequence primer 67 ccttgggaga acagaagcgg agcc
24 68 470 PRT homo sapiens 68 Met Arg Ile Phe Ala Val Phe Ile Phe
Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val
Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr
Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala
Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile
Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70
75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly
Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala
Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr
Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile
Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His
Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu
Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190
Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195
200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu
Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu
Glu Pro Arg 225 230 235 240 Ser Pro Thr Ile Lys Pro Cys Pro Pro Cys
Lys Cys Pro Ala Pro Asn 245 250 255 Leu Glu Gly Gly Pro Ser Val Phe
Ile Phe Pro Pro Lys Ile Lys Asp 260 265 270 Val Leu Met Ile Ser Leu
Ser Pro Ile Val Thr Cys Val Val Val Asp 275 280 285 Val Ser Glu Asp
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn 290 295 300 Val Glu
Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn 305 310 315
320 Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp
325 330 335 Met Ser Gly Lys Ala Phe Ala Cys Ala Val Asn Asn Lys Asp
Leu Pro 340 345 350 Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
Ser Val Arg Ala 355 360 365 Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
Glu Glu Met Thr Lys Lys 370 375 380 Gln Val Thr Leu Thr Cys Met Val
Thr Asp Phe Met Pro Glu Asp Ile 385 390 395
400 Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn
405 410 415 Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
Ser Lys 420 425 430 Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
Ser Tyr Ser Cys 435 440 445 Ser Val Val His Glu Gly Leu His Asn His
His Thr Thr Lys Ser Phe 450 455 460 Ser Arg Thr Pro Gly Lys 465 470
69 371 PRT murine 69 Met His Gly Trp Leu Leu Leu Val Trp Val Gln
Gly Leu Ile Gln Ala 1 5 10 15 Ala Phe Leu Ala Thr Gly Ala Thr Ala
Gly Thr Ile Asp Thr Lys Arg 20 25 30 Asn Ile Ser Ala Glu Glu Gly
Gly Ser Val Ile Leu Gln Cys His Phe 35 40 45 Ser Ser Asp Thr Ala
Glu Val Thr Gln Val Asp Trp Lys Gln Gln Asp 50 55 60 Gln Leu Leu
Ala Ile Tyr Ser Val Asp Leu Gly Trp His Val Ala Ser 65 70 75 80 Val
Phe Ser Asp Arg Val Val Pro Gly Pro Ser Leu Gly Leu Thr Phe 85 90
95 Gln Ser Leu Thr Met Asn Asp Thr Gly Glu Tyr Phe Cys Thr Tyr His
100 105 110 Thr Tyr Pro Gly Gly Ile Tyr Lys Gly Arg Ile Phe Leu Lys
Val Gln 115 120 125 Glu Ser Ser Val Ala Gln Phe Gln Thr Ala Glu Pro
Arg Ser Pro Thr 130 135 140 Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro
Ala Pro Asn Leu Glu Gly 145 150 155 160 Gly Pro Ser Val Phe Ile Phe
Pro Pro Lys Ile Lys Asp Val Leu Met 165 170 175 Ile Ser Leu Ser Pro
Ile Val Thr Cys Val Val Val Asp Val Ser Glu 180 185 190 Asp Asp Pro
Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val 195 200 205 His
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu 210 215
220 Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly
225 230 235 240 Lys Ala Phe Ala Cys Ala Val Asn Asn Lys Asp Leu Pro
Ala Pro Ile 245 250 255 Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
Arg Ala Pro Gln Val 260 265 270 Tyr Val Leu Pro Pro Pro Glu Glu Glu
Met Thr Lys Lys Gln Val Thr 275 280 285 Leu Thr Cys Met Val Thr Asp
Phe Met Pro Glu Asp Ile Tyr Val Glu 290 295 300 Trp Thr Asn Asn Gly
Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro 305 310 315 320 Val Leu
Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val 325 330 335
Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val 340
345 350 His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg
Thr 355 360 365 Pro Gly Lys 370
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