U.S. patent application number 14/887151 was filed with the patent office on 2016-02-04 for multivariable antigens complexed with targeting humanized monoclonal antibody.
The applicant listed for this patent is BAYLOR RESEARCH INSTITUTE. Invention is credited to Anne-Laure Flamar, Eynav Klechevsky, Gerard Zurawski.
Application Number | 20160031988 14/887151 |
Document ID | / |
Family ID | 39682338 |
Filed Date | 2016-02-04 |
United States Patent
Application |
20160031988 |
Kind Code |
A1 |
Zurawski; Gerard ; et
al. |
February 4, 2016 |
MULTIVARIABLE ANTIGENS COMPLEXED WITH TARGETING HUMANIZED
MONOCLONAL ANTIBODY
Abstract
The present invention includes compositions and methods for
designing, making and using modular recombinant antibodies or
fragments thereof with one half of a cohesin-dockerin pair that
permits the rapid assembly of multivariant antigen conjugates.
Inventors: |
Zurawski; Gerard;
(Midlothian, TX) ; Flamar; Anne-Laure; (New York,
NY) ; Klechevsky; Eynav; (Dallas, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYLOR RESEARCH INSTITUTE |
Dallas |
TX |
US |
|
|
Family ID: |
39682338 |
Appl. No.: |
14/887151 |
Filed: |
October 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12819618 |
Jun 21, 2010 |
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14887151 |
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12024036 |
Jan 31, 2008 |
7786267 |
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12819618 |
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60888029 |
Feb 2, 2007 |
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Current U.S.
Class: |
424/178.1 ;
424/193.1; 424/196.11; 424/197.11; 435/196; 435/320.1; 435/328;
435/7.21; 514/1.1; 514/19.3; 530/389.1; 530/391.7; 530/412;
536/23.4 |
Current CPC
Class: |
C07K 14/33 20130101;
C07K 16/00 20130101; C07K 2317/24 20130101; A61P 31/00 20180101;
C07K 2319/33 20130101; C07K 16/28 20130101; A61P 37/02 20180101;
C07K 2317/35 20130101; C07K 2317/54 20130101; C07K 2319/00
20130101; C07K 2317/55 20130101; A61K 39/44 20130101; A61P 35/00
20180101; A61P 37/06 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/44 20060101 A61K039/44 |
Goverment Interests
STATEMENT OF FEDERALLY FUNDED RESEARCH
[0002] This invention was made with U.S. Government support under
Contract No. 1U19AI057234-0100003 awarded by the NIH. The
government has certain rights in this invention.
Claims
1. A modular rAb carrier comprising an antigen-specific binding
domain linked to one or more antigen carrier domains comprising one
half of a cohesin-dockerin binding pair.
2. The rAb of claim 1, wherein the antigen-specific binding domain
comprises at least a portion of an antibody.
3. The rAb of claim 1, wherein the antigen-specific binding domain
comprises at least a portion of an antibody in a fusion protein
with the one half of the cohesin-dockerin binding pair.
4. The rAb of claim 1, further comprising a complementary half of
the cohesin-dockerin binding pair bound to an antigen that forms a
complex with the modular rAb carrier.
5. The rAb of claim 1, further comprising a complementary half of
the cohesin-dockerin binding pair that is a fusion protein with an
antigen.
6. The rAb of claim 1, wherein the antigen specific domain
comprises a full length antibody, an antibody variable region
domain, an Fab fragment, a Fab' fragment, an F(ab).sub.2 fragment,
and Fv fragment, and Fabc fragment and/or a Fab fragment with
portions of the Fc domain.
7. The rAb of claim 1, wherein the cohesin-dockerin are selected
from Clostridium thermocellum, Clostridium josui, Clostridium
cellulolyticum and Bacteroides cellulosolvens and combinations
thereof.
8. The rAb of claim 1, wherein the antigen-specific binding domain
binds a cell surface marker selected from MHC class I, MHC class
II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20,
CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83,
CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2,
MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,
IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma. receptor
or other receptor relatively specifically expressed by antigen
presenting cells.
9. The rAb of claim 1, wherein the rAb is further defined as: an
rAb.Doc; an rAb.Coh; an rAb.(Coh).sub.x; an rAb.(Doc).sub.x; an
rAb.(Coh.Doc).sub.x; or an rAb.(Coh).sub.x(Doc).sub.x; wherein x is
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
10. The rAb of claim 1, wherein the rAb is further defined as being
part of a complex: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen;
an rAb.(Coh).sub.x:(Doc.antigen).sub.x; an
rAb.(Doc).sub.x:(Coh.antigen).sub.x; an
rAb.(Coh.Doc).sub.x:(Doc.antigen.sup.1)(Coh.antigen.sup.2); or an
rAb.(Coh).sub.x(Doc).sub.x:(Doc.antigen.sup.1).sub.x(Coh.antigen.sup.2).s-
ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
11. A vaccine comprising a modular rAb carrier comprising an
antigen specific domain linked to one or more domains comprising
one half of the cohesin-dockerin binding pair bound to a
complementary half of the cohesin-dockerin binding pair bound to an
antigen.
12. The vaccine of claim 11, wherein the antigen specific domain is
specific for an immune cell surface protein selected from MHC class
I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16,
CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57,
CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40,
BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1,
B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fcy receptor
or other receptor relatively specifically expressed by antigen
presenting cells.
13. The vaccine of claim 11, wherein the antigen comprises a
bacterial, viral, fungal, protozoan or cancer protein.
14. The vaccine of claim 11, wherein the modular rAb carrier is
further defined: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an
rAb.(Coh).sub.x:(Doc.antigen).sub.x; an
rAb.(Doc).sub.x:(Coh.antigen).sub.x; an
rAb.(Coh.Doc).sub.x:(Doc.antigen.sup.1)(Coh.antigen.sup.2); or an
rAb.(Coh).sub.x(Doc).sub.x:(Doc.antigen.sup.1).sub.x(Coh.antigen.sup.2).s-
ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
15. An isolated nucleic acid comprising a coding segment for an
target specific domain and one or more domains and one half of a
cohesin-dockerin binding pair.
16. The nucleic acid of claim 15, wherein the target is an antigen
and the specific domain encodes at least a portion of an
antibody.
17. The nucleic acid of claim 15, wherein the one or more domains
encodes one or more cohesin domains, one or more dockerin domains
or a combination of one or more cohesin and dockerin domains.
18. The nucleic acid of claim 15, wherein the target specific
domain comprises an rAb is further defined as: an rAb.Doc; an
rAb.Coh; an rAb.(Coh).sub.x; an rAb.(Doc).sub.x; an
rAb.(Coh.Doc).sub.x; or an rAb.(Coh).sub.x(Doc).sub.x; wherein x is
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
19. A vector comprising a nucleic acid encoding an antigen specific
domain and one or more domains that comprise one half of a
cohesin-dockerin binding pair, a one half of a cohesin-dockerin
binding pair with a protein molecule to be carried and combinations
thereof.
20. The vector of claim 19, wherein the one half of a
cohesin-dockerin binding pair, a one half of a cohesin-dockerin
binding pair with a protein molecule to be carried and combinations
thereof are under the control of the same promoter, different
promoters, transcribed in-line, transcribed in opposite
directions.
21. A host cell comprising a vector comprising a nucleic acid
encoding an antigen specific domain and one or more domains and one
half of a cohesin-dockerin binding pair.
22. A method of making a modular rAb carrier comprising: combining
an antigen specific domain linked to one or more domains comprising
one half of a cohesin-dockerin binding pair.
23. The method of claim 22, wherein the rAb is further defined as:
an rAb.Doc; an rAb.Coh; an rAb.(Coh).sub.x; an rAb.(Doc).sub.x; an
rAb.(Coh.Doc).sub.x; or an rAb.(Coh).sub.x(Doc).sub.x; wherein x is
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
24. The method of claim 22, wherein the rAb is complexed with a
complementary half of a cohesion:dokerin pair bound to an antigen
and is selected from: an rAb.Doc:Coh.antigen; an
rAb.Coh:Doc.antigen; an rAb.(Coh).sub.x:(Doc.antigen).sub.x; an
rAb.(Doc).sub.x:(Coh.antigen).sub.x; an
rAb.(Coh.Doc).sub.x:(Doc.antigen.sup.1)(Coh.antigen.sup.2); or an
rAb.(Coh).sub.x(Doc).sub.x:(Doc.antigen.sup.1).sub.x(Coh.antigen.sup.2).s-
ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
25. An immunotoxin comprising an rAb.Doc:Coh.toxin self-assembled
conjugate, wherein the rAb is specific for a cell target.
26. The immunotoxin of claim 25, wherein the toxin is selected from
wherein the toxin is selected from the group consisting of a
radioactive isotope, metal, enzyme, botulin, tetanus, ricin,
cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins,
shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin,
saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin,
aconotoxin, snake venom and spider venom.
27. The immunotoxin of claim 25, wherein the cell target comprises
a cancer cell selected from hematological cancers, leukemias,
lymphomas, neurological tumors, astrocytomas or glioblastomas,
melanoma, breast cancer, lung cancer, head and neck cancer,
gastrointestinal tumors such as gastric or colon cancer, liver
cancer, pancreatic cancer, genitourinary tumors such cervix,
uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate
cancer or penile cancer, bone tumors, vascular tumors, or cancers
of the lip, nasopharynx, pharynx and oral cavity, esophagus,
rectum, gall bladder, biliary tree, larynx, lung and bronchus,
bladder, kidney, brain and other parts of the nervous system,
thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple
myeloma and leukemia.
28. The immunotoxin of claim 25, wherein the cell target comprises
a pathogen selected from a bacteria, a protozoan, a helminth, a
virally-infected cell or a fungus.
29. A method for protein purification, comprising: separating a
cohesin or dockerin fusion protein by interacting the fusion
protein with a rAb that is conjugated to the complementary cohesin
or dockerin bound to a substrate.
30. The method of claim 29, further comprising the step of
administering the protein in a therapeutic application comprising
transplantation, autoimmune disease, infectious disease or
cancer.
31. The use of the cohesin as a fusion partner for toxins for
conferring beneficial biochemical properties favoring ready
purification of active cohesin.toxin fusion protein.
32. The use of anti-DC rAb.Doc to target DC for therapeutic
applications where ablating DC.
33. An anti-DC-SIGN/L antibody provided in an amount that is
sufficient to enhance the survival of dendritic cells, wherein the
antibody matures and activates the dendritic cells for
immunization.
34. The antibody of claim 33, wherein the antibody is targeted in
vivo to dendritic cells as an adjuvant in vaccines.
35. A bivalent and multivalent (rAb.sup.1.Doc:Coh.rAb.sup.2)
self-assembled conjugates as therapeutic, diagnostic, and
industrial agents.
36. A bivalent and multivalent (rAb.Doc:Coh.cytokine),
(rAb.Coh:Doc.cytokine) or (cytokine.sup.1.Coh:cytokine.sup.2.Doc)
self-assembled conjugates as therapeutic, cell proliferation or
maturing agents.
37. A method for making modular rAb comprising: screening one or
more multivalent rAb and/or rAb.cytokine and/or cytokine.cytokine
combinations that are capable of specifically binding to a target
cell and delivering the cytokine such that it exerts its effect on
the target cell.
38. The method of claim 37, wherein the cytokine comprises
interleukins, transforming growth factors (TGFs), fibroblast growth
factors (FGFs), platelet derived growth factors (PDGFs), epidermal
growth factors (EGFs), connective tissue activated peptides
(CTAPs), osteogenic factors, and biologically active analogs,
fragments, and derivatives of such growth factors, B/T-cell
differentiation factors, B/T-cell growth factors, mitogenic
cytokines, chemotactic cytokines, colony stimulating factors,
angiogenesis factors, IFN-.alpha., IFN-.beta., IFN-.gamma., IL1,
IL2, IL3, IL4, ILS, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13,
IL14, IL15, IL16, IL17, IL18, etc., leptin, myostatin, macrophage
stimulating protein, platelet-derived growth factor, TNF-.alpha.,
TNF-.beta., NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-.beta.,
G-CSF, M-CSF, GM-CSF, PDGF, IL-1.alpha., IL1-.beta., IP-10, PF4,
GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF,
transforming growth factor (TGF) supergene family include the beta
transforming growth factors (for example TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3); bone morphogenetic proteins (for example, BMP-1,
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);
heparin-binding growth factors (fibroblast growth factor (FGF),
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), insulin-like growth factor (IGF)); Inhibins (for example,
Inhibin A, Inhibin B); growth differentiating factors (for example,
GDF-1); and Activins (for example, Activin A, Activin B, Activin
AB).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 60/888,029, filed Feb. 2, 2007, the contents
of which is incorporated by reference herein in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates in general to the field of
novel vaccines, and more particularly, to the design, manufacture
and use of multivariable antigens complexed with targeting
humanized monoclonal antibodies.
BACKGROUND OF THE INVENTION
[0004] Without limiting the scope of the invention, its background
is described in connection with vaccine development.
[0005] Protein engineering technology relating to monoclonal
antibodies is highy advanced regarding humanization (i.e.,
rendering e.g., a rodent mAb sequence into a human mAb sequence
while preserving specific antigen combining sites of the the
original mAb) and production (typically secreted from mammalian
cell lines). In research and development are new applications of
rAbs related to vaccination and are presently based on engineered
rAb-antigen fusion proteins (typically with the antigen coding
region placed in-frame with the C-terminal codon of the rAb heavy
or H chain). A roadblock to this technology is the successful
expression and production of fully functional rAb-antigen. In many,
perhaps most, cases the desired antigen confounds secretion of the
engineered rAb-antigen. Also, the likelihood of poor or null
expression is increased if the desired entity includes multiple
antigen coding regions.
SUMMARY OF THE INVENTION
[0006] The invention provides methods for the assembly of rAb
antigen complexes in a controlled manner by simple mixing
components and accomodates the ability to express and produce the
rAb and antigen(s) in different expression--production systems that
are best suited to the individual rAb and particular antigen. In
addition, the invention demonstrates the novel application of the
high affinity and high specificity cohesin-dockerin interaction to
secreted mammalian expression systems, thus permitting the
development of unique protein engineering formats and production of
new protein tools for research and clinical application.
[0007] More particularly, the present invention uses the
cohesin-dockerin protein domains and their surrounding linker. For
example, the invention permits the controlled assembly of
recombinant monoclonal antibodies (rAbs) complexed to antigens,
toxins, or cellular activating agents. The invention has wide
potential application in vaccination and cancer therapy. Also
claimed are derivatives of this technology that permit the
production of novel proteins with specific affinities for other
proteins.
[0008] The invention is based on particular components of the well
studied bacterial cellulose degrading protein complex called the
cellulosome. Specifically, two protein domains (cohesin and
dockerin) and natural protein linker sequences are utilized via the
invention in novel contexts and applications.
[0009] The present invention is based on the discovery that
particular cohesin and dockerin domains can be sucessfully and
efficiently secreted from mammalian cells as fusion proteins while
maintaining the specific and high affinity cohesin-dockerin
protein-protein interaction. While the extensive cohesin-dockerin
literature teaches the expectation that such fusion proteins should
have this functionality, it does not describe production of such
fusion proteins in mammalian secretion systems. The state of
scientific knowledge does not allow the prediction of the discovery
since the rules (other than features such as signal peptide) for
successful secretion are not fully established. Furthermore, the
cohesin linker regions are known to be glycosylated in their native
bacteria, and the cohesin and dockerin domains contain predicted
glycosylation sites. While this may actually favor secretion from
mammalian cells, it is unclear if `unnatural` glyosylation will
perturb the cohesis-dockerin interaction.
[0010] While cohesin-dockerin interaction for various commercial
applications has been published, the present invention is based on
a previously unrealized potential for this interaction built around
assembling specific protein complexes unrelated to the controlled
assembly enzyme applications.
[0011] The invention includes the use of all cohesin-dockerin
sequences from diverse cellulose degrading microbes, but describes
the application of specific cohesin and dockerin and linker
sequences from the microbe Clostridium thermocellum. For example,
the sequence described herein encodes the H chain of a human IgG4
linked at the C-terminal codon to a Clostridium thermocellum
dockerin sequence (called rAb.doc). Other embodiments of rAb.doc
proteins are described similarly with examples that are engineered
by simply transferring the dockerin coding region as a DNA fragment
to vectors encoding the different H chain entities.
[0012] More particularly, the present invention includes a modular
rAb carrier that includes an antigen-specific binding domain linked
to one or more antigen carrier domains and one half of a
cohesin-dockerin binding pair. The antigen-specific binding domain
may be at least a portion of an antibody and the antibody is a
fusion protein with and the binding pair in a fusion protein with
one half of a cohesin-dockerin binding pair. The rAb may also
include a complementary half of the cohesin-dockerin binding pair
bound to an antigen that forms a complex with the modular rAb
carrier. The complementary half of the cohesin-dockerin binding
pair may itself be a fusion protein with the antigen carried as
part of the complex (modular rAb carrier (cohesin/dockerin) antigen
complex). Examples of antigen specific domain include a full length
antibody, an antibody variable region domain, an Fab fragment, a
Fab' fragment, an F(ab).sub.2 fragment, and Fv fragment, and Fabc
fragment and/or a Fab fragment with portions of the Fc domain. Non
limiting examples of sources for the cohesin-dockerin binding pair
include Clostridium thermocellum, Clostridium josui, Clostridium
cellulolyticum and Bacteroides cellulosolvens and combinations
thereof.
[0013] Non-limiting examples for targeting by the antigen-specific
binding domain include: cell surface marker selected from MHC class
I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16,
CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57,
CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40,
BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1,
B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma.
receptor or other receptor relatively specifically expressed by
antigen presenting cells.
[0014] The rAb of the present invention may also includes
combinations of the domains that are defined as: an rAb.Doc; an
rAb.Coh; an rAb.(Coh).sub.x; an rAb.(Doc).sub.x; an
rAb.(Coh.Doc).sub.x; or an rAb.(Coh).sub.x(Doc).sub.x; wherein x is
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Examples of the modular rAb
carrier in a complex include: [0015] an rAb.Doc:Coh.antigen; [0016]
an rAb.Coh:Doc.antigen; [0017] an
rAb.(Coh).sub.x:(Doc.antigen).sub.x; [0018] an
rAb.(Doc).sub.x:(Coh.antigen).sub.x; [0019] an
rAb.(Coh.Doc).sub.x:(Doc.antigen.sup.1)(Coh.antigen.sup.2); or
[0020] an
rAb.(Coh).sub.x(Doc).sub.x:(Doc.antigen.sup.1).sub.x(Coh.antigen.sup.2).s-
ub.x; [0021] wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0022] The present invention also include a vaccine of a modular
rAb carrier that includes an antigen specific domain linked to one
or more domains comprising one half of the cohesin-dockerin binding
pair bound to a complementary half of the cohesin-dockerin binding
pair bound to an antigen. Non-limiting examples for targeting the
rAb include immune cell surface protein selected from MHC class I,
MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD
19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57,
CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40,
BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1,
B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fc.gamma.
receptor or other receptor relatively specifically expressed by
antigen presenting cells. Targets for vaccination with the rAb
antigen carrier include, e.g., a bacterial, viral, fungal,
protozoan or cancer protein and fragments thereof. The vaccine of
claim 11, wherein the modular rAb carrier is further defined: an
rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an
rAb.(Coh).sub.x:(Doc.antigen).sub.x; an
rAb.(Doc).sub.x:(Coh.antigen).sub.x; an rAb.(Coh.Doc).sub.x:
(Doc.antigen.sup.1)(Coh.antigen.sup.2); or an
rAb.(Coh).sub.x(Doc).sub.x:(Doc.antigen.sup.1).sub.x(Coh.antigen.sup.2).s-
ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0023] The present invention also includes an isolated nucleic acid
comprising a coding segment for a target-specific domain and one or
more domains and one half of a cohesin-dockerin binding pair. For
example, the target may be an antigen and the target specific
domain may encode at least a portion of an antibody. The one or
more domains can encode one or more cohesin domains, one or more
dockerin domains or a combination of one or more cohesin and
dockerin domains. The rAb is further defined as: an rAb.Doc; an
rAb.Coh; an rAb.(Coh).sub.x; an rAb.(Doc).sub.x; an
rAb.(Coh.Doc).sub.x; or an rAb.(Coh).sub.x(Doc).sub.x; wherein x is
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0024] The present invention also includes a vector that includes a
nucleic acid encoding an antigen specific domain and one or more
domains that comprise one half of a cohesin-dockerin binding pair,
a one half of a cohesin-dockerin binding pair with a protein
molecule to be carried and combinations thereof. The one half of a
cohesin-dockerin binding pair, a one half of a cohesin-dockerin
binding pair with a protein molecule to be carried and combinations
thereof are under the control of the same promoter, different
promoters, transcribed in-line, transcribed in opposite
directions.
[0025] The present invention also includes a host cell comprising a
vector comprising a nucleic acid encoding an antigen specific
domain and one or more domains and one half of a cohesin-dockerin
binding pair.
[0026] A method of making a modular rAb carrier by combining an
antigen specific domain linked to one or more domains of one half
of a cohesin-dockerin binding pair. The rAb is further defined as:
an rAb.Doc; an rAb.Coh; an rAb.(Coh).sub.x; an rAb.(Doc).sub.x; an
rAb.(Coh.Doc).sub.x; or an rAb.(Coh).sub.x(Doc).sub.x; wherein x is
1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Examples of the rAb is complexed
with a complementary half of a cohesion:dokerin pair bound to an
antigen and is selected from: an rAb.Doc:Coh.antigen; an
rAb.Coh:Doc.antigen; an rAb.(Coh).sub.x:(Doc.antigen).sub.x; an
rAb.(Doc).sub.x:(Coh.antigen).sub.x; an
rAb.(Coh.Doc).sub.x:(Doc.antigen.sup.1)(Coh.antigen.sup.2); or an
rAb.(Coh).sub.x(Doc).sub.x:(Doc.antigen.sup.1).sub.x(Coh.antigen.sup.2).s-
ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0027] The present invention may also be an immunotoxin that
includes an rAb.Doc:Coh.toxin self-assembled conjugate, wherein the
rAb is specific for a cell target. Examples of toxins include a
radioactive isotope, metal, enzyme, botulin, tetanus, ricin,
cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins,
shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin,
saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin,
aconotoxin, snake venom and spider venom. Cell targets for the
immunotoxin include diseased or infected cells. Examples of
diseased cells for targeting include cancer cell for, e.g.,
hematological cancers such as leukemias and lymphomas, neurological
tumors such as astrocytomas or glioblastomas, melanoma, breast
cancer, lung cancer, head and neck cancer, gastrointestinal tumors
such as gastric or colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia. The
immunotoxin may target pathogens directly, e.g., bacteria, a
protozoan, a helminth, a virally-infected cell or a fungus.
[0028] The present invention also includes a method for protein
purification by separating a cohesin or dockerin fusion protein by
interacting the fusion protein with a rAb that is conjugated to the
complementary cohesin or dockerin bound to a substrate. The present
invention may also use the cohesin as a fusion partner for toxins
for conferring beneficial biochemical properties favoring ready
purification of active cohesin.toxin fusion protein. The present
invention may also use the anti-DC rAb.Doc to target DC for
therapeutic applications where ablating DC. Therapeutic
applications include, e.g., transplantation, autoimmune disease,
infectious disease or cancer. The invention also includes an
anti-DC-SIGN/L antibody provided in an amount that is sufficient to
enhance the survival of dendritic cells, wherein the antibody
matures and activates the dendritic cells for immunization. The
antibody may target cells in vivo, e.g., dendritic cells as an
adjuvant in vaccines.
[0029] Also invented is a bivalent and multivalent
(rAb.sup.1.Doc:Coh.rAb.sup.2) self-assembled conjugates as
therapeutic, diagnostic, and industrial agents. Alternatively, the
invention is a bivalent and multivalent (rAb.Doc:Coh.cytokine),
(rAb.Coh:Doc.cytokine) or (cytokine.sup.1.Coh:cytokine.sup.2.Doc)
self-assembled conjugates as therapeutic, cell proliferation or
maturing agents. The modular rAbs carrier may be made by method
that includes screening one or more multivalent rAb and/or
rAb.cytokine and/or cytokine.cytokine combinations that are capable
of specifically binding to a target cell and delivering the
cytokine such that it exerts its effect on the target cell.
Cytokines for use with the present invention include: interleukins,
transforming growth factors (TGFs), fibroblast growth factors
(FGFs), platelet derived growth factors (PDGFs), epidermal growth
factors (EGFs), connective tissue activated peptides (CTAPs),
osteogenic factors, and biologically active analogs, fragments, and
derivatives of such growth factors, B/T-cell differentiation
factors, B/T-cell growth factors, mitogenic cytokines, chemotactic
cytokines, colony stimulating factors, angiogenesis factors,
IFN-.alpha., IFN-.beta., IFN-.gamma., IL1, IL2, IL3, IL4, ILS, IL6,
IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17,
IL18, etc., leptin, myostatin, macrophage stimulating protein,
platelet-derived growth factor, TNF-.alpha., TNF-.beta., NGF,
CD40L, CD137L/4-1BBL, human lymphotoxin-.beta., G-CSF, M-CSF,
GM-CSF, PDGF, IL-1a, IL1-.beta., IP-10, PF4, GRO, 9E3,
erythropoietin, endostatin, angiostatin, VEGF, transforming growth
factor (TGF) supergene family include the beta transforming growth
factors (for example TGF-.beta.1, TGF-.beta.2, TGF-.beta.3); bone
morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4,
BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors
(fibroblast growth factor (FGF), epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF)); Inhibins (for example, Inhibin A, Inhibin B); growth
differentiating factors (for example, GDF-1); and Activins (for
example, Activin A, Activin B, Activin AB).
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures and in which:
[0031] FIG. 1 compares the prior art (top portion) with an example
of the multiple antigens targeted in a complex simultaneously with
the same engineered humanized mAb (MATCHMAB)(bottom portion).
[0032] FIG. 2 shows the use of the present invention to form
Bi-specific mAbs.
[0033] FIG. 3 shows Protein G affinity purified secreted rAb
proteins analyzed by reducing SDS.PAGE and Coomassie Brilliant Blue
staining. Lanes are from left to right.
[0034] FIGS. 4A and 4B show the measurement by anti-human IgFc
ELISA of levels of secretion of various rAb.fusion proteins.
[0035] FIG. 5 shows the measurement by anti-human IgFc ELISA (HRP
activity) and LOX-1.alkaline phoshatase binding (AP activity) of
secreted anti-LOX1.sub.--15C4 rAb.(blue symbols) and
anti-LOX1.sub.--15C4.doc rAb (red symbols) proteins.
[0036] FIG. 6 shows that when co-transfected with a mIgG kappa
expression plasmid, rAB-pCMV(mIgG2bH-Dockerin) plasmid directs the
efficient secretion of rAB-mIgG2b.Dockerin fusion protein.
[0037] FIGS. 7A and 7B show that the secreted coh.alkaline
phosphatase (coh.AP) but not AP binds efficiently and specifically
to rAb.Doc immobilized on plastic.
[0038] FIGS. 8A and 8B shows various dilutions of a supernatant
containing secreted G.AP bound to immobilized mIgG2a and mIgG2b,
but not rAb.doc, while coh.AP bound rAb.doc specifically.
[0039] FIG. 9 shows the differential stability of complexes between
a fixed amount of proG.AP or coh.AP or coh2.AP (0.1 ug) and
immobilized mIgG2b or rAb.doc (0.25 ug) assembled by incubation for
1 hr in a micro-titre plate.
[0040] FIG. 10 shows the differential stability in human serum of
complexes between a fixed amount of proG.AP or coh.AP (0.1 ug) and
immobilized mIgG2b or rAb.doc (0.25 ug) were assembled by
incubation for 1 hr in a micro-titre plate.
[0041] FIG. 11 is a gel that shows the reduced vs. non-reduced
SDS.PAGE analysis of rAb.doc:Coh2.AP complexes produced by
sequential application of rAb.doc supernatant and coh.AP
supernatant to the same protein G affinity column.
[0042] FIG. 12 is a non-reduced SDS.PAGE analysis of
rAb.doc:Coh.Flu HA5-1 complexes produced by sequential application
of rAb.doc supernatant and coh.Flu HA5-1 supernatant to the same
protein G affinity column.
[0043] FIG. 13 shows that anti-DC_rAb.doc:coh.Flu M1 complex formed
by mixing the individual purified components was effective in vitro
in expanding Flu M1-specific T cells.
[0044] FIG. 14 shows that Anti-DC_rAb.doc:coh.Flu M1 but not
mIgG2b.doc:coh.Flu M1 complexes formed by mixing the individual
purified components was effective in vitro in expanding Flu
M1-specific T cells.
[0045] FIG. 15 shows CD34+ human DC were sorted into CD1a+ and
CD14+ subtypes and cultured with and without 3 nM Anti-DC_rAb.Flu
M1 PEP or Anti-DC rAb.
[0046] FIG. 16 shows E. coli harboring expression plasmids
directing the synthesis of coh.pep proteins were grown and induced
for specific protein production. Cells were harvested and broken by
sonication.
[0047] FIG. 17 shows that the DCIR.Doc rAb alone had no effect upon
the survival of DCs, but DC-SIGN/L.Doc rAb ehnaces their
survival.
[0048] FIG. 18 shows that Coh.PE38 alone slightly increase the
number of 7-AAD scored apoptotic cells (from 22.1-29.8%), but when
linked to DCIR or DC-SIGN/L.Doc rAbs, Coh.PE38 greatly enhanced the
number of 7-AAD scored apoptotic cells.
[0049] FIG. 19 shows the expression of anti-DC-SIGN/L and
Anti-DC-ASPGR rAb.Coh and rAb.Doc were efficiently secreted.
[0050] FIG. 20 shows the effect of IL-21 and Coh.IL-21 on the
proliferation of human B cells.
DETAILED DESCRIPTION OF THE INVENTION
[0051] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0052] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0053] At present, protein engineering technology enables the ready
and controlled addition of an antigen (or different antigens to one
of the chains) of a recombinant mAb (H or L, usually the C-terminus
of H is often used). If different antigens or different antigen
sets need to be linked to the mAb, then the mAb needs to be
re-engineered, expressed, and purified as a different entity.
[0054] The present invention provides for the complexing of
multiple antigens or proteins (engineered, expressed, and purified
independently from the primary mAb) in a controlled, multivariable
fashion, to one single primary recombinant mAb. Presently, there
are methods for engineering site-specific biotinylation sites that
provide for the addition of different proteins (each engineered
separately linked to streptavidin) to the one primary mAb. However,
the present invention provides for addition to the primary mAb of
multiple combinations, in fixed equimolar ratios and locations, of
separately engineered proteins.
[0055] As used herein, the term "modular rAb carrier" is used to
describe a recombinant antibody system that has been engineered to
provide the controlled modular addition of diverse antigens,
activating proteins, or other antibodies to a single recombinant
monoclonal antibody (mAb). The rAb may be a monoclonal antibody
made using standard hybridoma techniques, recombinant antibody
display, humanized monoclonal antibodies and the like. The modular
rAb carrier can be used to, e.g., target (via one primary
recombinant antibody against an internalizing receptor, e.g., a
human dendritic cell receptor) multiple antigens and/or antigens
and an activating cytokine to dendritic cells (DC). The modular rAb
carrier may also be used to join two different recombinant mAbs
end-to-end in a controlled and defined manner.
[0056] The antigen binding portion of the "modular rAb carrier" may
be one or more variable domains, one or more variable and the first
constant domain, an Fab fragment, a Fab' fragment, an F(ab).sub.2
fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment
with portions of the Fc domain to which the cognate modular binding
portions are added to the amino acid sequence and/or bound. The
antibody for use in the modular rAb carrier can be of any isotype
or class, subclass or from any source (animal and/or
recombinant).
[0057] In one non-limiting example, the modular rAb carrier is
engineered to have one or more modular cohesin-dockerin protein
domains for making specific and defined protein complexes in the
context of engineered recombinant mAbs. The mAb is a portion of a
fusion protein that includes one or more modular cohesin-dockerin
protein domains carboxy from the antigen binding domains of the
mAb. The cohesin-dockerin protein domains may even be attached
post-translationally, e.g., by using chemical cross-linkers and/or
disulfide bonding.
[0058] The modular rAb carrier will be used to carry a separate
molecule, e.g., a peptide, protein, lipid, carbohydrate, nucleic
acid (oligonucleotide, aptamer, vector with or without base or
backbone modifications) or combinations thereof by binding that
separate molecule to the complementary half of the
cohesion:dockerin pair. For example, either the dockerin or cohesin
made be made into a fusion protein or chemically bound to an
antigen, a peptide, a protein, a toxin, a cytokine, an enzyme, a
structural protein, an extracellular matrix protein, another
antibody, a cell or fragments thereof. The modular rAb carrier may
have one or more cohesin, dockerin or both cohesin and dockerin
domains that allow the formation of a complex with one or more
complementary cohesin/dockerin-molecules for delivery via the
antigen recognition domain of the modular rAb carrier.
[0059] The term "antigen" as used herein refers to a molecule that
can initiate a humoral and/or cellular immune response in a
recipient of the antigen. Antigen may be used in two different
contexts with the present invention: as a target for the antibody
or other antigen recognition domain of the rAb or as the molecule
that is carried to and/or into a cell or target by the rAb as part
of a dockerin/cohesin-molecule complement to the modular rAb
carrier. The antigen is usually an agent that causes a disease for
which a vaccination would be advantageous treatment. When the
antigen is presented on MHC, the peptide is often about 8 to about
25 amino acids. Antigens include any type of biologic molecule,
including, for example, simple intermediary metabolites, sugars,
lipids and hormones as well as macromolecules such as complex
carbohydrates, phospholipids, nucleic acids and proteins. Common
categories of antigens include, but are not limited to, viral
antigens, bacterial antigens, fungal antigens, protozoal and other
parasitic antigens, tumor antigens, antigens involved in autoimmune
disease, allergy and graft rejection, and other miscellaneous
antigens.
[0060] The modular rAb carrier is able to carry any number of
active agents, e.g., antibiotics, anti-infective agents, antiviral
agents, anti-tumoral agents, antipyretics, analgesics,
anti-inflammatory agents, therapeutic agents for osteoporosis,
enzymes, cytokines, anticoagulants, polysaccharides, collagen,
cells, and combinations of two or more of the foregoing active
agents. Examples of antibiotics for delivery using the present
invention include, without limitation, tetracycline,
aminoglycosides, penicillins, cephalosporins, sulfonamide drugs,
chloramphenicol sodium succinate, erythromycin, vancomycin,
lincomycin, clindamycin, nystatin, amphotericin B, amantidine,
idoxuridine, p-amino salicyclic acid, isoniazid, rifampin,
antinomycin D, mithramycin, daunomycin, adriamycin, bleomycin,
vinblastine, vincristine, procarbazine, imidazole carboxamide, and
the like.
[0061] Examples of anti-tumor agents for delivery using the present
invention include, without limitation, doxorubicin, Daunorubicin,
taxol, methotrexate, and the like. Examples of antipyretics and
analgesics include aspirin, Motrin.RTM., Ibuprofen.RTM., naprosyn,
acetaminophen, and the like.
[0062] Examples of anti-inflammatory agents for delivery using the
present invention include, without limitation, include NSAIDS,
aspirin, steroids, dexamethasone, hydrocortisone, prednisolone,
Diclofenac Na, and the like.
[0063] Examples of therapeutic agents for treating osteoporosis and
other factors acting on bone and skeleton include for delivery
using the present invention include, without limitation, calcium,
alendronate, bone GLa peptide, parathyroid hormone and its active
fragments, histone H4-related bone formation and proliferation
peptide and mutations, derivatives and analogs thereof.
[0064] Examples of enzymes and enzyme cofactors for delivery using
the present invention include, without limitation, pancrease,
L-asparaginase, hyaluronidase, chymotrypsin, trypsin, tPA,
streptokinase, urokinase, pancreatin, collagenase, trypsinogen,
chymotrypsinogen, plasminogen, streptokinase, adenyl cyclase,
superoxide dismutase (SOD), and the like.
[0065] Examples of cytokines for delivery using the present
invention include, without limitation, interleukins, transforming
growth factors (TGFs), fibroblast growth factors (FGFs), platelet
derived growth factors (PDGFs), epidermal growth factors (EGFs),
connective tissue activated peptides (CTAPs), osteogenic factors,
and biologically active analogs, fragments, and derivatives of such
growth factors. Cytokines may be B/T-cell differentiation factors,
B/T-cell growth factors, mitogenic cytokines, chemotactic
cytokines, colony stimulating factors, angiogenesis factors,
IFN-.alpha., IFN-.beta., IFN-.gamma., IL1, IL2, IL3, IL4, ILS, IL6,
IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17,
IL18, etc., leptin, myostatin, macrophage stimulating protein,
platelet-derived growth factor, TNF-.alpha., TNF-.beta., NGF,
CD40L, CD137L/4-1BBL, human lymphotoxin-.beta., G-CSF, M-CSF,
GM-CSF, PDGF, IL-1.alpha., IL1-.beta., IP-10, PF4, GRO, 9E3,
erythropoietin, endostatin, angiostatin, VEGF or any fragments or
combinations thereof. Other cytokines include members of the
transforming growth factor (TGF) supergene family include the beta
transforming growth factors (for example TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3); bone morphogenetic proteins (for example, BMP-1,
BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);
heparin-binding growth factors (for example, fibroblast growth
factor (FGF), epidermal growth factor (EGF), platelet-derived
growth factor (PDGF), insulin-like growth factor (IGF)); Inhibins
(for example, Inhibin A, Inhibin B); growth differentiating factors
(for example, GDF-1); and Activins (for example, Activin A, Activin
B, Activin AB).
[0066] Examples of growth factors for delivery using the present
invention include, without limitation, growth factors that can be
isolated from native or natural sources, such as from mammalian
cells, or can be prepared synthetically, such as by recombinant DNA
techniques or by various chemical processes. In addition, analogs,
fragments, or derivatives of these factors can be used, provided
that they exhibit at least some of the biological activity of the
native molecule. For example, analogs can be prepared by expression
of genes altered by site-specific mutagenesis or other genetic
engineering techniques.
[0067] Examples of anticoagulants for delivery using the present
invention include, without limitation, include warfarin, heparin,
Hirudin, and the like. Examples of factors acting on the immune
system include for delivery using the present invention include,
without limitation, factors which control inflammation and
malignant neoplasms and factors which attack infective
microorganisms, such as chemotactic peptides and bradykinins.
[0068] Examples of viral antigens and/or viral antigenic targets
include, but are not limited to, e.g., retroviral antigens such as
retroviral antigens from the human immunodeficiency virus (HIV)
antigens such as gene products of the gag, pol, and env genes, the
Nef protein, reverse transcriptase, and other HIV components;
hepatitis viral antigens such as the S, M, and L proteins of
hepatitis B virus, the pre-S antigen of hepatitis B virus, and
other hepatitis, e.g., hepatitis A, B, and C, viral components such
as hepatitis C viral RNA; influenza viral antigens such as
hemagglutinin and neuraminidase and other influenza viral
components; measles viral antigens such as the measles virus fusion
protein and other measles virus components; rubella viral antigens
such as proteins El and E2 and other rubella virus components;
rotaviral antigens such as VP7sc and other rotaviral components;
cytomegaloviral antigens such as envelope glycoprotein B and other
cytomegaloviral antigen components; respiratory syncytial viral
antigens such as the RSV fusion protein, the M2 protein and other
respiratory syncytial viral antigen components; herpes simplex
viral antigens such as immediate early proteins, glycoprotein D,
and other herpes simplex viral antigen components; varicella zoster
viral antigens such as gpl, gpII, and other varicella zoster viral
antigen components; Japanese encephalitis viral antigens such as
proteins E, M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese
encephalitis viral antigen components; rabies viral antigens such
as rabies glycoprotein, rabies nucleoprotein and other rabies viral
antigen components. See Fundamental Virology, Second Edition, eds.
Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for
additional examples of viral antigens.
[0069] Antigens and/or antigenic targets that may be delivered
using the rAb-DC/DC-antigen vaccines of the present invention
include genes encoding antigens such as viral antigens, bacterial
antigens, fungal antigens or parasitic antigens. Viruses include
picornavirus, coronavirus, togavirus, flavirvirus, rhabdovirus,
paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus,
retrovirus, papilomavirus, parvovirus, herpesvirus, poxvirus,
hepadnavirus, and spongiform virus. Other viral targets include
influenza, herpes simplex virus 1 and 2, measles, dengue, smallpox,
polio or HW. Pathogens include trypanosomes, tapeworms, roundworms,
helminthes, malaria. Tumor markers, such as fetal antigen or
prostate specific antigen, may be targeted in this manner. Other
examples include: HIV env proteins and hepatitis B surface antigen.
Administration of a vector according to the present invention for
vaccination purposes would require that the vector-associated
antigens be sufficiently non-immunogenic to enable long term
expression of the transgene, for which a strong immune response
would be desired. In some cases, vaccination of an individual may
only be required infrequently, such as yearly or biennially, and
provide long term immunologic protection against the infectious
agent. Specific examples of organisms, allergens and nucleic and
amino sequences for use in vectors and ultimately as antigens with
the present invention may be found in U.S. Pat. No. 6,541,011,
relevant portions incorporated herein by reference, in particular,
the tables that match organisms and specific sequences that may be
used with the present invention.
[0070] Bacterial antigens for use with the rAb vaccine disclosed
herein include, but are not limited to, e.g., bacterial antigens
such as pertussis toxin, filamentous hemagglutinin, pertactin,
FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen
components; diptheria bacterial antigens such as diptheria toxin or
toxoid and other diptheria bacterial antigen components; tetanus
bacterial antigens such as tetanus toxin or toxoid and other
tetanus bacterial antigen components; streptococcal bacterial
antigens such as M proteins and other streptococcal bacterial
antigen components; gram-negative bacilli bacterial antigens such
as lipopolysaccharides and other gram-negative bacterial antigen
components, Mycobacterium tuberculosis bacterial antigens such as
mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major
secreted protein, antigen 85A and other mycobacterial antigen
components; Helicobacter pylori bacterial antigen components;
pneumococcal bacterial antigens such as pneumolysin, pneumococcal
capsular polysaccharides and other pneumococcal bacterial antigen
components; haemophilus influenza bacterial antigens such as
capsular polysaccharides and other haemophilus influenza bacterial
antigen components; anthrax bacterial antigens such as anthrax
protective antigen and other anthrax bacterial antigen components;
rickettsiae bacterial antigens such as rompA and other rickettsiae
bacterial antigen component. Also included with the bacterial
antigens described herein are any other bacterial, mycobacterial,
mycoplasmal, rickettsial, or chlamydial antigens. Partial or whole
pathogens may also be: haemophilus influenza; Plasmodium
falciparum; neisseria meningitidis; streptococcus pneumoniae;
neisseria gonorrhoeae; salmonella serotype typhi; shigella; vibrio
cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis; lyme
disease; Yersinia pestis; west nile virus; yellow fever; tularemia;
hepatitis (viral; bacterial); RSV (respiratory syncytial virus);
HPIV 1 and HPIV 3; adenovirus; small pox; allergies and
cancers.
[0071] Fungal antigens for use with compositions and methods of the
invention include, but are not limited to, e.g., candida fungal
antigen components; histoplasma fungal antigens such as heat shock
protein 60 (HSP60) and other histoplasma fungal antigen components;
cryptococcal fungal antigens such as capsular polysaccharides and
other cryptococcal fungal antigen components; coccidiodes fungal
antigens such as spherule antigens and other coccidiodes fungal
antigen components; and tinea fungal antigens such as trichophytin
and other coccidiodes fungal antigen components.
[0072] Examples of protozoal and other parasitic antigens include,
but are not limited to, e.g., plasmodium falciparum antigens such
as merozoite surface antigens, sporozoite surface antigens,
circumsporozoite antigens, gametocyte/gamete surface antigens,
blood-stage antigen pf 155/RESA and other plasmodial antigen
components; toxoplasma antigens such as SAG-1, p30 and other
toxoplasmal antigen components; schistosomae antigens such as
glutathione-S-transferase, paramyosin, and other schistosomal
antigen components; leishmania major and other leishmaniae antigens
such as gp63, lipophosphoglycan and its associated protein and
other leishmanial antigen components; and trypanosoma cruzi
antigens such as the 75-77 kDa antigen, the 56 kDa antigen and
other trypanosomal antigen components.
[0073] Target antigens on immune cell surfaces that can be targeted
using the antigen recognition site of the antibody portion of the
rAb of the present invention will generally be selected based on a
number of factors, including: likelihood of internalization, level
of immune cell specificity, type of immune cell targeted, level of
immune cell maturity and/or activation and the like. Examples of
cell surface markers for dendritic cells include, but are not
limited to, MHC class I, MHC Class II, CD1, CD2, CD3, CD4, CD8,
CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44,
CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR,
DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor,
Langerin, DECTIN-1, B7-1, B7-2, IFN-.gamma. receptor and IL-2
receptor, ICAM-1, Fc.gamma. receptor or other receptor relatively
specifically expressed by antigen presenting cells. Examples of
cell surface markers for antigen presenting cells include, but are
not limited to, MHC class I, MHC Class II, CD1, CD2, CD3, CD4, CD8,
CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44,
CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR,
DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor,
Langerin, DECTIN-1, B7-1, B7-2, IFN-.gamma. receptor and IL-2
receptor, ICAM-1, Fc.gamma. receptor or other receptor relatively
specifically expressed by antigen presenting cells. Examples of
cell surface markers for T cells include, but are not limited to,
CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 and HLA-DR.
[0074] Target antigens on cell surfaces for delivery includes those
characteristic of tumor antigens typically will be derived from the
cell surface, cytoplasm, nucleus, organelles and the like of cells
of tumor tissue. Examples of tumor targets for the antibody portion
of the present invention include, without limitation, hematological
cancers such as leukemias and lymphomas, neurological tumors such
as astrocytomas or glioblastomas, melanoma, breast cancer, lung
cancer, head and neck cancer, gastrointestinal tumors such as
gastric or colon cancer, liver cancer, pancreatic cancer,
genitourinary tumors such cervix, uterus, ovarian cancer, vaginal
cancer, testicular cancer, prostate cancer or penile cancer, bone
tumors, vascular tumors, or cancers of the lip, nasopharynx,
pharynx and oral cavity, esophagus, rectum, gall bladder, biliary
tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease,
non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0075] Examples of antigens that may be delivered alone or in
combination to immune cells for antigen presentation using the
present invention include tumor proteins, e.g., mutated oncogenes;
viral proteins associated with tumors; and tumor mucins and
glycolipids. The antigens may be viral proteins associated with
tumors would be those from the classes of viruses noted above.
Certain antigens may be characteristic of tumors (one subset being
proteins not usually expressed by a tumor precursor cell), or may
be a protein which is normally expressed in a tumor precursor cell,
but having a mutation characteristic of a tumor. Other antigens
include mutant variant(s) of the normal protein having an altered
activity or subcellular distribution, e.g., mutations of genes
giving rise to tumor antigens.
[0076] Specific non-limiting examples of tumor antigens include:
CEA, prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE
1-4, 6 and 12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2
gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), Pmel
17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase
V intron V sequence), Prostate Ca psm, PRAME (melanoma antigen),
.beta.-catenin, MUM-1-B (melanoma ubiquitous mutated gene product),
GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2
(Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75,
human papilloma virus (HPV) E6 and E7, p53, lung resistance protein
(LRP), Bcl-2, and Ki-67. In addition, the immunogenic molecule can
be an autoantigen involved in the initiation and/or propagation of
an autoimmune disease, the pathology of which is largely due to the
activity of antibodies specific for a molecule expressed by the
relevant target organ, tissue, or cells, e.g., SLE or MG. In such
diseases, it can be desirable to direct an ongoing
antibody-mediated (i.e., a Th2-type) immune response to the
relevant autoantigen towards a cellular (i.e., a Th1-type) immune
response. Alternatively, it can be desirable to prevent onset of or
decrease the level of a Th2 response to the autoantigen in a
subject not having, but who is suspected of being susceptible to,
the relevant autoimmune disease by prophylactically inducing a Th1
response to the appropriate autoantigen. Autoantigens of interest
include, without limitation: (a) with respect to SLE, the Smith
protein, RNP ribonucleoprotein, and the SS-A and SS-B proteins; and
(b) with respect to MG, the acetylcholine receptor.Examples of
other miscellaneous antigens involved in one or more types of
autoimmune response include, e.g., endogenous hormones such as
luteinizing hormone, follicular stimulating hormone, testosterone,
growth hormone, prolactin, and other hormones.
[0077] Antigens involved in autoimmune diseases, allergy, and graft
rejection can be used in the compositions and methods of the
invention. For example, an antigen involved in any one or more of
the following autoimmune diseases or disorders can be used in the
present invention: diabetes, diabetes mellitus, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
myasthenia gravis, systemic lupus erythematosis, autoimmune
thyroiditis, dermatitis (including atopic dermatitis and eczematous
dermatitis), psoriasis, Sjogren's Syndrome, including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome,
alopecia areata, allergic responses due to arthropod bite
reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Crohn's disease, Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis
posterior, and interstitial lung fibrosis. Examples of antigens
involved in autoimmune disease include glutamic acid decarboxylase
65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid
protein, acetylcholine receptor components, thyroglobulin, and the
thyroid stimulating hormone (TSH) receptor. Examples of antigens
involved in allergy include pollen antigens such as Japanese cedar
pollen antigens, ragweed pollen antigens, rye grass pollen
antigens, animal derived antigens such as dust mite antigens and
feline antigens, histocompatiblity antigens, and penicillin and
other therapeutic drugs. Examples of antigens involved in graft
rejection include antigenic components of the graft to be
transplanted into the graft recipient such as heart, lung, liver,
pancreas, kidney, and neural graft components. The antigen may be
an altered peptide ligand useful in treating an autoimmune
disease.
[0078] As used herein, the term "epitope(s)" refer to a peptide or
protein antigen that includes a primary, secondary or tertiary
structure similar to an epitope located within any of a number of
pathogen polypeptides encoded by the pathogen DNA or RNA. The level
of similarity will generally be to such a degree that monoclonal or
polyclonal antibodies directed against such polypeptides will also
bind to, react with, or otherwise recognize, the peptide or protein
antigen. Various immunoassay methods may be employed in conjunction
with such antibodies, such as, for example, Western blotting,
ELISA, RIA, and the like, all of which are known to those of skill
in the art. The identification of pathogen epitopes, and/or their
functional equivalents, suitable for use in vaccines is part of the
present invention. Once isolated and identified, one may readily
obtain functional equivalents. For example, one may employ the
methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated
herein by reference, which teaches the identification and
preparation of epitopes from amino acid sequences on the basis of
hydrophilicity. The methods described in several other papers, and
software programs based thereon, can also be used to identify
epitopic core sequences (see, for example, Jameson and Wolf, 1988;
Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid
sequence of these "epitopic core sequences" may then be readily
incorporated into peptides, either through the application of
peptide synthesis or recombinant technology.
[0079] As used herein, the term "promoter" describes a control
sequence that is a region of a nucleic acid sequence at which
initiation and rate of transcription are controlled. It may contain
genetic elements at which regulatory proteins and molecules may
bind such as RNA polymerase and other transcription factors. The
phrases "operatively positioned," "operatively linked," "under
control," and "under transcriptional control" mean that a promoter
is in a correct functional location and/or orientation in relation
to a nucleic acid sequence (i.e., ORF) to control transcriptional
initiation and/or expression of that sequence. A promoter may or
may not be used in conjunction with an "enhancer," which refers to
a cis-acting regulatory sequence involved in the transcriptional
activation of a nucleic acid sequence. A listing of promoters
and/or enhancers that may be used with the present invention is
described in, e.g., U.S. Pat. No. 6,410,241, relevant descriptions
and tables incorporated herein by reference.
[0080] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations,
in vivo, ex vivo or in vitro. It is understood that all progeny may
not be identical due to deliberate or inadvertent mutations. In the
context of expressing a heterologous nucleic acid sequence, "host
cell" refers to a prokaryotic or eukaryotic cell, and it includes
any transformable organism that is capable of expressing a
heterologous gene encoded by a vector as delivered using the rAb
protein vector of the present invention. A host cell can, and has
been, used as a recipient for vectors. A host cell may be
"transfected" or "transformed," which refers to a process by which
the exogenous nucleic acid expressing an antigen, as disclosed
herein, is transferred or introduced into the host cell. A
transformed cell includes the primary subject cell and its
progeny.
[0081] The preparation of vaccine compositions that includes the
nucleic acids that encode antigens of the invention as the active
ingredient, may be prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to infection can also be prepared. The
preparation may be emulsified, encapsulated in liposomes. The
active immunogenic ingredients are often mixed with carriers which
are pharmaceutically acceptable and compatible with the active
ingredient.
[0082] The term "pharmaceutically acceptable carrier" refers to a
carrier that does not cause an allergic reaction or other untoward
effect in subjects to whom it is administered. Suitable
pharmaceutically acceptable carriers include, for example, one or
more of water, saline, phosphate buffered saline, dextrose,
glycerol, ethanol, or the like and combinations thereof. In
addition, if desired, the vaccine can contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, and/or adjuvants which enhance the effectiveness
of the vaccine. Examples of adjuvants that may be effective include
but are not limited to: aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI,
which contains three components extracted from bacteria,
monophosporyl lipid A, trehalose dimycolate and cell wall skeleton
(MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Other examples of
adjuvants include DDA (dimethyldioctadecylammonium bromide),
Freund's complete and incomplete adjuvants and QuilA. In addition,
immune modulating substances such as lymphokines (e.g.,
IFN-.gamma., IL-2 and IL-12) or synthetic IFN-.gamma. inducers such
as poly I:C can be used in combination with adjuvants described
herein.
[0083] Pharmaceutical products that may include a naked
polynucleotide with a single or multiple copies of the specific
nucleotide sequences that bind to specific DNA-binding sites of the
apolipoproteins present on plasma lipoproteins as described in the
current invention. The polynucleotide may encode a biologically
active peptide, antisense RNA, or ribozyme and will be provided in
a physiologically acceptable administrable form. Another
pharmaceutical product that may spring from the current invention
may include a highly purified plasma lipoprotein fraction, isolated
according to the methodology, described herein from either the
patients blood or other source, and a polynucleotide containing
single or multiple copies of the specific nucleotide sequences that
bind to specific DNA-binding sites of the apolipoproteins present
on plasma lipoproteins, prebound to the purified lipoprotein
fraction in a physiologically acceptable, administrable form.
[0084] Yet another pharmaceutical product may include a highly
purified plasma lipoprotein fraction which contains recombinant
apolipoprotein fragments containing single or multiple copies of
specific DNA-binding motifs, prebound to a polynucleotide
containing single or multiple copies of the specific nucleotide
sequences, in a physiologically acceptable administrable form. Yet
another pharmaceutical product may include a highly purified plasma
lipoprotein fraction which contains recombinant apolipoprotein
fragments containing single or multiple copies of specific
DNA-binding motifs, prebound to a polynucleotide containing single
or multiple copies of the specific nucleotide sequences, in a
physiologically acceptable administrable form.
[0085] The dosage to be administered depends to a great extent on
the body weight and physical condition of the subject being treated
as well as the route of administration and frequency of treatment.
A pharmaceutical composition that includes the naked polynucleotide
prebound to a highly purified lipoprotein fraction may be
administered in amounts ranging from 1 .mu.g to 1 mg polynucleotide
and 1 .mu.g to 100 mg protein.
[0086] Administration of the therapeutic virus particle to a
patient will follow general protocols for the administration of
chemotherapeutics, taking into account the toxicity, if any, of the
vector. It is anticipated that the treatment cycles would be
repeated as necessary. It also is contemplated that various
standard therapies, as well as surgical intervention, may be
applied in combination with the described gene therapy.
[0087] Where clinical application of a gene therapy is
contemplated, it will be necessary to prepare the complex as a
pharmaceutical composition appropriate for the intended
application. Generally this will entail preparing a pharmaceutical
composition that is essentially free of pyrogens, as well as any
other impurities that could be harmful to humans or animals. One
also will generally desire to employ appropriate salts and buffers
to render the complex stable and allow for complex uptake by target
cells.
[0088] Aqueous compositions of the present invention may include an
effective amount of the compound, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such
compositions can also be referred to as inocula. The use of such
media and agents for pharmaceutical active substances is well known
in the art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions. The compositions of the
present invention may include classic pharmaceutical preparations.
Dispersions also can be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0089] Disease States. Depending on the particular disease to be
treated, administration of therapeutic compositions according to
the present invention will be via any common route so long as the
target tissue is available via that route in order to maximize the
delivery of antigen to a site for maximum (or in some cases
minimum) immune response. Administration will generally be by
orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Other areas for delivery
include: oral, nasal, buccal, rectal, vaginal or topical. Topical
administration would be particularly advantageous for treatment of
skin cancers. Such compositions would normally be administered as
pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other
excipients.
[0090] Vaccine or treatment compositions of the invention may be
administered parenterally, by injection, for example, either
subcutaneously or intramuscularly. Additional formulations which
are suitable for other modes of administration include
suppositories, and in some cases, oral formulations or formulations
suitable for distribution as aerosols. In the case of the oral
formulations, the manipulation of T-cell subsets employing
adjuvants, antigen packaging, or the addition of individual
cytokines to various formulation that result in improved oral
vaccines with optimized immune responses. For suppositories,
traditional binders and carriers may include, for example,
polyalkylene glycols or triglycerides; such suppositories may be
formed from mixtures containing the active ingredient in the range
of 0.5% to 10%, preferably 1%-2%. Oral formulations include such
normally employed excipients as, for example, pharmaceutical grades
of mannitol, lactose, starch magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10%-95% of
active ingredient, preferably 25-70%.
[0091] The antigen encoding nucleic acids of the invention may be
formulated into the vaccine or treatment compositions as neutral or
salt forms. Pharmaceutically acceptable salts include the acid
addition salts (formed with free amino groups of the peptide) and
which are formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids, or with organic acids such as
acetic, oxalic, tartaric, maleic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0092] Vaccine or treatment compositions are administered in a
manner compatible with the dosage formulation, and in such amount
as will be prophylactically and/or therapeutically effective. The
quantity to be administered depends on the subject to be treated,
including, e.g., capacity of the subject's immune system to
synthesize antibodies, and the degree of protection or treatment
desired. Suitable dosage ranges are of the order of several hundred
micrograms active ingredient per vaccination with a range from
about 0.1 mg to 1000 mg, such as in the range from about 1 mg to
300 mg, and preferably in the range from about 10 mg to 50 mg.
Suitable regiments for initial administration and booster shots are
also variable but are typified by an initial administration
followed by subsequent inoculations or other administrations.
Precise amounts of active ingredient required to be administered
depend on the judgment of the practitioner and may be peculiar to
each subject. It will be apparent to those of skill in the art that
the therapeutically effective amount of nucleic acid molecule or
fusion polypeptides of this invention will depend, inter alia, upon
the administration schedule, the unit dose of antigen administered,
whether the nucleic acid molecule or fusion polypeptide is
administered in combination with other therapeutic agents, the
immune status and health of the recipient, and the therapeutic
activity of the particular nucleic acid molecule or fusion
polypeptide.
[0093] The compositions can be given in a single dose schedule or
in a multiple dose schedule. A multiple dose schedule is one in
which a primary course of vaccination may include, e.g., 1-10
separate doses, followed by other doses given at subsequent time
intervals required to maintain and or reinforce the immune
response, for example, at 1-4 months for a second dose, and if
needed, a subsequent dose(s) after several months. Periodic
boosters at intervals of 1-5 years, usually 3 years, are desirable
to maintain the desired levels of protective immunity. The course
of the immunization can be followed by in vitro proliferation
assays of peripheral blood lymphocytes (PBLs) co-cultured with
ESAT6 or ST-CF, and by measuring the levels of IFN-y released from
the primed lymphocytes. The assays may be performed using
conventional labels, such as radionuclides, enzymes, fluorescent
labels and the like. These techniques are known to one skilled in
the art and can be found in U.S. Pat. Nos. 3,791,932, 4,174,384 and
3,949,064, relevant portions incorporated by reference.
[0094] The modular rAb carrier and/or conjugated rAb
carrier-(cohesion/dockerin and/or dockerin-cohesin)-antigen complex
(rAb-DC/DC-antigen vaccine) may be provided in one or more "unit
doses" depending on whether the nucleic acid vectors are used, the
final purified proteins, or the final vaccine form is used. Unit
dose is defined as containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired responses
in association with its administration, i.e., the appropriate route
and treatment regimen. The quantity to be administered, and the
particular route and formulation, are within the skill of those in
the clinical arts. The subject to be treated may also be evaluated,
in particular, the state of the subject's immune system and the
protection desired. A unit dose need not be administered as a
single injection but may include continuous infusion over a set
period of time. Unit dose of the present invention may conveniently
may be described in terms of DNA/kg (or protein/Kg) body weight,
with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10,
50, 100, 1,000 or more mg/DNA or protein/kg body weight are
administered. Likewise the amount of rAb-DC/DC-antigen vaccine
delivered can vary from about 0.2 to about 8.0 mg/kg body weight.
Thus, in particular embodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg,
1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5
mg, 7.0 mg and 7.5 mg of the vaccine may be delivered to an
individual in vivo. The dosage of rAb-DC/DC-antigen vaccine to be
administered depends to a great extent on the weight and physical
condition of the subject being treated as well as the route of
administration and the frequency of treatment. A pharmaceutical
composition that includes a naked polynucleotide prebound to a
liposomal or viral delivery vector may be administered in amounts
ranging from 1 .mu.g to 1 mg polynucleotide to 1 .mu.g to 100 mg
protein. Thus, particular compositions may include between about 1
.mu.g, 5 .mu.g, 10 .mu.g, 20 .mu.g, 30 .mu.g, 40 .mu.g, 50 .mu.g,
60 .mu.g, 70 .mu.g, 80 .mu.g, 100 .mu.g, 150 .mu.g, 200 .mu.g, 250
.mu.g, 500 .mu.g, 600 .mu.g, 700 .mu.g, 800 .mu.g, 900 .mu.g or
1,000 .mu.g polynucleotide or protein that is bound independently
to 1 .mu.g, 5 .mu.g, 10 .mu.g, 20 .mu.g, 3.0 .mu.g, 40 .mu.g 50
.mu.g, 60 .mu.g, 70 .mu.g, 80 .mu.g, 100 .mu.g, 150 .mu.g, 200
.mu.g, 250 .mu.g, 500 .mu.g, 600 .mu.g, 700 .mu.g, 800 .mu.g, 900
.mu.g, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60
mg, 70 mg, 80 mg, 90 mg or 100 mg vector.
[0095] The present invention was tested in an in vitro cellular
system that measures immune stimulation of human Flu-specific T
cells by dendritic cells to which Flu antigen has been targeted.
The results shown herein demonstrate the specific expansion of such
antigen specific cells at doses of the antigen which are by
themselves ineffective in this system.
[0096] The present invention may also be used to make a modular rAb
carrier that is, e.g., a recombinant humanized mAb (directed to a
specific human dendritic cell receptor) complexed with protective
antigens from Ricin, Anthrax toxin, and Staphylococcus B
enterotoxin. The potential market for this entity is vaccination of
all military personel and stored vaccine held in reserve to
administer to large population centers in response to any biothreat
related to these agents. The invention has broad application to the
design of vaccines in general, both for human and animal use.
Industries of interest is pharmaceutical and biotechnology
[0097] One commercial application of the invention is a recombinant
humanized mAb (directed to the specific human dendritic cell
receptor DCIR) fused through the Ab heavy chain to antigens known
or suspected to encode protective antigens. These include as
examples for vaccination against various agents--hemagglutinins
from Influenza H5N1; HIV gag from attenuated toxins from Ricin,
Anthrax toxin, and Staphylococcus B enterotoxin; `strings` of
antigenic peptides from melanona anigens, etc. The potential market
for this entity is preventative or therapeutic vaccination of at
risk or infected people. The invention has broad application for
vaccination against many diseases and cancers, both for human and
animal use. Industries of interest are pharmaceutical and
biotechnology. In addition, this invention has implications beyond
anti-DCIR application since it describes a method to identify
particularly favorable sequences to enhance secretion of
recombinant antibodies.
[0098] The application of anti-DCIR combining regions for making
engineered recombinant monoclonal antibodies fused to antigens as
potent therapeutic or preventative vaccination agents. Use of
different V-region sequences against the same combining specificity
to find those most compatible with efficient expression of a H
chain C-terminal linked antigen or other protein sequence.
EXAMPLE 1
Multiple Antigens Targeted in a Complex Simultaneously with the
Same Engineered Humanized mAb (MATCHMAB)
[0099] One type of therapeutic (in this case, vaccination) entity
envisioned is a humanized DC-targeting mAb-antigen fusion protein,
where the antibody variable region specificity is directed against
an internalizing human dendritic cell receptor. The present
state-of-the art is to engineer the fusion of the desired antigen
to the C-terminus of the mAb H chain. This paradigm obviously
allows different antigens (A1, A2, A3) to be engineered to the same
proven targeting mAb backbone (Y in the figure below), thus
extending the utility of the one mAb to immunizing against
different pathogenic agents. This concept can be further extended
by engineering, e.g., the A1, A2, A3 coding regions end-to-end
fused to the IgGFc C-terminal coding region.
[0100] The present invention disclosed a new paradigm for linking
the antigen to the targeting mAb that extends the concept for the
first time to multiple antigens targeted in a complex
simultaneously with the same engineered humanized mAb
(MATCHMAB).
[0101] FIG. 1 compares the prior art (top portion) with an example
of the multiple antigens targeted in a complex simultaneously with
the same engineered humanized mAb (MATCHMAB)(bottom portion). Y
represents the humanized anti-DC targeting mAb; A1, A2, A3 are
independent protective antigens, or any other desired protein
domains; C1, C2, C2 are specific high affinity capture domains for,
respectively, docking domains D1, D2. D3; and DnAn are the
corresponding docking-antigen fusion proteins. Note that the
various domains are not drawn to scale. The mAb itself is
.about.150 kDa, C is .about.17 Da, D is .about.8 kDa and A varies,
but is usually >20 kDa).
[0102] The MATCHMAB is based on using cellulosome-assembly
cohesin-dockerin sequences to form modular non-covalent targeting
mAb-antigen complexes. The relatively small and specific
cohesin-dockerin protein-protein interaction domains can allow
simple customized formulation of targeting mAb-antigen complexes.
Thus, a single manufactured humanized mAb (in the above notation:
Y.C1.C2.C3.Cn) can be use as the basis of delivering multiple
antigens in various, yet strictly defined, combinations.
[0103] Example of sequence encoding C1.C2.C3.Cn is taken from the
public sequence >gi|50656899|gb|AAT79550.1| of cellulosomal
anchoring scaffoldin B precursor (Bacteroides cellulosolvens).
Below with blue showing the leader secretion sequence and yellow
and grey highlighting various cohesin domains. Red regions are
linkers spacing some of the cohesin domains.
TABLE-US-00001 (SEQ ID NO.: 1) ##STR00001## ##STR00002##
##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024##
PTVTPNVASPTPTKVVAEPTSNQPAGPGITGTIPTATTTATATPTKASVATATPTATPIVVVEPTIVRP
GYNKDADLAVFISSDKSRYEESSIITYSIEYKNIGKVNATNVKIAAQIPKFTKVYDAAKGAVKGSEIVWM
IGNLAVGESYTKEYKVKVDSLTKSEEYTDNTVTISSDQTVDIPENITTGNDDKSTIRVMLYSNRFTPGSH
SSYILGYKDKTFKPKQNVTRAEVAAMFARIMGLTVKDGAKSSYKDVSNKHWALKYIEAVTKSGIFKGYKD
STFHPNAPITRAELSTVIFNYLHLNNIAPSKVHFTDINKHWAKNYIEEIYRFKLIQGYSDGSFKPNNNIT
RAEVVTMINRMLYRGPLKVKVGSFPDVSPKYWAYGDIEEASRNHKYTRDEKDGSEILIE
[0104] The cohesin domains (C) interact with small domains (e.g.,
56 residues) called dockerins (D). These are Ca++ containing
structures with two-fold symmetry and they can bind to a cognate
cohesin with various affinities (e.g., 6E6 M, 2E7M). Affinities
between dockerin and multiple cohesins (as found on scaffoldins)
can be much higher (e.g., >E9 M). The interaction is
non-covalent and is well defined (by structure analysis) for at
least one C-D pair. Dockerins are designed to be domains linked to
different domain (enzyme in nature), and cohesions are designed to
function in linear arrays (either directly end-to-end, or joined by
flexible PT-rich linkers of various sizes (e.g., 12, 17, 25, 28,
36). It is known that a particular dockerin can have specificity
for a particular cohesin (e.g., a C-D pair from one bacterial
species may not be interchangeable with a C-D pair from a different
species). This feature makes it is possible to ensure the specific
and precise interaction of various D-antigen fusion proteins with
an engineered mAb containing cohesin domains of various
specificities.
[0105] In practice, this invention includes adapting C-D pairs
known from the literature, newly gleamed from nature, or developed
with new specificities using phage display technology. The latter
technology can also be used to enhance (`mature`) the affinity of a
C-D interaction, should this be desired. Also, engineering cysteine
residues at opposing faces of the C-D interaction (based on
modeling from the published C-D structures) could be used to make a
covalent bond between C-D to strengthen the interaction.
Furthermore, the dimeric nature of the mAb (and therefore the
linked C-domains) can be used to advantage for affinity enhancement
purposes. In this embodiment, e.g., the D-antigen fusion protein is
engineered either with a second identical dockerin domain
(D-antigen-D, or D-D-antigen), or with a homodimerization domain.
This configuration, provided the linkers between domains were not
constraining, will result in the preferred simultaneous binding of
both D domains to the same mAb, with greatly enhanced stability
compared to the single interaction.
[0106] Based on the crystal structure of the cohesin-dockerin
complex (e.g., see PNAS 2003,13809-13814, Cellulosome assembly
revealed by the crystal structure of the cohesin-dockerin complex.
Ana L. Carvalho *, Fernando M. V. Dias, Jose A. M. Prates, Tibor
Nagy, Harry J. Gilbert, Gideon J. Davies, Luis M. A. Ferreira,
Maria J. Romao and Carlos M. G. A. Fonte), it is apparent that one
embodiment is an antigen-dockerin fusion proteins (i.e., antigen
fused to the N-terminus of a dockerin). However, both from the
structure and from the nature of cohesin domain organization within
scaffoldins, it is apparent that cohesions can be fused end-to-end,
even without spacer sequences. Furthermore, it is apparent that
well-described techniques are available to engineer miniaturized
versions of the cohesin and dockerin domains (see for example,
Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10080-10085, September
1997. Structural mimicry of a native protein by a minimized binding
domain. Melissa A. Starovasnik, Andrew C. Braisted, And James A.
Wells).
[0107] It is recognized herein that the linker sequences have a
propensity for O-linked glycosylation resulting from ST richness.
Also, both the C and D domains can have potential N-linked sites.
These features can be advantageous in enhancing the solubility of
the mammalian cell-expressed engineered mAb through decoration with
carbohydrates. Of course, the consequences of glycosylation of the
C domains needs to be check by function (binding to the cognate D),
and if needed rectified by site directed mutagenesis. An attractive
feature of this invention is that D-A can be expressed in whatever
system is known to be best. For example, the tumor antigen MART1 is
a membrane protein and is best prepared in high yield via E. coli
inclusion bodies. Schema using antigens directly fused to the mAb
are restricted to antigens that are compatible with mammalian-cell
expression.
[0108] Another embodiment of the invention is the use of the D-C
interaction to make bi-specific mAbs joined tail-to-tail. FIG. 2
shows the use of the present invention to form Bi-specific mAbs.
mAb1 (black) is expressed with C-terminal C1 and mAb2 (magenta) is
expressed with C-terminal D1. Mixing equimolar mAb1 and mAb2 will
result in a bi-specific 1:1 complex. Note that, since each mAb
molecule contains two molar equivalents of C or D (the mAb is
itself a dimeric structure), the bi-specific mAb will be greatly
stabilized by two concurrent C-D interactions. Especially at lower
(mAb), this will be the most stable configuration.
EXAMPLE 2
Combination of Antibody and Cohesion/Dockerin Domains and
Antigens
[0109] Example 2 shows that particular cohesin and dockerin domains
can be sucessfully and efficiently secreted from mammalian cells as
fusion proteins while maintaining the specific and high affinity
cohesin-dockerin protein-protein interaction. While the extensive
cohesin-dockerin literature teaches the expectation that such
fusion proteins should have this functionality, it does not
describe production of such fusion proteins in mammalian secretion
systems. The state of scientific knowledge does not allow the
prediction of the discovery since the rules (other than features
such as signal peptide) for successful secretion are not fully
established. Furthermore, the cohesin linker regions are known to
be glycosylated in their native bacteria, and the cohesin and
dockerin domains contain predicted glycosylation sites. While this
may actually favor secretion from mammalian cells, it is unclear if
`unnatural` glyosylation will perturb the cohesis-dockerin
interaction.
[0110] While cohesin-dockerin interaction for various commercial
applications has been published, the present invention is based on
a previously unrealized utility for this interaction built around
assembling specific protein complexes unrelated to the envisioned
controlled assembly enzyme applications.
[0111] The invention includes the use of all cohesin-dockerin
sequences from diverse cellulose degrading microbes, but describes
the application of specific cohesin and dockerin and linker
sequences from the microbe Clostridium thermocellum. For example,
the sequence described in Table 1 encodes the H chain of a human
IgG4 linked at the C-terminal codon to a Clostridium thermocellum
dockerin sequence (called rAb.doc). Other embodiments of rAb.doc
proteins are described similarly in Table 2 and these are
engineered by simply transferring the dockerin coding region as a
DNA fragment to vectors encoding the different H chain
entities.
[0112] TABLE 1 shows the nucleic acid and amino acid sequences for
rAB-pIRES2(hIgG4H-Dockerin) or C52. DNA (entire coding region) and
amino acid sequence (the predicted secreted product) of human
IgG4H.doc fusion protein is shown below. The dockerin domain (taken
from Clostridium thermocellum celD is highlighted in yellow and the
H chain and dockerin joining sequence is underlined. The highly
predicted N-linked glycosylation site within the dockerin domain is
highlighted in red.
TABLE-US-00002 TABLE 1 rAB-pIRES2(hIgG4H-Dockerin) or C52. (SEQ ID
NO.: 2)
ATGGACCTCCTGTGCAAGAACATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCT-
GTCCCGGCTGC
AGCTGCAGGAGTCGGGCCCAGGCCTGCTGAAGCCTTCGGTGACCCTGTCCCTCACCTGCACTGTCTCGGGTGAC-
TCCGTCGCCAG
TAGTTCTTATTACTGGGGCTGGGTCCGTCAGCCCCCAGGGAAGGGACTCGAGTGGATAGGGACTATCAATTTTA-
GTGGCAATATG
TATTATAGTCCGTCCCTCAGGAGTCGAGTGACCATGTCGGCAGACATGTCCGAGAACTCCTTCTATCTGAAATT-
GGACTCTGTGA
CCGCAGCAGACACGGCCGTCTATTATTGTGCGGCAGGACACCTCGTTATGGGATTTGGGGCCCACTGGGGACAG-
GGAAAACTGGT
CTCCGTCTCTCCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGA-
GCACAGCCGCC
CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG-
CGTGCACACCT
TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC-
ACGAAGACCTA
CACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCAT-
GCCCACCCTGC
CCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTC-
CCGGACCCCTG
AGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG-
GAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC-
AGGACTGGCTG
AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGC-
CAAAGGGCAGC
CCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC-
CTGGTCAAAGG
CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC-
CCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTC-
ATGCTCCGTGA
TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCT-
CAAAATGAAGT
ACTGTACGGAGATGTGAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTA-
AAGCCGTCTCA
ACTCTCCCTTCTTCCAAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCAC-
AATACTTTCAA GATATTTGATAAGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 3)
RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKGLEWIGTINFSGNMYYSPSLRSRVTMSAD-
MSENSFYLKLD
SVTAADTAVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV-
SWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFP-
PKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP-
SSIEKTISKAK
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK-
SRWQEGNVFSC ##STR00025##
[0113] TABLE 2 shows the nucleic acid and amino acid sequences for
rAB-pIRES2(mAnti-DCIR2C9H-LV-hIgG4H-C-Dockerin) or C82. DNA (entire
coding region) and amino acid sequence (the predicted secreted
product) is shown below. The dockerin domain is highlighted in
yellow and the H chain and dockerin joining sequence is underlined.
The IgG variable region is highlighted in blue. The highly
predicted N-linked glycosylation site within the dockerin domain is
highlighted in red.
TABLE-US-00003 TABLE 2
rAB-pIRES2(mAnti-DCIR2C9H-LV-hIgG4H-C-Dockerin) or C82. (SEQ ID
NO.: 4)
ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAGCTGCAGCA-
GTCTGGGGCTG
AGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAATGACTACTATATC-
CACTGGGTGAA
GCAGCGGCCTGAACAGGGCCTGGAGCGGATTGGATGGATTGATCCTGACAATGGTAATACTATATATGACCCGA-
AGTTCCAGGGC
AAGGCCAGTATAACAGCAGACACATCCCCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACAC-
TGCCGTCTATT
ACTGTGCTAGAACCCGATCTCCTATGGTTACGACGGGGTTTGTTTACTGGGGCCAAGGGACTGTGGTCACTGTC-
TCTGCAGCCAA
AACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCT-
GCCTGGTCAAG
GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC-
TGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGC-
AACGTAGATCA
CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCAC-
CTGAGTTCGAA
GGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCAC-
GTGCGTGGTGG
TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAG-
ACRAAGCCGCG
GGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCA-
AGGAGTACAAG
TGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA-
GCCACAGGTGT
ACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC-
CCCAGCGACAT
CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG-
GCTCCTTCTTC
CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGA-
GGCTCTGCACA
ACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTGTAC-
GGAGATGTGAA
TGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTCTCC-
CTTCTTCCAAA
GCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATATTT-
GATAAGGGTAA TCGAGAAATTACCAATATAA (SEQ ID NO.: 5) ##STR00026##
##STR00027##
VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLF-
PPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL-
PSSIEKTISKA
KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD-
KSRWQEGNVFS ##STR00028##
[0114] TABLE 3 shows the nucleic acid and amino acid sequences for
rAB-(mAnti-ASGPR.sub.--49C11.sub.--7H-SLAML-V-hIgG4H-C-Dockerin) or
C153. DNA (entire coding region) and amino acid sequence (the
predicted secreted product) is shown below. The dockerin domain is
highlighted in yellow and the H chain and dockerin joining sequence
is underlined. The IgG variable region is highlighted in blue. The
highly predicted N-linked glycosylation site within the dockerin
domain is highlighted in red.
TABLE-US-00004 TABLE 3
rAB-(mAnti-ASGPR_49C11_7H-SLAML-V-hIgG4H-C-Dockerin) or C153. (SEQ
ID NO.: 6)
ATGGACCCCAAAGGCTCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGTCGTACGGAGA-
TGTGCAGCTTC
AGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATC-
ACCAGTGGTTA
TAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACTCTTCAGTGGTAGCA-
CTAACTACAAC
CCATCTCTGAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGT-
GACTACTGAGG
ACACAGCCACATATTTCTGTGCAAGATCTAACTATGGTTCCTTTGCTTCCTGGGGCCAAGGGACTCTGGTCACT-
GTCTCTGCAGC
CAAAACAAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGG-
GCTGCCTGGTC
AAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC-
GGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACC-
TGCAACGTAGA
TCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAG-
CACCTGAGTTC
GAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGT-
CACGTGCGTGG
TGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC-
AAGACAAAGCC
GCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG-
GCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG-
AGAGCCACAGG
TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC-
TACCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG-
ACGGCTCCTTC
TTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCA-
TGAGGCTCTGC
ACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTG-
TACGGAGATGT
GAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTC-
TCCCTTCTTCC
AAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATA-
TTTGATAAGGG TAATCGAGAAATTACCAATATAA (SEQ ID NO.: 7) ##STR00029##
##STR00030##
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP-
KDTLMISRTPE
VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI-
EKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW-
QEGNVFSCSVM ##STR00031##
[0115] TABLE 4 shows the nucleic acid and amino acid sequences for
rAB-pIRES2(mAnti-DC-SIGNL16E7H-LV-hIgG4H-C-Dockerin) or C92. DNA
(entire coding region) and amino acid sequence (the predicted
secreted product) is shown below. The dockerin domain is
highlighted in yellow and the H chain and dockerin joining sequence
is underlined. The IgG variable region is highlighted in blue. The
highly predicted N-linked glycosylation site within the dockerin
domain is highlighted in red.
TABLE-US-00005 TABLE 4
rAB-pIRES2(mAnti-DC-SIGNL16E7H-LV-hIgG4H-C-Dockerin) or C92 (SEQ ID
NO.: 8)
ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTTTCAGTAACTGCAGGTGTCCACTCCCAGGTCCAGCTTCAGCA-
GTCTGGGGCTG
AGCTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGATG-
CACTGGGTAAA
ACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTATCACTGGTTATACTGAGTACAATCAGA-
AGTTCAAGGAC
AAGGCCACCTTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTC-
TGCAGTCTATT
ACTGTGCAAGAGAGGGTTTAAGTGCTATGGACTATTGGGGTCAGGGAACCTCAGTCACCGTCACCTCAGCCAAA-
ACAACGGGCCC
ATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGG-
ACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC-
CTCAGGACTCT
ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCAC-
AAGCCCAGCAA
CACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAG-
GGGGACCATCA
GTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGT-
GGACGTGAGCC
AGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG-
GAGGAGCAGTT
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT-
GCAAGGTCTCC
AACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA-
CACCCTGCCCC
CATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC-
GCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC-
TCTACAGCAGG
CTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA-
CCACTACACAC
AGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTGTACGGAGATGTGAAT-
GATGACGGAAA
AGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTCTCCCTTCTTCCAAAG-
CTGAAAAGAAC
GCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATATTTGATAAGGGTAAT-
CGAGAAATTAC CAATATAA (SEQ ID NO.: 9) ##STR00032## ##STR00033##
PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP-
KDTLMISRTPE
VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI-
EKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW-
QEGNVFSCSVM ##STR00034##
[0116] Mammalian expression plasmids encoding such rAb.doc IgG H
chain proteins are created using standard molecular biology
techniques and can be based on commercially available expression
plasmid vectors such as pIRES2-DsRed2 (BD Biosciences). To produce
secreted rAb.doc, mammalian cells are co-transfected with this
expression plasmid and an expression plasmid encoding a
complimentary IgG L chain (exemplified in Table 3). Standard
protocols (such as the FreeStyle.TM. 293 Expression System,
Invitrogen) are used as for mammalian cells, transfection reagents,
and culture media. Transfected cells are cultured for 3-7 days and
the culture supernatant is harvested by centrifugation, clarified
by filtration, and the rAB.doc protein purified by Protein G
affinity chromatography using protocols from the column
manufacturer (GE Pharmacia).
[0117] FIG. 3 shows analysis of typical secreted rAb.doc products
by reducing SDS.PAGE with staining by Coomassie Brilliant Blue.
This analysis shows that the rAb.doc is efficiently produced as a
secreted H+L chain dimer. Heterogeneity in the H chain likely
reflects N-linked glycosylation at a highly predicted (Potential
0.6426, NetNGlyc 1.0 Server--Technical University of Denmark) site
within the dockerin sequence.
[0118] TABLE 5 shows the nucleic acid and amino acid sequences for
rAB-pIRES2(mAnti-DC-SIGNL16E7K-LV-hIgGK-C) or C73. DNA (entire
coding region) and amino acid sequence (the predicted secreted
product) of IgG Kappa protein fusing the V region from the
mAnti-DC-SIGNL16E7 hybridoma (highlighted in blue)to a human C
region (highlighted in yellow).
TABLE-US-00006 TABLE 5 rAB-pIRES2(mAnti-DC-SIGNL16E7K-LV-hIgGK-C)
or C73. (SEQ ID NO.: 10)
ATGCATCGCACCAGCATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTC-
TGGTGTTGGCG
GAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAG-
GCCAGTCAGGA
TGTGACTTCTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCA-
CCCGGCACACT
GGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGGGCAGGCTGA-
AGACCTGGCAC
TTTATTACTGTCACCAATATTATAGCGCTCCTCGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACT-
GTGGCTGCACC
ATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA-
ACTTCTATCCC
AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA-
GGACAGCAAGG
ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGC-
GAAGTCACCCA TCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
(SEQ ID NO.: 11) ##STR00035## ##STR00036## ##STR00037##
[0119] FIG. 3 shows Protein G affinity purified secreted rAb
proteins analyzed by reducing SDS.PAGE and Coomassie Brilliant Blue
staining. Lanes are from left to right.
[0120] The invention enbodies the unanticipated presence and use of
this glycosylation site that likely confers onto mammalian
cell-secreted dockerin fusion proteins desirable solubility and
pharmacokinetic properties well known to be associated with
glycosylation. FIG. 2 shows that rAb.antigen fusion proteins
employing identical IgG H and L sequences can differ dramatically
in efficiency of secretion. In both sited examples, rAb.doc
entities are well expressed compared to rAb fused to Influenza HAS
sequences which typically express very poorly. The invention also
embodies the unanticipated capacity of the dockerin domain to not
significantly hinder the secretion of the associated rAb entity.
Furthermore, the invention embodies the property of the dockerin
domain to not hinder the functionality of the rAb specific antigen
combining regions. This property is exemplified in FIG. 5 which
shows concordance between IgFc reactivity and LOX-1 reactivity
between anti-LOX1.sub.--15C4 rAb proteins and
anti-LOX1.sub.--15C4.doc.
[0121] FIGS. 4A and 4B show the measurement by anti-human IgFc
ELISA of levels of secretion of various rAb.fusion proteins. 2.5 ug
each of the H and L chain expression plasmids were transfected into
293F cells and two-fold dilutions of supernatant samples were
tested after three days of culture. Y axis values are arbitrary HRP
activity.
[0122] FIG. 5 shows the measurement by anti-human IgFc ELISA (HRP
activity) and LOX-1.alkaline phoshatase binding (AP activity) of
secreted anti-LOX1.sub.--15C4 rAb.(blue symbols)and
anti-LOX1.sub.--15C4.doc rAb (red symbols) proteins. Different
ratios totalling 5 ug of the H and L chain expression plasmids were
transfected into 293F cells and supernatant samples were tested
after three days of culture.
[0123] The invention embodies the property of the dockerin domain
to be efficiently and functionally expressed in the context of
fusion proteins other than hIgG4 and its close derivatives. For
example, Table 6 shows the sequence of a rAb.doc entity based on a
mouse IgG2b H chain fusion protein.
[0124] TABLE 6 shows the nucleic acid and amino acid sequences for
rAB-pCMV(mIgG2bH-Dockerin) or C19. DNA (entire coding region) and
amino acid sequence (the predicted secreted product) is shown. The
dockerin domain is highlighted in yellow and the H chain and
dockerin joining sequence is underlined. The highly predicted
N-linked glycosylation site within the dockerin domain is
highlighted in red.
TABLE-US-00007 TABLE 6 rAB-pCMV(mIgG2bH-Dockerin) or C19. (SEQ ID
NO.: 12)
ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTCCAACTGCAGCA-
GCCTGGGGCTG
AGCTGGTGAGGCCTGGGACTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGTTACATCTTTACCAGCTACTGGATG-
CACTGGGTAAA
GCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGACTGATTGATCCTTCTGATAGTTATAGTAAGTACAATCAAA-
AGTTCAAGGGC
AAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC-
TGCGGTCTATT
ACTGTGCAAGAGGGGAGCTCAGTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACA-
CCCCCATCAGT
CTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGGTCAAGGGCTACT-
TCCCTGAGTCA
GTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACT-
CTACACTATGA
GCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGC-
AGCACCACGGT
GGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAAT-
GCCCAGCTCCT
AACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCTGACACC-
CAAGGTCACGT
GTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACAC-
ACAGCTCAGAC
ACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGA-
TGAGTGGCAAG
GAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCT-
AGTCAGAGCTC
CACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTG-
GGCTTCAACCC
TGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGG-
ACTCTGACGGT
TCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGT-
GAGACACGAGG
GTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAAGCTAGCAATTCTCCTCAAAATGAA-
GTACTGTACGG
AGATGTGAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCT-
CAACTCTGCCT
TCTTCCAAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTC-
AAGATATTTGA TAAGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 13)
QVQLQQPGAELVRPGTSVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGLIDPSDSYSKYNQKFKGKATLTVDT-
SSSTAYMQLSS
LTSEDSAVYYCARGELSDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSG-
SLSSSVHTFPA
LLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSV-
FIFPPNIKDVL
MISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNN-
KDLPSPIERTI
SKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKL-
DIKTSKWEKTD ##STR00038##
[0125] FIG. 6 shows that when co-transfected with a mIgG kappa
expression plasmid, rAB-pCMV(mIgG2bH-Dockerin) plasmid directs the
efficient secretion of rAB-mIgG2b.Dockerin fusion protein. In FIG.
6, a Protein G affinity purified rAb proteins secreted from
transfected 293 F cells analyzed by reducing SDS.PAGE and Coomassie
Brilliant Blue staining. Lanes 11 and 12 show mIgG2b.doc
products.
[0126] The use of the rAb.doc invention detailed above is the
assembly of rAb-antigen or toxin or activator or enzyme complexes
via the specificity and tenacity of the dockerin-cohesin
interaction. Table 5 shows one embodiment of the invention in the
form of a cohesin.alkaline phosphatase fusion protein (coh.AP).
Also described are additional embodiments such as an alkaline
phosphatase fusion protein containing two cohesion domains
(coh.coh.AP) and other proteins are examples of the generality of
the invention such as the single cohesin domain fused to other
sequences such as the mature sequence of human prostate specific
antigen (coh.hPSA) and to the HA1 domain of influenza A HAS
(coh.Flu HA5-1).
[0127] TABLE 7 shows the nucleic acid and amino acid sequences for
Mam-pCDM8(Cohesin-SLAML-AP-6.times. His) or C16. DNA (entire coding
region) and amino acid sequence (the predicted secreted product) is
shown below. The cohesin domain is highlighted in yellow and the
cohesin and alkaline phosphatase joining sequence is underlined.
The highly predicted (G-score>0.5, NetOGlyc 3.1
Server--Technical University of Denmark) O-linked glycosylation
sites within the cohesin domain and the linker distal to the
cohesin domain are highlighted in red. Residues highlighted grey
are a C-terminal His tag to facilitate purification via metal
affinity chromatography.
TABLE-US-00008 TABLE 7 Mam-pCDM8(Cohesin-SLAML-AP-6xHis) or C16.
(SEQ ID NO.: 14)
ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT-
CGACGATCTGG
ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC-
GGTATACCATC
CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAGAACCGG-
GAGACATAATA
GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC-
AGAAGACAGCG
GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCT-
AACGGACTCAG
TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG-
GTGGAGTAAAT
GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC-
ACTCGAGATCA
TCCCAGTTGAGGAGGAGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTG-
CAGCCTGCACA
GACAGCCGCCAAGAACCTCATCATCTTCCTGGGCGATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCC-
TAAAAGGGCAG
AAGAAGGACAAACTGGGGCCTGAGTTACCCCTGGCCATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATA-
CAATGTAGACA
AACATGTGCCAGACAGTGGAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGC-
TTGAGTGCAGC
CGCCCGCTTTAACCAGTGCAACACGACACGCGGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCAG-
GGAAGTCAGTG
GGAGTGGTAACCACCACACGAGTGCAGCACGCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAACTG-
GTACTCGGACG
CCGACGTGCCTGCCTCGGCCCGCCAGGAGGGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGACATT-
GACGTGATCCT
AGGTGGAGGCCGAAAGTACATGTTTCGCATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTG-
GGACCAGGCTG
GACGGGAAGAATCTGGTGCAGGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTACGTGTGGAACCGCACTGAGCT-
CATGCAGGCTT
CCCTGGACCCGTCTGTGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGAC-
TCCACACTGGA
CCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCG-
TGGAGGGTGGT
CGCATCGACCATGGTCATCATGAAAGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCAT-
TGAGAGGGCGG
GCCAGCTCACCAGCGAGGAGGACACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGC-
TACCCCCTGCG
AGGGAGCTCCATCTTCGGGCTGGCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGAA-
ACGGTCCAGGC
TATGTGCTCAAGGACGGCGCCCGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAGTC-
AGCAGTGCCCC
TGGACGAAGAGACCCACGCAGGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCACGGC-
GTGCAGGAGCA
GACCTTCATAGCGCACGTCATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCCCCCG-
CCGGCACCACC CACCATCACCATCACCATTGA (SEQ ID NO.: 15) ##STR00039##
##STR00040##
ALEIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPELPLAM-
DRFPYVALSKT
YNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASP-
AGTYAHTVNRN
WYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKR-
QGARYVWNRTE
LMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYR-
ALTETIMFDDA
IERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTE-
SESGSPEYRQQ ##STR00041##
[0128] TABLE 8 shows the nucleic acid and amino acid sequences for
Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6.times. His) or C17. DNA
(entire coding region) and amino acid sequence (the predicted
secreted product) is shown below. The cohesin domain is highlighted
in yellow and the cohesin and alkaline phosphatase joining sequence
is underlined. The highly predicted O-linked glycosylation sites
within the linker distal to the cohesin domains are highlighted in
red as is a single highy predicted N-linked glycosylation site
(NPT). Residues highlighted grey are a C-terminal His tag to
facilitate purificaytion via metal affinity chromatography.
TABLE-US-00009 TABLE 8 Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6xHis) or
C17.
ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT-
CGACGATCTGG
ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC-
GGTATACCATC
CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAAAACCGG-
GAGAATTGATA
GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC-
AGAAGACAGCG
GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAATCCGGAGCACCT-
AACGGACTCAG
TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAATAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG-
GTGGAGTAAAT
GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC-
AGTAAGGATTA
AAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAATATACCTGTAAGATTCAGTGGTATACCATCCAAG-
GGAATAGCAAA
CTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAAAACCGGGAGAATTGATAGTTG-
ACCCGAATCCT
ACCAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATGATAGTATTCCTGTTTGCGGAAGACAGCGGAAC-
AGGAGCGTATG
CAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCTAACGGACTCAGTGTA-
ATCAAATTTGT
AGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGAGTAAATGTTG-
GAGATACAACA
GAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGCACTCGAGATCATCCC-
AGTTGAGGAGG
AGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTGCAGCCTGCACAGACA-
GCCGCCAAGAA
CCTCATCATCTTCCTGGGCGATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCCTAAAAGGGCAGAAGA-
AGGACAAACTG
GGGCCTGAGTTACCCCTGGCCATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATACAATGTAGACAAACA-
TGTGCCAGACA
GTGGAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGCTTGAGTGCAGCCGCC-
CGCTTTAACCA
GTGCAACACGACACGCGGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCAGGGAAGTCAGTGGGAG-
TGGTAACCACC
ACACGAGTGCAGCACGCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAACTGGTACTCGGACGCCGA-
CGTGCCTGCCT
CGGCCCGCCAGGAGGGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGACATTGACGTGATCCTAGGT-
GGAGGCCGAAA
GTACATGTTTCGCATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTGGGACCAGGCTGGACG-
GGAAGAATCTG
GTGCAGGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTACGTGTGGAACCGCACTGAGCTCATGCAGGCTTCCCT-
GGACCCGTCTG
TGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGACTCCACACTGGACCCC-
TCCCTGATGGA
GATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCGTGGAGGGTGGTCGCA-
TCGACCATGGT
CATCATGAAAGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCATTGAGAGGGCGGGCCA-
GCTCACCAGCG
AGGAGGACACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGCTACCCCCTGCGAGGG-
AGCTCCATCTT
CGGGCTGGCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGAAACGGTCCAGGCTATG-
TGCTCAAGGAC
GGCGCCCGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAGTCAGCAGTGCCCCTGGA-
CGAAGAGACCC
ACGCAGGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCACGGCGTGCAGGAGCAGACC-
TTCATAGCGCA
CGTCATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCCCCCGCCGGCACCACCCACC-
ATCACCATCAC CATTGA (SEQ ID NO.:16) ##STR00042## ##STR00043##
##STR00044## ##STR00045##
VEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPELPLAMDRFPYV-
ALSKTYNVDKH
VPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAH-
TVRNWYSDAD
VPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYV-
WNRTELMQASL
DPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFGFVEGGRIDHGHHESRAYRALTETI-
MFDDAIERAGQ
LTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSP-
EYRQQSAVPLD ##STR00046##
[0129] TABLE 9 shows the nucleic acid and amino acid sequences for
Mam-pCDM8(SLAML-Cohesin-hPSA) or C 149. DNA (entire coding region)
and amino acid sequence (the predicted secreted product) is shown
below. The cohesin domain is highlighted in yellow and the cohesin
and hPSA joining sequence is underlined. The highly predicted
O-linked glycosylation sites within the linker distal to the
cohesin domains and a single highly predicted N-linked
glycosylation site within the cohesin domain are highlighted in
red.
TABLE-US-00010 TABLE 9 Mam-pCDM8(SLAML-Cohesin-hPSA)or C149.
ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT-
CGACGATCTGG
ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC-
GGTATACCATC
CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAGAACCGG-
GAGACATAATA
GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC-
AGAAGACAGCG
GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCT-
AACGGACTCAG
TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG-
GTGGAGTAAAT
GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC-
ACTCGAGGCGC
CCCTCATCCTGTCTCGGATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCC-
TCTCGTGGCAG
GGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCG-
TGATCTTGCTG
GGTCGGCACAGCCTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCT-
CTACGATATGA
GCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAG-
CCTGCCGAGCT
CACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCT-
GGGGCAGCATT
GAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGCGC-
GCAAGTTCACC
CTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCT-
GGGGGCCCACT
TGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCC-
TGTACACCAAG GTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCTGA
(SEQ ID NO.: 18) ##STR00047## ##STR00048##
ALEAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGV-
FQVSHSFPHP
LYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQ-
CVDLHVISNDVC
AQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTI-
VANP (SEQ ID NO.: 19)
[0130] TABLE 10 shows the nucleic acid and amino acid sequences for
Mam-pCDM8(SLAML-Cohesin-F1uHA5-1-6.times. His)or C24. DNA (entire
coding region) and amino acid sequence (the predicted secreted
product) is shown below. The cohesin domain is highlighted in
yellow and the cohesin and Flu HA5-1 joining sequence is
underlined. The highly predicted O-linked glycosylation sites
within the linker distal to the cohesin domains and a single highly
predicted N-linked glycosylation site within the cohesin domain are
highlighted in red. Residues highlighted grey are a C-terminal His
tag to facilitate purification via metal affinity
chromatography.
TABLE-US-00011 TABLE 10 Mam-pCDM8(SLAML-Cohesin-FluHA5-1-6xHis)or
C24.
ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT-
CGACGATCTGG
ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC-
GGTATACCATC
CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAGAACCGG-
GAGACATAATA
GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC-
AGAAGACAGCG
GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCT-
AACGGACTCAG
TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG-
GTGGAGTAAAT
GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC-
ACTCGAGGATC
AGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTT-
ACACATGCCCA
AGACATACTGGAAAAGAAACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTGAGAGATT-
GTAGCGTAGCT
GGATGGCTCCTCGGAAACCCAATGTGTGACGAATTCATCAATGTGCCGGAATGGTCTTACATAGTGGAGAAGGC-
CAATCCAGTCA
ATGACCTCTGTTACCCAGGGGATTTCAATGACTATGAAAAATTGAAACACCTATTGAGCAGAATAAACCATTTT-
GAGAAAATTCA
GATCATCCCCAAAAGTTCTTGGTCCAGTCATGAAGCCTCATTAGGGGTGAGCTCAGCATGTCCATACCAGGGAA-
AGTCCTCCTTT
TTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAGGAGCTACAATAATACCAA-
CCAAGAAGATC
TTTTGGTACTGTGGGGGATTCACCATCCTAATGATGCGGCAGAGCAGACAAAGCTCTATCAAAACCCAACCACC-
TATATTTCCGT
TGGGACATCAACACTAAACCAGAGATTGGTACCAAGAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAA-
GGATGGAGTTC
TTCTGGACAATTTTAAAGCCGAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGC-
ATACAAAATTG
TCAAGAAAGGGGACTCAACAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCA-
ATGGGGGCGAT
AAACTCTAGCATGCCATTCCACAATATACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACA-
GATTAGTCCTT GCGCACCATCACCATCACCATTGA (SEQ ID NO.: 20) ##STR00049##
##STR00050##
ALEDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFI-
NVPEWSYIVEK
ANPVNDLCYPGDFNDYEKLKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNST-
YPTIKRSYNNT
NQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINF-
ESNGNGIAPEY ##STR00051## No.: 21)
[0131] Similar to the above mentioned rAb.doc constructs, the
invention embodies the efficient secretion from mammalian cells of
functional cohesin fusion proteins (called herein coh.fusions). It
was not obvious that cohesin domains could be so successfully
secreted while retaining dockerin-binding function. FIG. 5
demonstrates that supernatant containing secreted coh.alkaline
phosphatase (coh.AP) binds specifically to a rAb.doc protein
immobilized on a plastic surface.
[0132] FIGS. 7A and 7B show that the Expression plasmids encoding
secreted alkaline phosphatase (AP) or coh.AP directed secretion of
functional proteins from transfected 293F cells. After 3 days of
culture supernatants were harvested and tested for their ability to
bind 0.25 ug of either rAb.doc (top panel) or rAb (lower panel)
bound to a 96 well micro-titre plate. After 1 hr of incubation the
plates were washed and developed with a chromogenic AP
substrate.
[0133] The invention embodies the application of assembly of
specific protein complexes based on the cohesin.dockerin
interaction. Specific antibody.antigen complexes can also be
assembled using the established interaction of protein A or protein
G IgFc binding domains. The invention embodies unique properties of
the cohesin.dockerin interaction that result in greatly superior
complex formation compared to the e.g., protein G interaction with
IgG. In FIGS. 6 and 7 the interaction of a cohesin.AP (called
Coh.AP) protein is shown to be specific for a rAb.Doc protein.
[0134] FIGS. 8A and 8B shows various dilutions of a supernatant
containing secreted G.AP were incubated for 1 hr in micro-titre
wells containing 0.25 ug of immobilized mIgG2a, mIgG2b, or a
mIgG2b-based rAb.doc. After washing the bound AP activity was
developed using chromogenic AP substrate. The proG.AP did not bind
to the rAb.doc since it was an isotype variant of mIgG2b that did
not interact with the particular protein G domain used in the
proG.AP construct.
[0135] FIG. 8B shows an identical study, but employing dilutions of
a supernatant containing secreted Coh.AP. Coh.AP binds only to
rAb.doc, again demonstrating the specificity of the coh.doc
interaction.
[0136] FIG. 9 demonstrates the vastly superior stability of
preassembled complexes based on coh.doc interaction compared to
proG.IgGFc interaction. FIG. 9 shows the formation of complexes
between a fixed amount of proG.AP or coh.AP or coh2.AP (0.1 ug) and
immobilized mIgG2b or rAb.doc (0.25 ug) were assembled by
incubation for 1 hr in a micro-titre plate. At various times a
20-fold excess of soluble mIgG2b or rAb.doc were added and
incubation continued for various times. Plates were then washed and
bound AP activity accessed by addition of chromogenic AP
substrate.
[0137] This example shows the use of such coh.doc complexes in
settings containing serum (e.g., tissue culture media and in vivo
administration). FIG. 10 demonstrates the vast superiority of
coh.doc complexes compared to proG.IgGFc complexes in such a
setting. Under the conditions used, .about.15 ug/ml Ig was
sufficient to completely displace bound proG.AP, while the coh.AP
remained stably bound to rAb.doc even in the presence of pure serum
(15 mg/ml Ig)
[0138] FIG. 10 shows the formation of complexes between a fixed
amount of proG.AP or coh.AP (0.1 ug) and immobilized mIgG2b or
rAb.doc (0.25 ug) were assembled by incubation for 1 hr in a
micro-titre plate. Various dilutions of human serum were added and
incubation continued for 4 hrs. Plates were then washed and bound
AP activity accessed by addition of chromogenic AP substrate.
[0139] The invention also embodies a particular utility of the
coh.doc interaction that permits a production process that ensures
complete complex formation and that can be concomitant with a
purification process for the coh.fusion protein entity. This
invention is exemplified in FIGS. 11 and 12, which illustrate this
process via sequential capture of rAb.doc from culture supernatant
by protein G affinity chromatography, followed by capture of
coh.antigen from culture supernatant by the proteinG:rAb.doc
column. Elution with low pH then releases pure rAb.doc:coh.antigen.
If there is an excess of coh.antigen over rAb.doc, them full and
complete complex should result. A related embodiment of this
invention would be application to the protein G captured rAb.doc of
excess pure or partially purified coh.fusion protein.
[0140] FIG. 11 shows a gel of reduced vs. non-reduced SDS.PAGE
analysis of rAb.doc:Coh2.AP complexes produced by sequential
application of rAb.doc supernatant and coh.AP supernatant to the
same protein G affinity column. Lanes 2 and 4 show that Coh2.AP
co-purifies with rAb.doc.
[0141] FIG. 12 is a non-reduced SDS.PAGE analysis of
rAb.doc:Coh.Flu HA5-1 complexes produced by sequential application
of rAb.doc supernatant and coh.Flu HA5-1 supernatant to the same
protein G affinity column. Lanes 1 to 4 left to right show that
Coh.Flu HA5-1 co-purifies with rAb.doc.
[0142] A well described feature of cohesin domains is their
compatibility with the standard E. coli bacterial expression
system. The invention embodies the novel use of expression of
dockerin fusion proteins in mammalian secretion systems, and it
also encompasses the formation of coh.doc complexes where the
different components (i.e., coh and doc) are expressed in different
systems. This is a great advantage since it affords the possibility
of using the most favorable expression system for each component.
For example, coh.Flu M1 expression constructs failed to efficiently
direct the synthesis of secreted product from transfected mammalian
cells. However, coh.Flu M1 was very efficiently expressed as a
soluble protein in E. coli. Table 6 shows the sequence of the
coh.Flu M1 used in this example.
[0143] TABLE 11 shows the nucleic and amino acid sequence for E
coli-pET28(Cohesin-FluM1-6.times. His) or C32 is shown below. In
the amino acid sequence the cohesin domain is highlighted in yellow
and the point of fusion between cohesion and influenza A M1 protein
is underlined. Residues highliighted grey are a C-terminal His tag
to facilitate purificaytion via metal affinity chromatography.
TABLE-US-00012 TABLE 11 E. coli-pET28(Cohesin-FluM1-6xHis) or C32.
ATGGATCTGGATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAATATACCTGT-
AAGATTCAGTG
GTATACCATCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAG-
ATAAAACCGGG
AGAATTGATAGTTGACCCGAATCCTACCAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATGATAGTAT-
TCCTGTTTGCG
GAAGACAGCGGAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGA-
AGGAGCACCTA
ACGGGCTCAGTGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAG-
TTCTTTGACGG
TGGAGTAAATGTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATG-
ATCTGGATGCA
GCTAGCCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGC-
ACAGAGACTTG
AAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCA-
CCTCTGACTAA
GGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGGGGACTGCAGCGTAGACGCTTTGTCCAAA-
ATGCTCTTAAT
GGGAACGGAGATCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTTAAGAGGGAGATAACATTCCA-
TGGGGCCAAAG
AAATAGCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTG-
ACCACTGAAGT
GGCATTTGGCCTGGTATGCGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGA-
CAACAACCAAT
CCACTAATCAGACATGAGAACAGAATGGTTCTAGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATC-
GAGTGAGCAAG
CAGCAGAGGCCATGGATATTGCTAGTCAGGCCAGGCAAATGGTGCAGGCGATGAGAACCATTGGGACTCATCCT-
AGCTCCAGTGC
TGGTCTAAAAGATGATCTTCTTGAAAATTTGCAGGCTTACCAGAAACGGATGGGGGTGCAGATGCAGCGATTCA-
AGCTCGAGCAC CACCACCACCACCACTGA (SEQ ID NO.: 22) ##STR00052##
##STR00053##
ASLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERG-
LQRRRFVQNALN
GNGDPNNMDKAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQ-
HRSHRQMVTTTN ##STR00054## ##STR00055##
[0144] The invention embodies the use of the dockerin.cohesin
interaction to assemble ordered and specific complexes for various
therapeutic or vaccination purposes. An example is the use of
rAb.doc with binding specificity to an internalizing human
Dendritic Cell (DC) receptor complexed with coh.Flu M1 protein.
FIG. 11 demonstrates this utility by an in vitro study. DC cultured
with anti-DC_rAb.doc:coh.Flu M1, then co-cultured with autologous T
cells, directed the expansion of T cells with specific memory of
Flu M1. Equivalent doses of coh.Flu M1 alone had no such effect.
The study shows at least a 50-fold enhancement of Flu M1-specific T
cell expansion via the anti-DC_rAb.doc:coh.Flu M1 compared to
coh.Flu M1 alone.
[0145] FIG. 13 shows that functional anti-DC_rAb.doc:coh.Flu M1
complex was formed by mixing the individual purified components.
Various amounts of the complex, or coh.Flu M1 alone, were incubated
in culture medium with 5E4 human DC (from a HLA201 donor) and 10E5
autologous T cells. After 24 hr, the DC were activated with CD4OL
and incubation was continued for an additional 9 days. Cells were
harvested and stained with a PE-labeled Flu M1 peptide GILGFVFTL
(SEQ ID NO.:24) HLA-A2 tetramer and analyzed for the frequency of
antigen-specific CD8+ cells.
[0146] FIG. 14 shows a similar example incorporating the additional
control of coh.Flu M1 complexed to an isotype-matched mAb.doc with
no binding to the human DC. FIG. 12 shows that Anti-DC_rAb directly
linked via an H chain fusion to a peptide fragment spanning the Flu
M1 GILGFVFTL epitope is also effective in eliciting DC targeted
antigen delivery resulting in expansion of Flu M1-specific T cells.
However the Anti-DC_rAb.Flu M1 PEP entity was secreted very poorly
from mammalian cells, likely precluding production of such a
vaccine. This problem illustrates the embodiment of the invention
that allows production issues to be solved by employing expression
systems appropriate for the (in this case) vaccine antigen.
[0147] FIG. 14 shows that Anti-DC_rAb.doc:coh.Flu M1 or
mIgG2b.doc:coh.Flu M1 complexes were formed by mixing the
individual purified components. Various amounts of the complexes,
or coh.Flu M1 alone, were incubated in culture medium with 5E4
human DC (from a HLA201 donor) and 10E5 autologous T cells. After
24 hr, the DC were activated with CD40L and incubation was
continued for an additional 9 days. Cells were harvested and
stained with a PE-labeled Flu M1 peptide GILGFVFTL (SEQ ID NO.:24)
HLA-A2 tetramer and analyzed for the frequency of antigen-specific
CD8+ cells. Concentrations for mIgG2.doc complexes were the same as
those for Anti-DC_rAb complexes.
[0148] FIG. 15 shows CD34+ human DC were sorted into CD1a+ and
CD14+ subtypes and cultured with and without 3 nM Anti-DC_rAb.Flu
M1 PEP or Anti-DC_rAb. Autologous T cells were added after 1 day
and culture continued for a further 8 days. Analysis was as
described above. The CD1a+ cells were very efficient in expanding
Flu M1-specific CD8+ cells only with Anti-DC_rAb.Flu M1 PEP
treatment.
[0149] While one type of embodiment of the invention is a vaccine
composed of an Anti-DC-rAb.doc:coh.antigen complex, it is
envisioned that in some cases a preferred DC-targeting vaccine will
be Anti-DC-rAb.antigen where antigen is likely a string of
protective antigens. Identification of such antigens in efficacious
combinations compatible with efficient expression in production
systems is extremely problematic. One embodiment of the invention
affords a method to streamline testing of antigen epitope
combinations for the development of such vaccines. Specifically,
the invention teaches a method to screen likely antigen epitopes
alone and in combinations for efficacy as a prelude to addressing
production of the desired Anti-DC-rAb.antigen. For example, TABLE
13 shows the sequences of exemplative cohesin.peptide constructs
which can be readily expressed via E. coli systems. Using
techniques similar to those described in FIG. 11, diverse
collections of coh.pep proteins can be readily tested for efficacy
as complexes with a single anti-DC_rAb.doc entity. The most
efficacious coh.pep compounds can then be engineered directly as
anti-DC_rAb.peptide fusion proteins. FIG. 16 shows examples of
purified coh.PEP proteins expressed in E. coli.
[0150] TABLE 12 shows the amino acid sequence of the
melanoma-associated antigen gp100. Well known HLA-A201-restricted
dominant peptides are shaded and detailed below the sequence.
Peptide sequences labeled M are variants with enhanced affinity for
HLA-A201. C180 is an E. coli expression construct that encodes the
sequence shown below in which the cohesin domain is shaded blue and
the gp100 peptide is shaded grey. Underlined residues bounding the
peptide are native to gp100. C-terminal His tags are to facilitate
purification via metal affinity chromatography.
[0151] Shown below is the gp100 sequence and the associated
peptides referred to above.
TABLE-US-00013 (SEQ ID NO.: 25)
MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDG-
PTLIGANASFSI ##STR00056## ##STR00057## ##STR00058##
PSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQV-
TTTEWVETTARE
LPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPS-
GEGDAFELTVSC
QGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQE-
AGLGQVPLTVGI
LLVLMAVVLASLTYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV
[0152] The HLA-A0201 restricted peptide sequences are:
[0153] GP100 WT: 154-162: KTWGQYWQV (SEQ ID NO.:26)
[0154] GP100 M: 209-217 (2M): IMDQVPFSV (SEQ ID NO.:27); 209-217
WT: ITDQVPFSV (SEQ ID NO.:28)
[0155] GP100 M: 280-288 (9V): YLEPGPVTV (SEQ ID NO.:29) 280-288 WT:
YLEPGPVTA (SEQ ID NO.:30)
[0156] C180 is E. coli-pET28(Cohesin-hgp100-PeptideA-6.times.
His):
TABLE-US-00014 (SEQ ID NO.: 31) ##STR00059## ##STR00060##
##STR00061##
[0157] TABLE 13 shows the amino acid sequence of the melanoma
antigen MART-1. Well known HLA-A201-restricted dominant peptides
are shaded and detailed below the sequence. M peptides show peptide
sequence variants with enhanced affinity for HLA-A201. C181 is an
E. coli expression construct that encodes the sequence shown below
in which the cohesin domain is shaded yellow and the MART-1 peptide
is shaded grey. Underlined residues bounding the peptide are native
to MART-1. C172 and C174 are two constructs directing the
expression of anti-DC_rAb.MART-1 peptide and a matching control
rAb.MART-1 peptide H chain. Only the sequences appended to the
C-terminal residue are shown. C-terminal His tags are to facilitate
purification via metal affinity chromatography.
[0158] MART-1 is:
TABLE-US-00015 (SEQ ID NO.: 32) ##STR00062##
SLQEKNCEPVVPNAPPAYEKLSAEQSPPYSP
[0159] The HLA-A0201 restricted peptides sequences are:
TABLE-US-00016 (SEQ ID NO.: 33) ##STR00063## (SEQ ID NO.: 34)
##STR00064## (SEQ ID NO.: 35) ##STR00065##
[0160] C181 is E. coli-pET28(Cohesin-hMART-1-PeptideB-6.times.
His)
TABLE-US-00017 (SEQ ID NO.: 36) ##STR00066## ##STR00067##
##STR00068##
[0161] C186 is E. coli-pET28(Cohesin-Flex-hMART-1-PeptideA-6.times.
His)
TABLE-US-00018 (SEQ ID NO.: 37) ##STR00069## ##STR00070##
##STR00071##
[0162] C172 is
rAB-pIRES2(mAnti-ASGPR.sub.--49C11.sub.--7H-LV-hIgG4H-hMART-1-PeptideA)
[0163] C174 is rAB-pIRES2(hIgG4H-hMART-1-PeptideA)
TABLE-US-00019 (SEQ ID NO.: 38) ##STR00072##
[0164] FIG. 16 shows E. coli harboring expression plasmids
directing the synthesis of coh.pep proteins were grown and induced
for specific protein production. Cells were harvested and broken by
sonication. The supernatant fractions were applied purified by
metal affinity chromatography. Analysis was by reducing SDS.PAGE
gel stained by Coomassie Brilliant Blue. The figure shows typical
product coh.pep proteins labeled from left to right.
[0165] This Example shows the successful use of cohesin and
dockerin fusion proteins secreted from mammalian cells. If both
fusion partners are rAbs with different specificities (i.e.,
rAb1.doc and rAb2.coh), then simple mixing results in
rAb1.doc:rAb2.coh which is a bi-specific antibody. Bispecific
antibodies have many potential therapeutic and technical
applications. The invention provides a simple and predictable means
to assemble such entities through the doc:coh interaction.
Alternately, if rAb1.doc:rAb1.coh were assembled such entities
represent controlled cross-linked mAbs with potentially unique
biological properties.
[0166] Cohesin.dockerin modules exist in diverse cellulose
degrading species. While they have sequence similarities, they can
have specificities that do not cross between species. This affords
an opportunity to build novel scaffolds composed of cohesins with
different specificities and use this scaffold to assemble high
order complexes in a spatially and numerically controlled manner.
Others have described the core technology for using this notion for
biotechnology applications (see Fierobe, H.-P., Mechaly, A.,
Tardif, C., Belaich, A., Lamed, R., Shoham, Y., Belaich, J.-P., and
Bayer, E. A. (2001) Design and production of active cellulosome
chimeras: Selective incorporation of dockerin-containing enzymes
into defined functional complexes. J. Biol. Chem. 276,
21257-21261.). The invention embodies the specific use of this
technology for applications related to manufacture of
rAb.(doc:coh.fusion)n complexes where n represents >1 pairings
of doc:coh interactions with unique specificities. Thus, the
invention envisions the assembly (by simple mixing of components)
of spatially ordered complexes between rAb.doc1.doc2.doc3.etc. and
coh1.fusionA, coh2.fusionB, coh3.fusion3, etc. The coh.fusion
proteins could represent different antigens, or combinations of
antigens and activating agents like cytokines.
[0167] By extension multiple coh:doc specificities could also be
used to make bivalent rAbs with higher order antigen specificities.
Cellulose degrading bacteria and similar organisms also use
cellulose binding domains (CBD) to organize the degradation
machinery. The structure of a CBD from Clostridium thermocellum
shows that the N and C-termini are in close proximity and are not
an integral part of the CBD functional structure. In fact CBD
typically occurs linked to other domains such as coh.CBD.coh in
cipA. The invention encompasses the use of entities such as
coh.CBD.coh to assemble spatially and numerically ordered complexes
mimicking antibodies and multi subunit receptors. For example, a
IgG kappa chain v region fused to doc1 and a IgG H chain V region
linked to doc2 can assemble with coh1.CBD.coh2 to yield
VL.doc1:coh1.CBD.coh2:VH.doc2 to yield an entity with affinity and
binding specificity analogous to the original mAb. Such entities
should be e.g., very useful screening tools for refining mAb
specificities through mutagenesis procedures, particularly since
the VL and VH component could be mutated independently and combined
by mixing in various combinations. As described above, this
technology can be readily extended to multiple controlled coh:V.doc
combinations potentially yielding binding entities with extremely
high specificities and affinities. An extension of this would be
using e.g., coh1.coh2.CBD.coh3 as a template for assembly of
cytoR1.doc+cytoR2.doc+cytoR3.doc (where cytoR represents the
ectodomain of one subunit of a complex cytokine receptor). Such
entities will have utility for blocking cytokine interactions for
therapy and in biotechnology for measuring cytokines in complex
supernatants.
EXAMPLE 3
Using Cohesin-Dockerin Technology for Immunotoxin Therapy
[0168] Currently 1.2 million Americans develop cancer each year and
about 500,000 die from the disease, because most cancers cannot be
cured once they have metastasized. To develop a new treatment for
metastatic cancer, genetic engineering has been used to modify a
powerful bacterial toxin, Pseudomonas exotoxin A (PE), so that
instead of killing normal cells it selectively kills cancer cells.
PE is a three domain protein composed of 613 amino acids.
Anti-cancer agents are produced by deleting its binding domain (aa
1-252) and replacing it with the Fv fragment of an antibody or with
a growth factor that binds to antigens present on cancer cells.
These agents are termed recombinant immunotoxins (RITs). RITs have
been made that target Ley present on colon, breast, lung and other
epithelial cancers (B3(Fv)-PE38), that target the EGF receptor
overexpressed on glioblastomas (TGF-alpha-PE38), that target mutant
EGF receptors present on glioblastomas (MR-1(Fv)-PE38KDEL), and
that target the IL-2 receptor present on many T and B cell
leukemias and lymphomas LMB-2 or anti-Tac(Fv)-PE38 and that target
CD22 on B cell malignancies and that target BL22 or RFB4(dsFv)-PE38
ovarian cancers and mesotheliomas (SS1P). These agents are produced
in E. coli because large amounts can be readily purified from this
source and because the toxin itself would kill mammalian cells
expressing it. When administered to mice with the appropriate human
cancer xenograft, all these RITs produce complete tumor
regressions. Most of these agents are now in clinical trials in
humans and several have produced complete and partial remissions in
humans with cancer.
[0169] An ideal immunotoxin should be very active so that only
small amounts need to be given to cause tumor regressions, stable
so it remains functional during the 5-10 hours required to reach
the interior of a tumor, and non immunogenic so it can be given
repeatedly. Initially, recombinant immunotoxins contained amino
acids 253-613 of PE (domains II and III). It has been determined
that amino acids 364-395 can be deleted without loss of activity.
Increased stability can be addressed by linking the toxin to a
whole antibody, which are well known to have long half-lives and
the technology in the invention provides this solution.
[0170] While the rAb.Doc:Coh.toxin technology can be applied to
known cancer antigens, it can also be tested to kill intra-tumoral
DC that are suspected to foster escape of the tumor from immune
surveillance. In this latter case, anti-DC toxin therapy could be
doubly advantageous since build up of immunity against the
administered toxin itself should be suppressed (that is because DC
themselves are key to the initiation of this immune response via
uptake and processing of the antigen. In this therapy, the DCs that
uptake the antigen die and cannot mount the anti-toxin
response).
[0171] Frankel (Clinical Cancer Research, 8, 942-944, 2002)
describes issues hindering the wider application of immunotoxins.
These include production problems which often require refolding of
E. coli inclusion body expressed material where misfolding
contaminents are problematic. Also, affinity of the immunotoxin for
its target is often difficult to obtain in sufficient strength. The
technology basis of this invention addresses both these
issues--firstly, we found that cohesin. PE38 fusion protein is
expressed in E. coli as a soluble protein that can be purified in a
fully functional state (with both cohesin and toxin activities in
tact) by simple biochemical means without complex refolding.
Secondly, high affinity monoclonal antibodies against target
antigens can be routinely obtained by one practiced in the art.
What is difficult is engineering the antibody variable regions in a
form that is fused with toxin and fully functional for target
binding. The usual means (e.g., sFv forms) of engineering invarably
lead to significant loss of affinity against the target compared to
the initial monoclonal antibody. The rAb.Doc:Coh.toxin technology
circumvents this issue affording a means to preserve both the high
affinity binding sites of the initial mAb (note that humanization
of mouse mAb V regions while maintaining high and specific binding
activity is routine to one practiced in the art), as well as the
beneficial properties of long half-life and non-antigenicity of a
full recombinant hIgG context.
[0172] Furthermore since the cohesin.toxin is produced
independently, one formulation of the toxin can be conjugated to
any number of separately produced targeting rAb.Doc proteins by
simple mixing of the component prior to injection of the patient.
This greatly simplifies manufacturing as well as research
development time. The technology described in the invention can be
readily applied to any toxin and any rAb specificity.
[0173] Details of the rAb.Doc:Coh.toxin technology. pRB 391 (from
Dr. Pastan) Pastan, Chief of the Laboratory of Molecular Biology,
Division of Basic Sciences. NCI, NIH) was used as a template for
PCR with primers
TABLE-US-00020 PE38-N3
(cacggtcaccgtctccaaagcttccggagctagcGAGGGCGGCAGCCTG GCCGCGCT (SEQ ID
NO.: 39)) and PE38-C3
(GGCCGGCTCCTGCGAAGGGAGCCGGCCGGTCGCGGCCGCTTACTTCAGG
TCCTCGCGCGGCGGTTTGCCG (SEQ ID NO.: 40)).
[0174] Cloning was into the previously established construct C21 or
E. coli-pET28(Cohesin-6.times. His) to generate a fusion protein
encoding Cohesin-PE38 corresponding to the amino acid sequence
shown below (grey residues are cohesin; yellow residues are PE38,
separated by a linker sequence native to the cohesin domain).
TABLE-US-00021 (SEQ ID NO.: 41) ##STR00073## ##STR00074##
##STR00075## ##STR00076## ##STR00077## ##STR00078##
##STR00079##
[0175] Expression and purification of recombinant Coh.PE38
protein--E. coli cells from each 1 L fermentation were resuspended
in 25 ml ice-cold 50 mM Tris, 1 mM EDTA pH 8.0 with 0.1 ml of
protease inhibitor Cocktail II (Calbiochem). The cells were
sonicated on ice 2.times.5 min at setting 18 (Fisher Sonic
Dismembrator 60) with a 5 min rest period and then spun at 17,000
r.p.m. (Sorvall SA-600) for 20 min at 4.degree. C. The supernatant
was passed through 1 ml ANX Sepharose column equilibrated in 50 mM
Tris, 1 mM EDTA pH 8.0 and and eluted with a 0-1 M NaCl gradient in
Buffer B. Fractions containing Cohesin.PE38 sere identified by
SDS.PAGE and pooled fractions were further purified by purification
via anti-cohesin mAb affinity chromatography with elution by 0.1 M
glycine pH 2.7.
[0176] Selective killing of human DC by rAb.Doc targeted
Coh.PE38--Human DC were prepared from blood monocytes by culture
for 6 days in with GM-CSF and IL-4. The DCs were then cultured with
either Coh.PE38 alone, anti-DC-SIGN/L 16E7 rAb.Doc alone, anti-DCIR
24A5.Doc alone, or the rAb.Docs together with Coh.PE38 (1.25 ug/ml
of agents were added). After 48 hr the cells were stained with a
reagent (7-AAD) that detects apoptotic cells and analyzed by FACS
scoring forward versus side scatter and 7-AAD fluorescence.
[0177] FIG. 17 shows that the DCIR.Doc rAb alone had no effect upon
the survival of DCs. However, DC-SIGN/L alone has a survival
enhancing effect upon the DC (evidenced both by the scatter
analysis and the 7-AAD staining. FIG. 18 shows that Coh.PE38 alone
slightly increase the number of 7-AAD scored apoptotic cells (from
22.1-29.8%). However, targeting the Coh.PE38 toxin via DCIR.Doc
increased the 7-AAD positive population to 55.3%. The scatter
analysis even more dramatically revealed an almost complete loss of
the population characteristic of viable DC. Targeting the Coh.PE38
toxin via DC-SIGN/L.Doc increased the 7-AAD positive population to
53.7%.with a similar loss of the viable DC scatter population.
[0178] However, this latter result should be viewed in the context
of the survival effect of the DC-SIGN/L.Doc rAb, meaning that the
killing can be viewed as from 3.1-53.1% 7-AAD positive.
[0179] Using Cohesin-Dockerin Technology to make Multivalent
Antibodies. A Cohesin domain was engineered in-frame with the
C-terminus of a rAb H chain using PCR based on C17
(Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6.times. His))as template. The
resulting secreted H chain sequence is shown below (the cohesin
domain is highlighted in grey and the C-terminal H chain residue is
in bold):
TABLE-US-00022 (SEQ ID NO.: 42)
QIQLVQSGPELKKPGETVKISCKASGYSFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFAFSLET-
SASTAYLQISH
LKNEDMATYFCARGDFRYYYFDYWGQGTTLTGSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVIVS-
WNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP-
KPKDTLMISRI
PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS-
SIEKTISKAKG
QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYGRLTVDKS-
RWQEGNVFSCS ##STR00080## ##STR00081##
[0180] This expression construct was co-transfected with the
appropriate rAb L chain into 293F cells and expression of secreted
rAb was appraised by anti-hIgGFc ELISA at day 3. FIG. 19 shows the
expression of anti-DC-SIGN/L and Anti-DC-ASPGR rAb.Coh were
efficiently secreted.
[0181] Thus both Cohesin and Dockerin domains are readily expressed
as rAb fusion proteins. This property is essential for the use of
(rAb1.Coh:rAb2.Doc) complexes as bivalent antibodies (i.e., having
two different combining specificities in one protein). Bivalent
antibodies have many desirable features suited to industrial,
analytic, and therapeutic applications. They are, however,
difficult to develop and molecular tools used to engineer them
typically adulterate desirable features of high affinity and
specificity inherent to the parent monoclonal antibodies. The
(rAb1.Coh:rAb2.Doc) technology circumvents this obstacle and is,
moreover, extensible to higher (than 2) valency of combining power
by incorporating multiple Cohesin or Dockerin strings with pair
wise specificities as described elsewhere in this application.
Furthermore, this technology can be extended to using, e.g., a
cytokine to provide the additional valency (i.e.,
rAb1.Doc:Coh.cytokine).
[0182] For example, a fusion protein between a Cohesin domain and
IL-21 was engineered as an expression construct and the Coh.IL-21
protein was efficiently secreted from transiently transfected 293F
cells and easily purified by sequential Q Sepharose and
anti-Cohesin affinity chromatography. The sequence of the secreted
product is shown below with the cohesin domain shown in grey and
the IL-21 domain in yellow. This product was fully functional as
determined by it's efficacy in sustaining proliferation of human B
cells.
[0183] Mam-pCDM8(SLAML-Cohesin-hIL-21)
TABLE-US-00023 (SEQ ID NO.: 43) ##STR00082## ##STR00083##
##STR00084## ##STR00085##
[0184] Thus rAb.Doc:Coh.IL-21 can deliver concomitant proliferation
and activation signals to a B cel (i.e., if the rAb itself has
activation properties). This notion can be extended to any rAb with
biological properties directed to a particular cell type and any
cytokine with activity directed to the same cell type. FIG. 20
shows the effect of IL-21 and Coh:IL-21 on the proliferation of
human B cells.
[0185] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0186] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims.
[0187] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0188] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The use of
the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0189] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0190] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so
forth. The skilled artisan will understand that typically there is
no limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0191] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Sequence CWU 1
1
4312299PRTBacteroides cellulosolvens 1Met Gln Ser Pro Arg Leu Lys
Arg Lys Ile Leu Ser Val Ile Leu Ala 1 5 10 15 Val Cys Tyr Ile Ile
Ser Ser Phe Ser Ile Gln Phe Ala Ala Thr Pro 20 25 30 Gln Val Asn
Ile Ile Ile Gly Ser Ala Gln Gly Ile Pro Gly Ser Thr 35 40 45 Val
Lys Val Pro Ile Asn Leu Gln Asn Val Pro Glu Ile Gly Ile Asn 50 55
60 Asn Cys Asp Phe Thr Ile Lys Phe Asp Ser Asp Ile Leu Asp Phe Asn
65 70 75 80 Ser Val Glu Ala Gly Asp Ile Val Pro Leu Pro Val Ala Ser
Phe Ser 85 90 95 Ser Asn Asn Ser Lys Asp Ile Ile Lys Phe Leu Phe
Ser Asp Ala Thr 100 105 110 Gln Gly Asn Met Pro Ile Asn Glu Asn Gly
Leu Phe Ala Val Ile Ser 115 120 125 Phe Lys Ile Lys Asp Asn Ala Gln
Lys Gly Ile Ser Asn Ile Lys Val 130 135 140 Ser Ser Tyr Gly Ser Phe
Ser Gly Met Ser Gly Lys Glu Met Gln Ser 145 150 155 160 Leu Ser Pro
Thr Phe Phe Ser Gly Ser Ile Asp Val Ser Asp Val Ser 165 170 175 Thr
Ser Lys Leu Asp Val Lys Val Gly Asn Val Glu Gly Ile Ala Gly 180 185
190 Thr Glu Val Asn Val Pro Ile Thr Phe Glu Asn Val Pro Asp Asn Gly
195 200 205 Ile Asn Asn Cys Asn Phe Thr Leu Ser Tyr Asp Ser Asn Ala
Leu Glu 210 215 220 Phe Leu Thr Thr Glu Ala Gly Asn Ile Ile Pro Leu
Ala Ile Ala Asp 225 230 235 240 Tyr Ser Ser Tyr Arg Ser Met Glu Gly
Lys Ile Lys Phe Leu Phe Ser 245 250 255 Asp Ser Ser Gln Gly Thr Arg
Ser Ile Lys Asn Asp Gly Val Phe Ala 260 265 270 Asn Ile Lys Phe Lys
Ile Lys Gly Asn Ala Ile Arg Asp Thr Tyr Arg 275 280 285 Ile Asp Leu
Ser Glu Leu Gly Ser Phe Ser Ser Lys Gln Asn Asn Asn 290 295 300 Leu
Lys Ser Ile Ala Thr Gln Phe Leu Ser Gly Ser Val Asn Val Lys 305 310
315 320 Asp Ile Glu Ser Ser Val Ser Pro Thr Thr Ser Val His Pro Thr
Pro 325 330 335 Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser Pro Gly
Asn Lys Met 340 345 350 Lys Ile Gln Ile Gly Asp Val Lys Ala Asn Gln
Gly Asp Thr Val Ile 355 360 365 Val Pro Ile Thr Phe Asn Glu Val Pro
Val Met Gly Val Asn Asn Cys 370 375 380 Asn Phe Thr Leu Ala Tyr Asp
Lys Asn Ile Met Glu Phe Ile Ser Ala 385 390 395 400 Asp Ala Gly Asp
Ile Val Thr Leu Pro Met Ala Asn Tyr Ser Tyr Asn 405 410 415 Met Pro
Ser Asp Gly Leu Val Lys Phe Leu Tyr Asn Asp Gln Ala Gln 420 425 430
Gly Ala Met Ser Ile Lys Glu Asp Gly Thr Phe Ala Asn Val Lys Phe 435
440 445 Lys Ile Lys Gln Ser Ala Ala Phe Gly Lys Tyr Ser Val Gly Ile
Lys 450 455 460 Ala Ile Gly Ser Ile Ser Ala Leu Ser Asn Ser Lys Leu
Ile Pro Ile 465 470 475 480 Glu Ser Ile Phe Lys Asp Gly Ser Ile Thr
Val Thr Asn Lys Pro Ile 485 490 495 Val Asn Ile Glu Ile Gly Lys Val
Lys Val Lys Ala Gly Asp Lys Ile 500 505 510 Lys Val Pro Val Glu Ile
Lys Asp Ile Pro Ser Ile Gly Ile Asn Asn 515 520 525 Cys Asn Phe Thr
Leu Lys Tyr Asn Ser Asn Val Leu Lys Tyr Val Ser 530 535 540 Asn Glu
Ala Gly Thr Ile Val Pro Ala Pro Leu Ala Asn Leu Ser Ile 545 550 555
560 Asn Lys Pro Asp Glu Gly Ile Ile Lys Leu Leu Phe Ser Asp Ala Ser
565 570 575 Gln Gly Gly Met Pro Ile Lys Asp Asn Gly Ile Phe Val Asn
Leu Glu 580 585 590 Phe Gln Ala Val Asn Asp Ala Asn Ile Gly Val Tyr
Gly Leu Glu Leu 595 600 605 Asp Thr Ile Gly Ala Phe Ser Gly Ile Ser
Ser Ala Lys Met Thr Ser 610 615 620 Ile Glu Pro Gln Phe Asn Asn Gly
Ser Ile Glu Ile Phe Asn Ser Ala 625 630 635 640 Gln Thr Pro Val Pro
Ser Asn Thr Glu Val Gln Thr Pro Thr Asn Thr 645 650 655 Ile Ser Val
Thr Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Thr 660 665 670 Pro
Lys Pro Asn Pro Leu Tyr Asn Leu Asn Val Asn Ile Gly Glu Ile 675 680
685 Ser Gly Glu Ala Gly Gly Val Ile Glu Val Pro Ile Glu Phe Lys Asn
690 695 700 Val Pro Asp Phe Gly Ile Asn Asn Cys Asp Phe Ser Val Lys
Tyr Asp 705 710 715 720 Lys Ser Ile Phe Glu Tyr Val Thr Tyr Glu Ala
Gly Ser Ile Val Lys 725 730 735 Asp Ser Ile Val Asn Leu Ala Cys Met
Glu Asn Ser Gly Ile Ile Asn 740 745 750 Leu Leu Phe Asn Asp Ala Thr
Gln Ser Ser Ser Pro Ile Lys Asn Asn 755 760 765 Gly Val Phe Ala Lys
Leu Lys Phe Lys Ile Asn Ser Asn Ala Ala Ser 770 775 780 Gly Thr Tyr
Gln Ile Asn Ala Glu Gly Tyr Gly Lys Phe Ser Gly Asn 785 790 795 800
Leu Asn Gly Lys Leu Thr Ser Ile Asn Pro Ile Phe Glu Asn Gly Ile 805
810 815 Ile Asn Ile Gly Asn Val Thr Val Lys Pro Thr Ser Thr Pro Ala
Asp 820 825 830 Ser Ser Thr Ile Thr Pro Thr Ala Thr Pro Thr Ala Thr
Pro Thr Ile 835 840 845 Lys Gly Thr Pro Thr Val Thr Pro Ile Tyr Trp
Met Asn Val Leu Ile 850 855 860 Gly Asn Met Asn Ala Ala Ile Gly Glu
Glu Val Val Val Pro Ile Glu 865 870 875 880 Phe Lys Asn Val Pro Pro
Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu 885 890 895 Val Tyr Asp Ser
Asn Ala Leu Glu Leu Lys Lys Val Glu Ala Gly Asp 900 905 910 Ile Val
Pro Glu Pro Leu Ala Asn Leu Ser Ser Asn Lys Ser Glu Gly 915 920 925
Lys Ile Gln Phe Leu Phe Asn Asp Ala Ser Gln Gly Ser Met Gln Ile 930
935 940 Glu Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val Lys Ser
Thr 945 950 955 960 Ala Ala Ser Gly Ile Tyr Asn Ile Arg Lys Asp Ser
Val Gly Ser Phe 965 970 975 Ser Gly Leu Ile Asp Asn Lys Met Thr Ser
Ile Gly Pro Lys Phe Thr 980 985 990 Asp Gly Ser Ile Val Val Gly Thr
Val Thr Pro Thr Ala Thr Ala Thr 995 1000 1005 Pro Ser Ala Ile Val
Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys 1010 1015 1020 Pro Ile Ala
Thr Pro Thr Ile Lys Gly Thr Pro Thr Ala Thr Pro 1025 1030 1035 Met
Tyr Trp Met Asn Val Val Ile Gly Lys Met Asn Ala Glu Val 1040 1045
1050 Gly Gly Glu Val Val Val Pro Ile Glu Phe Asn Asn Val Pro Ser
1055 1060 1065 Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu Val Tyr Asp
Ala Thr 1070 1075 1080 Ala Leu Glu Leu Lys Asn Val Glu Ala Gly Asp
Ile Ile Lys Thr 1085 1090 1095 Pro Leu Ala Asn Phe Ser Asn Asn Lys
Ser Glu Glu Gly Lys Ile 1100 1105 1110 Ser Phe Leu Phe Asn Asp Ala
Ser Gln Gly Ser Met Gln Ile Glu 1115 1120 1125 Asn Gly Gly Val Phe
Ala Lys Ile Thr Phe Lys Val Lys Ser Thr 1130 1135 1140 Thr Ala Thr
Gly Val Tyr Asp Leu Arg Lys Asp Leu Val Gly Ser 1145 1150 1155 Phe
Ser Gly Leu Lys Asp Asn Lys Met Thr Ser Ile Gly Ala Glu 1160 1165
1170 Phe Thr Asn Gly Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val
1175 1180 1185 Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala Thr Pro
Thr Val 1190 1195 1200 Thr Pro Thr Ala Thr Ala Thr Pro Ser Val Thr
Ile Pro Thr Val 1205 1210 1215 Thr Pro Thr Ala Thr Ala Thr Pro Ser
Val Thr Ile Pro Thr Val 1220 1225 1230 Thr Pro Thr Ala Thr Ala Thr
Pro Ser Ala Ala Thr Pro Thr Val 1235 1240 1245 Thr Pro Thr Ala Thr
Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1250 1255 1260 Thr Pro Thr
Val Thr Ala Thr Pro Ser Asp Thr Ile Pro Thr Val 1265 1270 1275 Thr
Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr Ile 1280 1285
1290 Thr Pro Thr Ala Thr Ala Lys Pro Ile Ala Thr Pro Thr Ile Lys
1295 1300 1305 Gly Thr Pro Thr Ala Thr Pro Met Tyr Trp Met Asn Val
Val Ile 1310 1315 1320 Gly Lys Met Asn Ala Glu Val Gly Gly Glu Val
Val Val Pro Ile 1325 1330 1335 Glu Phe Lys Asn Val Pro Ser Phe Gly
Ile Asn Asn Cys Asp Phe 1340 1345 1350 Lys Leu Val Tyr Asp Ala Thr
Ala Leu Glu Leu Lys Asn Val Glu 1355 1360 1365 Ala Gly Asp Ile Ile
Lys Thr Pro Leu Ala Asn Phe Ser Asn Asn 1370 1375 1380 Lys Ser Glu
Glu Gly Lys Ile Ser Phe Leu Phe Asn Asp Ala Ser 1385 1390 1395 Gln
Gly Ser Met Gln Ile Glu Asn Gly Gly Val Ser Ala Lys Ile 1400 1405
1410 Thr Phe Lys Val Lys Ser Thr Thr Ala Ile Gly Val Tyr Asp Ile
1415 1420 1425 Arg Lys Asp Leu Ile Gly Ser Phe Ser Gly Leu Lys Asp
Ser Lys 1430 1435 1440 Met Thr Ser Ile Gly Ala Glu Phe Thr Asn Gly
Ser Ile Thr Val 1445 1450 1455 Ala Thr Thr Ala Pro Thr Val Thr Pro
Thr Ala Thr Ala Thr Pro 1460 1465 1470 Ser Val Thr Ile Pro Thr Val
Thr Pro Thr Ala Thr Ala Thr Pro 1475 1480 1485 Gly Thr Ala Thr Pro
Gly Thr Ala Thr Pro Thr Ala Thr Ala Thr 1490 1495 1500 Pro Gly Ala
Ala Thr Pro Thr Glu Thr Ala Thr Pro Ser Val Met 1505 1510 1515 Ile
Pro Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Thr Ala Thr 1520 1525
1530 Ala Thr Pro Thr Val Lys Gly Thr Pro Thr Ile Lys Pro Val Tyr
1535 1540 1545 Lys Met Asn Val Val Ile Gly Arg Val Asn Val Val Ala
Gly Glu 1550 1555 1560 Glu Val Val Val Pro Val Glu Phe Lys Asn Ile
Pro Ala Ile Gly 1565 1570 1575 Val Asn Asn Cys Asn Phe Val Leu Glu
Tyr Asp Ala Asn Val Leu 1580 1585 1590 Glu Val Lys Lys Val Asp Ala
Gly Glu Ile Val Pro Asp Ala Leu 1595 1600 1605 Ile Asn Phe Gly Ser
Asn Asn Ser Asp Glu Gly Lys Val Tyr Phe 1610 1615 1620 Leu Phe Asn
Asp Ala Leu Gln Gly Arg Met Gln Ile Ala Asn Asp 1625 1630 1635 Gly
Ile Phe Ala Asn Ile Thr Phe Lys Val Lys Ser Ser Ala Ala 1640 1645
1650 Ala Gly Ile Tyr Asn Ile Arg Lys Asp Ser Val Gly Ala Phe Ser
1655 1660 1665 Gly Leu Val Asp Lys Leu Val Pro Ile Ser Ala Glu Phe
Thr Asp 1670 1675 1680 Gly Ser Ile Ser Val Glu Ser Ala Lys Ser Thr
Pro Thr Ala Thr 1685 1690 1695 Ala Thr Gly Thr Asn Val Thr Pro Thr
Val Ala Ala Thr Val Thr 1700 1705 1710 Pro Thr Ala Thr Pro Ala Ser
Thr Thr Pro Thr Ala Thr Pro Thr 1715 1720 1725 Ala Thr Ser Thr Val
Lys Gly Thr Pro Thr Ala Thr Pro Leu Tyr 1730 1735 1740 Ser Met Asn
Val Ile Ile Gly Lys Val Asn Ala Glu Ala Ser Gly 1745 1750 1755 Glu
Val Val Val Pro Val Glu Phe Lys Asp Val Pro Ser Ile Gly 1760 1765
1770 Ile Asn Asn Cys Asn Phe Ile Leu Glu Tyr Asp Ala Ser Ala Leu
1775 1780 1785 Glu Leu Asp Ser Ala Glu Ala Gly Glu Ile Val Pro Val
Pro Leu 1790 1795 1800 Gly Asn Phe Ser Ser Asn Asn Lys Asp Glu Gly
Lys Ile Tyr Phe 1805 1810 1815 Leu Phe Ser Asp Gly Thr Gln Gly Arg
Met Gln Ile Val Asn Asp 1820 1825 1830 Gly Ile Phe Ala Lys Ile Lys
Phe Lys Val Lys Ser Thr Ala Ser 1835 1840 1845 Asp Gly Thr Tyr Tyr
Ile Arg Lys Asp Ser Val Gly Ala Phe Ser 1850 1855 1860 Gly Leu Ile
Glu Lys Lys Ile Ile Lys Ile Gly Ala Glu Phe Thr 1865 1870 1875 Asp
Gly Ser Ile Thr Val Arg Ser Leu Thr Pro Thr Pro Thr Val 1880 1885
1890 Thr Pro Asn Val Ala Ser Pro Thr Pro Thr Lys Val Val Ala Glu
1895 1900 1905 Pro Thr Ser Asn Gln Pro Ala Gly Pro Gly Pro Ile Thr
Gly Thr 1910 1915 1920 Ile Pro Thr Ala Thr Thr Thr Ala Thr Ala Thr
Pro Thr Lys Ala 1925 1930 1935 Ser Val Ala Thr Ala Thr Pro Thr Ala
Thr Pro Ile Val Val Val 1940 1945 1950 Glu Pro Thr Ile Val Arg Pro
Gly Tyr Asn Lys Asp Ala Asp Leu 1955 1960 1965 Ala Val Phe Ile Ser
Ser Asp Lys Ser Arg Tyr Glu Glu Ser Ser 1970 1975 1980 Ile Ile Thr
Tyr Ser Ile Glu Tyr Lys Asn Ile Gly Lys Val Asn 1985 1990 1995 Ala
Thr Asn Val Lys Ile Ala Ala Gln Ile Pro Lys Phe Thr Lys 2000 2005
2010 Val Tyr Asp Ala Ala Lys Gly Ala Val Lys Gly Ser Glu Ile Val
2015 2020 2025 Trp Met Ile Gly Asn Leu Ala Val Gly Glu Ser Tyr Thr
Lys Glu 2030 2035 2040 Tyr Lys Val Lys Val Asp Ser Leu Thr Lys Ser
Glu Glu Tyr Thr 2045 2050 2055 Asp Asn Thr Val Thr Ile Ser Ser Asp
Gln Thr Val Asp Ile Pro 2060 2065 2070 Glu Asn Ile Thr Thr Gly Asn
Asp Asp Lys Ser Thr Ile Arg Val 2075 2080 2085 Met Leu Tyr Ser Asn
Arg Phe Thr Pro Gly Ser His Ser Ser Tyr 2090 2095 2100 Ile Leu Gly
Tyr Lys Asp Lys Thr Phe Lys Pro Lys Gln Asn Val 2105 2110 2115 Thr
Arg Ala Glu Val Ala Ala Met Phe Ala Arg Ile Met Gly Leu 2120 2125
2130 Thr Val Lys Asp Gly Ala Lys Ser Ser Tyr Lys Asp Val Ser Asn
2135 2140 2145 Lys His Trp Ala Leu Lys Tyr Ile Glu Ala Val Thr Lys
Ser Gly 2150 2155 2160 Ile Phe Lys Gly Tyr Lys Asp Ser Thr Phe His
Pro Asn Ala Pro 2165 2170 2175 Ile Thr Arg Ala Glu Leu Ser Thr Val
Ile Phe Asn Tyr Leu His 2180 2185 2190 Leu Asn Asn Ile Ala Pro Ser
Lys Val His Phe Thr Asp Ile Asn 2195 2200 2205 Lys His Trp Ala Lys
Asn Tyr Ile Glu Glu Ile Tyr Arg Phe Lys 2210 2215 2220 Leu Ile Gln
Gly Tyr Ser Asp Gly Ser Phe Lys Pro Asn Asn Asn 2225 2230 2235
Ile Thr Arg Ala Glu Val Val Thr Met Ile Asn Arg Met Leu Tyr 2240
2245 2250 Arg Gly Pro Leu Lys Val Lys Val Gly Ser Phe Pro Asp Val
Ser 2255 2260 2265 Pro Lys Tyr Trp Ala Tyr Gly Asp Ile Glu Glu Ala
Ser Arg Asn 2270 2275 2280 His Lys Tyr Thr Arg Asp Glu Lys Asp Gly
Ser Glu Ile Leu Ile 2285 2290 2295 Glu 21653DNAArtificial
SequenceSynthetic oligonucleotide 2atggacctcc tgtgcaagaa catgaagcac
ctgtggttct tcctcctgct ggtggcggct 60cccagatggg tcctgtcccg gctgcagctg
caggagtcgg gcccaggcct gctgaagcct 120tcggtgaccc tgtccctcac
ctgcactgtc tcgggtgact ccgtcgccag tagttcttat 180tactggggct
gggtccgtca gcccccaggg aagggactcg agtggatagg gactatcaat
240tttagtggca atatgtatta tagtccgtcc ctcaggagtc gagtgaccat
gtcggcagac 300atgtccgaga actccttcta tctgaaattg gactctgtga
ccgcagcaga cacggccgtc 360tattattgtg cggcaggaca cctcgttatg
ggatttgggg cccactgggg acagggaaaa 420ctggtctccg tctctccagc
ttccaccaag ggcccatccg tcttccccct ggcgccctgc 480tccaggagca
cctccgagag cacagccgcc ctgggctgcc tggtcaagga ctacttcccc
540gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca
caccttcccg 600gctgtcctac agtcctcagg actctactcc ctcagcagcg
tggtgaccgt gccctccagc 660agcttgggca cgaagaccta cacctgcaac
gtagatcaca agcccagcaa caccaaggtg 720gacaagagag ttgagtccaa
atatggtccc ccatgcccac cctgcccagc acctgagttc 780gaagggggac
catcagtctt cctgttcccc ccaaaaccca aggacactct catgatctcc
840cggacccctg aggtcacgtg cgtggtggtg gacgtgagcc aggaagaccc
cgaggtccag 900ttcaactggt acgtggatgg cgtggaggtg cataatgcca
agacaaagcc gcgggaggag 960cagttcaaca gcacgtaccg tgtggtcagc
gtcctcaccg tcctgcacca ggactggctg 1020aacggcaagg agtacaagtg
caaggtctcc aacaaaggcc tcccgtcctc catcgagaaa 1080accatctcca
aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc
1140caggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg
cttctacccc 1200agcgacatcg ccgtggagtg ggagagcaat gggcagccgg
agaacaacta caagaccacg 1260cctcccgtgc tggactccga cggctccttc
ttcctctaca gcaggctaac cgtggacaag 1320agcaggtggc aggaggggaa
tgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1380cactacacac
agaagagcct ctccctgtct ctgggtaaag ctagcaattc tcctcaaaat
1440gaagtactgt acggagatgt gaatgatgac ggaaaagtaa actccactga
cttgactttg 1500ttaaaaagat atgttcttaa agccgtctca actctccctt
cttccaaagc tgaaaagaac 1560gcagatgtaa atcgtgacgg aagagttaat
tccagtgatg tcacaatact ttcaagatat 1620ttgataaggg taatcgagaa
attaccaata taa 16533524PRTArtificial SequenceSynthetic peptide.
3Arg Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Leu Lys Pro Ser Val 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Val Ala Ser
Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Val Arg Gln Pro Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Ile Gly Thr Ile Asn Phe Ser Gly Asn Met
Tyr Tyr Ser Pro Ser 50 55 60 Leu Arg Ser Arg Val Thr Met Ser Ala
Asp Met Ser Glu Asn Ser Phe 65 70 75 80 Tyr Leu Lys Leu Asp Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly His
Leu Val Met Gly Phe Gly Ala His Trp Gly Gln 100 105 110 Gly Lys Leu
Val Ser Val Ser Pro Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260
265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385
390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Lys Ala 435 440 445 Ser Asn Ser Pro Gln Asn Glu Val
Leu Tyr Gly Asp Val Asn Asp Asp 450 455 460 Gly Lys Val Asn Ser Thr
Asp Leu Thr Leu Leu Lys Arg Tyr Val Leu 465 470 475 480 Lys Ala Val
Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala Asp 485 490 495 Val
Asn Arg Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile Leu Ser 500 505
510 Arg Tyr Leu Ile Arg Val Ile Glu Lys Leu Pro Ile 515 520 4
1635DNAArtificial SequenceSynthetic oligonucleotide. 4atgaaatgca
gctgggtcat cttcttcctg atggcagtgg ttacaggggt caattcagag 60gttcagctgc
agcagtctgg ggctgagctt gtgaggccag gggccttagt caagttgtcc
120tgcaaagctt ctggcttcaa cattaatgac tactatatcc actgggtgaa
gcagcggcct 180gaacagggcc tggagcggat tggatggatt gatcctgaca
atggtaatac tatatatgac 240ccgaagttcc agggcaaggc cagtataaca
gcagacacat cccccaacac agcctacctg 300cagctcagca gcctgacatc
tgaggacact gccgtctatt actgtgctag aacccgatct 360cctatggtta
cgacggggtt tgtttactgg ggccaaggga ctgtggtcac tgtctctgca
420gccaaaacga agggcccatc cgtcttcccc ctggcgccct gctccaggag
cacctccgag 480agcacagccg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 540tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 600ggactctact ccctcagcag
cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660tacacctgca
acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc
720aaatatggtc ccccatgccc accctgccca gcacctgagt tcgaaggggg
accatcagtc 780ttcctgttcc ccccaaaacc caaggacact ctcatgatct
cccggacccc tgaggtcacg 840tgcgtggtgg tggacgtgag ccaggaagac
cccgaggtcc agttcaactg gtacgtggat 900ggcgtggagg tgcataatgc
caagacraag ccgcgggagg agcagttcaa cagcacgtac 960cgtgtggtca
gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag
1020tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc
caaagccaaa 1080gggcagcccc gagagccaca ggtgtacacc ctgcccccat
cccaggagga gatgaccaag 1140aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat cgccgtggag 1200tgggagagca atgggcagcc
ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260gacggctcct
tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg
1320aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 1380ctctccctgt ctctgggtaa agctagcaat tctcctcaaa
atgaagtact gtacggagat 1440gtgaatgatg acggaaaagt aaactccact
gacttgactt tgttaaaaag atatgttctt 1500aaagccgtct caactctccc
ttcttccaaa gctgaaaaga acgcagatgt aaatcgtgac 1560ggaagagtta
attccagtga tgtcacaata ctttcaagat atttgataag ggtaatcgag
1620aaattaccaa tataa 16355525PRTArtificial SequenceSynthetic
peptide. 5Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
Gly Ala 1 5 10 15 Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn
Ile Asn Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Glu
Gln Gly Leu Glu Arg Ile 35 40 45 Gly Trp Ile Asp Pro Asp Asn Gly
Asn Thr Ile Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Ser Ile
Thr Ala Asp Thr Ser Pro Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Thr Arg Ser Pro Met Val Thr Thr Gly Phe Val Tyr Trp Gly 100 105 110
Gln Gly Thr Val Val Thr Val Ser Ala Ala Lys Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220 Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser 225 230 235
240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 Ala Ser Asn Ser Pro
Gln Asn Glu Val Leu Tyr Gly Asp Val Asn Asp 450 455 460 Asp Gly Lys
Val Asn Ser Thr Asp Leu Thr Leu Leu Lys Arg Tyr Val 465 470 475 480
Leu Lys Ala Val Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala 485
490 495 Asp Val Asn Arg Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile
Leu 500 505 510 Ser Arg Tyr Leu Ile Arg Val Ile Glu Lys Leu Pro Ile
515 520 525 6 1638DNAArtificial SequenceSynthetic oligonucleotide.
6atggacccca aaggctccct ttcctggaga atacttctgt ttctctccct ggcttttgag
60ttgtcgtacg gagatgtgca gcttcaggag tcaggacctg acctggtgaa accttctcag
120tcactttcac tcacctgcac tgtcactggc tactccatca ccagtggtta
tagctggcac 180tggatccggc agtttccagg aaacaaactg gaatggatgg
gctacatact cttcagtggt 240agcactaact acaacccatc tctgaaaagt
cgaatctcta tcactcgaga cacatccaag 300aaccagttct tcctgcagtt
gaattctgtg actactgagg acacagccac atatttctgt 360gcaagatcta
actatggttc ctttgcttcc tggggccaag ggactctggt cactgtctct
420gcagccaaaa caaagggccc atccgtcttc cccctggcgc cctgctccag
gagcacctcc 480gagagcacag ccgccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 540tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 600tcaggactct actccctcag
cagcgtggtg accgtgccct ccagcagctt gggcacgaag 660acctacacct
gcaacgtaga tcacaagccc agcaacacca aggtggacaa gagagttgag
720tccaaatatg gtcccccatg cccaccctgc ccagcacctg agttcgaagg
gggaccatca 780gtcttcctgt tccccccaaa acccaaggac actctcatga
tctcccggac ccctgaggtc 840acgtgcgtgg tggtggacgt gagccaggaa
gaccccgagg tccagttcaa ctggtacgtg 900gatggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagtt caacagcacg 960taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaacgg caaggagtac
1020aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat
ctccaaagcc 1080aaagggcagc cccgagagcc acaggtgtac accctgcccc
catcccagga ggagatgacc 1140aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct accccagcga catcgccgtg 1200gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260tccgacggct
ccttcttcct ctacagcagg ctaaccgtgg acaagagcag gtggcaggag
1320gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacacagaag 1380agcctctccc tgtctctggg taaagctagc aattctcctc
aaaatgaagt actgtacgga 1440gatgtgaatg atgacggaaa agtaaactcc
actgacttga ctttgttaaa aagatatgtt 1500cttaaagccg tctcaactct
cccttcttcc aaagctgaaa agaacgcaga tgtaaatcgt 1560gacggaagag
ttaattccag tgatgtcaca atactttcaa gatatttgat aagggtaatc
1620gagaaattac caatataa 16387521PRTArtificial SequenceSynthetic
peptide. 7Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro
Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
Ile Thr Ser Gly 20 25 30 Tyr Ser Trp His Trp Ile Arg Gln Phe Pro
Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Leu Phe Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg
Ser Asn Tyr Gly Ser Phe Ala Ser Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ala Ala Lys Thr Lys Gly Pro Ser Val Phe Pro Leu 115
120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys
Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235
240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val
Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360
365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
370
375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys Ala Ser Asn Ser 435 440 445 Pro Gln Asn Glu Val Leu
Tyr Gly Asp Val Asn Asp Asp Gly Lys Val 450 455 460 Asn Ser Thr Asp
Leu Thr Leu Leu Lys Arg Tyr Val Leu Lys Ala Val 465 470 475 480 Ser
Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala Asp Val Asn Arg 485 490
495 Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile Leu Ser Arg Tyr Leu
500 505 510 Ile Arg Val Ile Glu Lys Leu Pro Ile 515 520 8
1623DNAArtificial SequenceSynthetic oligonucleotide. 8atggaaaggc
actggatctt tctcttcctg ttttcagtaa ctgcaggtgt ccactcccag 60gtccagcttc
agcagtctgg ggctgagctg gcaaaacctg gggcctcagt gaagatgtcc
120tgcaaggctt ctggctacac ctttactacc tactggatgc actgggtaaa
acagaggcct 180ggacagggtc tggaatggat tggatacatt aatcctatca
ctggttatac tgagtacaat 240cagaagttca aggacaaggc caccttgact
gcagacaaat cctccagcac agcctacatg 300caactgagca gcctgacatc
tgaggactct gcagtctatt actgtgcaag agagggttta 360agtgctatgg
actattgggg tcagggaacc tcagtcaccg tcacctcagc caaaacaacg
420ggcccatccg tcttccccct ggcgccctgc tccaggagca cctccgagag
cacagccgcc 480ctgggctgcc tggtcaagga ctacttcccc gaaccggtga
cggtgtcgtg gaactcaggc 540gccctgacca gcggcgtgca caccttcccg
gctgtcctac agtcctcagg actctactcc 600ctcagcagcg tggtgaccgt
gccctccagc agcttgggca cgaagaccta cacctgcaac 660gtagatcaca
agcccagcaa caccaaggtg gacaagagag ttgagtccaa atatggtccc
720ccatgcccac cctgcccagc acctgagttc gaagggggac catcagtctt
cctgttcccc 780ccaaaaccca aggacactct catgatctcc cggacccctg
aggtcacgtg cgtggtggtg 840gacgtgagcc aggaagaccc cgaggtccag
ttcaactggt acgtggatgg cgtggaggtg 900cataatgcca agacaaagcc
gcgggaggag cagttcaaca gcacgtaccg tgtggtcagc 960gtcctcaccg
tcctgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc
1020aacaaaggcc tcccgtcctc catcgagaaa accatctcca aagccaaagg
gcagccccga 1080gagccacagg tgtacaccct gcccccatcc caggaggaga
tgaccaagaa ccaggtcagc 1140ctgacctgcc tggtcaaagg cttctacccc
agcgacatcg ccgtggagtg ggagagcaat 1200gggcagccgg agaacaacta
caagaccacg cctcccgtgc tggactccga cggctccttc 1260ttcctctaca
gcaggctaac cgtggacaag agcaggtggc aggaggggaa tgtcttctca
1320tgctccgtga tgcatgaggc tctgcacaac cactacacac agaagagcct
ctccctgtct 1380ctgggtaaag ctagcaattc tcctcaaaat gaagtactgt
acggagatgt gaatgatgac 1440ggaaaagtaa actccactga cttgactttg
ttaaaaagat atgttcttaa agccgtctca 1500actctccctt cttccaaagc
tgaaaagaac gcagatgtaa atcgtgacgg aagagttaat 1560tccagtgatg
tcacaatact ttcaagatat ttgataaggg taatcgagaa attaccaata 1620taa
16239521PRTArtificial SequenceSynthetic peptide. 9Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30
Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asn Pro Ile Thr Gly Tyr Thr Glu Tyr Asn Gln Lys
Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Leu Ser Ala Met Asp
Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Thr Ser Ala Lys
Thr Thr Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165
170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly
Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290
295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410
415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser
Asn Ser 435 440 445 Pro Gln Asn Glu Val Leu Tyr Gly Asp Val Asn Asp
Asp Gly Lys Val 450 455 460 Asn Ser Thr Asp Leu Thr Leu Leu Lys Arg
Tyr Val Leu Lys Ala Val 465 470 475 480 Ser Thr Leu Pro Ser Ser Lys
Ala Glu Lys Asn Ala Asp Val Asn Arg 485 490 495 Asp Gly Arg Val Asn
Ser Ser Asp Val Thr Ile Leu Ser Arg Tyr Leu 500 505 510 Ile Arg Val
Ile Glu Lys Leu Pro Ile 515 520 10732DNAArtificial
SequenceSynthetic oligonucleotide. 10atgcatcgca ccagcatggg
catcaagatg gagtcacaga ttcaggcatt tgtattcgtg 60tttctctggt tgtctggtgt
tggcggagac attgtgatga cccagtctca caaattcatg 120tccacatcag
taggagacag ggtcagcgtc acctgcaagg ccagtcagga tgtgacttct
180gctgtagcct ggtatcaaca aaaaccaggg caatctccta aactactgat
ttactgggca 240tccacccggc acactggagt ccctgatcgc ttcacaggca
gtggatctgg gacagattat 300actctcacca tcagcagtgg gcaggctgaa
gacctggcac tttattactg tcaccaatat 360tatagcgctc ctcggacgtt
cggtggaggc accaagctcg agatcaaacg aactgtggct 420gcaccatctg
tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct
480gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg
gaaggtggat 540aacgccctcc aatcgggtaa ctcccaggag agtgtcacag
agcaggacag caaggacagc 600acctacagcc tcagcagcac cctgacgctg
agcaaagcag actacgagaa acacaaagtc 660tatgcctgcg aagtcaccca
tcagggcctg agctcgcccg tcacaaagag cttcaacagg 720ggagagtgtt ag
73211214PRTArtificial SequenceSynthetic peptide. 11Asp Ile Val Met
Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg
Val Ser Val Thr Cys Lys Ala Ser Gln Asp Val Thr Ser Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35
40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Gly Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys His Gln Tyr
Tyr Ser Ala Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
121644DNAArtificial SequenceSynthetic oligonucleotide. 12atgggatggt
catgtatcat cctttttcta gtagcaactg caactggagt acattcacag 60gtccaactgc
agcagcctgg ggctgagctg gtgaggcctg ggacttcagt gaagttgtcc
120tgcaaggctt ctggttacat ctttaccagc tactggatgc actgggtaaa
gcagaggcct 180ggacaaggcc ttgagtggat cggactgatt gatccttctg
atagttatag taagtacaat 240caaaagttca agggcaaggc cacattgact
gtagacacat cctccagcac agcctacatg 300cagctcagca gcctgacatc
tgaggactct gcggtctatt actgtgcaag aggggagctc 360agtgacttct
ggggccaagg caccactctc acagtctcct cagccaaaac aacaccccca
420tcagtctatc cactggcccc tgggtgtgga gatacaactg gttcctctgt
gactctggga 480tgcctggtca agggctactt ccctgagtca gtgactgtga
cttggaactc tggatccctg 540tccagcagtg tgcacacctt cccagctctc
ctgcagtctg gactctacac tatgagcagc 600tcagtgactg tcccctccag
cacctggcca agtcagaccg tcacctgcag cgttgctcac 660ccagccagca
gcaccacggt ggacaaaaaa cttgagccca gcgggcccat ttcaacaatc
720aacccctgtc ctccatgcaa ggagtgtcac aaatgcccag ctcctaacct
cgagggtgga 780ccatccgtct tcatcttccc tccaaatatc aaggatgtac
tcatgatctc cctgacaccc 840aaggtcacgt gtgtggtggt ggatgtgagc
gaggatgacc cagacgtccg gatcagctgg 900tttgtgaaca acgtggaagt
acacacagct cagacacaaa cccatagaga ggattacaac 960agtactatcc
gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag
1020gagttcaaat gcaaggtcaa caacaaagac ctcccatcac ccatcgagag
aaccatctca 1080aaaattaaag ggctagtcag agctccacaa gtatacatct
tgccgccacc agcagagcag 1140ttgtccagga aagatgtcag tctcacttgc
ctggtcgtgg gcttcaaccc tggagacatc 1200agtgtggagt ggaccagcaa
tgggcataca gaggagaact acaaggacac cgcaccagtc 1260ctggactctg
acggttctta cttcatatac agcaagctcg atataaaaac aagcaagtgg
1320gagaaaacag attccttctc atgcaacgtg agacacgagg gtctgaaaaa
ttactacctg 1380aagaagacca tctcccggtc tccgggtaaa gctagcaatt
ctcctcaaaa tgaagtactg 1440tacggagatg tgaatgatga cggaaaagta
aactccactg acttgacttt gttaaaaaga 1500tatgttctta aagccgtctc
aactctgcct tcttccaaag ctgaaaagaa cgcagatgta 1560aatcgtgacg
gaagagttaa ttccagtgat gtcacaatac tttcaagata tttgataagg
1620gtaatcgaga aattaccaat ataa 164413528PRTArtificial
SequenceSynthetic peptide. 13Gln Val Gln Leu Gln Gln Pro Gly Ala
Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile
Asp Pro Ser Asp Ser Tyr Ser Lys Tyr Asn Gln Lys Phe 50 55 60 Lys
Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Gly Glu Leu Ser Asp Phe Trp Gly Gln Gly Thr
Thr Leu Thr 100 105 110 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val
Tyr Pro Leu Ala Pro 115 120 125 Gly Cys Gly Asp Thr Thr Gly Ser Ser
Val Thr Leu Gly Cys Leu Val 130 135 140 Lys Gly Tyr Phe Pro Glu Ser
Val Thr Val Thr Trp Asn Ser Gly Ser 145 150 155 160 Leu Ser Ser Ser
Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu 165 170 175 Tyr Thr
Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 180 185 190
Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val 195
200 205 Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro
Cys 210 215 220 Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn
Leu Glu Gly 225 230 235 240 Gly Pro Ser Val Phe Ile Phe Pro Pro Asn
Ile Lys Asp Val Leu Met 245 250 255 Ile Ser Leu Thr Pro Lys Val Thr
Cys Val Val Val Asp Val Ser Glu 260 265 270 Asp Asp Pro Asp Val Arg
Ile Ser Trp Phe Val Asn Asn Val Glu Val 275 280 285 His Thr Ala Gln
Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile 290 295 300 Arg Val
Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly 305 310 315
320 Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile
325 330 335 Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro
Gln Val 340 345 350 Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg
Lys Asp Val Ser 355 360 365 Leu Thr Cys Leu Val Val Gly Phe Asn Pro
Gly Asp Ile Ser Val Glu 370 375 380 Trp Thr Ser Asn Gly His Thr Glu
Glu Asn Tyr Lys Asp Thr Ala Pro 385 390 395 400 Val Leu Asp Ser Asp
Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asp Ile 405 410 415 Lys Thr Ser
Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg 420 425 430 His
Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser 435 440
445 Pro Gly Lys Ala Ser Asn Ser Pro Gln Asn Glu Val Leu Tyr Gly Asp
450 455 460 Val Asn Asp Asp Gly Lys Val Asn Ser Thr Asp Leu Thr Leu
Leu Lys 465 470 475 480 Arg Tyr Val Leu Lys Ala Val Ser Thr Leu Pro
Ser Ser Lys Ala Glu 485 490 495 Lys Asn Ala Asp Val Asn Arg Asp Gly
Arg Val Asn Ser Ser Asp Val 500 505 510 Thr Ile Leu Ser Arg Tyr Leu
Ile Arg Val Ile Glu Lys Leu Pro Ile 515 520 525 14
2061DNAArtificial SequenceSynthetic oligonucleotide. 14atggatccca
aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag 60ttgagctacg
gactcgacga tctggatgca gtaaggatta aagtggacac agtaaatgca
120aaaccgggag acacagtaag aatacctgta agattcagcg gtataccatc
caagggaata 180gcaaactgtg actttgtata cagctatgac ccgaatgtac
ttgagataat agagatagaa 240ccgggagaca taatagttga cccgaatcct
gacaagagct ttgatactgc agtatatcct 300gacagaaaga taatagtatt
cctgtttgca gaagacagcg gaacaggagc gtatgcaata 360actaaagacg
gagtatttgc tacgatagta gcgaaagtaa aagaaggagc acctaacgga
420ctcagtgtaa tcaaatttgt agaagtaggc ggatttgcga acaatgacct
tgtagaacag 480aagacacagt tctttgacgg tggagtaaat gttggagata
caacagaacc tgcaacacct 540acaacacctg taacaacacc gacaacaaca
gatgatctgg atgcactcga gatcatccca 600gttgaggagg agaacccgga
cttctggaac cgcgaggcag ccgaggccct gggtgccgcc 660aagaagctgc
agcctgcaca gacagccgcc aagaacctca tcatcttcct gggcgatggg
720atgggggtgt ctacggtgac agctgccagg atcctaaaag ggcagaagaa
ggacaaactg 780gggcctgagt tacccctggc catggaccgc ttcccatatg
tggctctgtc caagacatac 840aatgtagaca aacatgtgcc agacagtgga
gccacagcca cggcctacct gtgcggggtc 900aagggcaact tccagaccat
tggcttgagt gcagccgccc gctttaacca gtgcaacacg 960acacgcggca
acgaggtcat ctccgtgatg aatcgggcca agaaagcagg gaagtcagtg
1020ggagtggtaa ccaccacacg agtgcagcac gcctcgccag ccggcaccta
cgcccacacg 1080gtgaaccgca actggtactc ggacgccgac gtgcctgcct
cggcccgcca ggaggggtgc 1140caggacatcg ctacgcagct catctccaac
atggacattg acgtgatcct aggtggaggc 1200cgaaagtaca tgtttcgcat
gggaacccca gaccctgagt acccagatga ctacagccaa 1260ggtgggacca
ggctggacgg gaagaatctg gtgcaggaat ggctggcgaa gcgccagggt
1320gcccggtacg tgtggaaccg cactgagctc atgcaggctt ccctggaccc
gtctgtgacc 1380catctcatgg
gtctctttga gcctggagac atgaaatacg agatccaccg agactccaca
1440ctggacccct ccctgatgga gatgacagag gctgccctgc gcctgctgag
caggaacccc 1500cgcggcttct tcctcttcgt ggagggtggt cgcatcgacc
atggtcatca tgaaagcagg 1560gcttaccggg cactgactga gacgatcatg
ttcgacgacg ccattgagag ggcgggccag 1620ctcaccagcg aggaggacac
gctgagcctc gtcactgccg accactccca cgtcttctcc 1680ttcggaggct
accccctgcg agggagctcc atcttcgggc tggcccctgg caaggcccgg
1740gacaggaagg cctacacggt cctcctatac ggaaacggtc caggctatgt
gctcaaggac 1800ggcgcccggc cggatgttac cgagagcgag agcgggagcc
ccgagtatcg gcagcagtca 1860gcagtgcccc tggacgaaga gacccacgca
ggcgaggacg tggcggtgtt cgcgcgcggc 1920ccgcaggcgc acctggttca
cggcgtgcag gagcagacct tcatagcgca cgtcatggcc 1980ttcgccgcct
gcctggagcc ctacaccgcc tgcgacctgg cgccccccgc cggcaccacc
2040caccatcacc atcaccattg a 206115662PRTArtificial
SequenceSynthetic peptide. 15Leu Asp Asp Leu Asp Ala Val Arg Ile
Lys Val Asp Thr Val Asn Ala 1 5 10 15 Lys Pro Gly Asp Thr Val Arg
Ile Pro Val Arg Phe Ser Gly Ile Pro 20 25 30 Ser Lys Gly Ile Ala
Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35 40 45 Val Leu Glu
Ile Ile Glu Ile Glu Pro Gly Glu Leu Ile Val Asp Pro 50 55 60 Asn
Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met 65 70
75 80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala
Ile 85 90 95 Thr Glu Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val
Lys Ser Gly 100 105 110 Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val
Glu Val Gly Gly Phe 115 120 125 Ala Asn Asn Asp Leu Val Glu Gln Lys
Thr Gln Phe Phe Asp Gly Gly 130 135 140 Val Asn Val Gly Asp Thr Thr
Glu Pro Ala Thr Pro Thr Thr Pro Val 145 150 155 160 Thr Thr Pro Thr
Thr Thr Asp Asp Leu Asp Ala Leu Glu Ile Ile Pro 165 170 175 Val Glu
Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala 180 185 190
Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala Lys Asn 195
200 205 Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr
Ala 210 215 220 Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly
Pro Glu Leu 225 230 235 240 Pro Leu Ala Met Asp Arg Phe Pro Tyr Val
Ala Leu Ser Lys Thr Tyr 245 250 255 Asn Val Asp Lys His Val Pro Asp
Ser Gly Ala Thr Ala Thr Ala Tyr 260 265 270 Leu Cys Gly Val Lys Gly
Asn Phe Gln Thr Ile Gly Leu Ser Ala Ala 275 280 285 Ala Arg Phe Asn
Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser 290 295 300 Val Met
Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val Val Thr 305 310 315
320 Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr
325 330 335 Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser
Ala Arg 340 345 350 Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile
Ser Asn Met Asp 355 360 365 Ile Asp Val Ile Leu Gly Gly Gly Arg Lys
Tyr Met Phe Arg Met Gly 370 375 380 Thr Pro Asp Pro Glu Tyr Pro Asp
Asp Tyr Ser Gln Gly Gly Thr Arg 385 390 395 400 Leu Asp Gly Lys Asn
Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly 405 410 415 Ala Arg Tyr
Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp 420 425 430 Pro
Ser Val Thr His Leu Met Gly Leu Phe Glu Pro Gly Asp Met Lys 435 440
445 Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met
450 455 460 Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly
Phe Phe 465 470 475 480 Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly
His His Glu Ser Arg 485 490 495 Ala Tyr Arg Ala Leu Thr Glu Thr Ile
Met Phe Asp Asp Ala Ile Glu 500 505 510 Arg Ala Gly Gln Leu Thr Ser
Glu Glu Asp Thr Leu Ser Leu Val Thr 515 520 525 Ala Asp His Ser His
Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly 530 535 540 Ser Ser Ile
Phe Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala 545 550 555 560
Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp 565
570 575 Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro Glu
Tyr 580 585 590 Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His
Ala Gly Glu 595 600 605 Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala
His Leu Val His Gly 610 615 620 Val Gln Glu Gln Thr Phe Ile Ala His
Val Met Ala Phe Ala Ala Cys 625 630 635 640 Leu Glu Pro Tyr Thr Ala
Cys Asp Leu Ala Pro Pro Ala Gly Thr Thr 645 650 655 His His His His
His His 660 16 2556DNAArtificial SequenceSynthetic oligonucleotide.
16atggatccca aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag
60ttgagctacg gactcgacga tctggatgca gtaaggatta aagtggacac agtaaatgca
120aaaccgggag acacagtaag aatacctgta agattcagcg gtataccatc
caagggaata 180gcaaactgtg actttgtata cagctatgac ccgaatgtac
ttgagataat agagataaaa 240ccgggagaat tgatagttga cccgaatcct
gacaagagct ttgatactgc agtatatcct 300gacagaaaga taatagtatt
cctgtttgca gaagacagcg gaacaggagc gtatgcaata 360actaaagacg
gagtatttgc tacgatagta gcgaaagtaa aatccggagc acctaacgga
420ctcagtgtaa tcaaatttgt agaagtaggc ggatttgcga ataatgacct
tgtagaacag 480aagacacagt tctttgacgg tggagtaaat gttggagata
caacagaacc tgcaacacct 540acaacacctg taacaacacc gacaacaaca
gatgatctgg atgcagtaag gattaaagtg 600gacacagtaa atgcaaaacc
gggagacaca gtaaatatac ctgtaagatt cagtggtata 660ccatccaagg
gaatagcaaa ctgtgacttt gtatacagct atgacccgaa tgtacttgag
720ataatagaga taaaaccggg agaattgata gttgacccga atcctaccaa
gagctttgat 780actgcagtat atcctgacag aaagatgata gtattcctgt
ttgcggaaga cagcggaaca 840ggagcgtatg caataactaa agacggagta
tttgctacga tagtagcgaa agtaaaagaa 900ggagcaccta acggactcag
tgtaatcaaa tttgtagaag taggcggatt tgcgaacaat 960gaccttgtag
aacagaagac acagttcttt gacggtggag taaatgttgg agatacaaca
1020gaacctgcaa cacctacaac acctgtaaca acaccgacaa caacagatga
tctggatgca 1080ctcgagatca tcccagttga ggaggagaac ccggacttct
ggaaccgcga ggcagccgag 1140gccctgggtg ccgccaagaa gctgcagcct
gcacagacag ccgccaagaa cctcatcatc 1200ttcctgggcg atgggatggg
ggtgtctacg gtgacagctg ccaggatcct aaaagggcag 1260aagaaggaca
aactggggcc tgagttaccc ctggccatgg accgcttccc atatgtggct
1320ctgtccaaga catacaatgt agacaaacat gtgccagaca gtggagccac
agccacggcc 1380tacctgtgcg gggtcaaggg caacttccag accattggct
tgagtgcagc cgcccgcttt 1440aaccagtgca acacgacacg cggcaacgag
gtcatctccg tgatgaatcg ggccaagaaa 1500gcagggaagt cagtgggagt
ggtaaccacc acacgagtgc agcacgcctc gccagccggc 1560acctacgccc
acacggtgaa ccgcaactgg tactcggacg ccgacgtgcc tgcctcggcc
1620cgccaggagg ggtgccagga catcgctacg cagctcatct ccaacatgga
cattgacgtg 1680atcctaggtg gaggccgaaa gtacatgttt cgcatgggaa
ccccagaccc tgagtaccca 1740gatgactaca gccaaggtgg gaccaggctg
gacgggaaga atctggtgca ggaatggctg 1800gcgaagcgcc agggtgcccg
gtacgtgtgg aaccgcactg agctcatgca ggcttccctg 1860gacccgtctg
tgacccatct catgggtctc tttgagcctg gagacatgaa atacgagatc
1920caccgagact ccacactgga cccctccctg atggagatga cagaggctgc
cctgcgcctg 1980ctgagcagga acccccgcgg cttcttcctc ttcgtggagg
gtggtcgcat cgaccatggt 2040catcatgaaa gcagggctta ccgggcactg
actgagacga tcatgttcga cgacgccatt 2100gagagggcgg gccagctcac
cagcgaggag gacacgctga gcctcgtcac tgccgaccac 2160tcccacgtct
tctccttcgg aggctacccc ctgcgaggga gctccatctt cgggctggcc
2220cctggcaagg cccgggacag gaaggcctac acggtcctcc tatacggaaa
cggtccaggc 2280tatgtgctca aggacggcgc ccggccggat gttaccgaga
gcgagagcgg gagccccgag 2340tatcggcagc agtcagcagt gcccctggac
gaagagaccc acgcaggcga ggacgtggcg 2400gtgttcgcgc gcggcccgca
ggcgcacctg gttcacggcg tgcaggagca gaccttcata 2460gcgcacgtca
tggccttcgc cgcctgcctg gagccctaca ccgcctgcga cctggcgccc
2520cccgccggca ccacccacca tcaccatcac cattga 255617826PRTArtificial
SequenceSynthetic peptide. 17Leu Asp Leu Asp Ala Val Arg Ile Lys
Val Asp Thr Val Asn Ala Lys 1 5 10 15 Pro Gly Asp Thr Val Arg Ile
Pro Val Arg Phe Ser Gly Ile Pro Ser 20 25 30 Lys Gly Ile Ala Asn
Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn Val 35 40 45 Leu Glu Ile
Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50 55 60 Pro
Asp Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Ile Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile
Thr 85 90 95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys
Ser Gly Ala 100 105 110 Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu
Val Gly Gly Phe Ala 115 120 125 Asn Asn Asp Leu Val Glu Gln Lys Thr
Gln Phe Phe Asp Gly Gly Val 130 135 140 Asn Val Gly Asp Thr Thr Glu
Pro Ala Thr Pro Thr Thr Pro Val Thr 145 150 155 160 Thr Pro Thr Thr
Thr Asp Asp Leu Asp Ala Val Arg Ile Lys Val Asp 165 170 175 Thr Val
Asn Ala Lys Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe 180 185 190
Ser Gly Ile Pro Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser 195
200 205 Tyr Asp Pro Asn Val Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu
Leu 210 215 220 Ile Val Asp Pro Asn Pro Thr Lys Ser Phe Asp Thr Ala
Val Tyr Pro 225 230 235 240 Asp Arg Lys Met Ile Val Phe Leu Phe Ala
Glu Asp Ser Gly Thr Gly 245 250 255 Ala Tyr Ala Ile Thr Lys Asp Gly
Val Phe Ala Thr Ile Val Ala Lys 260 265 270 Val Lys Glu Gly Ala Pro
Asn Gly Leu Ser Val Ile Lys Phe Val Glu 275 280 285 Val Gly Gly Phe
Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe 290 295 300 Phe Asp
Gly Gly Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro 305 310 315
320 Thr Thr Pro Val Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu
325 330 335 Glu Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn
Arg Glu 340 345 350 Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln
Pro Ala Gln Thr 355 360 365 Ala Ala Lys Asn Leu Ile Ile Phe Leu Gly
Asp Gly Met Gly Val Ser 370 375 380 Thr Val Thr Ala Ala Arg Ile Leu
Lys Gly Gln Lys Lys Asp Lys Leu 385 390 395 400 Gly Pro Glu Leu Pro
Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu 405 410 415 Ser Lys Thr
Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr 420 425 430 Ala
Thr Ala Tyr Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly 435 440
445 Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn
450 455 460 Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys
Ser Val 465 470 475 480 Gly Val Val Thr Thr Thr Arg Val Gln His Ala
Ser Pro Ala Gly Thr 485 490 495 Tyr Ala His Thr Val Asn Arg Asn Trp
Tyr Ser Asp Ala Asp Val Pro 500 505 510 Ala Ser Ala Arg Gln Glu Gly
Cys Gln Asp Ile Ala Thr Gln Leu Ile 515 520 525 Ser Asn Met Asp Ile
Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met 530 535 540 Phe Arg Met
Gly Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln 545 550 555 560
Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala 565
570 575 Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met
Gln 580 585 590 Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu
Phe Glu Pro 595 600 605 Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser
Thr Leu Asp Pro Ser 610 615 620 Leu Met Glu Met Thr Glu Ala Ala Leu
Arg Leu Leu Ser Arg Asn Pro 625 630 635 640 Arg Gly Phe Phe Leu Phe
Val Glu Gly Gly Arg Ile Asp His Gly His 645 650 655 His Glu Ser Arg
Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp 660 665 670 Asp Ala
Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu 675 680 685
Ser Leu Val Thr Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr 690
695 700 Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala
Arg 705 710 715 720 Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn
Gly Pro Gly Tyr 725 730 735 Val Leu Lys Asp Gly Ala Arg Pro Asp Val
Thr Glu Ser Glu Ser Gly 740 745 750 Ser Pro Glu Tyr Arg Gln Gln Ser
Ala Val Pro Leu Asp Glu Glu Thr 755 760 765 His Ala Gly Glu Asp Val
Ala Val Phe Ala Arg Gly Pro Gln Ala His 770 775 780 Leu Val His Gly
Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala 785 790 795 800 Phe
Ala Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro 805 810
815 Ala Gly Thr Thr His His His His His His 820 825 18
1326DNAArtificial SequenceSynthetic oligonucleotide. 18atggatccca
aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag 60ttgagctacg
gactcgacga tctggatgca gtaaggatta aagtggacac agtaaatgca
120aaaccgggag acacagtaag aatacctgta agattcagcg gtataccatc
caagggaata 180gcaaactgtg actttgtata cagctatgac ccgaatgtac
ttgagataat agagatagaa 240ccgggagaca taatagttga cccgaatcct
gacaagagct ttgatactgc agtatatcct 300gacagaaaga taatagtatt
cctgtttgca gaagacagcg gaacaggagc gtatgcaata 360actaaagacg
gagtatttgc tacgatagta gcgaaagtaa aagaaggagc acctaacgga
420ctcagtgtaa tcaaatttgt agaagtaggc ggatttgcga acaatgacct
tgtagaacag 480aagacacagt tctttgacgg tggagtaaat gttggagata
caacagaacc tgcaacacct 540acaacacctg taacaacacc gacaacaaca
gatgatctgg atgcactcga ggcgcccctc 600atcctgtctc ggattgtggg
aggctgggag tgcgagaagc attcccaacc ctggcaggtg 660cttgtggcct
ctcgtggcag ggcagtctgc ggcggtgttc tggtgcaccc ccagtgggtc
720ctcacagctg cccactgcat caggaacaaa agcgtgatct tgctgggtcg
gcacagcctg 780tttcatcctg aagacacagg ccaggtattt caggtcagcc
acagcttccc acacccgctc 840tacgatatga gcctcctgaa gaatcgattc
ctcaggccag gtgatgactc cagccacgac 900ctcatgctgc tccgcctgtc
agagcctgcc gagctcacgg atgctgtgaa ggtcatggac 960ctgcccaccc
aggagccagc actggggacc acctgctacg cctcaggctg gggcagcatt
1020gaaccagagg agttcttgac cccaaagaaa cttcagtgtg tggacctcca
tgttatttcc 1080aatgacgtgt gcgcgcaagt tcaccctcag aaggtgacca
agttcatgct gtgtgctgga 1140cgctggacag ggggcaaaag cacctgctcg
ggtgattctg ggggcccact tgtctgtaat 1200ggtgtgcttc aaggtatcac
gtcatggggc agtgaaccat gtgccctgcc cgaaaggcct 1260tccctgtaca
ccaaggtggt gcattaccgg aagtggatca aggacaccat cgtggccaac 1320ccctga
132619417PRTArtificial SequenceSynthetic peptide. 19Leu Asp Asp Leu
Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala 1 5 10 15 Lys Pro
Gly Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro 20 25 30
Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35
40 45 Val Leu Glu Ile Ile Glu Ile Glu Pro Gly Glu Leu Ile Val Asp
Pro 50 55
60 Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met
65 70 75 80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr
Ala Ile 85 90 95 Thr Glu Asp Gly Val Phe Ala Thr Ile Val Ala Lys
Val Lys Ser Gly 100 105 110 Ala Pro Asn Gly Leu Ser Val Ile Lys Phe
Val Glu Val Gly Gly Phe 115 120 125 Ala Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly 130 135 140 Val Asn Val Gly Asp Thr
Thr Glu Pro Ala Thr Pro Thr Thr Pro Val 145 150 155 160 Thr Thr Pro
Thr Thr Thr Asp Asp Leu Asp Ala Leu Glu Ala Pro Leu 165 170 175 Ile
Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln 180 185
190 Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly
195 200 205 Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His Cys
Ile Arg 210 215 220 Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu
Phe His Pro Glu 225 230 235 240 Asp Thr Gly Gln Val Phe Gln Val Ser
His Ser Phe Pro His Pro Leu 245 250 255 Tyr Asp Met Ser Leu Leu Lys
Asn Arg Phe Leu Arg Pro Gly Asp Asp 260 265 270 Ser Ser His Asp Leu
Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu 275 280 285 Thr Asp Ala
Val Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu 290 295 300 Gly
Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu 305 310
315 320 Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile
Ser 325 330 335 Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr
Lys Phe Met 340 345 350 Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser
Thr Cys Ser Gly Asp 355 360 365 Ser Gly Gly Pro Leu Val Cys Asn Gly
Val Leu Gln Gly Ile Thr Ser 370 375 380 Trp Gly Ser Glu Pro Cys Ala
Leu Pro Glu Arg Pro Ser Leu Tyr Thr 385 390 395 400 Lys Val Val His
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn 405 410 415 Pro
201554DNAArtificial SequenceSynthetic oligonucleotide. 20atggatccca
aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag 60ttgagctacg
gactcgacga tctggatgca gtaaggatta aagtggacac agtaaatgca
120aaaccgggag acacagtaag aatacctgta agattcagcg gtataccatc
caagggaata 180gcaaactgtg actttgtata cagctatgac ccgaatgtac
ttgagataat agagatagaa 240ccgggagaca taatagttga cccgaatcct
gacaagagct ttgatactgc agtatatcct 300gacagaaaga taatagtatt
cctgtttgca gaagacagcg gaacaggagc gtatgcaata 360actaaagacg
gagtatttgc tacgatagta gcgaaagtaa aagaaggagc acctaacgga
420ctcagtgtaa tcaaatttgt agaagtaggc ggatttgcga acaatgacct
tgtagaacag 480aagacacagt tctttgacgg tggagtaaat gttggagata
caacagaacc tgcaacacct 540acaacacctg taacaacacc gacaacaaca
gatgatctgg atgcactcga ggatcagatt 600tgcattggtt accatgcaaa
caactcgaca gagcaggttg acacaataat ggaaaagaac 660gttactgtta
cacatgccca agacatactg gaaaagaaac acaacgggaa gctctgcgat
720ctagatggag tgaagcctct aattttgaga gattgtagcg tagctggatg
gctcctcgga 780aacccaatgt gtgacgaatt catcaatgtg ccggaatggt
cttacatagt ggagaaggcc 840aatccagtca atgacctctg ttacccaggg
gatttcaatg actatgaaaa attgaaacac 900ctattgagca gaataaacca
ttttgagaaa attcagatca tccccaaaag ttcttggtcc 960agtcatgaag
cctcattagg ggtgagctca gcatgtccat accagggaaa gtcctccttt
1020ttcagaaatg tggtatggct tatcaaaaag aacagtacat acccaacaat
aaagaggagc 1080tacaataata ccaaccaaga agatcttttg gtactgtggg
ggattcacca tcctaatgat 1140gcggcagagc agacaaagct ctatcaaaac
ccaaccacct atatttccgt tgggacatca 1200acactaaacc agagattggt
accaagaata gctactagat ccaaagtaaa cgggcaaagt 1260ggaaggatgg
agttcttctg gacaatttta aagccgaatg atgcaatcaa cttcgagagt
1320aatggaaatt tcattgctcc agaatatgca tacaaaattg tcaagaaagg
ggactcaaca 1380attatgaaaa gtgaattgga atatggtaac tgcaacacca
agtgtcaaac tccaatgggg 1440gcgataaact ctagcatgcc attccacaat
atacaccctc tcaccattgg ggaatgcccc 1500aaatatgtga aatcaaacag
attagtcctt gcgcaccatc accatcacca ttga 155421493PRTArtificial
SequenceSynthetic peptide. 21Leu Asp Asp Leu Asp Ala Val Arg Ile
Lys Val Asp Thr Val Asn Ala 1 5 10 15 Lys Pro Gly Asp Thr Val Arg
Ile Pro Val Arg Phe Ser Gly Ile Pro 20 25 30 Ser Lys Gly Ile Ala
Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35 40 45 Val Leu Glu
Ile Ile Glu Ile Glu Pro Gly Glu Leu Ile Val Asp Pro 50 55 60 Asn
Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met 65 70
75 80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala
Ile 85 90 95 Thr Glu Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val
Lys Ser Gly 100 105 110 Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val
Glu Val Gly Gly Phe 115 120 125 Ala Asn Asn Asp Leu Val Glu Gln Lys
Thr Gln Phe Phe Asp Gly Gly 130 135 140 Val Asn Val Gly Asp Thr Thr
Glu Pro Ala Thr Pro Thr Thr Pro Val 145 150 155 160 Thr Thr Pro Thr
Thr Thr Asp Asp Leu Asp Ala Leu Glu Asp Gln Ile 165 170 175 Cys Ile
Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile 180 185 190
Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu Glu Lys 195
200 205 Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu
Ile 210 215 220 Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
Pro Met Cys 225 230 235 240 Asp Glu Phe Ile Asn Val Pro Glu Trp Ser
Tyr Ile Val Glu Lys Ala 245 250 255 Asn Pro Val Asn Asp Leu Cys Tyr
Pro Gly Asp Phe Asn Asp Tyr Glu 260 265 270 Lys Leu Lys His Leu Leu
Ser Arg Ile Asn His Phe Glu Lys Ile Gln 275 280 285 Ile Ile Pro Lys
Ser Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val 290 295 300 Ser Ser
Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe Arg Asn Val 305 310 315
320 Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile Lys Arg Ser
325 330 335 Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly
Ile His 340 345 350 His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr
Gln Asn Pro Thr 355 360 365 Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu
Asn Gln Arg Leu Val Pro 370 375 380 Arg Ile Ala Thr Arg Ser Lys Val
Asn Gly Gln Ser Gly Arg Met Glu 385 390 395 400 Phe Phe Trp Thr Ile
Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser 405 410 415 Asn Gly Asn
Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys 420 425 430 Gly
Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn 435 440
445 Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe
450 455 460 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr
Val Lys 465 470 475 480 Ser Asn Arg Leu Val Leu Ala His His His His
His His 485 490 221293DNAArtificial SequenceSynthetic
oligonucleotide. 22atggatctgg atgcagtaag gattaaagtg gacacagtaa
atgcaaaacc gggagacaca 60gtaaatatac ctgtaagatt cagtggtata ccatccaagg
gaatagcaaa ctgtgacttt 120gtatacagct atgacccgaa tgtacttgag
ataatagaga taaaaccggg agaattgata 180gttgacccga atcctaccaa
gagctttgat actgcagtat atcctgacag aaagatgata 240gtattcctgt
ttgcggaaga cagcggaaca ggagcgtatg caataactaa agacggagta
300tttgctacga tagtagcgaa agtaaaagaa ggagcaccta acgggctcag
tgtaatcaaa 360tttgtagaag taggcggatt tgcgaacaat gaccttgtag
aacagaagac acagttcttt 420gacggtggag taaatgttgg agatacaaca
gaacctgcaa cacctacaac acctgtaaca 480acaccgacaa caacagatga
tctggatgca gctagccttc taaccgaggt cgaaacgtac 540gttctctcta
tcatcccgtc aggccccctc aaagccgaga tcgcacagag acttgaagat
600gtctttgcag ggaagaacac cgatcttgag gttctcatgg aatggctaaa
gacaagacca 660atcctgtcac ctctgactaa ggggatttta ggatttgtgt
tcacgctcac cgtgcccagt 720gagcggggac tgcagcgtag acgctttgtc
caaaatgctc ttaatgggaa cggagatcca 780aataacatgg acaaagcagt
taaactgtat aggaagctta agagggagat aacattccat 840ggggccaaag
aaatagcact cagttattct gctggtgcac ttgccagttg tatgggcctc
900atatacaaca ggatgggggc tgtgaccact gaagtggcat ttggcctggt
atgcgcaacc 960tgtgaacaga ttgctgactc ccagcatcgg tctcataggc
aaatggtgac aacaaccaat 1020ccactaatca gacatgagaa cagaatggtt
ctagccagca ctacagctaa ggctatggag 1080caaatggctg gatcgagtga
gcaagcagca gaggccatgg atattgctag tcaggccagg 1140caaatggtgc
aggcgatgag aaccattggg actcatccta gctccagtgc tggtctaaaa
1200gatgatcttc ttgaaaattt gcaggcttac cagaaacgga tgggggtgca
gatgcagcga 1260ttcaagctcg agcaccacca ccaccaccac tga
129323430PRTArtificial SequenceSynthetic peptide. 23Met Asp Leu Asp
Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5 10 15 Pro Gly
Asp Thr Val Asn Ile Pro Val Arg Phe Ser Gly Ile Pro Ser 20 25 30
Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn Val 35
40 45 Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro
Asn 50 55 60 Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg
Lys Met Ile 65 70 75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90 95 Lys Asp Gly Val Phe Ala Thr Ile Val
Ala Lys Val Lys Glu Gly Ala 100 105 110 Pro Asn Gly Leu Ser Val Ile
Lys Phe Val Glu Val Gly Gly Phe Ala 115 120 125 Asn Asn Asp Leu Val
Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135 140 Asn Val Gly
Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr 145 150 155 160
Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Leu Leu Thr Glu 165
170 175 Val Glu Thr Tyr Val Leu Ser Ile Ile Pro Ser Gly Pro Leu Lys
Ala 180 185 190 Glu Ile Ala Gln Arg Leu Glu Asp Val Phe Ala Gly Lys
Asn Thr Asp 195 200 205 Leu Glu Val Leu Met Glu Trp Leu Lys Thr Arg
Pro Ile Leu Ser Pro 210 215 220 Leu Thr Lys Gly Ile Leu Gly Phe Val
Phe Thr Leu Thr Val Pro Ser 225 230 235 240 Glu Arg Gly Leu Gln Arg
Arg Arg Phe Val Gln Asn Ala Leu Asn Gly 245 250 255 Asn Gly Asp Pro
Asn Asn Met Asp Lys Ala Val Lys Leu Tyr Arg Lys 260 265 270 Leu Lys
Arg Glu Ile Thr Phe His Gly Ala Lys Glu Ile Ala Leu Ser 275 280 285
Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg 290
295 300 Met Gly Ala Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala
Thr 305 310 315 320 Cys Glu Gln Ile Ala Asp Ser Gln His Arg Ser His
Arg Gln Met Val 325 330 335 Thr Thr Thr Asn Pro Leu Ile Arg His Glu
Asn Arg Met Val Leu Ala 340 345 350 Ser Thr Thr Ala Lys Ala Met Glu
Gln Met Ala Gly Ser Ser Glu Gln 355 360 365 Ala Ala Glu Ala Met Asp
Ile Ala Ser Gln Ala Arg Gln Met Val Gln 370 375 380 Ala Met Arg Thr
Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys 385 390 395 400 Asp
Asp Leu Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val 405 410
415 Gln Met Gln Arg Phe Lys Leu Glu His His His His His His 420 425
430 249PRTArtificial SequenceSynthetic peptide. 24Gly Ile Leu Gly
Phe Val Phe Thr Leu 1 5 25661PRTArtificial SequenceSynthetic
peptide. 25Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val
Ile Gly 1 5 10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro Arg
Asn Gln Asp Trp 20 25 30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys
Ala Trp Asn Arg Gln Leu 35 40 45 Tyr Pro Glu Trp Thr Glu Ala Gln
Arg Leu Asp Cys Trp Arg Gly Gly 50 55 60 Gln Val Ser Leu Lys Val
Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala 65 70 75 80 Asn Ala Ser Phe
Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln Lys Val 85 90 95 Leu Pro
Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100 105 110
Ser Gln Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115
120 125 Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp
Ser 130 135 140 Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp Gly
Gln Tyr Trp 145 150 155 160 Gln Val Leu Gly Gly Pro Val Ser Gly Leu
Ser Ile Gly Thr Gly Arg 165 170 175 Ala Met Leu Gly Thr His Thr Met
Glu Val Thr Val Tyr His Arg Arg 180 185 190 Gly Ser Arg Ser Tyr Val
Pro Leu Ala His Ser Ser Ser Ala Phe Thr 195 200 205 Ile Thr Asp Gln
Val Pro Phe Ser Val Ser Val Ser Gln Leu Arg Ala 210 215 220 Leu Asp
Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe 225 230 235
240 Ala Leu Gln Leu His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu
245 250 255 Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile
Ser Arg 260 265 270 Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly
Pro Val Thr Ala 275 280 285 Gln Val Val Leu Gln Ala Ala Ile Pro Leu
Thr Ser Cys Gly Ser Ser 290 295 300 Pro Val Pro Gly Thr Thr Asp Gly
His Arg Pro Thr Ala Glu Ala Pro 305 310 315 320 Asn Thr Thr Ala Gly
Gln Val Pro Thr Thr Glu Val Val Gly Thr Thr 325 330 335 Pro Gly Gln
Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val Gln 340 345 350 Val
Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355 360
365 Ala Glu Ser Thr Gly Met Thr Pro Glu Lys Val Pro Val Ser Glu Val
370 375 380 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr
Gly Met 385 390 395 400 Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser
Gly Thr Thr Ala Ala 405 410 415 Gln Val Thr Thr Thr Glu Trp Val Glu
Thr Thr Ala Arg Glu Leu Pro 420 425 430 Ile Pro Glu Pro Glu Gly Pro
Asp Ala Ser Ser Ile Met Ser Thr Glu 435 440 445 Ser Ile Thr Gly Ser
Leu Gly Pro Leu Leu Asp Gly Thr Ala Thr Leu 450 455 460 Arg Leu Val
Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr 465 470 475 480
Gly Ser Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 485
490 495 Glu Ile Leu Gln Ala Val Pro Ser Gly Glu Gly Asp Ala Phe Glu
Leu 500 505 510 Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys
Met Glu Ile 515 520 525 Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg
Leu Cys Gln Pro Val 530
535 540 Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys
Gly 545 550 555 560 Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala
Asp Thr Asn Ser 565 570 575 Leu Ala Val Val Ser Thr Gln Leu Ile Met
Pro Gly Gln Glu Ala Gly 580 585 590 Leu Gly Gln Val Pro Leu Ile Val
Gly Ile Leu Leu Val Leu Met Ala 595 600 605 Val Val Leu Ala Ser Leu
Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610 615 620 Phe Ser Val Pro
Gln Leu Pro His Ser Ser Ser His Trp Leu Arg Leu 625 630 635 640 Pro
Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu 645 650
655 Ser Gly Gln Gln Val 660 269PRTArtificial SequenceSynthetic
peptide. 26Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5 279PRTArtificial
SequenceSynthetic peptide. 27Ile Met Asp Gln Val Pro Phe Ser Val 1
5 289PRTArtificialChemically synthesized peptide 28Ile Thr Asp Gln
Val Pro Phe Ser Val 1 5 299PRTArtificial SequenceSynthetic peptide.
29Tyr Leu Glu Pro Gly Pro Val Thr Val 1 5
309PRTArtificialChemically synthesized peptide 30Tyr Leu Glu Pro
Gly Pro Val Thr Ala 1 5 31204PRTArtificial SequenceSynthetic
peptide. 31Met Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn
Ala Lys 1 5 10 15 Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe Ser
Gly Ile Pro Ser 20 25 30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr
Ser Tyr Asp Pro Asn Val 35 40 45 Leu Glu Ile Ile Glu Ile Lys Pro
Gly Glu Leu Ile Val Asp Pro Asn 50 55 60 Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70 75 80 Val Phe Leu Phe
Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr 85 90 95 Lys Asp
Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala 100 105 110
Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly
Val 130 135 140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr
Pro Val Thr 145 150 155 160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala
Ala Arg Ser Ala Phe Thr 165 170 175 Ile Met Asp Gln Val Pro Phe Ser
Val Ser Val Ser Ala Ser Arg Lys 180 185 190 Gly Ala Ala Ala Leu Glu
His His His His His His 195 200 32118PRTArtificial
SequenceSynthetic peptide. 32Met Pro Arg Glu Asp Ala His Phe Ile
Tyr Gly Tyr Pro Lys Lys Gly 1 5 10 15 His Gly His Ser Tyr Thr Thr
Ala Glu Glu Ala Ala Gly Ile Gly Ile 20 25 30 Leu Thr Val Ile Leu
Gly Val Leu Leu Leu Ile Gly Cys Trp Tyr Cys 35 40 45 Arg Arg Arg
Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val 50 55 60 Gly
Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp 65 70
75 80 His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu Pro
Val 85 90 95 Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala
Glu Gln Ser 100 105 110 Pro Pro Pro Tyr Ser Pro 115
339PRTArtificial SequenceSynthetic peptide. 33Ala Ala Gly Ile Gly
Ile Leu Thr Val 1 5 3410PRTArtificial SequenceSynthetic peptide.
34Glu Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5 10 3510PRTArtificial
SequenceSynthetic peptide. 35Glu Leu Ala Gly Ile Gly Ile Leu Thr
Val 1 5 10 36204PRTArtificial SequenceSynthetic peptide. 36Met Asp
Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5 10 15
Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe Ser Gly Ile Pro Ser 20
25 30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val
Asp Pro Asn 50 55 60 Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro
Asp Arg Lys Met Ile 65 70 75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly
Thr Gly Ala Tyr Ala Ile Thr 85 90 95 Lys Asp Gly Val Phe Ala Thr
Ile Val Ala Lys Val Lys Glu Gly Ala 100 105 110 Pro Asn Gly Leu Ser
Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115 120 125 Asn Asn Asp
Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135 140 Asn
Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr 145 150
155 160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Arg Thr Ala Glu
Glu 165 170 175 Leu Ala Gly Ile Gly Ile Leu Thr Val Ile Leu Gly Ala
Ser Arg Lys 180 185 190 Gly Ala Ala Ala Leu Glu His His His His His
His 195 200 37241PRTArtificial SequenceSynthetic peptide. 37Met Asp
Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5 10 15
Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe Ser Gly Ile Pro Ser 20
25 30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val
Asp Pro Asn 50 55 60 Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro
Asp Arg Lys Met Ile 65 70 75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly
Thr Gly Ala Tyr Ala Ile Thr 85 90 95 Lys Asp Gly Val Phe Ala Thr
Ile Val Ala Lys Val Lys Glu Gly Ala 100 105 110 Pro Asn Gly Leu Ser
Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115 120 125 Asn Asn Asp
Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135 140 Asn
Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr 145 150
155 160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Asp Thr Thr
Glu 165 170 175 Ala Arg His Pro His Pro Pro Val Thr Thr Pro Thr Thr
Asp Arg Lys 180 185 190 Gly Thr Thr Ala Glu Glu Leu Ala Gly Ile Gly
Ile Leu Thr Val Ile 195 200 205 Leu Gly Gly Lys Arg Thr Asn Asn Ser
Thr Pro Thr Lys Gly Glu Phe 210 215 220 Cys Arg Tyr Pro Ser His Trp
Arg Pro Leu Glu His His His His His 225 230 235 240 His
3866PRTArtificial SequenceSynthetic peptide. 38Ala Ser Asp Thr Thr
Glu Ala Arg His Pro His Pro Pro Val Thr Thr 1 5 10 15 Pro Thr Thr
Thr Asp Arg Lys Gly Thr Thr Ala Glu Glu Leu Ala Gly 20 25 30 Ile
Gly Ile Leu Thr Val Ile Leu Gly Gly Lys Arg Thr Asn Asn Ser 35 40
45 Thr Pro Thr Lys Gly Glu Phe Cys Arg Tyr Pro Ser His Trp Arg Pro
50 55 60 Arg Leu 65 3957DNAArtificial SequenceSynthetic
oligonucleotide. 39cacggtcacc gtctccaaag cttccggagc tagcgagggc
ggcagcctgg ccgcgct 574070DNAArtificial SequenceSynthetic
oligonucleotide. 40ggccggctcc tgcgaaggga gccggccggt cgcggccgct
tacttcaggt cctcgcgcgg 60cggtttgccg 7041518PRTArtificial
SequenceSynthetic peptide. 41Met Asp Leu Asp Ala Val Arg Ile Lys
Val Asp Thr Val Asn Ala Lys 1 5 10 15 Pro Gly Asp Thr Val Asn Ile
Pro Val Arg Phe Ser Gly Ile Pro Ser 20 25 30 Lys Gly Ile Ala Asn
Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn Val 35 40 45 Leu Glu Ile
Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50 55 60 Pro
Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile
Thr 85 90 95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys
Glu Gly Ala 100 105 110 Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu
Val Gly Gly Phe Ala 115 120 125 Asn Asn Asp Leu Val Glu Gln Lys Thr
Gln Phe Phe Asp Gly Gly Val 130 135 140 Asn Val Gly Asp Thr Thr Glu
Pro Ala Thr Pro Thr Thr Pro Val Thr 145 150 155 160 Thr Pro Thr Thr
Thr Asp Asp Leu Asp Ala Ala Ser Glu Gly Gly Ser 165 170 175 Leu Ala
Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr 180 185 190
Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys 195
200 205 Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg
Leu 210 215 220 Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu
Ala Ser Pro 225 230 235 240 Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
Arg Glu Gln Pro Glu Gln 245 250 255 Ala Arg Leu Ala Leu Thr Leu Ala
Ala Ala Glu Ser Glu Arg Phe Val 260 265 270 Arg Gln Gly Thr Gly Asn
Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala 275 280 285 Asp Ser Gly Asp
Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu 290 295 300 Phe Leu
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln 305 310 315
320 Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu
325 330 335 Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu
Ala Ala 340 345 350 Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser
Gln Asp Leu Asp 355 360 365 Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
Asp Pro Ala Leu Ala Tyr 370 375 380 Gly Tyr Ala Gln Asp Gln Glu Pro
Asp Ala Arg Gly Arg Ile Arg Asn 385 390 395 400 Gly Ala Leu Leu Arg
Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe 405 410 415 Tyr Arg Thr
Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val 420 425 430 Glu
Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr 435 440
445 Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro
450 455 460 Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr
Asp Pro 465 470 475 480 Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser
Ile Pro Asp Lys Glu 485 490 495 Gln Ala Ile Ser Ala Leu Pro Asp Tyr
Ala Ser Gln Pro Gly Lys Pro 500 505 510 Pro Arg Glu Asp Leu Lys 515
42614PRTArtificial SequenceSynthetic peptide. 42Gln Ile Gln Leu Val
Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Gly
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40
45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe
50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr
Ala Tyr 65 70 75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Met Ala
Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Asp Phe Arg Tyr Tyr Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Gly Ser Ser Ala
Lys Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu
Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420
425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala
Ser 435 440 445 Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr Thr
Pro Thr Thr 450 455 460 Thr Asp Asp Leu Asp Ala Val Arg Ile Lys Val
Asp Thr Val Asn Ala 465 470 475 480 Lys Pro Gly Asp Thr Val Asn Ile
Pro Val Arg Phe Ser Gly Ile Pro 485 490 495 Ser Lys Gly Ile Ala Asn
Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 500 505 510 Val Leu Glu Ile
Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro 515 520 525 Asn Pro
Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met 530 535 540
Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile 545
550 555 560 Thr Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys
Glu Gly 565 570 575 Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu
Val Gly Gly Phe 580 585 590 Ala Asn Asn Asp Leu Val Glu Gln Lys Thr
Gln Phe Phe Asp Gly Gly 595 600 605 Val Asn Val Gly
Asp Thr 610 43308PRTArtificial SequenceSynthetic peptide. 43Leu Asp
Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala 1 5 10 15
Lys Pro Gly Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro 20
25 30 Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro
Asn 35 40 45 Val Leu Glu Ile Ile Glu Ile Glu Pro Gly Asp Ile Ile
Val Asp Pro 50 55 60 Asn Pro Asp Lys Ser Phe Asp Thr Ala Val Tyr
Pro Asp Arg Lys Ile 65 70 75 80 Ile Val Phe Leu Phe Ala Glu Asp Ser
Gly Thr Gly Ala Tyr Ala Ile 85 90 95 Thr Lys Asp Gly Val Phe Ala
Thr Ile Val Ala Lys Val Lys Glu Gly 100 105 110 Ala Pro Asn Gly Leu
Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe 115 120 125 Ala Asn Asn
Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly 130 135 140 Val
Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val 145 150
155 160 Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu Glu Ala Asp
Gln 165 170 175 Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile
Asp Ile Val 180 185 190 Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val
Pro Glu Phe Leu Pro 195 200 205 Ala Pro Glu Asp Val Glu Thr Asn Cys
Glu Trp Ser Ala Phe Ser Cys 210 215 220 Phe Gln Lys Ala Gln Leu Lys
Ser Ala Asn Thr Gly Asn Asn Glu Arg 225 230 235 240 Ile Ile Asn Val
Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr 245 250 255 Asn Ala
Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp 260 265 270
Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser 275
280 285 Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His
Gly 290 295 300 Ser Glu Asp Ser 305
* * * * *