U.S. patent application number 13/496696 was filed with the patent office on 2012-11-29 for coiled coil and/or tether containing protein complexes and uses thereof.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Erin H. Christensen, Dan L. Eaton, Andrew C. Vendel, Bernd Wranik.
Application Number | 20120302737 13/496696 |
Document ID | / |
Family ID | 43759225 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120302737 |
Kind Code |
A1 |
Christensen; Erin H. ; et
al. |
November 29, 2012 |
COILED COIL AND/OR TETHER CONTAINING PROTEIN COMPLEXES AND USES
THEREOF
Abstract
The invention provides engineered protein complexes constructed
using a coiled coil and/or a tether and methods for making, using,
and purifying such complexes, such as multispecific antibodies or
other multispecific Fc containing complexes.
Inventors: |
Christensen; Erin H.;
(Tiburon, CA) ; Eaton; Dan L.; (San Rafael,
CA) ; Vendel; Andrew C.; (San Mateo, CA) ;
Wranik; Bernd; (South San Francisco, CA) |
Assignee: |
Genentech, Inc.
San Francisco
CA
|
Family ID: |
43759225 |
Appl. No.: |
13/496696 |
Filed: |
September 16, 2010 |
PCT Filed: |
September 16, 2010 |
PCT NO: |
PCT/US2010/002546 |
371 Date: |
August 9, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61243105 |
Sep 16, 2009 |
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61266992 |
Dec 4, 2009 |
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Current U.S.
Class: |
530/387.3 ;
435/69.6; 530/387.1; 530/391.7 |
Current CPC
Class: |
C07K 16/283 20130101;
C07K 2317/53 20130101; C07K 2317/92 20130101; C07K 2317/51
20130101; C07K 2319/00 20130101; C07K 16/468 20130101; C07K 16/2863
20130101; C07K 2317/73 20130101; C07K 2319/73 20130101; C07K 16/32
20130101; C07K 2317/31 20130101 |
Class at
Publication: |
530/387.3 ;
530/387.1; 435/69.6; 530/391.7 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C07K 19/00 20060101 C07K019/00; C07K 16/46 20060101
C07K016/46; C12P 21/02 20060101 C12P021/02 |
Claims
1. An antibody comprising: (a) a first polypeptide comprising a VH
domain and a first coiled coil domain (CC), wherein the first CC
comprises a heptad repeat of Formula I: TABLE-US-00010 (SEQ ID NO:
29) (Xi X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7).sub.n
(Formula I)
Xi is a hydrophobic amino acid residue or Asparagine, X.sub.2,
X.sub.3, and X.sub.6 are each any amino acid residue, X.sub.4 is a
hydrophobic amino acid residue, and X.sub.5 and X.sub.7 are each a
charged amino acid residue; and (b) a second polypeptide comprising
a VH domain and a second coiled coil domain (CC), wherein the
second CC comprises a heptad repeat of Formula II: TABLE-US-00011
(SEQ ID NO: 30) X', X'.sub.2 X'.sub.3 X'.sub.4 X's X'.sub.6
X'.sub.7).sub.n (Formula II)
X' i is a hydrophobic amino acid residue or Asparagine, X'.sub.2,
X'.sub.3, and X'.sub.6 are each any amino acid residue, X'.sub.4 is
a hydrophobic amino acid residue, and X'.sub.5 and X'.sub.7 are
each a charged amino acid residue; wherein n in Formula I and II is
greater than or equal to 2; and wherein, in each heptad repeat, the
first CC comprises an X.sub.5 residue that is opposite in charge to
the X'.sub.7 residue in the second CC and the first CC comprises an
X.sub.7 residue that is opposite in charge to the X' 5 residue in
the second CC.
2. The antibody of claim 1, wherein the first and second
polypeptides each comprise a VH and a CH 1 domain.
3. The antibody of claim 2, wherein the first and second
polypeptides each further comprise a hinge domain.
4. The antibody of claim 1, wherein said first and second
polypeptides each further comprise a CH2 and a CH3 domain.
5. The antibody of claim 1, wherein the first and second
polypeptides each comprise VH, CHI, hinge, CH2, and CH3 domains
positioned relative to each other in an N-terminal to C-terminal
direction: VH-CH1-hinge-CH2-CH3.
6. The antibody of claim 1, wherein said antibody further comprises
a third and a fourth polypeptide, wherein said third polypeptide
comprises a first VL domain and said fourth polypeptide comprises a
second VL domain.
7. The antibody of claim 6, wherein said VH domain of the first
polypeptide is linked to the VL domain of the third polypeptide by
a tether and the VH domain of the second polypeptide is linked to
the VL domain of the fourth polypeptide by a tether.
8. The antibody of claim 6, wherein the third polypeptide further
comprises a first CL domain wherein said first VL and CL domains
are positioned relative to each other within the third polypeptide
in an N-terminal to C-terminal direction: VL-CL, and the fourth
polypeptide further comprises a second CL domain, and wherein said
second VL and CL domains are positioned relative to each other
within the fourth polypeptide in an N-terminal to C-terminal
direction: VL-CL.
9. The antibody of claim 1, wherein the sequences of said first VL
domain and said second VL domain are the same.
10. The antibody of claim 1, wherein the N-terminus of the VH of at
least one of said first or said second polypeptides is connected to
the C-terminus of a CL with a tether.
11. An antibody comprising: (a) a first polypeptide comprising a VH
domain and a first coiled coil domain (CC), wherein the first CC
comprises a heptad repeat of Formula I: TABLE-US-00012 (SEQ ID NO:
29) (X, X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7).sub.n
(Formula I)
Xi is a hydrophobic amino acid residue or Asparagine, X.sub.2,
X.sub.3, and X are each any amino acid residue, X.sub.4 is a
hydrophobic amino acid residue, and X.sub.5 and X.sub.7 are each a
charged amino acid residue; and (b) a second polypeptide comprising
a CH2 and CH3 domain and a second coiled coil (CC), wherein the
second CC comprises a heptad repeat of Formula II: TABLE-US-00013
(SEQ ID NO: 30) (X', X'.sub.2 X'.sub.3 X'.sub.4 X's X'.sub.6
X'.sub.7).sub.n (Formula II)
X' I is a hydrophobic amino acid residue or Asparagine, X'.sub.2,
X'.sub.3, and X'.sub.6 are each any amino acid residue, X'4 is a
hydrophobic amino acid residue, and X'.sub.5 and X'.sub.7 are each
a charged amino acid residue; wherein n in Formula I and II is
greater than or equal to 2; and wherein, in each heptad repeat, the
first CC comprises an X.sub.5 residue that is opposite in charge to
the X'.sub.7 residue in the second CC and the first CC comprises an
X.sub.7 residue that is opposite in charge to the X'.sub.5 residue
in the second CC.
12. The antibody of claim 11, wherein the first polypeptide
comprises a VH and CHI domain.
13. The antibody of claim 12, wherein the first polypeptide further
comprises a hinge domain.
14. The antibody of claim 12, wherein the first polypeptide further
comprises a CH2 and a CH3 domain.
15. The antibody of claim 11, wherein the first polypeptide
comprises VH, CH I, hinge, CH2, and CH3 domains positioned relative
to each other in an N-terminal to C-terminal direction: VH-CH
1-hinge-CH2-CH3.
16. The antibody of claim 11, wherein the antibody further
comprises a third polypeptide, wherein the third polypeptide
comprises a VL domain.
17. The antibody of claim 16, wherein said third polypeptide
further comprises a CL domain, and the VL and CL domains are
positioned relative to each other in an N-terminal to C-terminal
direction: VL-CL.
18. The antibody of claim 11, wherein the N-terminus of the VH of
said first polypeptide is connected to the C-terminus of a CL with
a tether.
19. The antibody of claim 1, wherein said hydrophobic amino acid
residue in any of Xi, X'I, X4, and X'.sub.4 is selected from the
group consisting of Alanine, Valine, Leucine, Isoleucine,
Tryptophan, Phenylalanine, and Methionine.
20. The antibody of claim 1, wherein said charged amino acid
residue in any of X.sub.5, X'.sub.5) X.sub.7, and X'.sub.7 is
selected from the group consisting of Lysine, Arginine, Histidine,
Aspartic Acid, and Glutamic Acid.
21. The antibody of claim 1, wherein, in at least one heptad repeat
of said first CC, Xi is Asparagine, and wherein the respective X' 1
is Asparagine in at least one heptad repeat of said second CC.
22. The antibody of claim 1, wherein (a) the first CC comprises a
heptad repeat wherein Xi is Leucine or Asparagine, X.sub.2 is
Alanine or Glutamine, X.sub.3 is Alanine or Glutamine, X.sub.4 is
Leucine, X.sub.5 is Glutamic Acid, X is Lysine or Tryptophan, and
X.sub.7 is Glutamic Acid; and (b) the second CC comprises a heptad
repeat wherein X' i is Leucine or Asparagine, X'.sub.2 is Alanine
or Glutamine, X'.sub.3 is Alanine or Glutamine, X'.sub.4 is
Leucine, X'.sub.5 is Lysine, X'.sub.6 is Lysine or Tryptophan, and
X'.sub.7 is Lysine.
23. The antibody of claim 1, wherein n is greater than or equal to
3.
24. The antibody of claim 23, wherein n is greater than or equal to
4.
25. The antibody of claim 1, wherein at least one of said first or
said second CC is linked C-terminal to a constant domain of the
antibody.
26. The antibody of claim 25, wherein said constant domain is a CH3
domain and the first CC is linked C-terminal to a CH3 domain of the
first polypeptide and the second CC is linked C-terminal to a CH3
domain of the second polypeptide.
27. The antibody of claim 25, wherein linkage is by a cleavable
linker sequence.
28. The antibody of claim 1, wherein a Lys-C endopeptidase cleavage
site is located N-terminal to at least one of said first or said
second CC.
29. An antibody comprising a first polypeptide comprising a VL, CL,
tether, VH, CH1, CH2, and CH3 domain positioned relative to each
other in an N-terminal to C-terminal direction:
VL-CL-tether-VH-CH1-CH2-CH3 (Formula III).
30. The antibody of claim 29, wherein said antibody further
comprises a second polypeptide of Formula EL
31. The antibody of claim 1, wherein the antibody is
multispecific.
32. The antibody of claim 31, wherein the antibody is capable of
binding at least 2 antigens.
33. The antibody of claim 31, wherein the antibody a capable of
binding at least 2 epitopes on the same antigen.
34. The antibody of claim 1, wherein said antibody is
bispecific.
35. The antibody of claim 7, wherein said tether comprises Glycine
(G) and Serine (S) residues.
36. The antibody of claim 7, wherein said tether is between 15 and
50 amino acids in length.
37. The antibody of claim 36, wherein said tether is between 20 and
26 amino acids in length.
38. The antibody of claim 7, wherein said tether comprises GGS
repeats.
39. The antibody of claim 7, wherein said tether is cleavable.
40. The antibody of claim 28, wherein said antibody comprises a
mutation that removes a Lys-C endopeptidase cleavage site.
41. The antibody of claim 40, wherein said mutation that removes a
Lys-C endopeptidase cleavage site is in a hinge domain.
42. The antibody of claim 41, wherein said antibody has a K222A
substitution (EU numbering system).
43. The antibody of claim 27, wherein said tether or said linker is
cleavable by one or more of the following endopeptidases: Furin,
Thrombin, Genenase, Lys-C, Arg-C, Asp-N, Glu-C, Factor Xa, Tobacco
Etch Virus Protease (TEV), Enterokinase, Human Rhinovirus C3
protease (HRV C3), and Kininogenase.
44. The antibody of claim 27, wherein said tether or said linker
comprises an Asparagine-Glycine peptide bond.
45. The antibody of claim 44, wherein said Asparagine-Glycine
peptide bond is cleavable by hydroxylamine.
46. The antibody of claim 1, wherein said antibody comprises a
constant region conjugated to a cytotoxic agent.
47. The antibody of claim 1, wherein said antibody is expressed by
a mammalian cell.
48. The antibody of claim 47, wherein said mammalian cell is a CHO
cell.
49. The antibody of claim 1, wherein said antibody is expressed by
a prokaryotic cell.
50. The antibody of claim 49, wherein said prokaryotic cell is an
E. coli cell.
51. A method of producing an antibody, said method comprising the
step of culturing a cell comprising a vector encoding the antibody
of claim 1 in a culture medium.
52. The method of claim 51, wherein said method further comprises
recovering said antibody from said cell or said culture medium.
53. The method of claim 52, further comprising the steps of (a)
capturing said antibody on a column comprising Protein A, (b)
eluting said antibody from said column, and (c) diluting said
eluted antibody into a solution containing a chaotropic agent or
mild detergent.
54. A method of maintaining a coiled coil containing antibody in
solution, said method comprising maintaining said antibody in the
presence of a chaotropic agent or mild detergent.
55. The method of claim 53, wherein said chaotropic agent or mild
detergent is Arginine, Guanidine-HCl, urea, lithium perchlorate,
Histidine, Sodium Dodecyl Sulfate (SDS), Tween, Triton, or NP-40.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel engineered proteins,
multispecific protein complexes, including multispecific
antibodies, methods of constructing them and producing them. This
invention also relates to the new application of technologies
useful in obtaining the multispecific protein complexes.
BACKGROUND OF THE INVENTION
[0002] Finding technologies for building multispecific antibodies
that are useful and scalable for commercial and therapeutic
purposes has been elusive. Many methods have been tried, but nearly
all suffer significant drawbacks such as being poorly soluble;
inexpressible in mammalian cells, demonstrating low yield of
heterodimer formation, technically challenging to manufacture,
immunogenic, short half-life in vivo, unstable among other problems
(e.g., Hollinger et al., (1993) PNAS 90:6444-6448; U.S. Pat. Nos.
5,932,448; 6,833,441; 5,591,828; 7,129,330; 7,507,796; Fischer et
al., (2007) Pathobiology 74:3-14; Booy (2006) Arch. Immunol. Ther.
Exp. 54:85-101; Cao et al (2003) 55:171-197; and Marvin et al.,
(2006) Current Opinion in Drug Discovery & Development
9(2):184-193. Thus, there is a need for improved technologies and
processes to make multispecific antibodies.
SUMMARY OF THE INVENTION
[0003] The present invention provides novel protein complexes and
methods of creating and manufacturing protein complexes. In one
aspect, the invention involves a coiled coil domain that is linked
to an Fc CH component, which coiled coil domain may or may not be
cleavable from the Fc containing protein if desired. In another
aspect, the invention involves a protein comprising a tether and an
Fc CH component complex, which tether may or may not be cleavable
from the protein. In another aspect, the invention involves a
protein comprising a coiled coil, a tether and an Fc CH component,
optimally able to form a protein complex, which tether and/or
coiled coil may or may not be cleavable from the protein depending
on the desired effect. In another aspect, the invention provides a
process of preparing the protein comprising a tether, wherein the
tether is cleaved by a host cell or cleaved by a chemical or
enzymatic reaction in vitro. In another aspect, the invention
involves a protein comprising a coiled coil, a tether and an Fc CH
component, optimally able to form a protein complex, which tether
and/or coiled coil are cleavable from the protein by a host cell
that expresses the protein and overexpresses enzymes capable of
cleaving the tether and/or coiled coil from the protein.
[0004] In another aspect, the invention provides a process of
making a protein or protein complex comprising a coiled coil and a
tether, wherein the tether and/or the coiled coil is cleaved by a
host cell or cleaved by a chemical or enzymatic reaction in vitro.
In one specific embodiment the protein complex further comprises an
Fc CH component. In another aspect, the invention involves a method
for manufacturing a heteromeric protein complex comprising the step
of culturing a host cell under conditions that express two
different proteins from the same or different recombinant nucleic
acid sequences, wherein each protein comprises a coiled coil domain
and a tether. In a further embodiment, the host cell comprises a
recombinant nucleic acid sequence encoding an enzyme capable of
cleaving the tether and/or the coiled coil. In one embodiment, the
manufacturing method further comprises the step of isolating the
proteins made by the host cell. In another embodiment, the
manufacturing method further comprises the step of cleaving the
tether and/or the coild coil from a protein produced by the host
cell.
[0005] In another aspect, the invention involves the protein
complexes described herein with or without the tether and/or the
coiled coil. In addition to the many advances and advantages
provided herein, the invention provides a simple, efficient, high
yield production process for manufacturing substantially homogenous
heteromultimeric complexes.
[0006] In one preferred embodiment, the present invention provides
a protein complex comprising two or more polypeptides, wherein
[0007] a first polypeptide comprises a first coiled coil domain
(CC) and a first Fc CH component (FcCH); and
[0008] a second polypeptide comprises (1) a second coiled coil
domain (CC) and a second FcCH,
[0009] wherein the first CC and the second CC complex with each
other; and the first FcCH and second FcCH complex with each
other.
[0010] In one embodiment, the first CC comprises the sequence of
Formula I herein and the second CC comprises the sequence of
Formula II herein.
[0011] In a second aspect, the invention features a protein complex
comprising (a) a first polypeptide comprising a first coiled coil
domain (CC), where the first CC comprises a heptad repeat of
Formula I; and (b) a second polypeptide comprising an Fc CH
component and a second coiled coil (CC), where the second CC
comprises a heptad repeat of Formula where n in Formula I and II is
greater than or equal to 2, and where, in each heptad repeat, the
first CC comprises an X.sub.5 residue that is opposite in charge to
the X'.sub.7 residue in the second CC and the first CC comprises an
X.sub.7 residue that is opposite in charge to the X'.sub.5 residue
in the second CC.
[0012] In one embodiment, the first polypeptide further comprises a
VH domain and a VL domain and the second polypeptide further
comprises a VH and VL domain, wherein the VH and VL domains of each
polypeptide are linked to each other in the N-terminal to
C-terminal order: VL-CL-tether-VH.
[0013] In a further embodiment, the VH domain of each polypeptide
is different from each other. In another embodiment, the VL domain
of each polypeptide is different from each other.
[0014] In one embodiment, the protein complex of this invention
comprises a hinge region, wherein the hinge region comprises a
K222A mutation in its hinge region, a C220A mutation in its hinge
region or a K222A and a C220A mutation in its hinge region.
[0015] In one embodiment, the protein complex is selected from the
group consisting of an antibody, an immunoadhesin, a peptibody or
an affibody. Thus, according to a further embodiment, the first
and/or second polypeptides can further comprise a target binding
sequence of an antibody (e.g., VH or VL domain), peptibody (e.g.,
peptide), immunoadhesin (e.g., extracellular domain) or a scaffold
protein comprising a sequence that binds the target.
[0016] According to one embodiment, the protein complex is a one
armed antibody.
[0017] In one aspect, the invention provides a protein complex
comprising a coiled coil comprising (a) a first polypeptide
comprising a first coiled coil domain (CC), where the first CC
comprises a heptad repeat of Formula I:
TABLE-US-00001 (SEQ ID NO: 29) (X.sub.1 X.sub.2 X.sub.3 X.sub.4
X.sub.5 X.sub.6 X.sub.7).sub.n (Formula I)
[0018] X.sub.1 is a hydrophobic amino acid residue or Asparagine,
[0019] X.sub.2, X.sub.3, and X.sub.6 are each any amino acid
residue, [0020] X.sub.4 is a hydrophobic amino acid residue, and
[0021] X.sub.5 and X.sub.7 are each a charged amino acid residue;
and (b) a second polypeptide comprising a second coiled coil domain
(CC), where the second CC comprises a heptad repeat of Formula
II:
TABLE-US-00002 [0021] (SEQ ID NO: 30) (X'.sub.1 X'.sub.2 X'.sub.3
X'.sub.4 X'.sub.5 X'.sub.6 X'.sub.7).sub.n (Formula II)
[0022] X'.sub.1 is a hydrophobic amino acid residue or Asparagine,
[0023] X'.sub.2, X'.sub.3, and X'.sub.6 are each any amino acid
residue, [0024] X'.sub.4 is a hydrophobic amino acid residue, and
[0025] X'.sub.5 and X'.sub.7 are each a charged amino acid residue;
where n in Formula I and II is greater than or equal to 2; and
where, in each heptad repeat, the first CC comprises an X.sub.5
residue that is opposite in charge to the X'.sub.7 residue in the
second CC and the first CC comprises an X.sub.7 residue that is
opposite in charge to the X'.sub.5 residue in the second CC.
[0026] In an embodiment, the first and second polypeptides each
comprise a VH and a CH1 domain, and may each further comprise a
hinge domain. In another embodiment, the first and second
polypeptides each further comprise a CH2 and a CH3 domain. In yet
another embodiment, the first and second polypeptides each comprise
VH, CH1, hinge, CH2, and CH3 domains positioned relative to each
other in an N-terminal to C-terminal direction:
VH-CH1-hinge-CH2-CH3.
[0027] In one aspect, the invention provides an antibody comprising
(a) a first polypeptide comprising a VH domain and a first coiled
coil domain (CC), where the first CC comprises a heptad repeat of
Formula I:
(X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7).sub.n (Formula
I) [0028] X.sub.1 is a hydrophobic amino acid residue or
Asparagine, [0029] X.sub.2, X.sub.3, and X.sub.6 are each any amino
acid residue, [0030] X.sub.4 is a hydrophobic amino acid residue,
and [0031] X.sub.5 and X.sub.7 are each a charged amino acid
residue; and (b) a second polypeptide comprising a VH domain and a
second coiled coil domain (CC), where the second CC comprises a
heptad repeat of Formula II:
[0031]
(X'.sub.1X'.sub.2X'.sub.3X'.sub.4X'.sub.5X'.sub.6X'.sub.7).sub.n
(Formula II) [0032] X'.sub.1 is a hydrophobic amino acid residue or
Asparagine, [0033] X'.sub.2, X'.sub.3, and X'.sub.6 are each any
amino acid residue, [0034] X'.sub.4 is a hydrophobic amino acid
residue, and [0035] X'.sub.5 and X'.sub.7 are each a charged amino
acid residue; where n in Formula I and II is greater than or equal
to 2; and where, in each heptad repeat, the first CC comprises an
X.sub.5 residue that is opposite in charge to the X'.sub.7 residue
in the second CC and the first CC comprises an X.sub.7 residue that
is opposite in charge to the X'.sub.5 residue in the second CC.
[0036] In an embodiment, the first and second polypeptides each
comprise a VH and a CH1 domain, and may each further comprise a
hinge domain. In another embodiment, the first and second
polypeptides each further comprise a CH2 and a CH3 domain. In yet
another embodiment, the first and second polypeptides each comprise
VH, CH1, hinge, CH2, and CH3 domains positioned relative to each
other in an N-terminal to C-terminal direction:
VH-CH1-hinge-CH2-CH3.
[0037] In a particular embodiment the antibody further comprises a
third and a fourth polypeptide, where the third polypeptide
comprises a first VL domain and the fourth polypeptide comprises a
second VL domain. In an embodiment, the VH domain of the first
polypeptide is linked to the VL domain of the third polypeptide by
a tether and the VH domain of the second polypeptide is linked to
the VL domain of the fourth polypeptide by a tether. In another
embodiment, the third polypeptide further comprises a first CL
domain where the first VL and CL domains are positioned relative to
each other within the third polypeptide in an N-terminal to
C-terminal direction: VL-CL, and the fourth polypeptide further
comprises a second CL domain, and where the second VL and CL
domains are positioned relative to each other within the fourth
polypeptide in an N-terminal to C-terminal direction: VL-CL.
[0038] In an additional embodiment, the sequences of the first VL
domain and the second VL domain are the same. In a further
embodiment, the N-terminus of the VH of at least one of the first
or the second polypeptides is connected to the C-terminus of a CL
with a tether.
[0039] In a second aspect, the invention features an antibody
comprising (a) a first polypeptide comprising a VH domain and a
first coiled coil domain (CC), where the first CC comprises a
heptad repeat of Formula I; and (b) a second polypeptide comprising
a CH2 and CH3 domain and a second coiled coil (CC), where the
second CC comprises a heptad repeat of Formula II, where n in
Formula I and II is greater than or equal to 2, and where, in each
heptad repeat, the first CC comprises an X.sub.5 residue that is
opposite in charge to the X'.sub.7 residue in the second CC and the
first CC comprises an X.sub.7 residue that is opposite in charge to
the X'.sub.5 residue in the second CC.
[0040] In one embodiment of the second aspect of the invention, the
first polypeptide comprises a VH and CH1 domain, and may further
comprise a hinge domain. In another embodiment, the first
polypeptide further comprises a CH2 and a CH3 domain. In a further
embodiment of the second aspect of the invention, the first
polypeptide comprises. VH, CH1, hinge, CH2, and CH3 domains
positioned relative to each other in an N-terminal to C-terminal
direction: VH-CH1-hinge-CH2-CH3. In yet another embodiment of the
second aspect of the invention, the antibody further comprises a
third polypeptide, where the third polypeptide comprises a VL
domain. In one example, the third polypeptide further comprises a
CL domain, and the VL and CL domains are positioned relative to
each other in an N-terminal to C-terminal direction: VL-CL. In yet
another embodiment of the second aspect of the invention, the
N-terminus of the VH of the first polypeptide is connected to the
C-terminus of a CL with a tether.
[0041] In one embodiment, a two armed antibody of this invention
comprises one, not two tethers such that the antibody comprises (1)
a polypeptide comprising a coiled coil domain and a heavy chain
tethered to a light chain according to this invention, (2) a
polypeptide comprising a coiled coil domain and a heavy chain and
(3) a polypeptide comprising a light chain. In another embodiment,
a host cell that expresses such two armed antibody is
contemplated.
[0042] In other embodiments, the hydrophobic amino acid residue in
any of X.sub.1, X'.sub.1, X.sub.4, and X'.sub.4 is selected from
the group Alanine, Valine, Leucine, Isoleucine, Tryptophan,
Phenylalanine, and Methionine. In another embodiment, the charged
amino acid residue in any of X.sub.5, X'.sub.5, X.sub.7, and
X'.sub.7 is selected from the group Lysine, Arginine, Histidine,
Aspartic Acid, and Glutamic Acid. In a further embodiment, in at
least one heptad repeat of the first CC, X.sub.1 is Asparagine, and
the respective X', is Asparagine in at least one heptad repeat of
the second CC.
[0043] In yet other embodiment, the first CC comprises a heptad
repeat where X.sub.1 is Leucine or Asparagine, X.sub.2 is Alanine
or Glutamine, X.sub.3 is Alanine or Glutamine, X.sub.4 is Leucine,
X.sub.5 is Glutamic Acid, X.sub.6 is Lysine or Tryptophan, and
X.sub.7 is Glutamic Acid; and the second CC comprises a heptad
repeat where X', is Leucine or Asparagine, X'.sub.2 is Alanine or
Glutamine, X'.sub.3 is Alanine or Glutamine, X'.sub.4 is Leucine,
X'.sub.5 is Lysine, X'.sub.6 is Lysine or Tryptophan, and X'.sub.7
is Lysine.
[0044] In further embodiments, n in Formula I and II is greater
than or equal to 3, for example, greater than or equal to 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.
[0045] In additional embodiments, at least one of the first or the
second CC is linked C-terminal to a constant domain of the protein.
For example, the constant domain is a CH3 domain and the first CC
is linked C-terminal to a CH3 domain of the first polypeptide and
the second CC is linked C-terminal to a CH3 domain of the second
polypeptide. The linkage, for example, is by a cleavable linker
sequence. In other embodiments, a Lys-C endopeptidase cleavage site
is located N-terminal to at least one of the first or the second
CC.
[0046] In another aspect, the invention features an antibody
comprising a first polypeptide comprising a VL, CL, tether, VH,
CH1, CH2, and CH3 domain positioned relative to each other in an
N-terminal to C-terminal direction: VL-CL-tether-VH-CH1-CH2-CH3
(Formula In one embodiment, the antibody further comprises a second
polypeptide of Formula III.
[0047] In a particular embodiment, the antibody of the invention is
multispecific. For example, the antibody is capable of binding at
least 2 antigens, or the antibody a capable of binding at least 2
epitopes on the same antigen. In another embodiment, the antibody
is bispecific.
[0048] In an additional embodiment, the proteins of this invention
comprise a tether comprising Glycine (G) and Serine (S) residues.
In one embodiment, the tether, for example, is between 15 and 50
amino acids in length. In a particular embodiment, the tether is
between 20 and 32 amino acids in length, for example, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids in length. The
tether, in one embodiment, comprises GGS repeats. In another
embodiment, the tether is cleavable. In one preferred embodiment,
the tether is cleavable in two sites at or near the N and C
terminus of the tether by the same enzyme. In one embodiment, the
tether comprises the cleavage site for furin. In a further
embodiment, the furin cleavage site is RXRXRR (SEQ ID NO:25),
wherein X is any amino acid.
[0049] In a further embodiment, the antibody of the invention
comprises a mutation that removes a Lys-C endopeptidase cleavage
site. In one example, the mutation that removes a Lys-C
endopeptidase cleavage site is in a hinge domain. For instance, the
antibody has a K222A substitution (EU numbering system).
[0050] In another embodiment, the tether or the linker is cleavable
by one or more of the following endopeptidases: Furin, Thrombin,
Genenase, Lys-C, Arg-C, Asp-N, Glu-C, Factor Xa, Tobacco Etch Virus
Protease (TEV), Enterokinase, Human Rhinovirus C3 protease (HRV
C3), or Kininogenase. In a particular embodiment, the tether or the
linker comprises an Asparagine-Glycine peptide bond, for example, a
Asparagine-Glycine peptide bond that is cleavable by
hydroxylamine.
[0051] In one embodiment, an antibody of the invention further
comprises mutations in a CL/CH1 and or in a VH/VL interface using
KnH technology. In one embodiment, a multispecific antibody of this
invention was constructed using a coiled coil of this invention and
a knob and hole at a CL/CH1 interface.
[0052] In an additional embodiment, the antibody of the invention
comprises a constant region conjugated to a cytotoxic agent.
[0053] In yet another embodiment, the antibody of the invention is
expressed by eukaryotic cell, for example, a mammalian cell such as
a CHO cell. In an alternative embodiment, the antibody is expressed
by a prokaryotic cell, for example, an E. coli cell.
[0054] In a further aspect, the invention features method for
producing a protein complex, such as an antibody. Accordingly, the
invention provides several new aspects. In one embodiment, this
method comprises the step of culturing a cell comprising a vector
encoding a protein of this invention in a culture medium. In one
embodiment, the method further comprises recovering the protein
from the cell or the culture medium. In another embodiment, the
method further comprises the steps of (a) capturing the antibody on
a column comprising Protein A, (b) eluting the antibody from the
column, and (c) diluting the eluted antibody into a solution
containing a chaotropic agent or mild detergent.
[0055] In yet another aspect, the invention features a method of
maintaining a coiled coil containing antibody in solution. This
method comprises maintaining the antibody in the presence of a
chaotropic agent or mild detergent. Examples, of chaotropic agents
or mild detergents that may be used in this method include
Arginine, Guanidine-HCl, urea, lithium perchlorate, Histidine,
Sodium Dodecyl Sulfate (SDS), Tween, Triton, and NP-40.
[0056] In one embodiment, a heteromultimeric complex of this
invention binds to two or more target molecules. In another
embodiment, each polypeptide in the heteromultimeric complex binds
to a different target molecule. In yet another embodiment, the
heteromultimeric complex inhibits the biological activity of the
target molecule(s) to which it binds. In one embodiment, when a
desired biological effect is to bring a cell to be depleted or
inactivated in close proximity to an effector cell (e.g., T
lymphocyte, natural killer cell (NK), macrophage or other
mononuclear cells, one of the target molecules can be CD3, CD16, or
CD64.
[0057] According to one embodiment, a heteromultimeric complex of
this invention binds to at least two target molecules selected from
the group consisting of: IL-1alpha and IL-1beta, IL-12 and IL-18;
IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4;
IL-13 and IL-1beta; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC;
IL-13 and MEF; IL-13 and TGF-.beta.; IL-13 and LHR agonist; IL-12
and TWEAK, IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b;
IL-13 and ADAMS, IL-13 and PED2, IL17A and IL17F, CD3 and CD19,
CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38
and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and
IL-6; CD20 and BR3, TNFalpha and TGF-beta, TNFalpha and IL-1beta;
TNFalpha and IL-2, TNF alpha and IL-3, TNFalpha and IL-4, TNFalpha
and IL-5, TNFalpha and IL6, TNFalpha and IL8, TNFalpha and IL-9,
TNFalpha and IL-10, TNFalpha and IL-11, TNFalpha and IL-12,
TNFalpha and IL-13, TNFalpha and IL-14, TNFalpha and IL-15,
TNFalpha and IL-16, TNFalpha and IL-17, TNFalpha and IL-18,
TNFalpha and IL-19, TNFalpha and IL-20, TNFalpha and IL-23,
TNFalpha and IFNalpha, TNFalpha and CD4, TNFalpha and VEGF,
TNFalpha and MIF, TNFalpha and ICAM-1, TNFalpha and PGE4, TNFalpha
and PEG2, TNFalpha and RANK ligand, TNFalpha and Te38; TNFalpha and
BAFF; TNFalpha and CD22; TNFalpha and CTLA-4; TNFalpha and GP130;
TNF.alpha. and IL-12p40; VEGF and HER2, VEGF-A and HER2, VEGF-A and
PDGF, HER1 and HER2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and
DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, VEGFR and
EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3;
EGFR and HER2, EGFR and HER3, EGFR and HER4, IL-13 and CD40L, IL4
and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R,
EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A;
CTLA-4 and BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B; MAG and RGM A;
NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-I and CTLA-4;
and RGM A and RGM B.
[0058] In a further embodiment, the invention features an isolated
antibody comprising a first heavy chain comprising the sequence of
SEQ ID NO:1, a second heavy chain comprising the sequence of SEQ ID
NO:2, and a light chain comprising the sequence of SEQ ID NO:3,
where the antibody specifically binds Fc.epsilon.R1 and
Fc.gamma.R2b.
[0059] In another embodiment, the invention features an isolated
antibody comprising a first heavy chain comprising the sequence of
SEQ ID NO:4, a second heavy chain comprising the sequence of SEQ ID
NO:5, and a light chain comprising the sequence of SEQ ID NO:6,
where the antibody specifically binds HER2.
[0060] In yet another embodiment, the invention features an
isolated antibody comprising a first heavy chain comprising the
sequence of SEQ ID NO:7, a second heavy chain comprising the
sequence of SEQ ID NO:5, and a light chain comprising the sequence
of SEQ ID NO:8, where the antibody specifically binds EGFR.
[0061] In an additional embodiment, the invention features an
isolated antibody comprising a first light chain sequence and a
first heavy chain sequence comprising the sequence of SEQ ID NO:9,
and a second light chain sequence and a second heavy chain sequence
comprising the sequence of SEQ ID NO:10, where the antibody
specifically binds HER2 and EGFR.
[0062] In a further embodiment, the invention features an isolated
antibody comprising a first light chain sequence and a first heavy
chain sequence comprising the sequence of SEQ ID NO:11, and a
second light chain sequence and a second heavy chain sequence
comprising the sequence of SEQ ID NO:10, where the antibody
specifically binds HER2 and EGFR.
[0063] The invention also features use of antibodies made according
to the methods described herein in methods of treatment. In one
embodiment the invention features use of an antibody that
specifically binds Fc.epsilon.R1 and Fc.gamma.R2b in a method of
treating an allergic or inflammatory response (e.g., an autoimmune
disease) in a subject. This method includes administering an
antibody or antibody fragment to a subject for a time and in an
amount sufficient to treat the allergic or inflammatory respone in
the subject. In another embodiment, the invention features use of
an antibody that specifically binds HER2 or EGFR (or both HER2 and
EGFR) in a method of treating a tumor in a subject. This method
includes administering an antibody or antibody fragment to a
subject for a time and in an amount sufficient to treat the tumor
in the subject.
[0064] In particular embodiments, the methods of treatment
described herein involve the use of an antibody fragment that lacks
a coiled coil and/or a tether. In this embodiment, the coiled coil
and/or tether sequences are cleaved from the antibody following
production and the resultant engineered antibody used for
therapeutic administration. In further embodiments, the methods of
treatment involve administering to the subject an effective amount
of a second drug. The second drug may contain another antibody or
antibody fragment, a chemotherapeutic agent, a cytotoxic agent, an
anti-angiogenic agent, an immunosuppressive agent, a prodrug, a
cytokine, a cytokine antagonist, cytotoxic radiotherapy, a
corticosteroid, an anti-emetic, a cancer vaccine, an analgesic, or
a growth-inhibitory agent. The second drug can be administered
prior or subsequent to the administration of the first drug (e.g.,
the antibody or antibody fragment). In another embodiment, the
second drug is administered concurrently with the first drug.
[0065] In additional embodiments, the invention features an
isolated polynucleotide encoding the sequence of any one of SEQ ID
NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 17-18, 26, 31-32 or 35-36 or
a combination thereof, a vector comprising a polynucleotide
including the sequence of any one of SEQ ID NO:1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 17-18, 26, 31-32 or 35-36 or a combination thereof,
and a host cell comprising such a vector. The host cell can be a
eukaryotic cell, such as a yeast, insect, or mammalian cell. In one
emboditment the mammalian cell is a Chinese Hamster Ovary (CHO
cell). The host cell can also be a prokaryotic cell, such as an E.
coli cell. In other embodiments, the invention features an isolated
polypeptide comprising any one of the sequence of SEQ ID NO:1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 17-18, 26, 31-32 or 35-36 or a
combination thereof.
[0066] Other features and advantages of the invention will be
apparent from the following Detailed Description, the Drawings, and
the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] FIG. 1 is a schematic diagram showing ionic and hydrophobic
interactions between amino acids in an exemplary coiled coil (CC)
structure. The residues in the first CC are labeled X.sub.1 through
X.sub.7 and the residues in the second CC are labeled X'.sub.1
through X'.sub.7. Ionic interactions between the X.sub.5 residue of
the first CC and the X'.sub.7 residue of the second CC and the
X.sub.7 residue of the first CC and the X'.sub.5 residue of the
second CC are indicated. In addition, hydrophobic interactions
between the X.sub.4 and X'.sub.4 and X.sub.1 and X', residues are
shown.
[0068] FIG. 2A shows the amino acid sequences of the exemplary
ACID.p1 (SEQ ID NO:12) and BASE.p1 (SEQ ID NO:13) coiled coil
heterodimerization domains and DNA sequences encoding them (SEQ ID
NO:21 and SEQ ID NO:22, respectively).
[0069] FIG. 2B is a schematic diagram showing interactions between
the exemplary ACID.p1 and BASE.p1 coiled coil heterodimerization
domains and DNA sequences SEQ ID NO:21 and SEQ ID NO:22,
respectively.
[0070] FIG. 3 is a schematic diagram showing the structure of an
exemplary bispecific antibody containing a common light chain
(common LC), a heterodimeric coiled coil, and a mutation in the
hinge region (K222A; Kabat numbering system) of the first and
second heavy chains (HC1 and HC2) that removes a Lys-C
endopeptidase cleavage site.
[0071] FIG. 4A is a schematic diagram showing the structure of an
exemplary one-armed antibody containing a full-length heavy chain
(HC1), a partial heavy chain (HC2) lacking the VH and CH1 domains,
a light chain (common LC), a heterodimeric coiled coil, and a
mutation in the hinge region (K222A) of HC1 that removes a Lys-C
endopeptidase cleavage site.
[0072] FIG. 4B is a schematic diagram showing the structure of an
exemplary conjugated antibody containing two full-length heavy
chains, a common light chain, a coiled coil, and a cytotoxic agent
conjugated to one of the heavy chain constant regions. The
cytotoxic agent is indicated by the star.
[0073] FIG. 5 is a schematic diagram showing the structure of an
exemplary tethered bispecific antibody. The antibody contains two
heavy chains (HC1 and HC2) and two light chains (LC1 and LC2). A
tether links the N-terminus of the variable heavy chain of HC1 with
the C-terminus of the constant light chain of LC1 and a second
tether links the N-terminus of the variable heavy chain of HC2 with
the C-terminus of the constant light chain of LC2. In this example,
the tethers include Glycine Glycine Serine (GGS) repeats. In this
figure, the light chains (LC1 and LC2) are different, but a
tethered antibody could also contain a common light chain. The
exemplary tethered antibody further contains a heterodimeric coiled
coil and a mutation in the hinge region (K222A) of HC1 and HC2 that
removes a Lys-C endopeptidase cleavage site.
[0074] FIG. 6 is a schematic diagram showing the structure of an
exemplary heavy chain (HC) and light chain (LC), as well as an
exemplary tether linking the N-terminus of the variable heavy chain
with the C-terminus of the constant light chain. In this example,
the distance spanned by the tether is approximately 92 .ANG., or
approximately 22 amino acids in length. Tethers of 20, 23, and 26
amino acids in length were tested.
[0075] FIG. 7A is a schematic diagram showing the structure of an
exemplary antibody containing cleavable tethers and a heterodimeric
coiled coil. As indicated in the figure, the exemplary tether links
the C-terminus of the light chain (LC) to the N-terminus of the
heavy chain (HC). The tether can be cleaved from the antibody at
cleavage sites (X) using, for example, Lys-C endopeptidase, Furin
(PC1), or NH.sub.2OH (hydroxylamine). The exemplary cleavage sites
are located at the N- and C-termini of the tether. The exemplary
antibody shown in in FIG. 7A also contains a heterodimeric coiled
coil, which can be cleaved from the antibody at cleavage sites (X)
N-terminal to the coiled coil domains using, for example, Lys-C
endopeptidase, Furin (PC1), or NH.sub.2OH.
[0076] FIG. 7B is a series of schematic diagrams showing exemplary
cleavable tethers. The top diagram shows an exemplary 26 amino acid
tether sequence (SEQ ID NO:17) in SEQ ID NO:31 that can be cleaved
by Furin and links the N-terminus of the light chain (LC) and the
C-terminus of the heavy chain (HC). Furin can cleave the tether
sequence at di-basic sites (Arginine-Arginine) at the N- and
C-termini of the tether. The bottom diagram shows an exemplary 26
amino acid tether sequence (SEQ ID NO:18) in SEQ ID NO:32 that can
be cleaved by Lys-C endopeptidase at Lysine residues at the N- and
C-termini of the tether sequence.
[0077] FIG. 8 shows the sequences of the heavy chains (HC;
Anti-Fc.gamma.R2b--BASE.p1 sequence and Anti-Fc.epsilon.R1-ACID.p1
sequence) and common light chain (4d5 LC) of a bispecific antibody
that binds to both Fc.epsilon.R1 and Fc.gamma.R2b. The
Anti-Fc.gamma.R2b--BASE.p1 sequence (SEQ ID NO:1) contains the
heavy chain sequence of anti-human Fc.gamma.R2b with a BASE.p1
coiled coil heterodimerization domain sequence and K222A mutation
in the hinge region. The Anti-Fc.epsilon.R1-ACID.p1 sequence (SEQ
ID NO:2) contains the heavy chain sequence of anti-human
Fc.epsilon.R1 with an ACID.p1 coiled coil heterodimerization domain
sequence and K222A mutation in the hinge region. The 4d5 antibody
light chain (SEQ ID NO:3) is common to both the Fc.gamma.R2b and
Fc.epsilon.R1HCs of this bispecific antibody.
[0078] FIGS. 9-1 and 9-2 are the sequences of used to generate
exemplary one-armed antibodies. One exemplary one-armed antibody
specifically binds HER2 and contains the Anti-HER2 antibody
1.ACID.p1 sequence (Anti-HER2 antibody 1 HC with an ACID.p1 coiled
coiled heterodimerization domain sequence and K222A mutation; SEQ
ID NO:4), the truncFC.BASE.p1 sequence (a heavy chain lacking the
VH and CH1 domains with a BASE.p1 coiled coil heterodimerization
domain sequence; SEQ ID NO:5), and the anti-HER2 antibody 1 LC
sequence (SEQ ID NO:6). Another exemplary one-armed antibody
specifically binds EGFR and contains the Anti-EGFR (D1.5).ACID.p1
sequence (anti-EGFR (D1.5) HC with an ACID.p1 coiled coiled
heterodimerization domain sequence and K222A mutation in the hinge
region; SEQ ID NO:7), the truncFC.BASE.p1 sequence (a heavy chain
lacking the VH and CH1 domains with a BASE.p1 coiled coil
heterodimerization domain sequence; SEQ ID NO:5), and anti-EGFR
(D1.5) antibody LC sequence (SEQ ID NO:8).
[0079] FIG. 10 shows the sequences of the tethered HC and LC
(Anti-HER2 (antibody 1)26.ACID.p1 and D1.5.26.BASE.p1) of a
bispecific antibody that binds both HER2 and EGFR/HER1. The
Anti-HER2 (antibody 1)26.ACID.p1 sequence contains the anti-HER2
antibody 1 LC sequence tethered to the anti-HER2 antibody 1 HC
sequence by a 26 amino acid Glycine Glycine Serine (GGS) tether
with an ACID.p1 coiled coil heterodimerization domain and K222A
mutation (SEQ ID NO:9). The D1.5.26.BASE.p1 sequence contains the
D1.5 anti-EGFR antibody LC sequence tethered to the D1.5 anti-EGFR
antibody HC sequence by a 26 amino acid GGS tether with a BASE.p1
coiled coil heterodimerization domain and K222A mutation (SEQ ID
NO:10).
[0080] FIG. 11 shows the sequences of the tethered HC and LC
(anti-HER2 (antibody 2).26.ACID.p1 and D1.5.26.BASE.p1) of another
exemplary antibody that binds both HER2 and EGFR/HER1. The
anti-HER2 (antibody 2).26.ACID.p1 sequence contains the anti-HER2
antibody 2 LC sequence tethered to the anti-HER2 antibody 2 HC
sequence by a 26 amino acid GGS tether with a ACID.p1 coiled coil
heterodimerization domain and K222A mutation (SEQ ID NO:11). The
D1.5.26.BASE.p1 sequence contains the D1.5 anti-EGFR antibody LC
sequence tethered to the D1.5 anti-EGFR antibody HC sequence by a
26 amino acid GGS tether with a BASE.p1 coiled coil
heterodimerization domain and K222A mutation (SEQ ID NO:10).
[0081] FIGS. 12A-1 and 12A-2 and 12B-1, 12B-2, and 12B-3 are
partial HC (SEQ ID NO:15) and LC (SEQ ID NO:16) amino acid
sequences and DNA sequences SEQ ID NO:23 and SEQ ID NO:24,
respectively of the anti-HER2 antibody 1 used to construct coiled
coil heterodimerization domain containing antibodies. The start of
the anti-HER2 antibody 1 HC sequence is indicated in FIG. 12A, as
is the location of the K222A mutation in the sequence. The start of
the anti-HER2 antibody 1 variable light chain (VL), the end of the
anti-HER2 antibody 1 LC, the start of the anti-HER2 antibody 1
variable heavy chain (VH), the end of the anti-HER2 antibody 1 VH,
and the location of the K to A mutation is indicated in FIG. 12B.
The locations of ClaUBsp106, BamH1, and ApaI restriction sites
useful in constructing vectors containing these sequences are also
indicated in FIGS. 12A and 12B.
[0082] FIGS. 13A and 13B are a series of graphs of mass
spectrometry results and schematic diagrams showing that the
heterodimeric coiled coil can be cleaved from an exemplary
.alpha.-Fc.epsilon.R1/.alpha.-Fc.gamma.R2b bispecific antibody
using Lys-C endopeptidase. The theoretical masses of the antibody
with the coiled coil (left diagram) and the antibody without the
coiled coil (right diagram) are indicated and are within the margin
of error of the experimentally observed masses indicated in the
graphs of the mass spectrometry results above the respective
diagram, showing that the coiled coil was cleaved from the
antibody.
[0083] FIGS. 14A and 14B are a series of graphs of mass
spectrometry results and schematic diagrams showing that Lys-C
endopeptidase (right panels) does not cleave within the LC or HC of
an exemplary .alpha.-Fc.epsilon.R1/.alpha.-Fc.gamma.R2b bispecific
antibody, but does cleave the coiled coil from the HCs (comparison
of left two bottom panels and right two bottom panels). The
theoretical masses of the light chain (MW=26263), the heavy chain
with a coiled coil domain (MW=54917 or 55164), and the heavy chain
without a coiled coil domain (MW=50528 and 50767) are within the
margin of error of the experimentally observed masses indicated in
the graph of the mass spectrometry results for the respective
construct.
[0084] FIG. 15 is a series of graphs showing that an exemplary
.alpha.-Fc.epsilon.R1/.alpha.-Fc.gamma.R2b bispecific antibody
specifically and simultaneously binds both of its antigens.
[0085] FIG. 16 is a graph showing the results for a histamine
release assay with an exemplary common LC
.alpha.-Fc.epsilon.R1/.alpha.-Fc.gamma.R2b bispecific antibody. The
concentration of the antibody used in the assay (in .mu.g/ml) is
indicated along the x-axis and the amount in histamine release (in
ng/ml) is indicated along the y-axis.
[0086] FIGS. 17A and 17B are a series of graphs of mass
spectrometry results and schematic diagrams showing that the coiled
coil can be cleaved from an exemplary one-armed .alpha.-EGFR
antibody using Lys-C endopeptidase. The theoretical masses of the
one-armed antibody with a coiled coil (MW=109112), and the
one-armed antibody without a coiled coil (MW=100419) are within the
margin of error of the experimentally observed masses indicated in
the graph of the mass spectrometry results for the respective
construct.
[0087] FIGS. 18A, 18B, and 18C are a series of graphs of mass
spectrometry results and schematic diagrams showing that Lys-C
endopeptidase does not cleave the LC (One-armed Light Chain; left
panels), full-length HC (One-armed Heavy Chain; middle panels), or
HC lacking the VH and CH1 domains (One-armed Fc; right panels) of
an exemplary .alpha.-EGFR antibody, but does cleave the coiled coil
domain from the HC and the HC lacking the VH and CH1 domains. The
theoretical molecular mass for the respective constructs is
indicated below the graph showing the mass spectrometry results
and, in each case, is within the margin of error of the
experimentally observed molecular mass.
[0088] FIGS. 19A and 19B are a series of graphs of mass
spectrometry results and schematic diagrams showing that the coiled
coil can be cleaved from an exemplary tethered
.alpha.-EGFR/.alpha.-HER2 bispecific antibody using Lys-C
endopeptidase. The theoretical molecular mass of the cleaved and
uncleaved antibodies is also indicated in the figure and is within
the margin of error of the respective experimentally observed
molecular mass indicated in the mass spectrometry results.
[0089] FIGS. 20A and 20B are a series of graphs of mass
spectrometry results and schematic diagrams showing that the coiled
coil can be cleaved from an exemplary tethered
.alpha.-EGFR/.alpha.-HER2 bispecific antibody using Lys-C
endopeptidase where the antibody has first been treated with Lys-C
endopeptidase and the sample then subjected to mass spectrometry
analysis. The theoretical molecular masses of the cleaved and
uncleaved HC/LC complexes are also indicated in the figure and the
theoretical molecular mass for each construct is within the margin
of error of the experimentally observed molecular mass shown in the
mass spectrometry results.
[0090] FIG. 21 is a graph showing the results from an Octet
analysis indicating that the wild-type anti-HER2 antibody 1 and
wild-type .alpha.-EGFR antibody do not cross react with each
other's antigen, but do bind their respective antigen.
[0091] FIG. 22 is a graph showing the results from an Octet
analysis indicating that the one-armed anti-HER2 antibody 1 and
one-armed .alpha.-EGFR antibody do not cross react with each
other's antigen, but do bind their respective antigen.
[0092] FIG. 23A is a graph showing the results from an Octet
analysis indicating that the exemplary tethered bispecific
Anti-HER2 antibody 1/.alpha.-EGFR antibody (8323) binds both HER2
and EGFR simultaneously. In the top trace, the antibody was first
incubated with the EGFR extracellular domain (ECD) and then with
the HER2 receptor ECD and in the bottom trace, the antibody was
first incubated with the HER2 receptor ECD and then with the EGFR
ECD.
[0093] FIG. 23B is a series of graphs showing the binding
affinities of an exemplary bispecific Anti-HER2 antibody
1/.alpha.-EGFR antibody for HER2 (top) and EGFR1 (bottom).
[0094] FIG. 24 is an image of immunoblots showing that the
exemplary bispecific Anti-HER2 antibody 1/.alpha.-EGFR (D1.5)
antibody inhibits transforming growth factor alpha (TGF.alpha.)
mediated EGFR (epidermal growth factor receptor) phosphorylation in
a dose dependent manner in EGFR expressing NR6 cells (left side).
The D1.5 anti-EGFR antibody is used as a control (right side).
Phosphorylation levels are determined using an
anti-phospho-tyrosine (.alpha.-pTyr) antibody and an anti-tubulin
antibody (.alpha.-tubulin) is used as a loading control.
[0095] FIG. 25 is a series of graphs showing that the bispecific
Anti-HER2 antibody 1/.alpha.-EGFR(D1.5) antibody inhibits
TGF.alpha.-induced growth, over a three-day period, in NR6 cells
that are stably transfected to express EGFR.
[0096] FIG. 26 is a graph showing that the exemplary bispecific
Anti-HER2 antibody 1/.alpha.-EGFR(D1.5) antibody inhibits growth of
HER2 amplified BT474 cells over a five-day period in a manner
similar to the anti-HER2 antibody 1 control.
[0097] FIG. 27 is a series of graphs showing Fc-Fc assay and Fc-Fc
ELISA assay results of a ten-day pharmacokinetics (PK) analysis of
the D1.5 human IgG1 control antibody (anti-EGFR) using SCID Beige
mice.
[0098] FIGS. 28A and 28B are a series of graphs showing
EGFR-HER2ELISA and Fc-Fc ELISA assay results of a ten-day PK
analysis of the exemplary bispecific Anti-HER2 antibody
1/.alpha.-EGFR(D1.5) antibody using SCID Beige mice.
[0099] FIG. 29 is a graph showing a comparison of the exposure of
the exemplary bispecific Anti-HER2 antibody 1/.alpha.-EGFR(D1.5)
antibody to the control D1.5 (anti-EGFR) and control (anti-HER2
antibody 2) antibodies in mice. The exemplary bispecific Anti-HER2
antibody 1/.alpha.-EGFR(D1.5) antibody has an exposure in mice over
the tested time period that is similar to the control
antibodies.
[0100] FIGS. 30A-1 and 30A-2, 30B-1 and 30B-2, 30C-1, 30C-2, 30C-3,
30C-4, and 30C-5, 30D-1, 30D-2, and 30D-3 are mass spectroscopy
graphs showing the cleavage products of the heavy chain and the
light chain of an antibody after cleavage by furin by a cell
co-expressing furin.
[0101] FIG. 31 is a non-reduced mass spectroscopy graph showing a
bispecific antibody made by expressing a furin-cleavable, tethered
coiled-coil antibody in a CHO cell that coexpressed furin and
exposing the antibody to carboxypeptidase digestion.
[0102] FIG. 32(A) and (B) is a reduced mass spectroscopy graph
showing a bispecific antibody made by expressing a furin-cleavable,
tethered coiled-coil antibody in a CHO cell that coexpressed furin
and exposing the antibody to carboxypeptidase digestion.
DETAILED DESCRIPTION
[0103] Without being bound by theory, applicants believe that the
coiled coil dimerization domains described herein provide the
initial trigger that drives the binding of two or more molecules
together with a high degree of accuracy and efficiency surprisingly
even in the presence of Fc regions of an immunoglobulin, which Fc
regions are also naturally attracted to each other under cell
culture conditions.
[0104] By reducing homodimerization of heavy chains, use of the
coiled coil heterodimerization domains described herein provides a
breakthrough in the ability to produce a homogeneous population of
protein complexes comprising a Fc CH component (e.g., multispecific
or one-armed antibodies, etc.). Multispecific complexes are
advantageous for use in therapeutic applications because, for
example, they can direct the co-localization of a target (e.g., a
tumor cell) and an agent directed against the target (e.g., a T
cell) or they can eliminate the need for combination therapy and
the risk associated with providing two or more therapeutics to a
subject. Further, to facilitate the construction of antibodies,
including multispecific antibodies, tethers according to the
present invention can be used to link the light and heavy chains of
an antibody and thereby aid in the proper association of each light
chain to its cognate heavy chain.
I. DEFINITIONS
[0105] The term "antibody" herein is used in the broadest sense and
refers to any immunoglobulin (Ig) molecule comprising two heavy
chains and two light chains, and any fragment, mutant, variant or
derivation thereof which so long as they exhibit the desired
biological activity (e.g., epitope binding activity). Examples of
antibodies include monoclonal antibodies, polyclonal antibodies,
multispecific antibodies and antibody fragments.
[0106] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g,
Kabat et al., Sequences of Inzmunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra). The "EU index as in Kabat" refers to the residue numbering
of the human IgG1 EU antibody. Unless stated otherwise herein,
references to residue numbers in the variable domain of antibodies
means residue numbering by the Kabat numbering system. Unless
stated otherwise herein, references to residue numbers in the heavy
chain constant domain of antibodies means residue numbering by the
EU numbering system.
[0107] The term "multispecific antibody" is used in the broadest
sense and specifically covers an antibody that has polyepitopic
specificity. Such multispecific antibodies include, but are not
limited to, an antibody comprising a heavy chain variable domain
(V.sub.H) and a light chain variable domain (V.sub.L), where the
V.sub.HV.sub.L unit has polyepitopic specificity, antibodies having
two or more V.sub.L and V.sub.H domains with each V.sub.HV.sub.L
unit binding to a different epitope, antibodies having two or more
single variable domains with each single variable domain binding to
a different epitope, full length antibodies, antibody fragments
such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and
triabodies, antibody fragments that have been linked covalently or
non-covalently. "Polyepitopic specificity" refers to the ability to
specifically bind to two or more different epitopes on the same or
different target(s). "Monospecific" refers to the ability to bind
only one epitope. According to one embodiment the multispecific
antibody is an IgG antibody that binds to each epitope with an
affinity of 5 .mu.M to 0.001 pM, 3 .mu.M to 0.001 pM, 1 .mu.M to
0.001 pM, 0.5 .mu.M to 0.001 pM, or 0.1 .mu.M to 0.001 pM.
[0108] A naturally occurring basic 4-chain antibody unit is a
heterotetrameric glycoprotein composed of two identical light (L)
chains and two identical heavy (H) chains (an IgM antibody consists
of 5 of the basic heterotetramer units along with an additional
polypeptide called J chain, and therefore contains 10 antigen
binding sites, while secreted IgA antibodies can polymerize to form
polyvalent assemblages comprising 2-5 of the basic 4-chain units
along with J chain). In the case of IgGs, the 4-chain unit is
generally about 150,000 daltons. Each L chain is linked to an H
chain by one covalent disulfide bond, while the two H chains are
linked to each other by one or more disulfide bonds depending on
the H chain isotype. Each H and L chain also has regularly spaced
intrachain disulfide bridges. Each H chain has, at the N-terminus,
a variable domain (V.sub.H) followed by three constant domains
(C.sub.H) for each of the .alpha. and .gamma. chains and four
C.sub.H domains for .mu. and .epsilon. isotypes. Each L chain has,
at the N-terminus, a variable domain (V.sub.L) followed by a
constant domain (C.sub.L) at its other end. The V.sub.L is aligned
with the V.sub.H and the C.sub.L is aligned with the first constant
domain of the heavy chain (C.sub.H1). Particular amino acid
residues are believed to form an interface between the light chain
and heavy chain variable domains. The pairing of a V.sub.H and
V.sub.L together forms a single antigen-binding site. For the
structure and properties of the different classes of antibodies,
see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P.
Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton &
Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
[0109] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .gamma., .epsilon., and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0110] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a beta-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the beta-sheet
structure. The hypervariable regions in each chain are held
together in close proximity by the FRs and, with the hypervariable
regions from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). The constant
domains are not involved directly in binding an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody dependent cellular
cytotoxicity (ADCC).
[0111] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0112] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00003 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0113] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0114] "Framework regions" (FR) are those variable domain residues
other than the CDR residues. Each variable domain typically has
four FRs identified as FR1, FR2, FR3, and FR4. If the CDRs are
defined according to Kabat, the light chain FR residues are
positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88
(LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are
positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94
(HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the
CDRs comprise amino acid residues from hypervariable loops, the
light chain FR residues are positioned about at residues 1-25
(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the
light chain and the heavy chain FR residues are positioned about at
residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113
(HCFR4) in the heavy chain residues. In some instances, when the
CDR comprises amino acids from both a CDR as defined by Kabat and
those of a hypervariable loop, the FR residues will be adjusted
accordingly. For example, when CDRH1 includes amino acids H26-H35,
the heavy chain FR1 residues are at positions 1-25 and the FR2
residues are at positions 36-49.
[0115] A "human consensus framework" is a framework that represents
the most commonly occurring amino acid residues in a selection of
human immunoglobulin VL or VH framework sequences. Generally, the
selection of human immunoglobulin VL or VH sequences is from a
subgroup of variable domain sequences. Generally, the subgroup of
sequences is a subgroup as in Kabat. In one embodiment, for the VL,
the subgroup is subgroup kappa I as in Kabat. In one embodiment,
for the VH, the subgroup is subgroup III as in Kabat.
[0116] One example of an "intact" antibody is one that comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2, and C.sub.H3. The constant
domains can be native sequence constant domains (e.g., human native
sequence constant domains) or amino acid sequence variant
thereof.
[0117] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or a variable region of
the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2, and Fv fragments; diabodies (Db); tandem
diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870,
Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995));
one-armed antibodies, single variable domain antibodies,
minibodies, single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments (e.g., including but not
limited to, Db-Fc, taDb-Fc, taDb-CH3 and (scFV)-4-Fc).
[0118] The expression "single domain antibodies" (sdAbs) or "single
variable domain (SVD) antibodies" generally refers to antibodies in
which a single variable domain (VH or VL) can confer antigen
binding. In other words, the single variable domain does not need
to interact with another variable domain in order to recognize the
target antigen. Examples of single domain antibodies include those
derived from camelids (lamas and camels) and cartilaginous fish
(e.g., nurse sharks) and those derived from recombinant methods
from humans and mouse antibodies (Nature (1989) 341:544-546; Dev
Comp Immunol (2006) 30:43-56; Trend Biochem Sci (2001) 26:230-235;
Trends Biotechnol (2003):21:484-490; WO 2005/035572; WO 03/035694;
Febs Lett (1994) 339:285-290; WO00/29004; WO 02/051870).
[0119] The expression "linear antibodies" generally refers to the
antibodies described in Zapata et al., Protein Eng. 8(10):1057-1062
(1995). Briefly, these antibodies comprise a pair of tandem Fd
segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0120] The term "knob-into-hole" or "KnH" technology as mentioned
herein refers to the technology directing the pairing of two
polypeptides together in vitro or in vivo by introducing a
pertuberance (knob) into one polypeptide and a cavity (hole) into
the other polypeptide at an interface in which they interact. For
example, KnHs have been introduced in the Fc:Fc binding interfaces,
CL:CH1 interfaces or VH/VL interfaces of antibodies (e.g.,
US2007/0178552, WO 96/027011, WO 98/050431 and Zhu et al. (1997)
Protein Science 6:781-788). This is especially useful in driving
the pairing of two different heavy chains together during the
manufacture of multispecific antibodies. For example, multispecific
antibodies having KnH in their Fc regions can further comprise
single variable domains linked to each Fc region, or further
comprise different heavy chain variable domains that pair with
similar or different light chain variable domains. KnH technology
can be also be used to pair two different receptor extracellular
domains together or any other polypeptide sequences that comprises
different target recognition sequences (e.g., including affibodies,
peptibodies and other Fc fusions). Papain digestion of antibodies
produces two identical antigen-binding fragments, called "Fab"
fragments, and a residual "Fc" fragment, a designation reflecting
the ability to crystallize readily. The Fab fragment consists of an
entire L chain along with the variable region domain of the H chain
(V.sub.H), and the first constant domain of one heavy chain
(C.sub.H1). Pepsin treatment of an antibody yields a single large
F(ab').sub.2 fragment which roughly corresponds to two disulfide
linked Fab fragments having divalent antigen-binding activity and
is still capable of cross-linking antigen. Fab' fragments differ
from Fab fragments by having additional few residues at the carboxy
terminus of the C.sub.H1 domain including one or more cysteines
from the antibody hinge region. Fab'-SH is the designation herein
for Fab' in which the cysteine residue(s) of the constant domains
bear a free thiol group. F(ab').sub.2 antibody fragments originally
were produced as pairs of Fab' fragments which have hinge cysteines
between them. Other chemical couplings of antibody fragments are
also known.
[0121] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region; this
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0122] "Fv" consists of a dimer of one heavy- and one light-chain
variable region domain in tight, non-covalent association. From the
folding of these two domains emanate six hypervariable loops (3
loops each from the H and L chain) that contribute the amino acid
residues for antigen binding and confer antigen binding specificity
to the antibody. However, even a single variable domain (or half of
an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although often at a lower
affinity than the entire binding site.
[0123] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains, which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Malmborg et al., J. Immunol. Methods 183:7-13, 1995.
[0124] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).
[0125] The term "one-armed antibody" or "one-armed antibodies"
refers to an antibody that comprises (1) a variable domain joined
by a peptide bond to a polypeptide comprising a CH2 domain, a CH3
domain or a CH2-CH3 domain and (2) a second CH2, CH3 or CH2-CH3
domain, wherein a variable domain is not joined by a peptide bond
to a polypeptide comprising the second CH2, CH3 or CH2-CH3 domain.
In one embodiment, the one-armed antibody comprises 3 polypeptides
(1) a first polypeptide comprising a variable domain (e.g., VH),
CH1, CH2 and CH3, (2) a second polypeptide comprising a variable
domain (e.g., VL) and a CL domain, and (3) a third polypeptide
comprising a CH2 and CH3 domain. In an embodiment, the third
polypeptide does not comprise a variable domain. In another
embodiment, the one-armed antibody has a partial hinge region
containing the two cysteine residues which form disulphide bonds
linking the constant heavy chains. In one embodiment, the variable
domains of the one armed antibody form an antigen binding region.
In another embodiment, a variable domain of the one armed antibody
is a single variable domain, wherein each single variable domain is
an antigen binding region.
[0126] Antibodies of the invention can be "chimeric" antibodies in
which a portion of the heavy and/or light chain is identical with
or homologous to corresponding sequences in antibodies derived from
a particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, provided that they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; and
Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
Chimeric antibodies of interest herein include primatized
antibodies comprising variable domain antigen-binding sequences
derived from a non-human primate (e.g., Old World Monkey, Ape,
etc.) and human constant region sequences.
[0127] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0128] "Complex" or "complexed" as used here in refers to the
association of two or more molecules that interact with each other
through bonds and/or forces (e.g., van der waals, hydrophobic,
hydrophilic forces) that are not peptide bonds. In one embodiment,
the complex is heteromultimeric. It should be understood that the
term "protein complex" or "polypeptide complex" as used herein
includes complexes that have a non-protein entity conjugated to a
protein in the protein complex (e.g., including, but not limited
to, chemical molecules such as a toxin or a detection agent).
[0129] The term "heteromultimer" or "heteromultimeric" as used
herein describes two or more polypeptides that interact with each
other by a non-peptidic, covalent bond (e.g., disulfide bond)
and/or a non-covalent interaction (e.g., hydrogen bonds, ionic
bonds, Van der Waals forces, and hydrophobic interactions), wherein
at least two of the molecules have different sequences from each
other.
[0130] As used herein, the term "immunoadhesin" designates
molecules which combine the binding specificity of a heterologous
protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with a desired binding
specificity, which amino acid sequence is other than the antigen
recognition and binding site of an antibody (i.e., is
"heterologous" compared to a constant region of an antibody), and
an immunoglobulin constant domain sequence (e.g., CH2 and/or CH3
sequence of an IgG). Exemplary adhesin sequences include contiguous
amino acid sequences that comprise a portion of a receptor or a
ligand that binds to a protein of interest. Adhesin sequences can
also be sequences that bind a protein of interest, but are not
receptor or ligand sequences (e.g., adhesin sequences in
peptibodies). Such polypeptide sequences can be selected or
identified by various methods, include phage display techniques and
high throughput sorting methods. The immunoglobulin constant domain
sequence in the immunoadhesin can be obtained from any
immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA
(including IgA-1 and IgA-2), IgE, IgD, or IgM.
[0131] An antibody of this invention "which binds" an antigen of
interest is one that binds the antigen with sufficient affinity
such that the antibody is useful as a diagnostic and/or therapeutic
agent in targeting a protein or a cell or tissue expressing the
antigen, and does not significantly cross-react with other
proteins. In such embodiments, the extent of binding of the
antibody to a "non-target" protein will be less than about 10% of
the binding of the antibody to its particular target protein as
determined by fluorescence activated cell sorting (FACS) analysis
or radioimmunoprecipitation (RIA) or ELISA. With regard to the
binding of an antibody to a target molecule, the term "specific
binding" or "specifically binds to" or is "specific for" a
particular polypeptide or an epitope on a particular polypeptide
target means binding that is measurably different from a
non-specific interaction (e.g., a non-specific interaction may be
binding to bovine serum albumin or casein). Specific binding can be
measured, for example, by determining binding of a molecule
compared to binding of a control molecule. For example, specific
binding can be determined by competition with a control molecule
that is similar to the target, for example, an excess of
non-labeled target. In this case, specific binding is indicated if
the binding of the labeled target to a probe is competitively
inhibited by excess unlabeled target. The term "specific binding"
or "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a particular polypeptide target as
used herein can be exhibited, for example, by a molecule having a
Kd for the target of at least about 200 nM, alternatively at least
about 150 nM, alternatively at least about 100 nM, alternatively at
least about 60 nM, alternatively at least about 50 nM,
alternatively at least about 40 nM, alternatively at least about 30
nM, alternatively at least about 20 nM, alternatively at least
about 10 nM, alternatively at least about 8 nM, alternatively at
least about 6 nM, alternatively at least about 4 nM, alternatively
at least about 2 nM, alternatively at least about 1 nM, or greater.
In one embodiment, the term "specific binding" refers to binding
where a molecule binds to a particular polypeptide or epitope on a
particular polypeptide without substantially binding to any other
polypeptide or polypeptide epitope.
[0132] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd). For
example, the Kd can be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM,
40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or
stronger. Affinity can be measured by common methods known in the
art, including those described herein. Low-affinity antibodies
generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and
tend to remain bound longer. A variety of methods of measuring
binding affinity are known in the art, any of which can be used for
purposes of the present invention.
[0133] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by using surface plasmon resonance assays
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CM5
chips at .about.10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 .mu.g/ml (0.2 .mu.M) before injection at a flow rate of 5
.mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to
500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at
25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (k.sub.on) and dissociation rates (k.sub.off) are
calculated using a simple one-to-one Langmuir binding model
(BIAcore Evaluation Software version 3.2) by simultaneous fitting
the association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999). If the on-rate exceeds 10.sup.6 M.sup.-1
S.sup.-1 by the surface plasmon resonance assay above, then the
on-rate can be determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red
cuvette.
[0134] An "on-rate" or "rate of association" or "association rate"
or "k.sub.on" according to this invention can also be determined
with the same surface plasmon resonance technique described above
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CM5
chips at -10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate
of 5 .mu.l/minute to achieve approximately 10 response units (RU)
of coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to
500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at
25.degree. C. at a flow rate of approximately 25 .mu.l/min.
Association rates (k.sub.on) and dissociation rates (k.sub.on) are
calculated using a simple one-to-one Langmuir binding model
(BIAcore Evaluation Software version 3.2) by simultaneous fitting
the association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen et al., J. Mol. Biol.
293:865-881 (1999). However, if the on-rate exceeds 10.sup.6
M.sup.-1 S.sup.-1 by the surface plasmon resonance assay above,
then the on-rate is preferably determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophotometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stirred cuvette.
[0135] "Biologically active" and "biological activity" and
"biological characteristics" with respect to a polypeptide of this
invention, such as an antibody, fragment, or derivative thereof,
means having the ability to bind to a biological molecule, except
where specified otherwise.
[0136] "Peptibody" or "peptibodies" refers to a fusion of randomly
generated peptides with an Fc domain. See U.S. Pat. No. 6,660,843,
issued Dec. 9, 2003 to Feige et al. (incorporated by reference in
its entirety). They include one or more peptides linked to the
N-terminus, C-terminus, amino acid sidechains, or to more than one
of these sites. Peptibody technology enables design of therapeutic
agents that incorporate peptides that target one or more ligands or
receptors, tumor-homing peptides, membrane-transporting peptides,
and the like. Peptibody technology has proven useful in design of a
number of such molecules, including linear and
disulfide-constrained peptides, "tandem peptide multimers" (i.e.,
more than one peptide on a single chain of an Fc domain). See, for
example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156,
published Oct. 16, 2003 (corresponding to WO 02/092620, published
Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18,
2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S.
Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO
00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023,
published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003;
U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003
(corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser.
No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329,
published Apr. 1, 2004), each of which is hereby incorporated by
reference in its entirety.
[0137] "Affibodies" or "Affibody" refers to the use of a protein
liked by peptide bond to an Fc region, wherein the protein is used
as a scaffold to provide a binding surface for a target molecule.
The protein is often a naturally occurring protein such as
staphylococcal protein A or IgG-binding B domain, or the Z protein
derived therefrom (see Nilsson et al (1987), Prot Eng 1, 107-133,
and U.S. Pat. No. 5,143,844) or a fragment or derivative thereof.
For example, affibodies can be created from Z proteins variants
having altered binding affinity to target molecule(s), wherein a
segment of the Z protein has been mutated by random mutagenesis to
create a library of variants capable of binding a target molecule.
Examples of affibodies include U.S. Pat. No. 6,534,628, Nord K et
al, Prot Eng 8:601-608 (1995) and Nord K et al, Nat Biotech
15:772-777 (1997). Biotechnol Appl Biochem. 2008 June; 50(Pt
2):97-112.
[0138] "Isolated" heteromultimer or complex means a heteromultimer
or complex which has been separated and/or recovered from a
component of its natural cell culture environment. Contaminant
components of its natural environment are materials which would
interfere with diagnostic or therapeutic uses for the
heteromultimer, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the heteromultimer will be purified (1) to greater
than 95% by weight of protein as determined by the Lowry method,
and most preferably more than 99% by weight, (2) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence by use of a spinning cup sequenator, or (3) to
homogeneity by SDS-PAGE under reducing or nonreducing conditions
using Coomassie blue or, preferably, silver stain.
[0139] The heteromultimers of the present invention are generally
purified to substantial homogeneity. The phrases "substantially
homogeneous", "substantially homogeneous form" and "substantial
homogeneity" are used to indicate that the product is substantially
devoid of by-products originated from undesired polypeptide
combinations (e.g. homomultimers).
[0140] Expressed in terms of purity, substantial homogeneity means
that the amount of by-products does not exceed 10%, 9%, 8%, 7%, 6%,
4%, 3%, 2% or 1% by weight or is less than 1% by weight. In one
embodiment, the by-product is below 5%.
[0141] "Biological molecule" refers to a nucleic acid, a protein, a
carbohydrate, a lipid, and combinations thereof. In one embodiment,
the biologic molecule exists in nature.
[0142] "Isolated," when used to describe the various antibodies
disclosed herein, means an antibody that has been identified and
separated and/or recovered from a cell or cell culture from which
it was expressed. Contaminant components of its natural environment
are materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and can include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the antibody will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes antibodies in situ within recombinant
cells, because at least one component of the polypeptide natural
environment will not be present. Ordinarily, however, isolated
polypeptide will be prepared by at least one purification step.
[0143] By "linked" or "links" as used herein is meant either a
direct peptide bond linkage between a first and second amino acid
sequence or a linkage that involves a third amino acid sequence
that is peptide bonded to and between the first and second amino
acid sequences. For example, a linker peptide bonded to the
C-terminal end of one amino acid sequence and to the N-terminal end
of the other amino acid sequence.
[0144] By "linker" as used herein is meant an amino acid sequence
of two or more amino acids in length. The linker can consist of
neutral polar or nonpolar amino acids. A linker can be, for
example, 2 to 100 amino acids in length, such as between 2 and 50
amino acids in length, for example, 3, 5, 10, 15, 20, 25, 30, 35,
40, 45, or 50 amino acids in length. A linker can be "cleavable,"
for example, by auto-cleavage, or enzymatic or chemical cleavage.
Cleavage sites in amino acid sequences and enzymes and chemicals
that cleave at such sites are well known in the art and are also
described herein.
[0145] By a "tether" as used herein is meant an amino acid linker
that joins two other amino acid sequences. A tether as described
herein can link the N-terminus of an immunoglobulin heavy chain
variable domain with the C-terminus of an immunoglobulin light
chain constant domain. In particular embodiments, a tether is
between about 15 and 50 amino acids in length, for example, between
20 and 26 amino acids in length (e.g., 20, 21, 22, 23, 24, 25, or
26 amino acids in length). A tether may be "cleavable," for
example, by auto-cleavage, or enzymatic or chemical cleavage using
methods and reagents standard in the art.
[0146] Enzymatic cleavage of a "linker" or a "tether" may involve
the use of an endopeptidase such as, for example, Lys-C, Asp-N,
Arg-C, V8, Glu-C, chymotrypsin, trypsin, pepsin, papain, thrombin,
Genenase, Factor Xa, TEV (tobacco etch virus cysteine protease),
Enterokinase, HRV C3 (human rhinovirus C3 protease), Kininogenase,
as well as subtilisin-like proprotein convertases (e.g., Furin
(PC1), PC2, or PC3) or N-arginine dibasic convertase. Chemical
cleavage may involve use of, for example, hydroxylamine,
N-chlorosuccinimide, N-bromosuccinimide, or cyanogen bromide.
[0147] A "Lys-C endopeptidase cleavage site" as used herein is a
Lysine residue in an amino acid sequence that can be cleaved at the
C-terminal side by Lys-C endopeptidase. Lys-C endopeptidase cleaves
at the C-terminal side of a Lysine residue.
[0148] By a "heptad repeat" as used herein is meant a sequence of 7
consecutive amino acids that are repeated at least once in an amino
acid sequence. The heptad repeats may be arranged consecutively in
the amino acid sequence with the C-terminus of the first repeat
being immediately adjacent to the N-terminus of the second repeat.
In one embodiment, the heptad repeat has the sequence of Formula I
or Formula II as defined herein.
[0149] By a "coiled coil domain," "coiled coil heterodimerization
domain," "coil," or "coil heterodimerization domain" as used herein
is meant an amino acid sequence that forms an alpha-helical
structure that can interact with a second alpha-helical structure
(a second "coiled coil domain") to form a "coiled coil" or
"heterodimeric coiled coil." The alpha helical structures may be
right-handed alpha helices. In one embodiment, the alpha helical
structures are made up of heptad repeats. In one particular
example, the coil coil domain has a structure as shown in FIG. 1
where residues at the "X.sub.1" and "X.sub.1'" of a first and a
second alpha helical structure form hydrophobic interactions with
each other, residues at the "X.sub.4" and "X.sub.4'" positions of
the first and the second alpha helical structure form hydrophobic
interactions with each other, residues at the "X.sub.5" positions
of the first alpha helical structure form ionic interactions with
residues at the "X.sub.7'" position of the second alpha helical
structure, and residues at the "X.sub.7" positions of the first
alpha helical structure form ionic interactions with residues at
the "X.sub.5'" position of the second alpha helical structure. The
coiled coil domain may be made up of 2 or more heptad repeats of
Formula I or Formula II as defined herein.
[0150] By a "hydrophobic residue" is meant Alanine, Valine,
Leucine, Isoleucine, Tryptophan, Phenylalanine, Proline, or
Methionine. In a particular embodiment, the hydrophobic residue is
not Proline.
By a "charged residue" is meant an acidic or basic amino acid.
Lysine, Arginine, and Histidine are basic amino acids, and Aspartic
Acid and Glutamic Acid are acidic amino acids.
[0151] By a "chaotropic agent" is meant a water-soluble substance
which disrupts the three-dimensional structure of a protein (e.g.,
an antibody) by interfering with stabilizing intra-molecular
interactions (e.g., hydrogen bonds, van der Waals forces, or
hydrophobic effects). Exemplary chaotropic agents include, but are
not limited to, urea, Guanidine-HCl, lithium perchlorate,
Histidine, and Arginine.
[0152] By a "mild detergent" is meant a water-soluble substance
which disrupts the three-dimensional structure of a protein (e.g.,
an antibody) by interfering with stabilizing intra-molecular
interactions (e.g., hydrogen bonds, van der Waals forces, or
hydrophobic effects), but which does not permanently disrupt the
protein structure as to cause a loss of biological activity (i.e.,
does not denature the protein). Exemplary mild detergents include,
but are not limited to, Tween (e.g., Tween-20), Triton (e.g.,
Triton X-100), NP-40 (nonyl phenoxylpolyethoxylethanol), Nonidet
P-40 (octyl phenoxylpolyethoxylethanol), and Sodium Dodecyl Sulfate
(SDS).
[0153] "Hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206
(1985)). Hinge regions of other IgG isotypes may be aligned with
the IgG1 sequence by placing the first and last cysteine residues
forming inter-heavy chain S--S bonds in the same positions.
[0154] The "lower hinge region" of an Fc region is normally defined
as the stretch of residues immediately C-terminal to the hinge
region, i.e. residues 233 to 239 of the Fc region. Prior to the
present invention, FcgammaR binding was generally attributed to
amino acid residues in the lower hinge region of an IgG Fc
region.
[0155] The "CH2 domain" of a human IgG Fc region usually extends
from about residues 231 to about 340 of the IgG. The CH2 domain is
unique in that it is not closely paired with another domain.
Rather, two N-linked branched carbohydrate chains are interposed
between the two CH2 domains of an intact native IgG molecule. It
has been speculated that the carbohydrate may provide a substitute
for the domain-domain pairing and help stabilize the CH2 domain.
Burton, Molec. Immunol. 22:161-206 (1985).
[0156] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e. from about amino
acid residue 341 to about amino acid residue 447 of an IgG).
[0157] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue.
[0158] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down
regulation of cell surface receptors (e.g. B cell receptor; BCR),
etc. Such effector functions generally require the Fc region to be
combined with a binding domain (e.g., an antibody variable domain)
and can be assessed using various assays as disclosed, for example,
in definitions herein.
[0159] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. Native sequence human Fc regions include a native
sequence human IgG1 Fc region (non-A and A allotypes); native
sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0160] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g. from about
one to about ten amino acid substitutions, and preferably from
about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith.
[0161] "Fc complex" as used herein refers to two CH2 domains of an
Fc region interacting together and/or two CH3 domains of an Fc
region interacting together, wherein the CH2 domains and/or the CH3
domains interact through bonds and/or forces (e.g., van der waals,
hydrophobic, hydrophilic forces) that are not peptide bonds.
[0162] "Fc component" as used herein refers to a hinge region, a
CH2 domain or a CH3 domain of an Fc region.
[0163] "Fc CH component" or "FcCH" as used here in refers to a
polypeptide comprising a CH2 domain, a CH3 domain, or CH2 and CH3
domains of an Fc region.
[0164] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0165] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxic
agents. The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 can be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest can be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA
95:652-656 (1998).
[0166] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one that binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0167] "Human effector cells" are leukocytes that express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes that mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells, and neutrophils; with PBMCs and NK
cells being preferred. The effector cells can be isolated from a
native source, e.g., from blood.
[0168] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) that are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be
performed.
[0169] The term "therapeutically effective amount" refers to an
amount of an antibody, antibody fragment, or derivative to treat a
disease or disorder in a subject. In the case of tumor (e.g., a
cancerous tumor), the therapeutically effective amount of the
antibody or antibody fragment (e.g., a multispecific antibody or
antibody fragment) may reduce the number of cancer cells; reduce
the primary tumor size; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
disorder. To the extent the antibody or antibody fragment may
prevent growth and/or kill existing cancer cells, it may be
cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo
can, for example, be measured by assessing the duration of
survival, time to disease progression (TTP), the response rates
(RR), duration of response, and/or quality of life.
[0170] By "reduce or inhibit" is meant the ability to cause an
overall decrease preferably of 20% or greater, more preferably of
50% or greater, and most preferably of 75%, 85%, 90%, 95%, or
greater. Reduce or inhibit can refer to the symptoms of the
disorder being treated, the presence or size of metastases, the
size of the primary tumor, or the size or number of the blood
vessels in angiogenic disorders.
[0171] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Included in this
definition are benign and malignant cancers. Examples of cancer
include but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers
include squamous cell cancer, small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of
the lung, cancer of the peritoneum, hepatocellular cancer, gastric
or stomach cancer including gastrointestinal cancer, pancreatic
cancer, glioblastoma, glioma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, breast cancer, colon
cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer (e.g., renal cell
carcinoma), liver cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, and various types of head and neck cancer. By "early
stage cancer" is meant a cancer that is not invasive or metastatic
or is classified as a Stage 0, I, or II cancer. The term
"precancerous" refers to a condition or a growth that typically
precedes or develops into a cancer. By "non-metastatic" is meant a
cancer that is benign or that remains at the primary site and has
not penetrated into the lymphatic or blood vessel system or to
tissues other than the primary site. Generally, a non-metastatic
cancer is any cancer that is a Stage 0, I, or II cancer, and
occasionally a Stage III cancer.
[0172] A "non-malignant disease or disorder involving abnormal
activation of HER2" is a condition that does not involve a cancer
where abnormal activation of HER2 is occurring in cells or tissue
of the subject having, or predisposed to, the disease or disorder.
Examples of such diseases or disorders include autoimmune disease
(e.g., psoriasis), see definition below; endometriosis;
scleroderma; restenosis; polyps such as colon polyps, nasal polyps
or gastrointestinal polyps; fibroadenoma; respiratory disease
(e.g., chronic bronchitis, asthma including acute asthma and
allergic asthma, cystic fibrosis, bronchiectasis, allergic or other
rhinitis or sinusitis, .alpha.1-anti-trypsin deficiency, coughs,
pulmonary emphysema, pulmonary fibrosis or hyper-reactive airways,
chronic obstructive pulmonary disease, and chronic obstructive lung
disorder); cholecystitis; neurofibromatosis; polycystic kidney
disease; inflammatory diseases; skin disorders including psoriasis
and dermatitis; vascular disease; conditions involving abnormal
proliferation of vascular epithelial cells; gastrointestinal
ulcers; Menetrier's disease, secreting adenomas or protein loss
syndrome; renal disorders; angiogenic disorders; ocular disease
such as age related macular degeneration, presumed ocular
histoplasmosis syndrome, retinal, neovascularization from
proliferative diabetic retinopathy, retinal vascularization,
diabetic retinopathy, or age related macular degeneration; bone
associated pathologies such as osteoarthritis, rickets and
osteoporosis; damage following a cerebral ischemic event; fibrotic
or edemia diseases such as hepatic cirrhosis, lung fibrosis,
carcoidosis, throiditis, hyperviscosity syndrome systemic, Osler
Weber-Rendu disease, chronic occlusive pulmonary disease, or edema
following burns, trauma, radiation, stroke, hypoxia or ischemia;
hypersensitivity reaction of the skin; diabetic retinopathy and
diabetic nephropathy; Guillain-Barre syndrome; graft versus host
disease or transplant rejection; Paget's disease; bone or joint
inflammation; photoaging (e.g. caused by UV radiation of human
skin); benign prostatic hypertrophy; certain microbial infections
including microbial pathogens selected from adenovirus,
hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella
pertussis; thrombus caused by platelet aggregation; reproductive
conditions such as endometriosis, ovarian hyperstimulation
syndrome, preeclampsia, dysfunctional uterine bleeding, or
menometrorrhagia; synovitis; atheroma; acute and chronic
nephropathies (including proliferative glomerulonephritis and
diabetes-induced renal disease); eczema; hypertrophic scar
formation; endotoxic shock and fungal infection; familial
adenomatosis polyposis; neurodedenerative diseases (e.g.
Alzheimer's disease, AIDS-related dementia, Parkinson's disease,
amyotrophic lateral sclerosis, retinitis pigmentosa, spinal
muscular atrophy and cerebellar degeneration); myelodysplastic
syndromes; aplastic anemia; ischemic injury; fibrosis of the lung,
kidney or liver; T-cell mediated hypersensitivity disease;
infantile hypertrophic pyloric stenosis; urinary obstructive
syndrome; psoriatic arthritis; and Hashimoto's thyroiditis.
[0173] An "allergic or inflammatory disorder" herein is a disease
or disorder that results from a hyper-activation of the immune
system of an individual. Exemplary allergic or inflammatory
disorders include, but are not limited to, asthma, psoriasis,
rheumatoid arthritis, atopic dermatitis, multiple sclerosis,
systemic lupus, erythematosus, eczema, organ transplantation,
age-related mucular degeneration, Crohn's disease, ulcerative
colitis, eosinophilic esophagitis, and autoimmune diseases
associated with inflammation.
[0174] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis such as
acute arthritis, chronic rheumatoid arthritis, gouty arthritis,
acute gouty arthritis, chronic inflammatory arthritis, degenerative
arthritis, infectious arthritis, Lyme arthritis, proliferative
arthritis, psoriatic arthritis, vertebral arthritis, and
juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis
chronica progrediente, arthritis deformans, polyarthritis chronica
primaria, reactive arthritis, and ankylosing spondylitis),
inflammatory hyperproliferative skin diseases, psoriasis such as
plaque psoriasis, gutatte psoriasis, pustular psoriasis, and
psoriasis of the nails, dermatitis including contact dermatitis,
chronic contact dermatitis, allergic dermatitis, allergic contact
dermatitis, dermatitis herpetiformis, and atopic dermatitis,
x-linked hyper IgM syndrome, urticaria such as chronic allergic
urticaria and chronic idiopathic urticaria, including chronic
autoimmune urticaria, polymyositis/dermatomyositis, juvenile
dermatomyositis, toxic epidermal necrolysis, scleroderma (including
systemic scleroderma), sclerosis such as systemic sclerosis,
multiple sclerosis (MS) such as spino-optical MS, primary
progressive MS (PPMS), and relapsing remitting MS (RRMS),
progressive systemic sclerosis, atherosclerosis, arteriosclerosis,
sclerosis disseminata, and ataxic sclerosis, inflammatory bowel
disease (IBD) (for example, Crohn's disease, autoimmune-mediated
gastrointestinal diseases, colitis such as ulcerative colitis,
colitis ulcerosa, microscopic colitis, collagenous colitis, colitis
polyposa, necrotizing enterocolitis, and transmural colitis, and
autoimmune inflammatory bowel disease), pyoderma gangrenosum,
erythema nodosum, primary sclerosing cholangitis, episcleritis),
respiratory distress syndrome, including adult or acute respiratory
distress syndrome (ARDS), meningitis, inflammation of all or part
of the uvea, iritis, choroiditis, an autoimmune hematological
disorder, rheumatoid spondylitis, sudden hearing loss, IgE-mediated
diseases such as anaphylaxis and allergic and atopic rhinitis,
encephalitis such as Rasmussen's encephalitis and limbic and/or
brainstem encephalitis, uveitis, such as anterior uveitis, acute
anterior uveitis, granulomatous uveitis, nongranulomatous uveitis,
phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN or idiopathic membranous nephropathy,
membrano- or membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
allergic reaction, eczema including allergic or atopic eczema,
asthma such as asthma bronchiale, bronchial asthma, and auto-immune
asthma, conditions involving infiltration of T cells and chronic
inflammatory responses, chronic pulmonary inflammatory disease,
autoimmune myocarditis, leukocyte adhesion deficiency, systemic
lupus erythematosus (SLE) or systemic lupus erythematodes such as
cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal
lupus syndrome (NLE), lupus erythematosus disseminatus, lupus
(including nephritis, cerebritis, pediatric, non-renal,
extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes
mellitus, including pediatric insulin-dependent diabetes mellitus
(IDDM), adult onset diabetes mellitus (Type II diabetes),
autoimmune diabetes, idiopathic diabetes insipidus, immune
responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including lymphomatoid granulomatosis, Wegener's
granulomatosis, agranulocytosis, vasculitides, including vasculitis
(including large vessel vasculitis (including polymyalgia
rheumatica and giant cell (Takayasu's) arteritis), medium vessel
vasculitis (including Kawasaki's disease and polyarteritis nodosa),
microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous,
or hypersensitivity vasculitis, systemic necrotizing vasculitis,
and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or
syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
hemolytic anemia or immune hemolytic anemia including autoimmune
hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa),
Addison's disease, pure red cell anemia or aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome such as
those secondary to septicemia, trauma or hemorrhage,
antigen-antibody complex-mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet's or Behcet's disease, Castleman's
syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's
syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid
bullous and skin pemphigoid, pemphigus (including pemphigus
vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid,
and pemphigus erythematosus), autoimmune polyendocrinopathies,
Reiter's disease or syndrome, immune complex nephritis,
antibody-mediated nephritis, neuromyelitis optica,
polyneuropathies, chronic neuropathy such as IgM polyneuropathies
or IgM-mediated neuropathy, thrombocytopenia (as developed by
myocardial infarction patients, for example), including thrombotic
thrombocytopenic purpura (TTP) and autoimmune or immune-mediated
thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP)
including chronic or acute ITP, autoimmune disease of the testis
and ovary including autoimmune orchitis and oophoritis, primary
hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as autoimmune thyroiditis, Hashimoto's
disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, polyglandular syndromes such as autoimmune
polyglandular syndromes (or polyglandular endocrinopathy
syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome
or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,
encephalomyelitis such as allergic encephalomyelitis or
encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE), myasthenia gravis such as
thymoma-associated myasthenia gravis, cerebellar degeneration,
neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS),
and sensory neuropathy, multifocal motor neuropathy, Sheehan's
syndrome, autoimmune hepatitis, chronic hepatitis, lupoid
hepatitis, giant cell hepatitis, chronic active hepatitis or
autoimmune chronic active hepatitis, lymphoid interstitial
pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP,
Guillain-Barre syndrome, Berger's disease (IgA nephropathy),
idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary
cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome,
Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy),
refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic
lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery
disease, autoimmune ear disease such as autoimmune inner ear
disease (AIED), autoimmune hearing loss, opsoclonus myoclonus
syndrome (OMS), polychondritis such as refractory or relapsed
polychondritis, pulmonary alveolar proteinosis, amyloidosis,
scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis,
which includes monoclonal B cell lymphocytosis (e.g., benign
monoclonal gammopathy and monoclonal garnmopathy of undetermined
significance, MGUS), peripheral neuropathy, paraneoplastic
syndrome, channelopathies such as epilepsy, migraine, arrhythmia,
muscular disorders, deafness, blindness, periodic paralysis, and
channelopathies of the CNS, autism, inflammatory myopathy, focal
segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy,
uveoretinitis, chorioretinitis, autoimmune hepatological disorder,
fibromyalgia, multiple endocrine failure, Schmidt's syndrome,
adrenalitis, gastric atrophy, presenile dementia, demyelinating
diseases such as autoimmune demyelinating diseases, diabetic
nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome
(calcinosis, Raynaud's phenomenon, esophageal dysmotility,
sclerodactyl), and telangiectasia), male and female autoimmune
infertility, mixed connective tissue disease, Chagas' disease,
rheumatic fever, recurrent abortion, farmer's lung, erythema
multiforme, post-cardiotomy syndrome, Cushing's syndrome,
bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis,
Sampter's syndrome, Caplan's syndrome, dengue, endocarditis,
endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis,
interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic
fibrosis, endophthalmitis, erythema elevatum et diutinum,
erythroblastosis fetalis, eosinophilic faciitis, Shulman's
syndrome, Felty's syndrome, flariasis, cyclitis such as chronic
cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's
cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus
(HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's
disease, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, giant cell polymyalgia, endocrine ophthamopathy,
chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca,
epidemic keratoconjunctivitis, idiopathic nephritic syndrome,
minimal change nephropathy, benign familial and
ischemia-reperfusion injury, retinal autoimmunity, joint
inflammation, bronchitis, chronic obstructive airway disease,
silicosis, aphthae, aphthous stomatitis, arteriosclerotic
disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica, enteritis allergica, erythema nodosum leprosum,
idiopathic facial paralysis, chronic fatigue syndrome, febris
rheumatica, Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary
idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis
granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma
gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,
infertility due to antispermatozoan antibodies, non-malignant
thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases,
acquired immune deficiency syndrome (AIDS), parasitic diseases such
as Leishmania, toxic-shock syndrome, food poisoning, conditions
involving infiltration of T cells, leukocyte-adhesion deficiency,
immune responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, diseases involving
leukocyte diapedesis, multiple organ injury syndrome,
antigen-antibody complex-mediated diseases, antiglomerular basement
membrane disease, allergic neuritis, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune
atrophic gastritis, sympathetic ophthalmia, rheumatic diseases,
mixed connective tissue disease, nephrotic syndrome, insulitis,
polyendocrine failure, peripheral neuropathy, autoimmune
polyglandular syndrome type I, adult-onset idiopathic
hypoparathyroidism (AOIH), alopecia totalis, dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA),
hemochromatosis, myocarditis, nephrotic syndrome, primary
sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or
chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid
sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,
Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary eosinophilia, bronchopneumonic aspergillosis,
aspergilloma, or granulomas containing eosinophils, anaphylaxis,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune disorders associated with collagen disease, rheumatism,
neurological disease, ischemic re-perfusion disorder, reduction in
blood pressure response, vascular dysfunction, antgiectasis, tissue
injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia,
and disease accompanying vascularization, allergic hypersensitivity
disorders, glomerulonephritides, reperfusion injury, reperfusion
injury of myocardial or other tissues, dermatoses with acute
inflammatory components, acute purulent meningitis or other central
nervous system inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated
syndromes, cytokine-induced toxicity, acute serious inflammation,
chronic intractable inflammation, pyelitis, pneumonocirrhosis,
diabetic retinopathy, diabetic large-artery disorder, endarterial
hyperplasia, peptic ulcer, valvulitis, and endometriosis.
[0175] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of a cell and/or
causes destruction of a cell. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212,
Ra.sup.223, P.sup.32, and radioactive isotopes of Lu),
chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca
alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents, enzymes and fragments thereof such as
nucleolytic enzymes, antibiotics, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof, and
the various antitumor, anticancer, and chemotherapeutic agents
disclosed herein. Other cytotoxic agents are described herein. A
tumoricidal agent causes destruction of tumor cells.
[0176] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma 1 (see, e.g., Agnew, Chem. Intl. Ed. Engl. 33: 183-186
(1994)); dynemicin, including dynemicin A; an esperamicin; as well
as neocarzinostatin chromophore and related chromoprotein enediyne
antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.)); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0177] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0178] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell either in
vitro or in vivo. Thus, the growth inhibitory agent may be one
which significantly reduces the percentage of cells in S phase.
Examples of growth inhibitory agents include agents that block cell
cycle progression (at a place other than S phase), such as agents
that induce G1 arrest and M-phase arrest. Classical M-phase
blockers include the vincas (e.g., vincristine and vinblastine),
taxanes, and topoisomerase II inhibitors such as doxorubicin,
epirubicin, daunorubicin, etoposide, and bleomycin. The agents that
arrest G1 also spill over into S-phase arrest, for example, DNA
alkylating agents such as tamoxifen, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and
ara-C. Further information can be found in The Molecular Basis of
Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell
cycle regulation, oncogenes, and antineoplastic drugs" by Murakami
et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The
taxanes (paclitaxel and docetaxel) are anticancer drugs both
derived from the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc
Rorer), derived from the European yew, is a semisynthetic analogue
of paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0179] "Anti-cancer therapy" as used herein refers to a treatment
that reduces or inhibits cancer in a subject. Examples of
anti-cancer therapy include cytotoxic radiotherapy as well as the
administration of a therapeutically effective amount of a cytotoxic
agent, a chemotherapeutic agent, a growth inhibitory agent, a
cancer vaccine, an angiogenesis inhibitor, a prodrug, a cytokine, a
cytokine antagonist, a corticosteroid, an immunosuppressive agent,
an anti-emetic, an antibody or antibody fragment, or an analgesic
to the subject.
[0180] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). Prodrugs include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs,
beta-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0181] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone (HGH), N-methionyl
human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin;
glycoprotein hormones such as follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH), and luteinizing hormone (LH);
epidermal growth factor (EGF); hepatic growth factor; fibroblast
growth factor (FGF); prolactin; placental lactogen; tumor necrosis
factor-alpha and -beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, -beta
and -gamma colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF
(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-1beta,
IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;
IL-18a tumor necrosis factor such as TNF-alpha or TNF-beta; and
other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cytokine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native sequence cytokines.
[0182] By "cytokine antagonist" is meant a molecule that partially
or fully blocks, inhibits, or neutralizes a biological activity of
at least one cytokine. For example, the cytokine antagonists may
inhibit cytokine activity by inhibiting cytokine expression and/or
secretion, or by binding to a cytokine or to a cytokine receptor.
Cytokine antagonists include antibodies, synthetic or
native-sequence peptides, immunoadhesins, and small-molecule
antagonists that bind to a cytokine or cytokine receptor. The
cytokine antagonist is optionally conjugated with or fused to a
cytotoxic agent. Exemplary TNF antagonists are etanercept
(ENBREL.RTM.), infliximab (REMICADE.RTM.), and adalimumab
(HUMIRA.TM.).
[0183] The term "immunosuppressive agent" as used herein refers to
substances that act to suppress or mask the immune system of the
subject being treated. This includes substances that suppress
cytokine production, downregulate or suppress self-antigen
expression, or mask the MHC antigens. Examples of immunosuppressive
agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S.
Pat. No. 4,665,077); mycophenolate mofetil such as CELLCEPT.RTM.;
azathioprine (IMURAN.RTM., AZASAN.RTM./6-mercaptopurine;
bromocryptine; danazol; dapsone; glutaraldehyde (which masks the
MHC antigens, as described in U.S. Pat. No. 4,120,649);
anti-idiotypic antibodies for MHC antigens and MHC fragments;
cyclosporin A; steroids such as corticosteroids and
glucocorticosteroids, e.g., prednisone, prednisolone such as
PEDIAPRED.RTM. (prednisolone sodium phosphate) or ORAPRED.RTM.
(prednisolone sodium phosphate oral solution), methylprednisolone,
and dexamethasone; methotrexate (oral or subcutaneous)
(RHEUMATREX.RTM., TREXALL.TM.); hydroxycloroquine/chloroquine;
sulfasalazine; leflunomide; cytokine or cytokine receptor
antagonists including anti-interferon-.gamma., -.gamma., or
-.alpha. antibodies, anti-tumor necrosis factor-.alpha. antibodies
(infliximab or adalimumab), anti-TNF.alpha. immunoadhesin
(ENBREL.RTM., etanercept), anti-tumor necrosis factor-.beta.
antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor
antibodies; anti-LFA-1 antibodies, including anti-CD11a and
anti-CD18 antibodies; anti-L3T4 antibodies; heterologous
anti-lymphocyte globulin; polyclonal or pan-T antibodies, or
monoclonal anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide
containing a LFA-3 binding domain (WO 90/08187); streptokinase;
TGF-.beta.; streptodornase; RNA or DNA from the host; FK506;
RS-61443; deoxyspergualin; rapamycin; T-cell receptor (Cohen et
al., U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner et
al. Science 251: 430-432 (1991); WO 90/11294; Ianeway, Nature
341:482 (1989); and WO 91/01133); T cell receptor antibodies (EP
340,109) such as T10B9; cyclophosphamide (CYTOXAN.RTM.); dapsone;
penicillamine (CUPRIMINE.RTM.); plasma exchange; or intravenous
immunoglobulin (IVIG). These may be used alone or in combination
with each other, particularly combinations of steroid and another
immunosuppressive agent or such combinations followed by a
maintenance dose with a non-steroid agent to reduce the need for
steroids.
[0184] An "analgesic" refers to a drug that acts to inhibit or
suppress pain in a subject. Exemplary analgesics include
non-steroidal anti-inflammatory drugs (NSAIDs) including ibuprofen
(MOTRIN.RTM.), naproxen (NAPROSYN.RTM.), acetylsalicylic acid,
indomethacin, sulindac, and tolmetin, including salts and
derivatives thereof, as well as various other medications used to
reduce the stabbing pains that may occur, including anticonvulsants
(gabapentin, phenyloin, carbamazepine) or tricyclic
antidepressants. Specific examples include acetaminophen, aspirin,
amitriptyline (ELAVIL.RTM.), carbamazepine (TEGRETOL.RTM.),
phenyltoin (DILANTIN.RTM.), gabapentin (NEURONTIN.RTM.),
(E)-N-Vanillyl-8-methyl-6-noneamid (CAPSAICIN.RTM.), or a nerve
blocker.
[0185] "Corticosteroid" refers to any one of several synthetic or
naturally occurring substances with the general chemical structure
of steroids that mimic or augment the effects of the naturally
occurring corticosteroids. Examples of synthetic corticosteroids
include prednisone, prednisolone (including methylprednisolone),
dexamethasone triamcinolone, and betamethasone.
[0186] A "cancer vaccine," as used herein is a composition that
stimulates an immune response in a subject against a cancer. Cancer
vaccines typically consist of a source of cancer-associated
material or cells (antigen) that may be autologous (from self) or
allogenic (from others) to the subject, along with other components
(e.g., adjuvants) to further stimulate and boost the immune
response against the antigen. Cancer vaccines can result in
stimulating the immune system of the subject to produce antibodies
to one or several specific antigens, and/or to produce killer T
cells to attack cancer cells that have those antigens.
[0187] "Cytotoxic radiotherapy" as used herein refers to radiation
therapy that inhibits or prevents the function of cells and/or
causes destruction of cells. Radiation therapy may include, for
example, external beam irradiation or therapy with a radioactive
labeled agent, such as an antibody. The term is intended to include
use of radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, Ra.sup.223, P.sup.32, and radioactive isotopes of
Lu).
[0188] "Target molecule" refers to a molecule which can bind to a
protein complex of this invention (preferably with affinity higher
than 1 uM Kd according to scatchard analysis). Examples of target
molecules include, but are not limited to, serum soluble proteins
and their receptors, such as cytokines and cytokine receptors,
adhesins, growth factors and their receptors, hormones, viral
particles (e.g., RSV F protein, CMV, StaphA, influenza, hepatitis C
virus), micoorganisms (e.g., bacterial cell proteins, fungal
cells), adhesins, CD proteins and their receptors.
[0189] An "anti-emetic" is a compound that reduces or prevents
nausea in a subject. Anti-emetic compounds include, for example,
neurokinin-1 receptor antagonists, 5HT3 receptor antagonists (such
as ondansetron, granisetron, tropisetron, and zatisetron), GABAB
receptor agonists, such as baclofen, a corticosteroid such as
dexamethasone, KENALOG.RTM., ARISTOCORT.RTM., or NASALIDE.RTM., an
antidopaminergic, phenothiazines (for example prochlorperazine,
fluphenazine, thioridazine and mesoridazine), dronabinol,
metroclopramide, domperidone, haloperidol, cyclizine, lorazepam,
prochlorperazine, and levomepromazine.
[0190] A "subject" is a vertebrate, such as a mammal, e.g., a
human. Mammals include, but are not limited to, farm animals (such
as cows), sport animals, pets (such as cats, dogs and horses),
primates, mice, and rats.
[0191] Commercially available reagents referred to in the Examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
Examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Unless otherwise noted, the present invention uses standard
procedures of recombinant DNA technology, such as those described
hereinabove and in the following textbooks: Sambrook et al., supra;
Ausubel et al., Current Protocols in Molecular Biology (Green
Publishing Associates and Wiley Interscience, NY, 1989); Innis et
al., PCR Protocols: A Guide to Methods and Applications (Academic
Press, Inc., NY, 1990); Harlow et al., Antibodies: A Laboratory
Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait,
Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney,
Animal Cell Culture, 1987; Coligan et al., Current Protocols in
Immunology, 1991.
[0192] Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising," will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
II. CONSTRUCTION OF COILED COIL CONTAINING AND TETHERED
ANTIBODIES
[0193] Protein complexes described herein may be constructed by
using a heterodimerizing domain (e.g., a coiled coil domain) and/or
a tether.
[0194] Use of a heterodimerizing domain enables the construction of
a relatively pure population of antibodies that have different
heavy chains within a single antibody. In particular, as described
above, antibodies typically include two identical heavy chains,
which are each paired with an identical light chain. Use of the
coiled coil heterodimerization domain technology of the invention
enables different antibody heavy chains to preferentially dimerize
with each other in the formation of a single antibody. The
resulting antibody thus includes two different heavy chains, each
of which is typically (but need not be) paired with an identical
light chain. Each pair of heavy and light chains within such an
antibody has different binding specificity, due to the presence of
the different heavy chains, and thus the antibody can be considered
as a multispecific antibody. Tethers can also be exploited to
engineer antibodies of the invention, either alone or in
combination with the coiled-coil technology. The tethers can
connect the C-terminus of a constant light chain to the N-terminus
of a variable heavy chain, thus enabling proper light chain and
heavy chain association, as well as recombinant antibody production
using a single antibody-encoding plasmid. Antibodies including
coiled coils and/or tethers are further described below.
[0195] A. Coiled Coil Domains
[0196] The heterodimerizing domain used to generate the protein
complexes described herein can be an alpha helix (e.g., a
right-handed alpha helix) that can form a coiled coil upon
association with a second alpha helix containing oppositely charged
residues. To generate homogeneous or nearly homogeneous populations
of heterodimeric molecules, the heterodimerization domain must have
a strong preference for forming heterodimers over homodimers. In
this respect, the heterodimerization domains described herein
provide a significant advantage over Fos/Jun leucine zipper domains
because Jun readily forms homodimers. Exemplary alpha-helical
heterodimerization domains are illustrated in FIGS. 1, 2A, and 2B.
In particular embodiments, the first coiled coil domain contains a
heptad repeat of Formula I:
(X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7).sub.n (Formula
I), where [0197] X.sub.1 is a hydrophobic amino acid residue or
Asparagine, [0198] X.sub.2, X.sub.3, and X.sub.6 are each any amino
acid residue, [0199] X.sub.4 is a hydrophobic amino acid residue,
and [0200] X.sub.5 and X.sub.7 are each a charged amino acid
residue, and the second coiled coil domain contains a heptad repeat
of Formula II:
[0200]
(X'.sub.1X'.sub.2X'.sub.3X'.sub.4X'.sub.5X'.sub.6X'.sub.7).sub.n
(Formula II), where [0201] X'.sub.1 is a hydrophobic amino acid
residue or Asparagine, [0202] X'.sub.2, X'.sub.3, and X'.sub.6 are
each any amino acid residue, [0203] X'.sub.4 is a hydrophobic amino
acid residue, and [0204] X'.sub.5 and X'.sub.7 are each a charged
amino acid residue. In both Formula I and Formula II, n is greater
than or equal to 2 (e.g., greater than or equal to 3 or 4), and
less than or equal to 100. In one embodiment, n is between 2 and
20.
[0205] The X.sub.5 and X.sub.7 residues of the first coiled coil
domain and the X'.sub.5 and X'.sub.7 residues of the second coiled
coil domain may have, but need not have, the same charge. Thus, in
one example, the X.sub.5 and X.sub.7 residues of the first coiled
coil domain are basic residues, and the X'.sub.5 and X'.sub.7
residues of the second coiled coil domain are acidic residues. In
another example, X.sub.5 in the first coiled coil domain is a basic
residue, and X.sub.7 of the first coiled coil domain is an acidic
residue. In this example, the second coiled coil domain has a basic
residue in the X'.sub.5 position, and an acidic residue in the
X'.sub.7 position. As shown in FIG. 1, an ionic interaction occurs
between the X.sub.5 residue of the first coiled coil domain and the
X'.sub.7 residue of the second coiled coil domain, as well as
between the X.sub.7 residue of the first coiled coil domain and the
X'.sub.5 residue of the second coiled coil domain. In a related
example, X.sub.5 in the first coiled coil domain is an acidic
residue, X.sub.7 in the first coiled coil domain is a basic
residue, X'.sub.5 in the second coiled coil domain is an acidic
residue, and X'.sub.7 in the second coiled coil domain is a basic
residue. In addition, inclusion of at least one heptad repeat with
an Asparagine at the X.sub.1/X'.sub.1 position of both the first
and second coiled coil domains may be used to ensure a parallel
orientation of the first and second coiled coil domains.
[0206] The hydrophobic residues in the heptad repeats are
preferably chosen from Alanine, Valine, Leucine, Isoleucine,
Tryptophan, Phenylalanine, and Methionine. Proline, while
hydrophobic, is in one embodiment not included in a coiled coil
domain of Formula I or Formula II because the presence of Proline
in an amino acid sequence can limit its ability to form an alpha
helical structure. In addition, in other embodiments, the coiled
coil domain of Formula I or Formula II does not contain a Glycine
residue because, due to its conformational flexibility, Glycine
does not readily adopt the constrained alpha helical structure.
Charged residues that may be included in a coiled coil domain of
Formula I or Formula II include Lysine, Arginine, Histidine,
Aspartic Acid, and Glutamic Acid, where Lysine, Arginine, and
Histidine are basic residues, and Aspartic Acid and Glutamic Acid
are acidic residues.
[0207] Construction of an antibody described herein may use a
coiled coil domain of Formula I and a coiled coil domain of Formula
II (a first and a second coiled coil domain) where the first coiled
coil domain is linked to a first constant domain of the antibody
(e.g., CH3 of a first heavy chain) and the second coiled coil
domain is linked to a second constant domain of the antibody (e.g.,
CH3 of a second heavy chain). The linkage may be a direct linkage
by a peptide bond or may be through a linker sequence. A linker can
be peptide bonded to the C-terminal end of one amino acid sequence
(e.g., the constant region) and to the N-terminal end of the other
amino acid sequence (e.g., the coiled coil domain). The linker can
be long enough to allow for cleavage of the coiled coil domain from
the antibody constant region, as described further elsewhere
herein, but short enough to confer heterodimeric association of two
antibody constant regions (e.g., two heavy chain constant regions).
As such, a linker may be an amino acid sequence of 2 to 100 amino
acids in length. In a particular embodiment, the linker is between
2 and 50 amino acids in length, for example, 3, 5, 10, 15, 20, 25,
30, 35, 40, 45, or 50 amino acids in length. The linker can consist
of, for example, neutral polar or nonpolar amino acids.
[0208] B. Multispecific Antibodies
[0209] It should be understood that the variable domains of such
antibodies can be derived from several methods. For example, the
variable domains of the antibodies of this invention can be the
same as existing antibodies known in the art.
[0210] A coiled coil domain may be used to generate a multispecific
antibody (an antibody that binds to at least two antigens or to at
least two epitopes on the same antigen). In one example, the
multispecific antibody is a bispecific antibody. Typically, in
naturally occurring IgG antibodies, the variable regions of each
pair of heavy and light chains in the antibody are identical. Use
of coiled coil domains according to the present invention enables
the two heavy chains within an antibody to be different, resulting
in antibodies having antigen binding domains with different binding
specificities. In particular, coiled coil heterodimerization
domains on each heavy chain (e.g., C-terminal to CH3) promote
binding between different heavy chains. Optionally the coiled coil
domains are linked to the heavy chain constant regions by a linker
that can be cleaved so that the coiled coil can be removed from the
antibody after assembly.
[0211] A schematic representation of an exemplary bispecific
antibody, which includes two different heavy chains (HC1 and HC2)
and two identical or common light chains, is shown in FIG. 3. The
exemplary bispecific antibody in FIG. 3 also contains a
heterodimeric coiled coil. The antibody may also contain a Lys-C
endopeptidase cleavage site N-terminal to each coiled coil
heterodimerization domain that allows for the removal of the coiled
coil from the antibody once the antibody has been assembled. Both
of the heavy chains in this exemplary bispecific antibody also
contain a K222A mutation in the hinge region to remove a Lys-C
endopeptidase cleavage site, so that Lys-C endopeptidase treatment
results only in removal of the coiled coil and not cleavage within
the heavy chain constant regions.
[0212] While the exemplary antibody contains a mutation that
removes a Lys-C endopeptidase cleavage site in the hinge region,
the location of Lys-C endopeptidase cleavage sites can vary
depending on the antibody sequence used. One skilled in the art can
readily scan the sequence of an antibody to determine whether there
are any cleavage sites (e.g., a Lys-C endopeptidase cleavage site)
in the heavy or light chain sequences that would need to be removed
to avoid cleavage of the antibody itself upon removal of the coiled
coil or tether sequences.
[0213] Further, multispecific antibodies may be constructed using
the methods described herein where the heavy chain lacks the CH1
domain (the VH is directly connected to the hinge-CH2 domain) and
the corresponding light chain lacks the CL domain. Such antibodies
can be used to bring to different antigens together or to associate
B and T cells.
[0214] C. One-Armed Antibodies
[0215] Heterodimerizing coiled coil domains can also be used to
generate one-armed antibodies A schematic diagram illustrating an
example of a one-armed antibody is shown in FIG. 4A. The exemplary
antibody shown in FIG. 4A includes a light chain (LC), one
full-length heavy chain (HC1), and a second heavy chain (HC2) lacks
the VH and CH1 domains and part of the hinge region. Both the HC1
and the HC2 include a coiled coil heterodimerization domain at the
C-terminus. The HC1 sequence in this example contains a K222A
mutation in the hinge region to remove a Lys-C endopeptidase
cleavage site, so that Lys-C cleavage only removes the coiled coil
and does not result in cleavage within the heavy chain.
[0216] D. Conjugated Protein Complexes
[0217] Coiled coil heterodimerization domains may also be used to
generate protein complexes such as antibodies (e.g., monospecific,
bispecific, multispecific, one-armed, or tethered antibodies) in
which a constant region is modified by conjugation to a cytotoxic
agent. For instance, the coiled coil heterodimerization domain
enables the construction of antibodies where one of the heavy chain
constant regions (HC1 or HC2) contains a modification that allows
for conjugation to a cytotoxic agent, while the other heavy chain
constant region does not. In one example, HC1 is conjugated to a
cytotoxic agent while HC2 is not. A schematic diagram illustrating
an example of a conjugated antibody is shown in FIG. 4B. The
exemplary antibody includes two full-length heavy chains and two
identical light chains (common light chain), as well as a coiled
coil. As indicated by the star, one of the heavy chains has been
conjugated to a cytotoxic agent (for example, a toxin). Similarly,
in an alternative antibody construct, one of the light chain
constant regions may be conjugated to a cytotoxic agent, while the
other light chain constant region is not (e.g., LC1 is conjugated
to a cytotoxic agent and LC2 is not).
[0218] In one particular example, a constant region of the antibody
may be modified to introduce electrophilic moieties which can react
with nucleophilic substituents on a linker reagent used to
conjugate the cytotoxic agent to the antibody or on the cytotoxic
agent itself. The sugars of glycosylated antibodies may be
oxidized, e.g., with periodate oxidizing reagents, to form aldehyde
or ketone groups which may react with the amine group of linker
reagents or a cytotoxic agent. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g., by
borohydride reagents, to form stable amine linkages. Nucleophilic
groups on a cytotoxic agent include, but are not limited to, amine,
thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,
hydrazine carboxylate, and arylhydrazide groups capable of reacting
to form covalent bonds with electrophilic groups on antibody
regions and linker reagents including: (i) active esters such as
NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl
and benzyl halides such as haloacetamides; and (iii) aldehydes,
ketones, carboxyl, and maleimide groups.
[0219] E. Tethered Protein Complexes
[0220] The invention also provides protein complexes constructed
using tethers, for example, an antibody can have a tether that
links the C-terminus of a constant light chain to the N-terminus of
a variable heavy chain. The tether aids in proper association of
the light chain and the heavy chain (i.e., association of the light
chain with the heavy chain to which it is tethered). Such a
tethered antibody can be constructed with or without a
heterodimerizing domain, as described above. A schematic diagram of
an exemplary tethered antibody containing a coiled coil is shown in
FIG. 5. The exemplary antibody shown in FIG. 5 contains two
different heavy chains (HC1 and HC2), as well as two different
light chains (LC1 and LC2). Tethered antibodies can also be
constructed to contain common light chains and/or common heavy
chains. In the exemplary antibody, HC1 and HC2 contain a K222A
mutation in the hinge region to remove a Lys-C endopeptidase
cleavage site, as described above, as well as coiled coil
heterodimerization domains at their C-termini.
[0221] The addition of a heterodimerizing domain to a tethered
antibody aids in bringing the heavy chain/light chain complexes
together and thereby reduces or eliminates homodimerization of such
complexes. In a particular embodiment, tethers are long enough to
span the distance between the N-terminus of the variable heavy
chain and the C-terminus of the constant light chain in the
assembled antibody (FIG. 6) to allow for the proper light
chain/heavy chain association, but are short enough to prevent
interchain association (i.e., association of the light chain with a
heavy chain to which it is not tethered). In the example shown in
FIG. 6, the distance between the N-terminus of the variable heavy
chain and the C-terminus of the constant light chain is
approximately 92 .ANG.. A peptide bond spans about 4.3 .ANG.. In
this example, a tether should be about 22 amino acids in length to
span the distance between the N-terminus of the variable heavy
chain and the C-terminus of the constant light chain. The distance
between the C-terminus of the constant light chain and the
N-terminus of the variable heavy chain can differ between
antibodies and the length of a tether therefore can also vary
between antibodies. Tethers of 20, 23, and 26 amino acids in length
were tested and, in general, tethers of 15-50 amino acids are
effective. A tether may remain flexible and not form secondary
structures, and for this purpose a tether containing Glycine (G)
and Serine (S) residues can be used. A tether may consist solely of
G and S residues, but also may include other residues, as long as
the tether remains flexible to allow for the assembly of the light
chain and heavy chain of the antibody. In a particular embodiment,
the tether contains GGS repeats (FIG. 5). For a tether of 15-30
amino acids in length, the tether, in one embodiment, contains at
least 5 GGS repeats. An exemplary tether described herein and
having the sequence of SEQ ID NO:14 contains 8 GGS repeats and
contains an additional Glycine residue at both the N- and
C-termini. Other exemplary tether sequences are show in in FIG. 7B
and contain either Furin or Lys-C endopeptidase cleavage sites at
their N- and C-termini.
[0222] F. Cleavage of Tether and Linker Sequences
[0223] Once a protein complex is assembled, the tether may no
longer be required and can, if desired, be cleaved, from the
antibody. Cleavage sites found in the tether, but not in the
antibody sequence, can be used to remove the tether. Similarly, the
coiled coil is also no longer required once the antibody has been
assembled and can also, if desired, be cleaved from the
antibody.
[0224] FIG. 7A illustrates the location of exemplary cleavage sites
in a tether as well as a linker sequence that joins the coiled coil
to the antibody. In general, cleavage sites in the tether are
located at or close to the C- and N-terminus of the tether sequence
or within the antibody sequence at or close to the site where the
antibody and tether are joined. A cleavage site for a linker
generally is located at the N-terminus of the linker sequence (or
coiled coil) or in the antibody sequence at or close to the site
where the antibody and linker (or coiled coil) are joined. If the
linker is cleaved using Lys-C endopeptidase (e.g., at a Lysine
residue at the C-terminus of the constant heavy chain), the
sequence of the antibody may need to be modified to remove Lys-C
endopeptidase cleavage sites. An example of such a modification is
the mutation of a Lysine in the hinge region to an Alanine (e.g.,
K222A, Kabat numbering system; K222A, EU numbering system in
exemplary antibodies described herein). Modifications of other
cleavage sites may be required and made in a similar manner when
different cleavage agents are selected for use in the
invention.
[0225] Cleavage of amino acid sequences at particular sites is
standard in the art and can involve enzymatic cleavage, chemical
cleavage, or auto-processing. For example, a tether or linker may
be cleaved from an protein using an endopeptidase. Exemplary
endopeptidases include, without limitation, Lys-C, Asp-N, Arg-C,
V8, Glu-C, Thrombin, Genenase (a variant of subtilisin BPN''
protease), Factor Xa, TEV (tobacco etch virus cysteine protease),
Enterokinase, HRV C3 (human rhinovirus C3 protease), Kininogenase,
chymotrypsin, trypsin, pepsin, and papain, all of which are
commercially available (e.g., from Boehringer Mannheim, Thermo
Scientific, or New England Biolabs). Lys-C cleaves at the carboxyl
side of Lysine residues, V8 and Glu-C cleave at the carboxyl side
of Glutamate residues, Arg-C cleaves at the carboxyl side of
Arginine residues, Asp-N cleaves at the amino side of Aspartate
residues, chymotropsin cleaves at the carboxyl side of Tyrosine,
Phenylalanine, Tryptophan, and Leucine residues, and trypsin
cleaves at the carboxyl side of Arginine and Lysine residues. TEV
cleaved the amino acid sequence GluAsnLeuTyrPheGlnGly (SEQ ID
NO:19) between the "Gln" and "Gly" residues. Use of such enzymes is
standard in the art and protocols are available from the
manufacturers.
[0226] Alternatively a tether or linker may be cleaved from an
protein using a chemical, such as hydroxylamine. Hydroxylamine
cleaves Asparagine-Glycine peptide bonds. If hydroxylamine is used
to cleave the tether and linker from a protein, several Glycine or
Asparagine residues in the protein may need to be mutated to avoid
fragmenting the protein.
[0227] Numerous other chemicals that cleave peptide bonds are known
in the art. For example, N-chlorosuccinimide cleaves at the
C-terminal side of Tryptophan residues (Shechter et al.,
Biochemistry 15:5071-5075 (1976)). N-bromosuccinimide and cyanogen
bromide also cleave at the C-terminal side of Tryptophan residues.
In addition, 2-nitrothiocyanobenzoic acid or organophosphines may
be used to cleave a protein at the N-terminal side of a Cysteine
residue (see, e.g., EP 0339217).
[0228] A linker or tether may also be cleaved at dibasic sites
(e.g., an Arginine-Arginine, Lysine-Arginine, or Lysine-Lysine
site). Enzymes that cleave at dibasic sites are known in the art
and include, for example, N-arginine dibasic convertase (Chow et
al., JBC 275:19545-19551 (2000)) and subtilisin-like proprotein
convertases such as Furin (PC1), PC2, and PC3 (Steiner (1991) in
Peptide Biosynthesis and Processing (Fricker ed.) pp. 1-16, CRC
Press, Boca Raton, Fla.; Muller et al., JBC 275:39213-39222,
(2000)).
[0229] Proteins are also known to auto-process. For example, the
Hedgehog protein is processed at a Gly.AspTrpAsnAlaArgTrp.CysPhe
cleavage site (SEQ ID NO:20) by a proteolytic activity within the
protein. An autoproteolytic cleavage site may also be included in a
linker or tether sequence.
[0230] G. Other Protein Features
[0231] Proteins according to the invention can include sequences
from any source, including human or murine sources, or combinations
thereof. The sequences of certain portions of the proteins (e.g.,
the hypervariable regions) can also be artificial sequences, such
as sequences identified by screening a library (e.g., a phage
display library) including random sequences.
[0232] In the case of antibodies including sequences from different
sources, the antibodies can be "chimeric" antibodies in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, provided that they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; and
Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
Such chimeric antibodies may, for example, include murine variable
regions (or portions thereof) and human constant regions.
[0233] The chimeric antibodies can optionally also be "humanized"
antibodies, which contain minimal sequence derived from the
non-human antibody. Humanized antibodies typically are human
antibodies (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0234] In more detail, a humanized antibody can have one or more
amino acid residues introduced into it from a source that is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0235] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence that is closest to that of the rodent
is then accepted as the human framework (FR) for the humanized
antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al.,
J. Mol. Biol. 196:901 (1987)). Another method uses a particular
framework derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285
(1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0236] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to an
exemplary method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available that illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
III. VECTORS, HOST CELLS, AND RECOMBINANT METHODS
[0237] For recombinant production of an antibody of the invention,
the nucleic acid encoding it is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody is readily isolated and
sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The choice of vector depends in part on the
host cell to be used. Generally, preferred host cells are of either
prokaryotic or eukaryotic (generally mammalian, but also including
fungi (e.g., yeast), insect, plant, and nucleated cells from other
multicellular organisms) origin. It will be appreciated that
constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD, and IgE constant regions, and that
such constant regions can be obtained from any human or animal
species.
[0238] a. Generating Antibodies Using Prokaryotic Host Cells
[0239] i. Vector Construction
[0240] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0241] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0242] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lamda.GEM.TM.-11 may be utilized in
making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0243] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. An inducible promoter is a promoter that
initiates increased levels of transcription of the cistron under
its control in response to changes in the culture condition, e.g.,
the presence or absence of a nutrient or a change in
temperature.
[0244] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0245] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .gamma.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker to ligate them to
cistrons encoding the target light and heavy chains (Siebenlist et
al., (1980) Cell 20:269) using linkers or adaptors to supply any
required restriction sites.
[0246] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e., cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, Pe1B, OmpA, and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0247] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB- strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits (Proba and Pluckthun, Gene, 159:203
(1995)).
[0248] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhuA (.DELTA.tonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA. (nmpc-fepE) degP41 kanR (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli .lamda..alpha.1776 (ATCC
31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These
examples are illustrative rather than limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria
having defined genotypes are known in the art and described in, for
example, Bass et al., Proteins 8:309-314 (1990). It is generally
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well-known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon. Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may
desirably be incorporated in the cell culture.
[0249] ii. Antibody Production
[0250] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0251] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0252] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include Luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0253] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol, and dithiothreitol.
[0254] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the preferred
temperature ranges from about 20.degree. C. to about 39.degree. C.,
more preferably from about 25.degree. C. to about 37.degree. C.,
even more preferably at about 30.degree. C. The pH of the medium
may be any pH ranging from about 5 to about 9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8
to about 7.4, and more preferably about 7.0.
[0255] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. Preferably,
the phosphate-limiting medium is the C.R.A.P medium (see, e.g.,
Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety
of other inducers may be used, according to the vector construct
employed, as is known in the art.
[0256] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0257] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, preferably about 1,000 to 100,000
liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small-scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0258] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0259] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD, and/or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells (Chen et al., (1999) J. Biol. Chem. 274:19601-19605; Georgiou
et al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun, (2000) J. Biol. Chem.
275:17106-17113; Arie et al., (2001) Mol. Microbiol.
39:199-210).
[0260] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI, and combinations thereof.
Some E. coli protease-deficient strains are available and described
in, for example, Joly et al., (1998), Proc. Natl. Acad. Sci. USA
95:2773-2777; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et
al., U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug
Resistance, 2:63-72 (1996).
[0261] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
iii. Antibody Purification
[0262] Standard protein purification methods known in the art can
be employed. The following procedures are exemplary of suitable
purification procedures: fractionation on immunoaffinity or
ion-exchange columns, ethanol precipitation, reverse phase HPLC,
chromatography on silica or on a cation-exchange resin such as
DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation,
and gel filtration using, for example, Sephadex G-75.
[0263] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the full length antibody
products of the invention. Protein A is a 41 kD cell wall protein
from Staphylococcus aureus which binds with a high affinity to the
Fc region of antibodies. Lindmark et al., (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
[0264] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
The antibody of interest may be recovered from the solid phase by
elution into a solution containing a chaotropic agent or mild
detergent. Exemplary chaotropic agents and mild detergents include,
but are not limited to, Guanidine-HCl, urea, lithium perclorate,
Arginine, Histidine, SDS (sodium dodecyl sulfate), Tween, Triton,
and NP-40, all of which are commercially available. Diluting the
antibody into a solution containing a chaotropic agent or mild
detergent after elution from the column (e.g., mAbSure column)
maintains the stability of the antibody post elution and allows for
the efficient removal of the coiled coil by Lys-C
endopeptidase.
[0265] b. Generating Antibodies Using Eukaryotic Host Cells
[0266] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0267] i. Signal Sequence Component
[0268] A vector for use in a eukaryotic host cell may contain a
signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected can be one that
is recognized and processed (i.e., cleaved by a signal peptidase)
by the host cell. In mammalian cell expression, mammalian signal
sequences as well as viral secretory leaders, for example, the
herpes simplex gD signal, are available. The DNA for such precursor
region is ligated in reading frame to DNA encoding the
antibody.
[0269] ii. Origin of Replication
[0270] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used, but only because it contains the early
promoter.
[0271] iii. Selection Gene Component
[0272] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0273] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid,
and hygromycin.
[0274] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0275] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity (e.g., ATCC CRL-9096).
[0276] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418.
See, for example, U.S. Pat. No. 4,965,199.
[0277] iv. Promoter Component
[0278] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody polypeptide nucleic acid. Promoter sequences are known
for eukaryotes. Virtually all eukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors.
[0279] Antibody polypeptide transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as, for example, polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B
virus, and Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
or from heat-shock promoters, provided such promoters are
compatible with the host cell systems.
[0280] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. Alternatively, the Rous
Sarcoma Virus long terminal repeat can be used as the promoter.
[0281] v. Enhancer Element Component
[0282] Transcription of DNA encoding an antibody polypeptide by
higher eukaryotes can be increased by inserting an enhancer
sequence into the vector. Many enhancer sequences are now known
from mammalian genes (e.g., globin, elastase, albumin,
.alpha.-fetoprotein, and insulin genes). Also, one may use an
enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) for a
description of elements for enhancing activation of eukaryotic
promoters. The enhancer may be spliced into the vector at a
position 5' or 3' to the antibody polypeptide-encoding sequence,
provided that enhancement is achieved, but is generally located at
a site 5' from the promoter.
[0283] vi. Transcription Termination Component
[0284] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO 94/11026 and the expression vector
disclosed therein.
[0285] vii. Selection and Transformation of Host Cells
[0286] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0287] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0288] viii. Culturing the Host Cells
[0289] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0290] ix. Purification of Antibody
[0291] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0292] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0293] In one embodiment, the antibody of interest is recovered
from the solid phase of a column by elution into a solution
containing a chaotropic agent or mild detergent. Exemplary
chaotropic agents and mild detergents include, but are not limited
to, Guanidine-HCl, urea, lithium perclorate, Arginine, Histidine,
SDS (sodium dodecyl sulfate), Tween, Triton, and NP-40, all of
which are commercially available.
[0294] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25 M salt).
[0295] x. Antibody Production Using Baculovirus
[0296] Recombinant baculovirus may be generated by co-transfecting
a plasmid encoding an antibody or antibody fragment and
BaculoGold.TM. virus DNA (Pharmingen) into an insect cell such as a
Spodoptera frugiperda cell (e.g., Sf9 cells; ATCC CRL 1711) or a
Drosophila melanogaster S2 cell using, for example, lipofectin
(commercially available from GIBCO-BRL). In a particular example,
an antibody sequence is fused upstream of an epitope tag contained
within a baculovirus expression vector. Such epitope tags include
poly-His tags. A variety of plasmids may be employed, including
plasmids derived from commercially available plasmids such as
pVL1393 (Novagen) or pAcGP67B (Pharmingen). Briefly, the sequence
encoding an antibody or a fragment thereof may be amplified by PCR
with primers complementary to the 5' and 3' regions. The 5' primer
may incorporate flanking (selected) restriction enzyme sites. The
product may then be digested with the selected restriction enzymes
and subcloned into the expression vector.
[0297] After tranfection with the expression vector, the host cells
(e.g., Sf9 cells) are incubated for 4-5 days at 28.degree. C. and
the released virus is harvested and used for further
amplifications. Viral infection and protein expression may be
performed as described, for example, by O'Reilley et al.
(Baculovirus expression vectors: A Laboratory Manual. Oxford:
Oxford University Press (1994)).
[0298] Expressed poly-His tagged antibody can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts can be prepared from recombinant virus-infected Sf9 cells
as described by Rupert et al. (Nature 362:175-179 (1993)). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL HEPES
pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40;
0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate; 300 mM NaCl;
10% glycerol pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water,
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl; 10% glycerol
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged antibody are pooled and dialyzed against loading
buffer.
[0299] Alternatively, purification of the antibody can be performed
using known chromatography techniques, including for instance,
Protein A or protein G column chromatography. The antibody of
interest may be recovered from the solid phase of the column by
elution into a solution containing a chaotropic agent or mild
detergent. Exemplary chaotropic agents and mild detergents include,
but are not limited to, Guanidine-HCl, urea, lithium perclorate,
Arginine, Histidine, SDS (sodium dodecyl sulfate), Tween, Triton,
and NP-40, all of which are commercially available.
[0300] c. Optimized Purification Technique
[0301] One particular purification approach that may be used for
coiled coil containing antibodies is shown below.
##STR00001##
[0302] In addition to Arginine, other chaotropic agents or mild
detergents that can be used in the above purification protocol
after the initial Protein A column step include, but are not
limited to, Guanidine-HCl, urea, lithium perclorate, Histidine, SDS
(sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which
are commercially available. Diluting the antibody into a solution
containing a chaotropic agent or mild detergent after elution from
the initial Protein A containing column (e.g., mAbSure column)
maintains the stability of the antibody post elution and allows for
the efficient removal of the coiled coil by Lys-C
endopeptidase.
IV. CONJUGATED PROTEINS
[0303] The invention also provides conjugated proteins such as
conjugated antibodies or immunoconjugates (for example,
"antibody-drug conjugates" or "ADC"), comprising any of the
antibodies described herein (e.g., a coiled coil containing
antibody, a tethered antibody, or an antibody made according to the
methods described herein) where one of the constant regions of the
light chain or the heavy chain is conjugated to a chemical molecule
such as a dye or cytotoxic agent such as a chemotherapeutic agent,
a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope (i.e., a
radioconjugate). In particular, as described herein, the use of
coiled coil domains enables the construction of antibodies
containing two different heavy chains (HC1 and HC2) as well as two
different light chains (LC1 and LC2). An immunoconjugate
constructed using the methods described herein may contain the
cytotoxic agent conjugated to a constant region of only one of the
heavy chains (HC1 or HC2) or only one of the light chains (LC1 or
LC2). Also, because the immunoconjugate can have the cytotoxic
agent attached to only one heavy or light chain, the amount of the
cytotoxic agent being administered to a subject is reduced relative
to administration of an antibody having the cytotoxic agent
attached to both heavy or light chains. Reducing the amount of
cytotoxic agent being administered to a subject limits adverse side
effects associated with the cytotoxic agent.
[0304] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos,
Anticancer Research 19:605-614 (1999); Niculescu-Duvaz and
Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No.
4,975,278) allows targeted delivery of the drug moiety to tumors,
and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., Lancet (Mar.
15, 1986):603-605 (1986); Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., Cancer Immunol. Immunother. 21:183-187 (1986)).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al., Jour. of the Nat. Cancer
Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic &
Med. Chem. Letters 10:1025-1028 (2000); Mandler et al.,
Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213;
Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and
calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et
al., Cancer Res. 53:3336-3342 (1993)). The toxins may effect their
cytotoxic and cytostatic effects by mechanisms including tubulin
binding, DNA binding, or topoisomerase inhibition. Some cytotoxic
drugs tend to be inactive or less active when conjugated to large
antibodies or protein receptor ligands.
[0305] Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein (e.g., above). Enzymatically
active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes. See,
e.g., WO 93/21232 published Oct. 28, 1993. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (TT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See, e.g., WO94/11026.
[0306] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
aurostatins, a trichothecene, and CC1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
[0307] i. Maytansine and Maytansinoids
[0308] In some embodiments, the immunoconjugate comprises an
antibody (full length or fragments) of the invention conjugated to
one or more maytansinoid molecules.
[0309] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0310] Maytansinoid drug moieties are attractive drug moieties in
antibody drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification,
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through the non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0311] Immunoconjugates containing maytansinoids, methods of making
same, and their therapeutic use are disclosed, for example, in U.S.
Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1,
the disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid
conjugate was tested in vitro on the human breast cancer cell line
SK- BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
[0312] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No.
5,208,020 (the disclosure of which is hereby expressly incorporated
by reference). An average of 3-4 maytansinoid molecules conjugated
per antibody molecule has shown efficacy in enhancing cytotoxicity
of target cells without negatively affecting the function or
solubility of the antibody, although even one molecule of
toxin/antibody would be expected to enhance cytotoxicity over the
use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S.
Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred to hereinabove. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0313] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1,
Chari et al., Cancer Research 52:127-131 (1992), and U.S. Patent
Application Publication No. 2005/0169933, the disclosures of which
are hereby expressly incorporated by reference.
Antibody-maytansinoid conjugates comprising the linker component
SMCC may be prepared as disclosed in U.S. Patent Application
Publication No. 2005/0169933. The linking groups include disulfide
groups, thioether groups, acid labile groups, photolabile groups,
peptidase labile groups, or esterase labile groups, as disclosed in
the above-identified patents, disulfide and thioether groups being
preferred. Additional linking groups are described and exemplified
herein.
[0314] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (TT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as
disuccinirnidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred
coupling agents include N-succinimidyl-3-(2-pyridyldithio) (SPDP)
(Carlsson et al., Biochem. J. 173:723-737 (1978)) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0315] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
ii. Auristatins and Dolastatins
[0316] In some embodiments, the immunoconjugate comprises an
antibody of the invention conjugated to dolastatins or dolostatin
peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos.
5,635,483 and 5,780,588). Dolastatins and auristatins have been
shown to interfere with microtubule dynamics, GTP hydrolysis, and
nuclear and cellular division (Woyke et al., Antimicrob. Agents and
Chemother. 45(12):3580-3584 (2001)) and have anticancer (U.S. Pat.
No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob.
Agents Chemother. 42:2961-2965 (1998)). The dolastatin or
auristatin drug moiety may be attached to the antibody through the
N-(amino) terminus or the C- (carboxyl) terminus of the peptidic
drug moiety (WO 02/088172).
[0317] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Monomethylvaline Compounds Capable of Conjugation to Ligands,"
U.S. Application Publication No. 2005/0238649, the disclosure of
which is expressly incorporated by reference in its entirety.
[0318] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides," volume 1, pp. 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. Nos. 5,635,483 and 5,780,588; Pettit et
al., J. Nat. Prod. 44:482-485 (1981); Pettit et al., Anti-Cancer
Drug Design 13:47-66 (1998); Poncet, Curr. Pharm. Des. 5:139-162
(1999); and Pettit, Fortschr. Chem. Org. Naturst. 70:1-79 (1997).
See also Doronina, Nat. Biotechnol. 21(7):778-784 (2003); and
"Monomethylvaline Compounds Capable of Conjugation to Ligands,"
U.S. Application Publication No. 2005/0238649, hereby incorporated
by reference in its entirety (disclosing, e.g., linkers and methods
of preparing monomethylvaline compounds such as MMAE and MMAF
conjugated to linkers).
[0319] iii. Calicheamicin
[0320] In other embodiments, the immunoconjugate comprises an
antibody of the invention conjugated to one or more calicheamicin
molecules. The calicheamicin family of antibiotics are capable of
producing double-stranded DNA breaks at sub-picomolar
concentrations. For the preparation of conjugates of the
calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and
5,877,296 (all to American Cyanamid Company). Structural analogues
of calicheamicin which may be used include, but are not limited to,
.gamma..sub.1.sup.1, .alpha..sub.2.sup.1, .alpha..sub.3.sup.1,
N-acetyl-.gamma..sub.1.sup.1, PSAG and .theta..sup.1.sub.1 (Hinman
et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer
Research 58:2925-2928 (1998) and the aforementioned U.S. patents to
American Cyanamid). Another anti-tumor drug that the antibody can
be conjugated is QFA, which is an antifolate. Both calicheamicin
and QFA have intracellular sites of action and do not readily cross
the plasma membrane. Therefore, cellular uptake of these agents
through antibody mediated internalization greatly enhances their
cytotoxic effects.
[0321] iv. Other Cytotoxic Agents
[0322] Other antitumor agents that can be conjugated to the
antibodies of the invention or made according to the methods
described herein include BCNU, streptozoicin, vincristine and
5-fluorouracil, the family of agents known collectively LL-E33288
complex described in U.S. Pat. Nos. 5,053,394 and 5,770,710, as
well as esperamicins (U.S. Pat. No. 5,877,296).
[0323] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes (see, for example, WO 93/21232, published Oct. 28,
1993).
[0324] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0325] For selective destruction of a tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, O.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0326] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.186, Re.sup.188 and
In.sup.111 can be attached via a cysteine residue in the peptide.
Yttrium-90 can be attached via a lysine residue. The IODOGEN method
(Fraker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978)) can
be used to incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0327] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See, e.g., WO94/11026. The linker
may be a "cleavable linker" facilitating release of the cytotoxic
drug in the cell. For example, an acid-labile linker,
peptidase-sensitive linker, photolabile linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Research
52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
[0328] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0329] v. Preparation of Conjugated Antibodies
[0330] In the conjugated antibodies of the invention, an antibody
is conjugated to one or more moieties (for example, drug moieties),
e.g. about 1 to about 20 moieties per antibody, optionally through
a linker. The conjugated antibodies may be prepared by several
routes, employing organic chemistry reactions, conditions, and
reagents known to those skilled in the art, including: (1) reaction
of a nucleophilic group of an antibody with a bivalent linker
reagent via a covalent bond, followed by reaction with a moiety of
interest; and (2) reaction of a nucleophilic group of a moiety with
a bivalent linker reagent via a covalent bond, followed by reaction
with the nucleophilic group of an antibody. Additional methods for
preparing conjugated antibodies are described herein.
[0331] The linker reagent may be composed of one or more linker
components. Exemplary linker components include 6-maleimidocaproyl
("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC`), and N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("STAB").
Additional linker components are known in the art and some are
described herein. See also "Monomethylvaline Compounds Capable of
Conjugation to Ligands," U.S. Application Publication No.
2005/0238649, the contents of which are hereby incorporated by
reference in its entirety.
[0332] In some embodiments, the linker may comprise amino acid
residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for
enzymatic cleavage by a particular enzymes, for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin
protease.
[0333] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e., cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues).
[0334] Conjugated antibodies of the invention may also be produced
by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic substituents on the
linker reagent or drug or other moiety. The sugars of glycosylated
antibodies may be oxidized, e.g., with periodate oxidizing
reagents, to form aldehyde or ketone groups which may react with
the amine group of linker reagents or drug or other moieties. The
resulting imine Schiff base groups may form a stable linkage, or
may be reduced, e.g., by borohydride reagents to form stable amine
linkages. In one embodiment, reaction of the carbohydrate portion
of a glycosylated antibody with either glactose oxidase or sodium
meta-periodate may yield carbonyl (aldehyde and ketone) groups in
the protein that can react with appropriate groups on the drug or
other moiety (Hermanson, Bioconjugate Techniques). In another
embodiment, proteins containing N-terminal serine or threonine
residues can react with sodium meta-periodate, resulting in
production of an aldehyde in place of the first amino acid
(Geoghegan and Stroh, Bioconjugate Chem. 3:138-146 (1992); U.S.
Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0335] Likewise, nucleophilic groups on a moiety (such as a drug
moiety) include, but are not limited to: amine, thiol, hydroxyl,
hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine
carboxylate, and arylhydrazide groups capable of reacting to form
covalent bonds with electrophilic groups on linker moieties and
linker reagents including: (i) active esters such as NHS esters,
HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl
halides such as haloacetamides; and (iii) aldehydes, ketones,
carboxyl, and maleimide groups.
[0336] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate. In
yet another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the individual, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
V. THERAPEUTIC USES
[0337] The protein complexes such as antibodies and antibody
fragments described herein (e.g., a coiled coil containing
antibody, a tethered antibody, or an antibody made according to the
methods described herein) may be used for therapeutic applications.
For example, such antibodies and antibody fragments can be used for
the treatment of tumors, including pre-cancerous, non-metastatic,
metastatic, and cancerous tumors (e.g., early stage cancer), for
the treatment of allergic or inflammatory disorders, or for the
treatment of autoimmune disease, or for the treatment of a subject
at risk for developing cancer (for example, breast cancer,
colorectal cancer, lung cancer, renal cell carcinoma, glioma, or
ovarian cancer), an allergic or inflammatory disorder, or an
autoimmune disease.
[0338] The term cancer embraces a collection of proliferative
disorders, including but not limited to pre-cancerous growths,
benign tumors, and malignant tumors. Benign tumors remain localized
at the site of origin and do not have the capacity to infiltrate,
invade, or metastasize to distant sites. Malignant tumors will
invade and damage other tissues around them. They can also gain the
ability to break off from where they started and spread to other
parts of the body (metastasize), usually through the bloodstream or
through the Jymphatic system where the lymph nodes are located.
Primary tumors are classified by the type of tissue from which they
arise; metastatic tumors are classified by the tissue type from
which the cancer cells are derived. Over time, the cells of a
malignant tumor become more abnormal and appear less like normal
cells. This change in the appearance of cancer cells is called the
tumor grade and cancer cells are described as being
well-differentiated, moderately-differentiated,
poorly-differentiated, or undifferentiated. Well-differentiated
cells are quite normal appearing and resemble the normal cells from
which they originated. Undifferentiated cells are cells that have
become so abnormal that it is no longer possible to determine the
origin of the cells.
[0339] The tumor can be a solid tumor or a non-solid or soft tissue
tumor. Examples of soft tissue tumors include leukemia (e.g.,
chronic myelogenous leukemia, acute myelogenous leukemia, adult
acute lymphoblastic leukemia, acute myelogenous leukemia, mature
B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia,
polymphocytic leukemia, or hairy cell leukemia), or lymphoma (e.g.,
non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's
disease). A solid tumor includes any cancer of body tissues other
than blood, bone marrow, or the lymphatic system. Solid tumors can
be further separated into those of epithelial cell origin and those
of non-epithelial cell origin. Examples of epithelial cell solid
tumors include tumors of the gastrointestinal tract, colon, breast,
prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral
cavity, stomach, duodenum, small intestine, large intestine, anus,
gall bladder, labium, nasopharynx, skin, uterus, male genital
organ, urinary organs, bladder, and skin. Solid tumors of
non-epithelial origin include sarcomas, brain tumors, and bone
tumors.
[0340] Epithelial cancers generally evolve from a benign tumor to a
preinvasive stage (e.g., carcinoma in situ), to a malignant cancer,
which has penetrated the basement membrane and invaded the
subepithelial stroma.
[0341] Multispecific protein complexes can also be used in these
therapeutic applications, and antibodies that bind HER2 can in
particular be used to treat breast cancer, colorectal cancer, lung
cancer, renal cell carcinoma, glioma, or ovarian cancer.
[0342] Other subjects that are candidates for receiving
compositions of this invention have, or are at risk for developing,
abnormal proliferation of fibrovascular tissue, acne rosacea,
acquired immune deficiency syndrome, artery occlusion, atopic
keratitis, bacterial ulcers, Bechets disease, blood borne tumors,
carotid obstructive disease, choroidal neovascularization, chronic
inflammation, chronic retinal detachment, chronic uveitis, chronic
vitritis, contact lens overwear, corneal graft rejection, corneal
neovascularization, corneal graft neovascularization, Crohn's
disease, Eales disease, epidemic keratoconjunctivitis, fungal
ulcers, Herpes simplex infections, Herpes zoster infections,
hyperviscosity syndromes, Kaposi's sarcoma, leukemia, lipid
degeneration, Lyme's disease, marginal keratolysis, Mooren ulcer,
Mycobacteria infections other than leprosy, myopia, ocular
neovascular disease, optic pits, Osler-Weber syndrome
(Osler-Weber-Rendu), osteoarthritis, Paget's disease, pars
planitis, pemphigoid, phylectenulosis, polyarteritis, post-laser
complications, protozoan infections, pseudoxanthoma elasticum,
pterygium keratitis sicca, radial keratotomy, retinal
neovascularization, retinopathy of prematurity, retrolental
fibroplasias, sarcoid, scleritis, sickle cell anemia, Sogren's
syndrome, solid tumors, Stargart's disease, Steven's Johnson
disease, superior limbic keratitis, syphilis, systemic lupus,
Terrien's marginal degeneration, toxoplasmosis, tumors of Ewing
sarcoma, tumors of neuroblastoma, tumors of osteosarcoma, tumors of
retinoblastoma, tumors of rhabdomyosarcoma, ulcerative colitis,
vein occlusion, Vitamin A deficiency, Wegener's sarcoidosis,
undesired angiogenesis associated with diabetes, parasitic
diseases, abnormal wound healing, hypertrophy following surgery,
injury or trauma (e.g., acute lung injury/ARDS), inhibition of hair
growth, inhibition of ovulation and corpus luteum formation,
inhibition of implantation, and inhibition of embryo development in
the uterus.
[0343] Examples of allergic or inflammatory disorders or autoimmune
diseases or disorders that may be treated using a coiled coil
containing antibody, a tethered antibody, or an antibody made
according to the methods described herein include, but are not
limited to arthritis (rheumatoid arthritis such as acute arthritis,
chronic rheumatoid arthritis, gouty arthritis, acute gouty
arthritis, chronic inflammatory arthritis, degenerative arthritis,
infectious arthritis, Lyme arthritis, proliferative arthritis,
psoriatic arthritis, vertebral arthritis, and juvenile-onset
rheumatoid arthritis, osteoarthritis, arthritis chronica
progrediente, arthritis deformans, polyarthritis chronica primaria,
reactive arthritis, and ankylosing spondylitis), inflammatory
hyperproliferative skin diseases, psoriasis such as plaque
psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of
the nails, dermatitis including contact dermatitis, chronic contact
dermatitis, allergic dermatitis, allergic contact dermatitis,
dermatitis herpetiformis, and atopic dermatitis, x-linked hyper IgM
syndrome, urticaria such as chronic allergic urticaria and chronic
idiopathic urticaria, including chronic autoimmune urticaria,
polymyositis/dermatomyositis, juvenile dermatomyositis, toxic
epidermal necrolysis, scleroderma (including systemic scleroderma),
sclerosis such as systemic sclerosis, multiple sclerosis (MS) such
as spino-optical MS, primary progressive MS (PPMS), and relapsing
remitting MS (RRMS), progressive systemic sclerosis,
atherosclerosis, arteriosclerosis, sclerosis disseminata, and
ataxic sclerosis, inflammatory bowel disease (IBD) (for example,
Crohn's disease, autoimmune-mediated gastrointestinal diseases,
colitis such as ulcerative colitis, colitis ulcerosa, microscopic
colitis, collagenous colitis, colitis polyposa, necrotizing
enterocolitis, and transmural colitis, and autoimmune inflammatory
bowel disease), pyoderma gangrenosum, erythema nodosum, primary
sclerosing cholangitis, episcleritis), respiratory distress
syndrome, including adult or acute respiratory distress syndrome
(ARDS), meningitis, inflammation of all or part of the uvea,
iritis, choroiditis, an autoimmune hematological disorder,
rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases
such as anaphylaxis and allergic and atopic rhinitis, encephalitis
such as Rasmussen's encephalitis and limbic and/or brainstem
encephalitis, uveitis, such as anterior uveitis, acute anterior
uveitis, granulomatous uveitis, nongranulomatous uveitis,
phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN or idiopathic membranous nephropathy,
membrano- or membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
allergic reaction, eczema including allergic or atopic eczema,
asthma such as asthma bronchiale, bronchial asthma, and auto-immune
asthma, conditions involving infiltration of T-cells and chronic
inflammatory responses, chronic pulmonary inflammatory disease,
autoimmune myocarditis, leukocyte adhesion deficiency, systemic
lupus erythematosus (SLE) or systemic lupus erythematodes such as
cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal
lupus syndrome (NLE), lupus erythematosus disseminatus, lupus
(including nephritis, cerebritis, pediatric, non-renal,
extra-renal, discoid, alopecia), juvenile onset (Type I) diabetes
mellitus, including pediatric insulin-dependent diabetes mellitus
(IDDM), adult onset diabetes mellitus (Type II diabetes),
autoimmune diabetes, idiopathic diabetes insipidus, immune
responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including lymphomatoid granulomatosis, Wegener's
granulomatosis, agranulocytosis, vasculitides, including vasculitis
(including large vessel vasculitis (including polymyalgia
rheumatica and giant cell (Takayasu's) arteritis), medium vessel
vasculitis (including Kawasaki's disease and polyarteritis nodosa),
microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous,
or hypersensitivity vasculitis, systemic necrotizing vasculitis,
and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or
syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
hemolytic anemia or immune hemolytic anemia including autoimmune
hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa),
Addison's disease, pure red cell anemia or aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome such as
those secondary to septicemia, trauma or hemorrhage,
antigen-antibody complex-mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet's or Behcet's disease, Castleman's
syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's
syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid
bullous and skin pemphigoid, pemphigus (including pemphigus
vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid,
and pemphigus erythematosus), autoimmune polyendocrinopathies,
Reiter's disease or syndrome, immune complex nephritis,
antibody-mediated nephritis, neuromyelitis optica,
polyneuropathies, chronic neuropathy such as IgM polyneuropathies
or IgM-mediated neuropathy, thrombocytopenia (as developed by
myocardial infarction patients, for example), including thrombotic
thrombocytopenic purpura (TTP) and autoimmune or immune-mediated
thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP)
including chronic or acute ITP, autoimmune disease of the testis
and ovary including autoimune orchitis and oophoritis, primary
hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases
including thyroiditis such as autoimmune thyroiditis, Hashimoto's
disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, polyglandular syndromes such as autoimmune
polyglandular syndromes (or polyglandular endocrinopathy
syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome
or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,
encephalomyelitis such as allergic encephalomyelitis or
encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE), myasthenia gravis such as
thymoma-associated myasthenia gravis, cerebellar degeneration,
neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS),
and sensory neuropathy, multifocal motor neuropathy, Sheehan's
syndrome, autoimmune hepatitis, chronic hepatitis, lupoid
hepatitis, giant cell hepatitis, chronic active hepatitis or
autoimmune chronic active hepatitis, lymphoid interstitial
pneumonitis, bronchiolitis obliterans (non-transplant) vs NSIP,
Guillain-Barre syndrome, Berger's disease (IgA nephropathy),
idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary
cirrhosis, pneumonocirrhosis, autoimmune enteropathy syndrome,
Celiac disease, Coeliac disease, celiac sprue (gluten enteropathy),
refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic
lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery
disease, autoimmune ear disease such as autoimmune inner ear
disease (AIED), autoimmune hearing loss, opsoclonus myoclonus
syndrome (OMS), polychondritis such as refractory or relapsed
polychondritis, pulmonary alveolar proteinosis, amyloidosis,
scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis,
which includes monoclonal B cell lymphocytosis (e.g., benign
monoclonal gammopathy and monoclonal gammopathy of undetermined
significance, MGUS), peripheral neuropathy, paraneoplastic
syndrome, channelopathies such as epilepsy, migraine, arrhythmia,
muscular disorders, deafness, blindness, periodic paralysis, and
channelopathies of the CNS, autism, inflammatory myopathy, focal
segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy,
uveoretinitis, chorioretinitis, autoimmune hepatological disorder,
fibromyalgia, multiple endocrine failure, Schmidt's syndrome,
adrenalitis, gastric atrophy, presenile dementia, demyelinating
diseases such as autoimmune demyelinating diseases, diabetic
nephropathy, Dressler's syndrome, alopecia greata, CREST syndrome
(calcinosis, Raynaud's phenomenon, esophageal dysmotility,
sclerodactyl), and telangiectasia), male and female autoimmune
infertility, mixed connective tissue disease, Chagas' disease,
rheumatic fever, recurrent abortion, farmer's lung, erythema
multiforme, post-cardiotomy syndrome, Cushing's syndrome,
bird-fancier's lung, allergic granulomatous angiitis, benign
lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis,
Sampter's syndrome, Caplan's syndrome, dengue, endocarditis,
endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis,
interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic
fibrosis, endophthalmitis, erythema elevatum et diutinum,
erythroblastosis fetalis, eosinophilic faciitis, Shulman's
syndrome, Felty's syndrome, flariasis, cyclitis such as chronic
cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's
cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus
(HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's
disease, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, giant cell polymyalgia, endocrine ophthamopathy,
chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca,
epidemic keratoconjunctivitis, idiopathic nephritic syndrome,
minimal change nephropathy, benign familial and
ischemia-reperfusion injury, retinal autoimmunity, joint
inflammation, bronchitis, chronic obstructive airway disease,
silicosis, aphthae, aphthous stomatitis, arteriosclerotic
disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica, enteritis allergica, erythema nodosum leprosum,
idiopathic facial paralysis, chronic fatigue syndrome, febris
rheumatica, Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary
idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis
granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma
gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,
infertility due to antispermatozoan antobodies, non-malignant
thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases,
acquired immune deficiency syndrome (AIDS), parasitic diseases such
as Leishmania, toxic-shock syndrome, food poisoning, conditions
involving infiltration of T-cells, leukocyte-adhesion deficiency,
immune responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, diseases involving
leukocyte diapedesis, multiple organ injury syndrome,
antigen-antibody complex-mediated diseases, antiglomerular basement
membrane disease, allergic neuritis, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune
atrophic gastritis, sympathetic ophthalmia, rheumatic diseases,
mixed connective tissue disease, nephrotic syndrome, insulitis,
polyendocrine failure, peripheral neuropathy, autoimmune
polyglandular syndrome type I, adult-onset idiopathic
hypoparathyroidism (AOIH), alopecia totalis, dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA),
hemochromatosis, myocarditis, nephrotic syndrome, primary
sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or
chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid
sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,
Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary eosinophilia, bronchopneumonic aspergillosis,
aspergilloma, or granulomas containing eosinophils, anaphylaxis,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune disorders associated with collagen disease, rheumatism,
neurological disease, ischemic re-perfusion disorder, reduction in
blood pressure response, vascular dysfunction, antgiectasis, tissue
injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia,
and disease accompanying vascularization, allergic hypersensitivity
disorders, glomerulonephritides, reperfusion injury, reperfusion
injury of myocardial or other tissues, dermatoses with acute
inflammatory components, acute purulent meningitis or other central
nervous system inflammatory disorders, ocular and orbital
inflammatory disorders, granulocyte transfusion-associated
syndromes, cytokine-induced toxicity, acute serious inflammation,
chronic intractable inflammation, pyelitis, pneumonocirrhosis,
diabetic retinopathy, diabetic large-artery disorder, endarterial
hyperplasia, peptic ulcer, valvulitis, and endometriosis.
[0344] In addition to therapeutic uses, the antibodies of the
invention can be used for other purposes, including diagnostic
methods, such as diagnostic methods for the diseases and conditions
described herein.
VI. DOSAGES, FORMULATIONS, AND DURATION
[0345] The proteins of this invention will be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual subject, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The "therapeutically
effective amount" of the proteins to be administered will be
governed by such considerations, and is the minimum amount
necessary to prevent, ameliorate, or treat a particular disorder
(for example, a cancer, allergic or inflammatory disorder, or
autoimmune disorder). The proteins need not be, but are optionally,
formulated with one or more agents currently used to prevent or
treat the disorder. The effective amount of such other agents
depends on the amount of proteins present in the formulation, the
type of disorder or treatment, and other factors discussed above.
These are generally used in the same dosages and with
administration routes as used hereinbefore or about from 1 to 99%
of the heretofore employed dosages. Generally, alleviation or
treatment of a cancer involves the lessening of one or more
symptoms or medical problems associated with the cancer. The
therapeutically effective amount of the drug can accomplish one or
a combination of the following: reduce (by at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the number of cancer
cells; reduce or inhibit the tumor size or tumor burden; inhibit
(i.e., to decrease to some extent and/or stop) cancer cell
infiltration into peripheral organs; reduce hormonal secretion in
the case of adenomas; reduce vessel density; inhibit tumor
metastasis; reduce or inhibit tumor growth; and/or relieve to some
extent one or more of the symptoms associated with the cancer. In
some embodiments, the proteins are used to prevent the occurrence
or reoccurrence of cancer or an autoimmune disorder in the
subject.
[0346] In one embodiment, the present invention can be used for
increasing the duration of survival of a human subject susceptible
to or diagnosed with a cancer or autoimmune disorder. Duration of
survival is defined as the time from first administration of the
drug to death. Duration of survival can also be measured by
stratified hazard ratio (HR) of the treatment group versus control
group, which represents the risk of death for a subject during the
treatment.
[0347] In yet another embodiment, the treatment of the present
invention significantly increases response rate in a group of human
subjects susceptible to or diagnosed with a cancer who are treated
with various anti-cancer therapies. Response rate is defined as the
percentage of treated subjects who responded to the treatment. In
one aspect, the combination treatment of the invention using
proteins of this invention and surgery, radiation therapy, or one
or more chemotherapeutic agents significantly increases response
rate in the treated subject group compared to the group treated
with surgery, radiation therapy, or chemotherapy alone, the
increase having a Chi-square p-value of less than 0.005. Additional
measurements of therapeutic efficacy in the treatment of cancers
are described in U.S. Patent Application Publication No.
20050186208.
[0348] Therapeutic formulations are prepared using standard methods
known in the art by mixing the active ingredient having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences
(20.sup.th edition), ed. A. Gennaro, 2000, Lippincott, Williams
& Wilkins, Philadelphia, Pa.). Acceptable carriers, include
saline, or buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagines, arginine or lysine; monosaccharides, disaccharides,
and other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM., PLURONICS.TM., or PEG.
[0349] Optionally, but preferably, the formulation contains a
pharmaceutically acceptable salt, preferably sodium chloride, and
preferably at about physiological concentrations. Optionally, the
formulations of the invention can contain a pharmaceutically
acceptable preservative. In some embodiments the preservative
concentration ranges from 0.1 to 2.0%, typically v/v. Suitable
preservatives include those known in the pharmaceutical arts.
Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben
are preferred preservatives. Optionally, the formulations of the
invention can include a pharmaceutically acceptable surfactant at a
concentration of 0.005 to 0.02%.
[0350] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0351] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
[0352] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0353] The proteins described herein (e.g., a coiled coil
containing antibody, a tethered antibody, or an antibody made
according to the methods described herein) are administered to a
human subject, in accord with known methods, such as intravenous
administration as a bolus or by continuous infusion over a period
of time, by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
topical, or inhalation routes. Local administration may be
particularly desired if extensive side effects or toxicity is
associated with antagonism to the target molecule recognized by the
proteins. An ex vivo strategy can also be used for therapeutic
applications. Ex vivo strategies involve transfecting or
transducing cells obtained from the subject with a polynucleotide
encoding a protein of this invention. The transfected or transduced
cells are then returned to the subject. The cells can be any of a
wide range of types including, without limitation, hemopoietic
cells (e.g., bone marrow cells, macrophages, monocytes, dendritic
cells, T cells, or B cells), fibroblasts, epithelial cells,
endothelial cells, keratinocytes, or muscle cells.
[0354] In one example, the protein complex is (e.g., a coiled coil
containing antibody, a tethered antibody, or an antibody made
according to the methods described herein) is administered locally,
e.g., by direct injections, when the disorder or location of the
tumor permits, and the injections can be repeated periodically. The
protein complex can also be delivered systemically to the subject
or directly to the tumor cells, e.g., to a tumor or a tumor bed
following surgical excision of the tumor, in order to prevent or
reduce local recurrence or metastasis.
VII. ARTICLES OF MANUFACTURE
[0355] Another embodiment of the invention is an article of
manufacture containing one or more protein complexes described
herein, and materials useful for the treatment or diagnosis of a
disorder (for example, an autoimmune disease or cancer). The
article of manufacture comprises a container and a label or package
insert on or associated with the container. Suitable containers
include, for example, bottles, vials, syringes, etc. The containers
may be formed from a variety of materials such as glass or plastic.
The container holds a composition that is effective for treating
the condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is an antibody or antibody fragment
antibody of the invention. The label or package insert indicates
that the composition is used for treating the particular condition.
The label or package insert will further comprise instructions for
administering the antibody composition to the subject. Articles of
manufacture and kits comprising combinatorial therapies described
herein are also contemplated.
[0356] Package insert refers to instructions customarily included
in commercial packages of therapeutic products that contain
information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such
therapeutic products. In certain embodiments, the package insert
indicates that the composition is used for treating breast cancer,
colorectal cancer, lung cancer, renal cell carcinoma, glioma, or
ovarian cancer.
[0357] Additionally, the article of manufacture may further
comprise a second container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
considered from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0358] Kits are also provided that are useful for various purposes,
e.g., for purification or immunoprecipitation of an antigen (e.g.,
HER2 or EGFR) from cells. For isolation and purification of an
antigen (e.g., HER2 or EGFR) the kit can contain an antibody (e.g.,
an EGFR/HER2 antibody) coupled to beads (e.g., sepharose beads).
Kits can be provided which contain the antibodies for detection and
quantitation of the antigen in vitro, e.g., in an ELISA or a
Western blot. As with the article of manufacture, the kit comprises
a container and a label or package insert on or associated with the
container. The container holds a composition comprising at least
one multispecific antibody or antibody fragment of the invention.
Additional containers may be included that contain, e.g., diluents
and buffers or control antibodies. The label or package insert may
provide a description of the composition as well as instructions
for the intended in vitro or diagnostic use.
[0359] The foregoing written description is considered to be
sufficient to enable one skilled in the art to practice the
invention. The following Examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
VII. TARGET MOLECULES
[0360] Examples of molecules that may be targeted by a complex of
this invention include, but are not limited to, soluble serum
proteins and their receptors and other membrane bound proteins
(e.g., adhesins).
[0361] In another embodiment the binding protein of the invention
is capable of binding one, two or more cytokines, cytokine-related
proteins, and cytokine receptors selected from the group consisting
of BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2
(GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3
(int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10,
FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21,
FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1,
IFNG, IFNW1, FEL1, FEL1 (EPSELON), FEL1 (ZETA), ILIA, IL1B, IL2,
IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13,
IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24,
IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA,
TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40
ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand),
TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL),
TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B,
TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF,
VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, LL1RL2, IL2RA, IL2RB, IL2RG,
IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB,
IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1,
IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN,
IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and
THPO.
[0362] In another embodiment, a target molecule is a chemokine,
chemokine receptor, or a chemokine-related protein selected from
the group consisting of CCL1 (1-309), CCL2 (MCP-1/MCAF), CCL3
(MIP-Ia), CCL4 (MIP-Ib), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2),
CCLH (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17
(TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3a), CCL21
(SLC/exodus-2), CCL22 (MDC/STC-I), CCL23 (MPIF-I), CCL24
(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27
(CTACK/ILC), CCL28, CXCL1 (GRO1), CXCL2 (GRO2), CXCL3 (GRO3), CXCL5
(ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL1O (IP 10), CXCLI1
(1-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP
(CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphotactin), XCL2 (SCM-Ib),
BLR1 (MDR15), CCBP2 (D6/JAB61), CCR1 (CKR1/HM145), CCR2
(mcp-1RB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6
(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBII), CCR8
(CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR),
XCR1 (GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4,
GPR2 (CCR10), GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6
(TYMSTR/STRL33/Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R
(GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6,
CKLFSF7, CKLFSF8, BDNF, C5R1, CSF3, GRCC1O (C10), EPO, FY (DARC),
GDF5, HDF1A, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1,
TREM2, and VHL.
[0363] In another embodiment the binding proteins of the invention
are capable of binding one or more targets selected from the group
consisting of ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A;
Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2;
ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1
(zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); BAG1; BAI1;
BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BMP1; BMP2; BMP3B (GDF10);
BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1;
C19orf1O (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1;
CCBP2 (D6/JAB61); CCL1 (1-309); CCLI1 (eotaxin); CCL13 (MCP-4);
CCL15 (MIP-Id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19
(MIP-3b); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21 (MTP-2); SLC;
exodus-2; CCL22 (MDC/STC-I); CCL23 (MPIF-1); CCL24
(MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27
(CTACK/ILC); CCL28; CCL3 (MTP-Ia); CCL4 (MDP-Ib); CCL5 (RANTES);
CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1
(CKR1/HM145); CCR2 (mcp-1RB/RA); CCR3 (CKR3/CMKBR3); CCR4; CCR5
(CMKBR5/ChemR13); CCR6 (CMKBR6/CKR-L3/STRL22/DRY6); CCR7
(CKR7/EBB); CCR8 (CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1
(VSHK1); CCRL2 (L-CCR); CD164; CD19; CD1C; CD20; CD200; CD22; CD24;
CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44;
CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81;
CD83; CD86; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19;
CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7;
CDK9; CDKN1A (p21Wap1/Cip1); CDKN1B (p27Kip1); CDKN1C; CDKN2A
(P16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB;
Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6;
CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin);
CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2;
CRP; CSF1 (M-CSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1
(b-catenin); CTSB (cathepsin B); CX3CL1 (SCYD1); CX3CR1 (V28);
CXCL1 (GRO1); CXCL10 (IP-10); CXCLI1 (I-TAC/IP-9); CXCL12 (SDF1);
CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5
(ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9/CKR-L2);
CXCR4; CXCR6 (TYMSTR/STRL33/Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP;
DES; DKFZp451J0118; DNCL1; DPP4; E2F1; ECGF1; EDG1; EFNA1; EFNA3;
EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2
(Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A;
FCER2; FCGR3A; FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B;
FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20;
FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2);
FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FEL1 (EPSILON); FIL1
(ZETA); FLJ 12584; FLJ25530; FLRT1 (fibronectin); FLT1; FOS; FOSL1
(FRA-I); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST;
GATA3; GDF5; GFI1; GGT1; GM-CSF; GNAS1; GNRH1; GPR2 (CCR1O); GPR31;
GPR44; GPR81 (FKSG80); GRCC1O (C1O); GRP; GSN (Gelsolin); GSTP1;
HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HDP1; histamine
and histamine receptors; HLA-A; HLA-DRA; HM74; HMOX1; HUMCYT2A;
ICEBERG; ICOSL; ID2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6;
IFNA7; IFNB1; IFNgamma; DFNW1; IGBP1; IGF1; IGF1R; IGF2; IGFBP2;
IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12;
IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13 .mu.l; IL13RA2; IL14;
IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP;
IL18R1; IL18RAP; IL19; IL1A; IL1B; ILIF10; IL1F5; IL1F6; IL1F7;
IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; RAPL1; IL1RAPL2; RL1;
IL1RL2, IL1RN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2;
IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB;
IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST
(glycoprotein 130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R;
DLK; INHA; INHBA; INSL3; INSL4; IRAKI; ERAK2; ITGA1; ITGA2; ITGA3;
ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAG1;
JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP); KLF6;
KLK1O; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9;
KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6 (hair-specific type H
keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA
(TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp;
MAP2K7 (c-Jun); MDK; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67);
MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-III); MTSS1; MUC1
(mucin); MYC; MYD88; NCK2; neurocan; NFKB1; NFKB2; NGFB (NGF);
NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1
(NM23A); N0X5; NPPB; NROB1; NROB2; NRID1; NR1D2; NR1H2; NR1H3;
NR1H4; NR112; NR113; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2;
NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1;
NRP1; NRP2; NT5E; NTN4; ODZ1; OPRD1; P2RX7; PAP; PART1; PATE; PAWR;
PCA3; PCNA; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR;
phosphacan; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7);
PPID; PR1; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN;
PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGS1; RGS13; RGS3; RNFI1O
(ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1
(mammaglobin2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial
Monocyte-activating cytokine); SDF2; SERPINA1; SERPINA3; SERP1NB5
(maspin); SERPINE1 (PAI-I); SERPDMF1; SHBG; SLA2; SLC2A2; SLC33A1;
SLC43A1; SL1T2; SPP1; SPRR1B (Spr1); ST6GAL1; STAB1; STAT6; STEAP;
STEAP2; TB4R2; TBX21; TCP1O; TDGF1; TEK; TGFA; TGFB1; TGFB1II;
TGFB2; TGFB3; TGFBI; TGFBR1; TGFBR2; TGFBR3; TH1L; THBS1
(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissue
factor; TLR1O; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF;
TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSF1A; TNFRSF1B;
TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9;
TNFSF10 (TRAIL); TNFSF1 1 (TRANCE); TNFSF12 (APO3L); TNFSF13
(April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18;
TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7
(CD27 ligand); TNFSF8 (CD30 ligand); TNFSF9 (4-1BB ligand); TOLLIP;
Toll-like receptors; TOP2A (topoisomerase Ea); TP53; TPM1; TPM2;
TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2;
TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4;
XCL1 (lymphotactin); XCL2 (SCM-Ib); XCR1 (GPR5/CCXCR1); YY1; and
ZFPM2.
[0364] Preferred molecular target molecules for antibodies
encompassed by the present invention include CD proteins such as
CD3, CD4, CD8, CD16, CD19, CD20, CD34; CD64, CD200 members of the
ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4
receptor; cell adhesion molecules such as LFA-1, Macl, p150.95,
VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin, and alphav/beta3
integrin including either alpha or beta subunits thereof (e.g.
anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors
such as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon
alphaIFN); TNFalpha, an interleukin, such as IL-1beta, IL-3, IL-4,
IL-5, IL-8, IL-9, IL-13, IL17A/F, IL-18, IL-13Ralpha1, IL13Ralpha2,
IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2/flt3 receptor;
obesity (OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F
protein, protein C etc.
[0365] In one embodiment, the heteromultimeric complexes of this
invention binds to at least two target molecules selected from the
group consisting of: IL-1alpha and IL-1beta, IL-12 and IL-18; IL-13
and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and
IL-1beta; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and
MEF; IL-13 and TGF-13; IL-13 and LHR agonist; IL-12 and TWEAK,
IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and
ADAM8, IL-13 and PED2, IL17A and IL17F, CD3 and CD19, CD138 and
CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38 and CD138;
CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CD20
and BR3, TNFalpha and TGF-beta, TNFalpha and IL-1beta; TNFalpha and
IL-2, TNF alpha and IL-3, TNFalpha and IL-4, TNFalpha and IL-5,
TNFalpha and IL6, TNFalpha and IL8, TNFalpha and IL-9, TNFalpha and
IL-10, TNFalpha and IL-11, TNFalpha and IL-12, TNFalpha and IL-13,
TNFalpha and IL-14, TNFalpha and IL-15, TNFalpha and IL-16,
TNFalpha and IL-17, TNFalpha and IL-18, TNFalpha and IL-19,
TNFalpha and IL-20, TNFalpha and IL-23, TNFalpha and IFNalpha,
TNFalpha and CD4, TNFalpha and VEGF, TNFalpha and MIF, TNFalpha and
ICAM-1, TNFalpha and PGE4, TNFalpha and PEG2, TNFalpha and RANK
ligand, TNFalpha and Te38; TNFalpha and BAFF; TNFalpha and CD22;
TNFalpha and CTLA-4; TNFalpha and GP130; TNF.alpha. and IL-12p40;
VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2,
VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8,
VEGF and MET, VEGFR and MET receptor, VEGFR and EGFR, HER2 and
CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR(HER1) and
HER2, EGFR and HER3, EGFR and HER4, IL-13 and CD40L, IL4 and CD40L,
TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and
CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and
BTNO2; IGF1 and IGF2; IGF1/2 and Erb2B; MAG and RGM A; NgR and RGM
A; NogoA and RGM A; OMGp and RGM A; PDL-I and CTLA-4; and RGM A and
RGM B.
[0366] Soluble antigens or fragments thereof, optionally conjugated
to other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g. the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
[0367] All patents, patent applications, patent application
publications, and other publications cited or referred to in this
specification are herein incorporated by reference to the same
extent as if each independent patent, patent application, patent
application publication or publication was specifically and
individually indicated to be incorporated by reference. Such patent
applications specifically include U.S. provisional application Nos.
61/243,105 and 61/266,992, filed on Sep. 16, 2009 and Dec. 4, 2009,
respectively, from which this application claims benefit.
EXAMPLES
Example 1
Construction of Vectors for the Expression of Coiled Coil
Containing Antibodies
[0368] The coiled coil heterodimerization domains described herein
can be linked to a constant chain (e.g., the C-terminus of the HC)
of any antibody. Numerous antibody sequences that can be used to
construct coiled coil containing antibodies are known in the art
and techniques required to manipulate DNA sequences are also well
known in the art. An exemplary method for constructing coiled coil
containing antibodies is described below.
[0369] The HC backbone for the generation of antibodies containing
a coiled coil was constructed as follows. Sense and anti-sense
oligonucleotides were designed and synthesized to encode either the
ACID.p1 (GGSAQLEKELQALEKENAQLEWELQALEKELAQGAT; SEQ ID NO:33) or
BASE.p1 (GGSAQLKKKLQALKKKNAQLKWKLQALKKKLAQGAT; SEQ ID NO:34) coiled
coil domain sequence with 5' AscI and 3' XbaI overhangs. The
oligonucleotides were annealed, phosphorylated, and ligated into a
digested and dephosphorylated pRK plasmid (Genentech Inc.; Eaton et
al., Biochemistry 25:8343-8347 (1986)). The C.sub.H1 through
C.sub.H3 domain of a hIgG1 was prepared using PCR (polymerase chain
reaction) to include a 5' multiple cloning site (MCS)
(ClaI-BamHI-KpnI-ApaI) and a 3' AscI site and cloned into the
previously prepared pRK-ACID.p1 or pRK-BASE.p1 vector using ClaI
and AscI. Finally, the Lysine residue at position H222 (Kabat
numbering scheme) was mutated into an Alanine residue using
Stratagene's Quikchange II XL site-directed mutagenesis kit to
prevent Fab release during Lys-C cleavage.
[0370] Antibodies containing a coiled coil domain were constructed
as follows. For common LC and one-armed antibodies, the V.sub.H
domain of the desired antibody was prepared using PCR to include 5'
ClaI and 3' ApaI restriction sites. The PCR fragments were digested
and cloned into a similarly prepared backbone vector. No changes
had to be made to the LC constructs already available for these
antibodies.
[0371] For tethered antibodies the V.sub.H domain (minus the signal
sequence) of the desired antibody was first prepared using PCR
where the 5' primer contained the 3' half of a GGS tether and
terminated in a 5' BamHI site and the 3' primer terminated in a 3'
ApaI site. The fragments were digested and cloned into a similarly
prepared backbone vector. The cognate LC of the desired antibody
was then prepared using PCR where the 5' primer terminated in a 5'
ClaI site and the 3' primer contained the 5' portion of the GGS
tether and terminated in a 3' BamHI. The LC fragment was joined to
its cognate HC (now in the backbone vector) by cloning the fragment
in front of the V.sub.H using ClaI and BamHI. The completed tether
sequence linking the LC to the V.sub.H was
GGGSGGSGGSGGSGGSGGSGGSGGSG (SEQ ID NO:14). The vectors were
transfected into mammalian cells (CHO or 293 cells) using standard
transfection techniques.
[0372] A bispecific antibody that specifically binds both
Fc.epsilon.R1 and Fc.gamma.R2b and having a common LC was prepared
using the methods described herein. This antibody has a "BASE.p1"
sequence containing an anti-human Fc.gamma.R2b HC sequence with a
BASE.p1 coiled coil domain sequence and the K222A mutation (SEQ ID
NO:1), an "ACID.p1" sequence containing an anti-human Fc.epsilon.R1
HC sequence with an ACID.p1 coiled coil domain sequence and the
K222A mutation (SEQ ID NO:2), and a common LC sequence (SEQ ID
NO:3) (FIG. 8).
[0373] One-armed antibodies that specifically bind either HER2 or
EGFR were also prepared. The antibody that specifically binds HER2
contains an anti-HER2 antibody 1 HC sequence with an ACID.p1 coiled
coil domain sequence and the K222A mutation (SEQ ID NO:4), an HC
region lacking the VH and CH1 domains with a BASE.p1 coiled coil
domain sequence (SEQ ID NO:5), and an antib-HER2 antibody 1 LC
sequence (SEQ ID NO:6). The antibody that specifically binds EGFR
contains an anti-EGFR HC sequence with an ACID.p1 coiled coil
domain sequence and the K222A mutation (SEQ ID NO:7), an HC region
lacking the VH and CH1 domains with a BASE.p1 coiled coil domain
sequence (SEQ ID NO:5), and an anti-EGFR (D1.5) LC sequence (SEQ ID
NO:8) (FIGS. 9-1 and 9-2).
[0374] Tethered antibodies that specifically bind HER2 and
EGFR/HER1 were also prepared (FIGS. 10 and 11). One antibody that
specifically binds HER2 and EGFR contains (1) an anti-HER2 antibody
1 LC sequence tethered to an anti-HER2 antibody 1 HC sequence by a
26 amino acid GGS tether, an ACID.p1 coiled coil domain sequence,
and the K222A mutation (SEQ ID NO:9) and (2) an anti-EGFR antibody
LC sequence tethered to an anti-EGFR antibody HC sequence by a 26
amino acid GGS tether, a BASE.p1 coiled coil domain sequence, and
the K222A mutation (SEQ ID NO:10) (FIG. 10). A second antibody that
specifically binds HER2 and EGFR contains (1) the anti-HER2
antibody 2 LC sequence tethered to the anti-HER2 antibody 2 HC
sequence by a 26 amino acid GGS tether, an ACID.p1 coiled coil
domain sequence, and the K222A mutation (SEQ ID NO:11) and (2) an
anti-EGFR antibody LC sequence tethered to an anti-EGFR antibody HC
sequence by a 26 amino acid GGS tether, a BASE.p1 coiled coil
domain sequence, and the K222A mutation (SEQ ID NO:10) (FIG. 11).
Anti-HER2 antibody 1 LC and HC sequences used in the construction
of the coiled coil containing antibodies are shown in FIGS. 12A and
12B (SEQ ID NOS:15 and 16). The location of various restriction
sites used in constructing the vectors encoding these antibodies is
also shown in FIGS. 12B1-3.
Example 2
Purification of Coiled Coil Containing Antibodies
[0375] An exemplary schema that can be used to purify coiled coil
containing antibodies is shown below.
##STR00002##
[0376] In particular, antibodies were purified from conditioned
media using mAbSure Select resin from GE Healthcare (Sweden)
overnight at 4.degree. C. The column was washed with two column
volumes (CV) of PBS (phosphate buffered saline), followed by 10 CV
of PBS+0.1% Triton X114 detergent, followed by 10 CV potassium
phosphate buffer. The columns were eluted with 10 mM Acetic Acid
(pH 2.9) and immediately diluted with Arginine (100 mM final
concentration) and Tris (200 mM final concentration), pH 8.0.
Coiled coils were removed from antibodies upon treatment with a
1:500 (weight:weight) ratio of Lys-C endopeptidase (Wako Pure
Chemical Laboratories) at 37.degree. C. for 1-5 hours. Cleaved
samples were loaded back over an mAbSure resin column to separate
cleaved coiled-coils from antibodies and eluted as above. Antibody
concentrations were adjusted to 10 mg/ml prior to separation via
size exclusion chromatography using a Sephacryl S200 column run in
PBS, 150 mM NaCl, 100 mM Arginine, and 1 mM NaN.sub.3. Peak
fractions were pooled and dialyzed against PBS overnight prior to
mass spectrum analysis to ensure identity and purity.
[0377] In addition to Arginine, other chaotropic agents or mild
detergents that can be used in the above purification protocol
after the initial mAbSure resin column step include, but are not
limited to, Guanidine-HCl, urea, lithium perclorate, Histidine, SDS
(sodium dodecyl sulfate), Tween, Triton, and NP-40, all of which
are commercially available. Diluting the antibody into a solution
containing a chaotropic agent or mild detergent after elution from
the initial Protein A containing column (e.g., mAbSure column)
maintains the stability of the antibody post elution and allows for
the efficient removal of the coiled coil by Lys-C
endopeptidase.
[0378] Table 1 shows a summary of the purification results for
Anti-HER2 antibody 1/.alpha.-EGFR (D1.5) antibodies.
TABLE-US-00004 TABLE 1 mAb Sure S200 Sample Column Column Volume
Recovery Recovery Yield Aggregation 40 L 200 mg 147 mg 73% 18% 50 L
246 mg 196 mg 80% 13% 50 L 280 mg 213 mg 76% 11%
[0379] The coiled coil was removed from the antibody by Lys-C
endopeptidase during the purification process.
[0380] An antibody constructed using coiled coil heterdimerization
domains, but which no longer contains the coiled coil, is referred
to as an "engineered antibody" in the following examples.
Example 3
Cleavage of Coiled Coil Containing Antibodies
[0381] The various coiled coil containing antibodies were subjected
to cleavage experiments to show that the coiled coil (and tether,
if present) could be cleaved from the antibody sequence while
leaving the antibody sequence intact. In particular, FIGS. 13A and
B show that the coiled coil was cleaved from an exemplary
.alpha.-Fc.epsilon.R1/.alpha.-Fc.gamma.R2b antibody using Lys-C
endopeptidase and that the antibody remained intact. The
theoretical mass for the antibody with the coiled coil is within
the margin of error of the mass experimentally observed by mass
spectrometry. Similary, the theoretical mass for the engineered
antibody without the coiled coil is within the margin of error of
that experimentally observed by mass spectrometry showing that
Lys-C cleaved the coiled coil from the antibody.
[0382] Mass spectrometry results also demonstrated that Lys-C
endopeptidase did not cleave the LC or HC of the exemplary
.alpha.-FcER1/.alpha.-Fc.gamma.R2b antibody (FIGS. 14A and B). In
particular, the molecular mass was determined both pre-Lys-C
endopeptidase treatment (left panels) and post-Lys-C endopeptidase
treatment (right panels) for the LC (top two panels) and the
.alpha.-Fc.epsilon.R1 and .alpha.-Fc.gamma.R2b HCs (bottom four
panels) using mass spectrometry. The experimentally observed
molecular masses are within the margin of error of the theoretical
masses for the various contructs showing that Lys-C endopeptidase
cleaved the coiled coil domain from the HC, but did not cleave the
LC or HC itself.
[0383] Similarly, mass spectrometry results demonstrated that the
coiled coil was cleaved from an exemplary one-armed .alpha.-EGFR
antibody using Lys-C endopeptidase (FIGS. 17A and B). In
particular, the experimentally observed molecular mass was within
the margin of error of the theoretical mass for both the one-armed
antibody with the coiled coil and for the one-armed antibody
without the coiled coil. As shown in FIGS. 18A-C, the theoretical
molecular mass was within the margin of error of the experimentally
observed molecular mass for each construct, indicating that Lys-C
endopeptidase did not cleave the LC, HC, or HC lacking the VH and
CH1 domains (one-armed Fc) of the exemplary .alpha.-EGFR antibody,
but did cleave the coiled coil domains from the HC and HC lacking
the VH anc CH1 domains.
[0384] In addition, mass spectrometry results showed that the
coiled coil was cleaved from an exemplary tethered
.alpha.-HER2/.alpha.-EGFR antibody using Lys-C endopeptidase (FIGS.
19A and B). As shown in FIG. 19B, the theoretical and
experimentally observed molecular masses are within the margin of
error for each construct. The coiled coil was also cleaved from the
exemplary tethered .alpha.-HER2/.alpha.-EGFR antibody using Lys-C
endopeptidase where the antibody had first treated with Lys-C
endopeptidase and the sample then was subjected to mass
spectrometry analysis (FIGS. 20A-B). The theoretical molecular mass
for each construct is within the margin of error of the
experimentally observed molecular mass, indicating that the coiled
coil is indeed cleaved from the antibody sequence and that the
antibody sequence itself is not cleaved. The mass spectrometry
results, including the molecular mass (MS), for exemplary coiled
coil containing antibodies are summarized in Table 2.
TABLE-US-00005 TABLE 2 MS MS LLS MS MS Cleaved, Cleaved, Sample
Conc. Agg. Intact Reduced FL Reduced Common LC 0.64 mg/ml 5.20%
156503 LC 23262 147800 LC 23263 anti-Fc.epsilon.R1/ HC-1 54918 HC-1
50525 anti-Fc.gamma.R2b HC-2 55165 HC-2 50763 One-Armed 1.0 mg/ml
109359 LC 23440 100665 LC 23440 Anti-HER2 FC 30907 FC 26568
(antibody 1) HC 55016 HC 50665 One-Armed 1.0 mg/ml 5.50% 109119 LC
23326 100419 LC 23326 EGFR FC 30910 FC 26568 HC 54881 HC 50532
Tethered anti- 10 mg/ml 1.80% 160057 EGFR 79903 151367 EGFR 75561
EGFR(D1.5)/ HER 80156 HER 75810 Anti-HER2 (antibody 1) FL = Full
Length; Conc. = Concentration; Agg. = Aggregation
Example 4
Characterization of Engineered Antibodies
[0385] To determine whether the exemplary engineered antibodies
constructed using coiled coil heterodimerization domains retained
the binding properties of the antibodies from which their sequences
were derived, binding assays were conducted. These binding assays
were run using the kinetics wizard program on the ForteBio Octet
system. All samples tested were at a concentration of 25 .mu.g/ml,
a concentration that indicates saturation of the anti-human IgG
probes in repeat experiments and among varying samples. The probes
were loaded with the first sample for 15 minutes and washed for 30
seconds in PBS. All associations for the second and third samples
were carried out for 10 minutes with 30-second PBS washes between
associations.
[0386] In particular, the common LC
anti-Fc.epsilon.R1/anti-Fc.gamma.R2b bispecific engineered antibody
was loaded onto an anti-human IgG probe (Octet) by incubating the
probe with 25 .mu.g/ml of the antibody for 15 minutes followed by a
PBS wash step. To evaluate binding, the loaded probe was incubated
with 25 .mu.g/ml of Fc.epsilon.R1 and subsequently 25 .mu.g/ml of
Fc.gamma.R2b. A PBS wash step was performed between the two binding
incubations. The data represented in FIG. 15 shows that the
bispecific, engineered antibody simultaneously bound both of its
antigens.
[0387] To test the functionality of the engineered antibodies, a
rat basophil leukemia (RBL) cell line created to express human
Fc.epsilon.RIa and human Fc.gamma.R2b1 was cultured for 72 hours at
37.degree. C. with 1 .mu.g/ml NP-specific human IgE (JW8.5.13) in
complete growth media (MEM with Earle's salts Gibco Cat#11090, 1 mM
glutamine (Genentech Inc.), 1 mM sodium pyruvate (Gibco
Cat#11360-070), 0.1 mM nonessential amino acids (Gibco
Cat#11140-050), 1.5 g/L sodium bicarbonate (Gibco Cat#25080-094),
15% fetal bovine serum (Hyclone Cat# SH30071.03). Cells were
trypsinized and plated onto a 96-well, flat bottom tissue culture
plate at 3.5.times.10.sup.5 cells/ml in 200 .mu.l of complete
growth media containing 1 .mu.g/ml NP-specific human IgE and
allowed to adhere for 2 hours. Next, the cells were washed three
times with fresh media to remove unbound NP-specific human IgE.
Cells were treated with 0-10 .mu.g/ml of bispecific antibody and
incubated for 1 hour at 37.degree. C., prior to activation with
antigen. Cells were activated by incubation with 0.1 .mu.g/ml
NP-conjugated ovalbumin (Biosearch Technologies, Inc. Cat.
N-5051-10) or 45 minutes at 37.degree. C. Following incubation, the
histamine levels in the cell supernatants (cell culture medium)
were measured by ELISA (enzyme linked immunosorbent assay) using a
Histamine ELISA kit (KMI Diagnostics, Minneapolis, Minn.).
Background histamine levels were obtained from cells treated with
NP-specific human IgE alone with no activation (FIG. 16).
[0388] Octet binding studies were also performed for exemplary
one-armed antibodies and tethered engineered antibodies. As a
control, octet analysis was used to show that the wild-type
anti-HER2 antibody 1 and wild-type .alpha.-EGFR antibody did not
cross react with each other's antigen, but do bind their respective
antigen (FIG. 21). To test the exemplary coiled coil containing
antibodies, a one-armed anti-HER2 antibody 1 was loaded at 25
.mu.g/ml onto an anti-human IgG antibody probe for 15 minutes, and
the probe was subsequently washed with PBS for 30 seconds. The
loaded probe was then incubated with EGFR ECD (extracellular
domain) at 25 .mu.g/ml, which showed no binding signal. The probe
was then washed for 30 seconds in PBS and incubated with HER2
receptor ECD at 25 .mu.g/ml, which showed a strong binding signal
(FIG. 22; top trace).
[0389] A one-armed EGFR engineered antibody was loaded at 25
.mu.g/ml onto an anti-human IgG antibody probe for 15 minutes and
subsequently washed with PBS for 30 seconds. The probe was then
incubated with HER2ECD at 25 .mu.g/ml, which showed no binding
signal. The probe was washed for 30 seconds in PBS and incubated
with EGFR ECD at 25 .mu.g/ml, which showed a strong binding signal
(FIG. 22; bottom trace).
[0390] A tethered bispecific anti-EGFR(D1.5)/anti-HER2 engineered
antibody was incubated with an anti-human IgG antibody probe at 25
.mu.g/ml for 15 minutes and subsequently washed with PBS for 30
seconds. This incubation loaded the probe with the bispecific
antibody. The probe was then incubated with EGFR ECD at 25 .mu.g/ml
for 3 minutes followed by a 30 second PBS wash then subsequently
incubated with the HER2 receptor ECD at 25 .mu.g/ml for 3 minutes
(FIG. 23A; top trace). For the results shown in the bottom trace of
FIG. 23A, the bispecific loaded probe was first incubated with the
HER2 receptor ECD then with the EGFR ECD. The data show that the
bispecific, engineered antibody bound both the EGF and HER2
receptors simultaneously. As shown in FIG. 23B, the bispecific
anti-EGFR(D1.5)/anti-HER2 antibody bound HER2 with a Kd of
approximately 0.06 nM and bound EGF receptor with a Kd of
approximately 0.660 nM.
[0391] To further analyze the binding characteristics of the
engineered antibodies, cell based assays were performed on two cell
lines, either NR6 expressing EGFR or HER2, or HCA7 cells which
co-express both EGFR and HER2. Prior to performing the binding
assays, cells were harvested and allowed to cool for 30 minutes on
ice in binding buffer (RPMI medium with 1% fetal bovine serum
(FBS), 10 mM HEPES, and 0.2% NaN.sub.3). Unlabeled antibody was
prepared at the desired starting concentration and diluted 1:1 with
binding buffer to give multiple data points. Labeled antibody was
prepared at one concentration to be used throughout the entire
assay. Equilibrium binding studies were carried out using
radiolabeled antibody competed with various concentrations of
unlabeled antibody. Unlabeled antibody was placed in a 96-well
plate, followed by labeled material, and cells were then added to
the mixture. The plate was incubated for 2 hours at room
temperature. After the incubation, the plate was harvested using
Millipore Membrane Multi-Screen Plates to separate the solution
from the cells. The cell-bound radiolabeled antibody was then
counted on a Perkin Elmer Gamma counter and the data was analyzed
using New Ligand software. The results of the affinity binding
studies for one-armed and tethered engineered antibody constructs
are summarized in Table 3.
TABLE-US-00006 TABLE 3 Antibody Cell Line Kd (nM) Wt .alpha.-EGFR
(D1.5) NR6 expressing EGFR 0.56 +/- 0.19 .alpha.-EGFR Fab NR6
expressing EGFR 2.20 +/- 0.23 1-armed .alpha.-EGFR NR6 expressing
EGFR 1.15 +/- 0.05 Tethered NR6 expressing EGFR 2.79 +/- 0.13
.alpha.-EGFR/Anti-HER2 (antibody 1) Wt Anti-HER2 (antibody 1) NR6
expressing HER2 0.94 +/- 0.17 Anti-HER2 (antibody 1) NR6 expressing
HER2 2.78 +/- 0.11 Fab 1-armed NR6 expressing HER2 1.70 +/- 0.09
Anti-HER2 (antibody 1) Tethered NR6 expressing HER2 5.13 +/- 0.36
.alpha.-EGFR/Anti-HER2 (antibody 1) Tethered HCA7 co-expressing
0.93 +/- 0.11 .alpha.-EGFR/Anti-HER2 EGFR and HER2 (antibody 1) Wt
.alpha.-EGFR (D1.5) HCA7 co-expressing 0.34 +/- 0.06 EGFR and HER2
Wt Anti-HER2 (antibody 1) HCA7 co-expressing 0.12 +/- 0.03 EGFR and
HER2
[0392] The functional properties of exemplary engineered antibodies
were also characterized biochemically. EGFR-expressing NR6 cells
were plated in 12-well plates. Following serum starvation cells
were pre-incubated with various concentrations of antibodies for 2
hours at 37.degree. C. Subsequently, cells were stimulated with the
TGF.alpha. for 12 minutes. Whole cell lysates were subjected to
SDS-PAGE analysis, and immunoblots were probed with
anti-phosphotyrosine, anti-phosphoAkt, or anti-tubulin as a loading
control (FIG. 24). These results show that the exemplary
.alpha.-EGFR(D1.5)/Anti-HER2 (antibody 1) engineered antibody, like
the D1.5 IgG1 control antibody, inhibited TGF.alpha.-induced
phosphorylation in EGFR-expressing NR6 cells in a dose-dependent
manner.
[0393] For cell proliferation assays, cells were plated in 96-well
plates (EGFR-NR6: 2,000 cells/well) (BT474: 10,000 cells/well) and
incubated overnight at 37.degree. C. The next day, the medium was
removed and cells were treated in 1% serum containing medium. To
compare the effect on cell growth of the
.alpha.-EGFR(D1.5)/Anti-HER2 (antibody 1) engineered antibody to
the D1.5 antibody on EGFR-NR6 cells, 3 nM TGF.alpha. was added to
the medium, and cells were treated with various concentrations of
antibodies. After 3 days AlamarBlue was added to the wells and
fluorescence was read using a 96-well fluorometer with excitation
at 530 nm and emission of 590 nm. The results are expressed in
relative fluorescence units (RFU) (FIG. 25). To compare the effect
on cell growth of the .alpha.-EGFR(D1.5)/Anti-HER2 (antibody 1)
engineered antibody to the anti-HER2 antibody 1, BT474 cells were
treated in 1% serum containing medium with various concentrations
of antibody (FIG. 26). After 5 days AlamarBlue assays were
performed as described above. These results show that the exemplary
.alpha.-EGFR(D1.5)/Anti-HER2 (antibody 1) engineered antibody, like
the D1.5 IgG1 control antibody, inhibited TGF.alpha.-induced
phosphorylation in EGFR-expressing NR6 cells in a dose-dependent
manner and, like the anti-HER2 antibody 1, inhibited growth of
BT474 cells.
Example 5
Pharmacokinetic Analysis of Engineered Antibodies
[0394] Pharmacokinetic studies were conducted to compare the
pharmacokinetics (PK) of a bispecific engineered antibody with
those of typical human IgG (hIgG) antibodies, and to determine the
dosing for efficacy experiments. Like the D1.5 hIgG1 control
antibody, the HER1/HER2 (D1.5/Anti-HER2 antibody 1) engineered
antibody also showed cross-reactivity with mice. The anti-HER2
antibody 2 hIgG1 control antibody did not show cross-reactivity
with mice.
[0395] The PK of the D1.5 hIgG1 positive control antibody was
determined over a 10-day period using SCID Beige mice. In
particular, the serum concentration of the antibody over time was
determined using an Fc-Fc assay after administration of the
antibody at various doses (0.5 mg/kg, 5 mg/kg, and 50 mg/kg). In
addition, the serum concentration relative to dose was monitored
for ten days using an Fc-Fc ELISA assay (FIG. 27). The area under
the curve (AUC), normalized by dose, was also determined and is
summarized in Table 4. The D1.5 hIgG1 antibody showed nonlinear PK
in mice in the tested dose range.
TABLE-US-00007 TABLE 4 Dose (mg/kg) AUC till day 10 normalized by
dose 0.5 11.8 5 53.8 50 135
[0396] In addition, the PK of the anti-HER2 antibody 2 hIgG1
positive control antibodies was also determined over a 10-day
period using SCID Beige mice. The serum concentration of the
antibody over time was determined using an Fc-Fc ELISA or a
HER2-ECD (extracellular domain) ELISA after administration of the
antibody at 10 mg/kg. The AUC normalized by dose was also
determined and is summarized in Table 5.
TABLE-US-00008 TABLE 5 AUC till day 10 Molecule Assay Format
normalized by dose 10 mg/kg anti-HER2 HER2-ECD 42.9 (antibody 2)
hIgG1 Fc-Fc 63.3
[0397] Similarly, the PK of the HER1(EGFR)/HER2 (D1.5/Anti-HER2
antibody 1) engineered antibody was determined over a ten-day
period in SCID Beige mice. The serum concentration of the antibody
over time was determined using an Fc-Fc ELISA or an EGFR-HER2ELISA
after administration of the antibody at various doses (0.5 mg/kg, 5
mg/kg, and 20 mg/kg). In addition, the serum concentration relative
to dose was monitored for ten days using an Fc-Fc ELISA or
EGFR-HER2ELISA (FIG. 28). The AUC normalized by dose was also
determined and is summarized in Table 6. The HER1(EGFR)/HER2
(D1.5/Anti-HER2 antibody 1) engineered antibody showed nonlinear PK
in mice in the tested dose range.
TABLE-US-00009 TABLE 6 AUC till day 10 Dose mg/kg Assay format
normalized by dose 0.5 EGFR-HER2 83.8 Fc-Fc 104 5 EGFR-HER2 42.6
Fc-Fc 53.2 20 EGFR-HER2 95.0 Fc-Fc 148
[0398] Based on the results of the PK assays, the HER1(EGFR)/HER2
(D1.5/Anti-HER2 antibody 1) engineered antibody was determined to
have similar or better exposure in mice over the tested time period
(until day 10) in comparison to the D1.5 hIgG1 control antibo
xzzzdy (FIG. 29).
Example 6
Producing Tethered Antibodies in Mammalian Cell Lines Engineered to
Express Enzymes to Cleave Tethers
[0399] For construction of the 26AA Furin cleavable tethered coiled
coil antibodies (FIG. 30A) the VH domain (minus the signal
sequence) of the desired antibody was first prepared using PCR
wherein the 5' primer contained the 3' half of a GGS-Furin tether
and terminated in a 5' BamHI site and the 3' primer terminated in a
3' ApaI site. The fragments were digested and cloned into a
similarly prepared antibody-coiled coil backbone vector. The
cognate LC of the desired antibody was then prepared using PCR
wherein the 5' primer terminated in a 5' ClaI site and the 3'
primer contained the 5' portion of the Furin-GGS tether and
terminated in a 3' BamHI. The LC fragment was joined to its cognate
HC (now in the antibody coiled coil backbone) by cloning the
fragment in front of the VH via ClaI and BamHI. The completed
tether sequence linking the CL to the VH was
RCRRGSGGSGGSGGSGGSGGSGRSRKRR (SEQ ID NO:35). For construction of
the 26AA Furin-cleavable tether (--C) (FIG. 30B), two mutations
were introduced into the above mentioned construct. The c-terminal
Cys residue of the LC was mutated into and Ala residue using
Stratagene's Quikchange II XL site-directed mutagenesis kit.
According to the Kabat numbering system, the Cys terminal residue
in the CL is at position 214. C220 of the HC was also mutated into
an A to eliminate possible mis-folding due to this newly
non-disulfide bonded Cys.
[0400] The methods used in constructing the 32AA Furin cleavable
tether (FIG. 30C) was identical to the construction of the 26AA
Furin cleavable tether except that the finished tether sequence was
RKRKRRGSGGSGGSGGSGGSGGSGRSRKRR (SEQ ID NO:36). For Furin
over-expression, human or murine Furin was cloned into the pRK
vector system and co-transfected with the antibody chain
plasmids.
[0401] Carboxypeptidase B digestion (FIG. 30D) was carried out in
50 mM Sodium Borate pH8.0 for 1 hr. at 37C with 1:20 wt:wt of
CpB.
[0402] FIGS. 30A1-2 is a diagram and reduced Mass Spec (MS) results
for the 26 amino acid FURIN cleavable tether. The heavy chain MS
trace or graph shows a heavy chain (1) which has fully native n-
and c-termini as well as a smaller amount of "full length antibody"
(i.e., for these studies, was not cleaved at either Furin site
(FL)). The light chain MS trace shows a peak corresponding to the
LC plus the entire length of tether (1) and three other peaks (2-4)
corresponding to the erosion of the 3' end of the tether,
presumably due to Carboxypeptidase B activity in the CHO media.
Evidenced by the lack of MS peaks within the region of the bottom
trace indicated by the purple oval, there is no cleavage at the
n-terminal Furin site. A cartoon of the resulting antibody is
provided showing the non-native residues (underlined "R") as well
as the 23-26 amino acid tether still attached to the c-terminus of
the LC
[0403] FIG. 30B1-2 is a diagram and reduced Mass Spec (MS) results
for the 26 amino acid FURIN cleavable tether ("--C"). In this
construct, the C residue was removed and replaced). The heavy chain
MS trace shows a heavy chain (1) which has fully native n- and
c-termini and no remaining "full length antibody" (FL). The light
chain MS trace shows a peak corresponding to the LC plus 2
additional R residues (peak 2) plus one additional R residue (peak
3) and with it's native c-terminus (peak 4), presumably due to
Carboxypeptidase B activity in the CHO media. A cartoon of the
resulting antibody is provided showing the non-native residues
(yellow) as well as the 0, 1 or 2 R residues still attached to the
c-terminus of the LC.
[0404] FIG. 30C1-5 is a diagram and reduced Mass Spec (MS) results
for the 32 amino acid FURIN cleavable tether. FIG. 30C3 shows a
Heavy chain (peak 1) which has fully native n- and c-termini as
well as a smaller amount of "full length antibody" (FL) which was
not cleaved at either Furin site. FIGS. 30C2 and 30C3 show the
resulting material obtained from CHO cells expressing native levels
of Furin whereas FIGS. 30C4 and 30C5 show the resulting material
obtained from CHO cells over-expressing Furin. FIG. 30C2 shows a
peak corresponding to the LC plus the entire length of tether (peak
1) and five other peaks (peaks 2-6) corresponding to the erosion of
the 3' end of the tether as well as an additional peak showing the
LC with only the Furin recognition sequence still attached (peak 7)
and five additional peaks (peaks 8-12) corresponding to the erosion
of the c-terminal basic residues, presumably due to
Carboxypeptidase B activity in the CHO media. FIG. 30C5 shows a
heavy chain (1) which has fully native n- and c-termini and no
remaining Full length antibody (FL) and FIG. 30C4 shows the LC now
fully cleaved at the n-terminal Furin site (7) and four additional
peaks (8-11) corresponding to the erosion of the c-terminal basic
residues.
[0405] FIG. 30D2 is the same as FIG. 30C4. After a 1 hr. incubation
at 37C with 1:20 wt:wt of CpB, the remaining residues
(corresponding to peaks 7-11) were completely removed resulting in
a LC with a native c-terminus (FIG. 30D3). A cartoon is provided
showing the only non-native residues to be the K222A mutation in
each HC and an otherwise completely native (compared to parentals)
bispecific antibody.
Example 7
Expression of Enzyme-Cleavable Tethered Coiled-Coil Multisecific
Antibody in Eukaryotic Cells and Production of Multispecific
Antibody Without Tethers or Coiled Coils
[0406] Tethered. coiled coil bispecific antibodies comprising two
different VH and VL, each arm recognizing a different target, was
produced in CHO cells overexpressing human furin as described
above. The antibody, which also contained a K222A mutation, was
treated with Lys-C endopeptidase to remove the coiled coil and with
Carboxypeptidase B. It was not necessary to mutate the antibody any
further in the hinge, and constant regions to achieve the final
product. FIG. 31 shows a non-reduced mass spec trace of the
finished product. Although a small amount of homodimer is
observable in the non-reduced MS, this is due to the imbalance in
the expression level of the two Ab chains and is easily corrected
by modulating their relative expression levels. FIG. 32 shows a
reduced mass spec trace of the finished product. The observed
masses of the LCs and HCs confirm that the Ab chains all have
native n- and c-termini.
[0407] These results show that this platform can be used for the
production of several types of one-armed and bispecific antibodies
in mammalian cells. In our hands, we have been able to generate
mature bispecific antibodies differing from their parental wt Abs
only by a single Lys-Ala mutation within the hinge region of each
HC. These antibodies retain their specificity, and bispecific
variants are able to bind both antigens simultaneously. These
antibodies bind their antigens with high affinity.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 39 <210> SEQ ID NO 1 <211> LENGTH: 490 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 1 Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25
30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Gly Ile Thr Pro Asp Gly Gly Ala Thr Asp Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asp Leu Gly Ser Arg Glu
Phe Tyr Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220 Asp Ala Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280
285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405
410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 435 440 445 Pro Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln
Leu Lys Lys Lys Leu 450 455 460 Gln Ala Leu Lys Lys Lys Asn Ala Gln
Leu Lys Trp Lys Leu Gln Ala 465 470 475 480 Leu Lys Lys Lys Leu Ala
Gln Gly Ala Thr 485 490 <210> SEQ ID NO 2 <211> LENGTH:
490 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 2
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Tyr Ala
Asn 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Tyr Ile Gly Pro Asn Phe Gly Arg Ser Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Trp Arg
Arg Ser Leu Met Ser Val Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr
Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Ala Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260
265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385
390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Trp Arg Ala Gly Gly
Ser Ala Gln Leu Glu Lys Glu Leu 450 455 460 Gln Ala Leu Glu Lys Glu
Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala 465 470 475 480 Leu Glu Lys
Glu Leu Ala Gln Gly Ala Thr 485 490 <210> SEQ ID NO 3
<211> LENGTH: 214 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 3 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 <210> SEQ ID NO 4
<211> LENGTH: 489 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 4 Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220 Ala Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu
Gln 450 455 460 Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu
Gln Ala Leu 465 470 475 480 Glu Lys Glu Leu Ala Gln Gly Ala Thr 485
<210> SEQ ID NO 5 <211> LENGTH: 262 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 5 Glu Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1 5 10 15 Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25 30 Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 35 40
45 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
50 55 60 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 65 70 75 80 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 85 90 95 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile 100 105 110 Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125 Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135 140 Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 145 150 155 160 Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 165 170
175 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
180 185 190 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 195 200 205 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys Trp 210 215 220 Arg Ala Gly Gly Ser Ala Gln Leu Lys Lys
Lys Leu Gln Ala Leu Lys 225 230 235 240 Lys Lys Asn Ala Gln Leu Lys
Trp Lys Leu Gln Ala Leu Lys Lys Lys 245 250 255 Leu Ala Gln Gly Ala
Thr 260 <210> SEQ ID NO 6 <211> LENGTH: 214 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 6 Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
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 <210>
SEQ ID NO 7 <211> LENGTH: 490 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 7 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Asn 20 25 30 Trp
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Glu Ile Ser Pro Ser Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Arg Val Ser Tyr Glu Ala
Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220 Asp Ala Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420
425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 435 440 445 Pro Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu Glu
Lys Glu Leu 450 455 460 Gln Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu
Trp Glu Leu Gln Ala 465 470 475 480 Leu Glu Lys Glu Leu Ala Gln Gly
Ala Thr 485 490 <210> SEQ ID NO 8 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Pro Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val 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
<210> SEQ ID NO 9 <211> LENGTH: 729 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 9 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr
Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Gly Ser Gly Gly
Ser Gly Gly Ser 210 215 220 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly 225 230 235 240 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 245 250 255 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 260 265 270 Tyr Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 275 280 285 Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 290 295
300 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
305 310 315 320 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 325 330 335 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 355 360 365 Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala 370 375 380 Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 385 390 395 400 Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 405 410 415
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 420
425 430 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys 435 440 445 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp 450 455 460 Ala Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly 465 470 475 480 Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile 485 490 495 Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu 500 505 510 Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 515 520 525 Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 530 535 540
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 545
550 555 560 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu 565 570 575 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 580 585 590 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu 595 600 605 Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp 610 615 620 Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 625 630 635 640 Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 645 650 655 Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 660 665
670 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
675 680 685 Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu
Leu Gln 690 695 700 Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu
Leu Gln Ala Leu 705 710 715 720 Glu Lys Glu Leu Ala Gln Gly Ala Thr
725 <210> SEQ ID NO 10 <211> LENGTH: 729 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 10 Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20
25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val 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 Gly Gly Gly
Ser Gly Gly Ser Gly Gly Ser 210 215 220 Gly Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly 225 230 235 240 Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 245 250 255 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 260 265 270
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 275
280 285 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
Val 290 295 300 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr 305 310 315 320 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 325 330 335 Ser Arg Trp Gly Gly Asp Gly Phe
Tyr Ala Met Asp Tyr Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 355 360 365 Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 370 375 380 Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 385 390 395
400 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
405 410 415 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 420 425 430 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 435 440 445 Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 450 455 460 Ala Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 465 470 475 480 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 485 490 495 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 500 505 510 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 515 520
525 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
530 535 540 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 545 550 555 560 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 565 570 575 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 580 585 590 Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 595 600 605 Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 610 615 620 Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 625 630 635 640
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 645
650 655 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 660 665 670 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 675 680 685 Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu
Glu Lys Glu Leu Gln 690 695 700 Ala Leu Glu Lys Glu Asn Ala Gln Leu
Glu Trp Glu Leu Gln Ala Leu 705 710 715 720 Glu Lys Glu Leu Ala Gln
Gly Ala Thr 725 <210> SEQ ID NO 11 <211> LENGTH: 729
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile
Gly 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr 85 90 95 Thr Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys
Gly Gly Gly Ser Gly Gly Ser Gly Gly 210 215 220 Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 225 230 235 240 Gly Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 245 250 255
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp 260
265 270 Tyr Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp 275 280 285 Val Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr
Asn Gln Arg 290 295 300 Phe Lys Gly Arg Phe Thr Leu Ser Val Asp Arg
Ser Lys Asn Thr Leu 305 310 315 320 Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr 325 330 335 Cys Ala Arg Asn Leu Gly
Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln 340 345 350 Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 355 360 365 Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 370 375 380
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 385
390 395 400 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val 405 410 415 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 420 425 430 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 435 440 445 Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp 450 455 460 Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 465 470 475 480 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 485 490 495 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 500 505
510 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
515 520 525 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 530 535 540 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 545 550 555 560 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 565 570 575 Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 580 585 590 Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 595 600 605 Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 610 615 620 Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 625 630
635 640 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 645 650 655 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 660 665 670 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 675 680 685 Gly Lys Trp Arg Ala Gly Gly Ser Ala
Gln Leu Glu Lys Glu Leu Gln 690 695 700 Ala Leu Glu Lys Glu Asn Ala
Gln Leu Glu Trp Glu Leu Gln Ala Leu 705 710 715 720 Glu Lys Glu Leu
Ala Gln Gly Ala Thr 725 <210> SEQ ID NO 12 <211>
LENGTH: 38 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 12 Arg Ala Gly Gly Ser Ala Gln Leu Glu Lys
Glu Leu Gln Ala Leu Glu 1 5 10 15 Lys Glu Asn Ala Gln Leu Glu Trp
Glu Leu Gln Ala Leu Glu Lys Glu 20 25 30 Leu Ala Gln Gly Ala Thr 35
<210> SEQ ID NO 13 <211> LENGTH: 38 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 13 Arg Ala Gly Gly Ser
Ala Gln Leu Lys Lys Lys Leu Gln Ala Leu Lys 1 5 10 15 Lys Lys Asn
Ala Gln Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys Lys 20 25 30 Leu
Ala Gln Gly Ala Thr 35 <210> SEQ ID NO 14 <211> LENGTH:
26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 14 Gly
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10
15 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 20 25 <210> SEQ ID
NO 15 <211> LENGTH: 291 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 15 Met Gly Trp Ser Cys Ile Ile
Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 35 40 45 Lys
Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn 85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly
Phe Tyr Ala Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185
190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Ala Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu
290 <210> SEQ ID NO 16 <211> LENGTH: 524 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 16 Met Gly
Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15
Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20
25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Val 35 40 45 Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys 50 55 60 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser
Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Arg Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser 85 90 95 Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln His Tyr Thr 100 105 110 Thr Pro Pro Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150
155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr 180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly
Glu Cys Gly Gly Gly Ser Gly Gly Ser 225 230 235 240 Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 245 250 255 Gly Ser
Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 260 265 270
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 275
280 285 Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 290 295 300 Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala 305 310 315 320 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn 325 330 335 Thr Ala Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val 340 345 350 Tyr Tyr Cys Ser Arg Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 355 360 365 Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 370 375 380 Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 385 390 395
400 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
405 410 415 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe 420 425 430 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val 435 440 445 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val 450 455 460 Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys 465 470 475 480 Ser Cys Asp Ala Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 485 490 495 Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 500 505 510 Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 515 520 <210> SEQ
ID NO 17 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 17 Arg Arg Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10 15 Gly Gly Ser Gly Arg Ser
Arg Lys Arg Arg 20 25 <210> SEQ ID NO 18 <211> LENGTH:
26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 18 Lys
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10
15 Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys 20 25 <210> SEQ ID
NO 19 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 19 Glu Asn Leu Tyr Phe Gln Gly 1 5
<210> SEQ ID NO 20 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Hedgehog peptide
<400> SEQUENCE: 20 Gly Asp Trp Asn Ala Arg Trp Cys Phe 1 5
<210> SEQ ID NO 21 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (3)..(116) <400> SEQUENCE: 21 gg
cgc gcc gga ggt tca gct caa ctt gag aag gag ctg caa gct ctg 47 Arg
Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln Ala Leu 1 5 10 15
gaa aag gag aac gct caa ctg gaa tgg gag ctg caa gct ctg gaa aag 95
Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys 20
25 30 gag ctg gct caa gga gct acc tga 119 Glu Leu Ala Gln Gly Ala
Thr 35 <210> SEQ ID NO 22 <211> LENGTH: 119 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polynucleotide <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (3)..(116) <400>
SEQUENCE: 22 gg cgc gcc gga ggt tcc gct caa ctt aag aag aag ctt caa
gct ctg 47 Arg Ala Gly Gly Ser Ala Gln Leu Lys Lys Lys Leu Gln Ala
Leu 1 5 10 15 aag aag aag aac gct caa ctt aag tgg aag ctg caa gct
ctg aag aag 95 Lys Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala
Leu Lys Lys 20 25 30 aag ctg gct caa gga gct acc tga 119 Lys Leu
Ala Gln Gly Ala Thr 35 <210> SEQ ID NO 23 <211> LENGTH:
900 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <400> SEQUENCE:
23 acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc
tttttctagt 60 agcaactgca actggagtac attcagaagt tcagctggtg
gagtctggcg gtggcctggt 120 gcagccaggg ggctcactcc gtttgtcctg
tgcagcttct ggcttcaaca ttaaagacac 180 ctatatacac tgggtgcgtc
aggccccggg taagggcctg gaatgggttg caaggattta 240 tcctacgaat
ggttatacta gatatgccga tagcgtcaag ggccgtttca ctataagcgc 300
agacacatcc aaaaacacag cctacctgca gatgaacagc ctgcgtgctg aggacactgc
360 cgtctattat tgttctagat ggggagggga cggcttctat gctatggact
actggggtca 420 aggaaccctg gtcaccgtct cctcggcctc caccaagggc
ccatcggtct tccccctggc 480 accctcctcc aagagcacct ctgggggcac
agcggccctg ggctgcctgg tcaaggacta 540 cttccccgaa ccggtgacgg
tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac 600 cttcccggct
gtcctacagt cctcaggact ctactccctc agcagcgtgg tgactgtgcc 660
ctctagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac
720 caaggtggac aagaaagttg agcccaaatc ttgtgacgca actcacacat
gcccaccgtg 780 cccagcacct gaactcctgg ggggaccgtc agtcttcctc
ttccccccaa aacccaagga 840 caccctcatg atctcccgga cccctgaggt
cacatgcgtg gtggtggacg tgagccacga 900 <210> SEQ ID NO 24
<211> LENGTH: 1600 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (29)..(1600) <400> SEQUENCE: 24
acctcggttc tatcgattga attccacc atg gga tgg tca tgt atc atc ctt 52
Met Gly Trp Ser Cys Ile Ile Leu 1 5 ttt cta gta gca act gca act gga
gta cat tca gat atc cag atg acc 100 Phe Leu Val Ala Thr Ala Thr Gly
Val His Ser Asp Ile Gln Met Thr 10 15 20 cag tcc ccg agc tcc ctg
tcc gcc tct gtg ggc gat agg gtc acc atc 148 Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile 25 30 35 40 acc tgc cgt gcc
agt cag gat gtg aat act gct gta gcc tgg tat caa 196 Thr Cys Arg Ala
Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln 45 50 55 cag aaa
cca gga aaa gct ccg aaa cta ctg att tac tcg gca tcc ttc 244 Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe 60 65 70
ctc tac tct gga gtc cct tct cgc ttc tct ggt tcc aga tct ggg acg 292
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr 75
80 85 gat ttc act ctg acc atc agc agt ctg cag ccg gaa gac ttc gca
act 340 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr 90 95 100 tat tac tgt cag caa cat tat act act cct ccc acg ttc
gga cag ggt 388 Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe
Gly Gln Gly 105 110 115 120 acc aag gtg gag atc aaa cga act gtg gct
gca cca tct gtc ttc atc 436 Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala Pro Ser Val Phe Ile 125 130 135 ttc ccg cca tct gat gag cag ttg
aaa tct gga act gcc tct gtt gtg 484 Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val Val 140 145 150 tgc ctg ctg aat aac ttc
tat ccc aga gag gcc aaa gta cag tgg aag 532 Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 155 160 165 gtg gat aac gcc
ctc caa tcg ggt aac tcc cag gag agt gtc aca gag 580 Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 170 175 180 cag gac
agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg 628 Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 185 190 195
200 agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc
676 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
205 210 215 cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg
gga gag 724 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu 220 225 230 tgt gga gga ggt tca gga ggt tct ggt ggt tcg gga
gga tcc gga gga 772 Cys Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly 235 240 245 tct gga ggt tca gga ggt tct ggt ggg tca
gga gaa gtt cag ctg gtg 820 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Glu Val Gln Leu Val 250 255 260 gag tct ggc ggt ggc ctg gtg cag
cca ggg ggc tca ctc cgt ttg tcc 868 Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser 265 270 275 280 tgt gca gct tct ggc
ttc aac att aaa gac acc tat ata cac tgg gtg 916 Cys Ala Ala Ser Gly
Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val 285 290 295 cgt cag gcc
ccg ggt aag ggc ctg gaa tgg gtt gca agg att tat cct 964 Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro 300 305 310 acg
aat ggt tat act aga tat gcc gat agc gtc aag ggc cgt ttc act 1012
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 315
320 325 ata agc gca gac aca tcc aaa aac aca gcc tac ctg cag atg aac
agc 1060 Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met
Asn Ser 330 335 340 ctg cgt gct gag gac act gcc gtc tat tat tgt tct
aga tgg gga ggg 1108 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ser Arg Trp Gly Gly 345 350 355 360 gac ggc ttc tat gct atg gac tac
tgg ggt caa gga acc ctg gtc acc 1156 Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 365 370 375 gtc tcc tcg gcc tcc
acc aag ggc cca tcg gtc ttc ccc ctg gca ccc 1204 Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 380 385 390 tcc tcc
aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc 1252 Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 395 400
405 aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc
1300 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala 410 415 420 ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag
tcc tca gga 1348 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 425 430 435 440 ctc tac tcc ctc agc agc gtg gtg act
gtg ccc tct agc agc ttg ggc 1396 Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly 445 450 455 acc cag acc tac atc tgc
aac gtg aat cac aag ccc agc aac acc aag 1444 Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 460 465 470 gtg gac aag
aaa gtt gag ccc aaa tct tgt gac gca act cac aca tgc 1492 Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Ala Thr His Thr Cys 475 480 485
cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc
1540 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu 490 495 500 ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg
acc cct gag 1588 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu 505 510 515 520 gtc aca tgc gtg 1600 Val Thr Cys
Val <210> SEQ ID NO 25 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(4) <223>
OTHER INFORMATION: Any amino acid <400> SEQUENCE: 25 Arg Xaa
Arg Xaa Arg Arg 1 5 <210> SEQ ID NO 26 <400> SEQUENCE:
26 000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000
<210> SEQ ID NO 28 <400> SEQUENCE: 28 000 <210>
SEQ ID NO 29 <211> LENGTH: 14 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic peptide <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (1)..(1) <223> OTHER
INFORMATION: Any hydrophobic amino acid residue or Asparagine
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(14) <223> OTHER INFORMATION: This sequence
consists of 2 heptad repeats; see specification as filed for
detailed description of preferred embodiments <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(3)
<223> OTHER INFORMATION: Any amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Any hydrophobic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Any
charged amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Any amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: Any hydrophobic amino acid
residue or Asparagine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(10) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Any charged
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (14)..(14)
<223> OTHER INFORMATION: Any charged amino acid residue
<400> SEQUENCE: 29 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 <210> SEQ ID NO 30 <211> LENGTH:
14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
or Asparagine <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (1)..(14) <223> OTHER
INFORMATION: This sequence consists of 2 heptad repeats; see
specification as filed for detailed description of preferred
embodiments <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (2)..(3) <223> OTHER INFORMATION: Any
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Any hydrophobic amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Any charged amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (6)..(6) <223> OTHER INFORMATION: Any
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Any charged amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (8)..(8)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
or Asparagine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(10) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Any charged
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (14)..(14)
<223> OTHER INFORMATION: Any charged amino acid residue
<400> SEQUENCE: 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 <210> SEQ ID NO 31 <211> LENGTH:
33 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 31
Arg Gly Arg Cys Arg Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5
10 15 Ser Gly Gly Ser Gly Gly Ser Gly Arg Ser Arg Lys Arg Arg Glu
Val 20 25 30 Gln <210> SEQ ID NO 32 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 32
Arg Gly Glu Cys Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5
10 15 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Glu
Val 20 25 30 Gln <210> SEQ ID NO 33 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 33
Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu 1 5
10 15 Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu
Ala 20 25 30 Gln Gly Ala Thr 35 <210> SEQ ID NO 34
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 34 Gly Gly Ser Ala Gln Leu Lys
Lys Lys Leu Gln Ala Leu Lys Lys Lys 1 5 10 15 Asn Ala Gln Leu Lys
Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala 20 25 30 Gln Gly Ala
Thr 35 <210> SEQ ID NO 35 <211> LENGTH: 28 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 35 Arg Cys Arg
Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly
Ser Gly Gly Ser Gly Arg Ser Arg Lys Arg Arg 20 25 <210> SEQ
ID NO 36 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 36 Arg Lys Arg Lys Arg Arg Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Ser Gly Gly Ser Gly
Gly Ser Gly Arg Ser Arg Lys Arg Arg 20 25 30 <210> SEQ ID NO
37 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 37 Arg Gly Arg Ala Arg Arg Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Ser Gly Gly Ser Gly
Gly Ser Gly Arg Ser Arg Lys Arg Arg Glu Val 20 25 30 Gln
<210> SEQ ID NO 38 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 38 Arg Gly Glu Cys Arg
Lys Arg Lys Arg Arg Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Arg Ser Arg Lys 20 25 30 Arg
Arg Glu Val Gln 35 <210> SEQ ID NO 39 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 39 Arg
Gly Glu Cys Arg Lys Arg Lys Arg Arg 1 5 10
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 39 <210>
SEQ ID NO 1 <211> LENGTH: 490 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 1 Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30 Trp
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Gly Ile Thr Pro Asp Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Asn Asp Leu Gly Ser Arg Glu Phe Tyr
Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220 Asp Ala Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295
300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420
425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser 435 440 445 Pro Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu Lys
Lys Lys Leu 450 455 460 Gln Ala Leu Lys Lys Lys Asn Ala Gln Leu Lys
Trp Lys Leu Gln Ala 465 470 475 480 Leu Lys Lys Lys Leu Ala Gln Gly
Ala Thr 485 490 <210> SEQ ID NO 2 <211> LENGTH: 490
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 2
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Tyr Ala
Asn 20 25 30 Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Tyr Ile Gly Pro Asn Phe Gly Arg Ser Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Trp Arg
Arg Ser Leu Met Ser Val Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr
Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Ala Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260
265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385
390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Trp Arg Ala Gly Gly
Ser Ala Gln Leu Glu Lys Glu Leu 450 455 460 Gln Ala Leu Glu Lys Glu
Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala 465 470 475 480 Leu Glu Lys
Glu Leu Ala Gln Gly Ala Thr 485 490 <210> SEQ ID NO 3
<211> LENGTH: 214 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 3 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 <210> SEQ ID NO 4 <211> LENGTH: 489 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <400> SEQUENCE: 4 Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe
Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220 Ala Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405
410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 435 440 445 Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu
Glu Lys Glu Leu Gln 450 455 460 Ala Leu Glu Lys Glu Asn Ala Gln Leu
Glu Trp Glu Leu Gln Ala Leu 465 470 475 480 Glu Lys Glu Leu Ala Gln
Gly Ala Thr 485 <210> SEQ ID NO 5 <211> LENGTH: 262
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 5
Glu Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 1 5
10 15 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 20 25 30 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 35 40 45 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 50 55 60 Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser 65 70 75 80 Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85 90 95 Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 100 105 110 Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 115 120 125 Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 130 135
140 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
145 150 155 160 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser 165 170 175 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg 180 185 190 Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 195 200 205 His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys Trp 210 215 220 Arg Ala Gly Gly Ser
Ala Gln Leu Lys Lys Lys Leu Gln Ala Leu Lys 225 230 235 240 Lys Lys
Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys Lys 245 250 255
Leu Ala Gln Gly Ala Thr 260 <210> SEQ ID NO 6 <211>
LENGTH: 214 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val 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 <210> SEQ ID NO 7 <211> LENGTH:
490 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide
<400> SEQUENCE: 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Thr Gly Asn 20 25 30 Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Ser Pro
Ser Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Glu Ser Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215
220 Asp Ala Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly
Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu 450 455 460
Gln Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala 465
470 475 480 Leu Glu Lys Glu Leu Ala Gln Gly Ala Thr 485 490
<210> SEQ ID NO 8 <211> LENGTH: 214 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 8 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Pro Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 <210> SEQ ID
NO 9 <211> LENGTH: 729 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 9 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Gly Ser Gly Gly Ser Gly
Gly Ser 210 215 220 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly 225 230 235 240 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 245 250 255 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Asn Ile Lys Asp Thr 260 265 270 Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 275 280 285 Ala Arg Ile
Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 290 295 300 Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 305 310
315 320 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 325 330 335 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 355 360 365 Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala 370 375 380 Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser 385 390 395 400 Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 405 410 415 Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 420 425 430
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 435
440 445 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp 450 455 460 Ala Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly 465 470 475 480
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 485
490 495 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 500 505 510 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His 515 520 525 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg 530 535 540 Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys 545 550 555 560 Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 565 570 575 Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 580 585 590 Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 595 600 605
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 610
615 620 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val 625 630 635 640 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 645 650 655 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 660 665 670 Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 675 680 685 Gly Lys Trp Arg Ala Gly
Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln 690 695 700 Ala Leu Glu Lys
Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu 705 710 715 720 Glu
Lys Glu Leu Ala Gln Gly Ala Thr 725 <210> SEQ ID NO 10
<211> LENGTH: 729 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 10 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val 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 Gly Gly Gly Ser Gly Gly Ser Gly
Gly Ser 210 215 220 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly 225 230 235 240 Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 245 250 255 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Asn Ile Lys Asp Thr 260 265 270 Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 275 280 285 Ala Arg Ile
Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 290 295 300 Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 305 310
315 320 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 325 330 335 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
Trp Gly Gln 340 345 350 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 355 360 365 Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala 370 375 380 Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser 385 390 395 400 Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 405 410 415 Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 420 425 430
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 435
440 445 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp 450 455 460 Ala Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly 465 470 475 480 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile 485 490 495 Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu 500 505 510 Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His 515 520 525 Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 530 535 540 Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 545 550 555
560 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
565 570 575 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 580 585 590 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 595 600 605 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 610 615 620 Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val 625 630 635 640 Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 645 650 655 Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 660 665 670 Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 675 680
685 Gly Lys Trp Arg Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln
690 695 700 Ala Leu Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln
Ala Leu 705 710 715 720 Glu Lys Glu Leu Ala Gln Gly Ala Thr 725
<210> SEQ ID NO 11 <211> LENGTH: 729 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 11 Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr
Ile Tyr Pro Tyr 85 90 95 Thr Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170
175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Ser Gly
Gly Ser Gly Gly 210 215 220
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 225
230 235 240 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly 245 250 255 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Asp 260 265 270 Tyr Thr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp 275 280 285 Val Ala Asp Val Asn Pro Asn Ser
Gly Gly Ser Ile Tyr Asn Gln Arg 290 295 300 Phe Lys Gly Arg Phe Thr
Leu Ser Val Asp Arg Ser Lys Asn Thr Leu 305 310 315 320 Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 325 330 335 Cys
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln 340 345
350 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
355 360 365 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 370 375 380 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 385 390 395 400 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 405 410 415 Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 420 425 430 Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 435 440 445 Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 450 455 460 Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 465 470
475 480 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 485 490 495 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 500 505 510 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 515 520 525 Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg 530 535 540 Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 545 550 555 560 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 565 570 575 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 580 585 590
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 595
600 605 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp 610 615 620 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val 625 630 635 640 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 645 650 655 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 660 665 670 Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 675 680 685 Gly Lys Trp Arg
Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln 690 695 700 Ala Leu
Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu 705 710 715
720 Glu Lys Glu Leu Ala Gln Gly Ala Thr 725 <210> SEQ ID NO
12 <211> LENGTH: 38 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 12 Arg Ala Gly Gly Ser Ala Gln
Leu Glu Lys Glu Leu Gln Ala Leu Glu 1 5 10 15 Lys Glu Asn Ala Gln
Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu 20 25 30 Leu Ala Gln
Gly Ala Thr 35 <210> SEQ ID NO 13 <211> LENGTH: 38
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 13
Arg Ala Gly Gly Ser Ala Gln Leu Lys Lys Lys Leu Gln Ala Leu Lys 1 5
10 15 Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala Leu Lys Lys
Lys 20 25 30 Leu Ala Gln Gly Ala Thr 35 <210> SEQ ID NO 14
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 14 Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser 1 5 10 15 Gly Gly Ser Gly Gly Ser Gly Gly
Ser Gly 20 25 <210> SEQ ID NO 15 <211> LENGTH: 291
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 15
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5
10 15 Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Asn Ile 35 40 45 Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Arg Ile Tyr Pro Thr
Asn Gly Tyr Thr Arg Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Ala Asp Thr Ser Lys Asn 85 90 95 Thr Ala Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 115 120 125 Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135
140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys
Asp Ala Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260
265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val 275 280 285 Ser His Glu 290 <210> SEQ ID NO 16
<211> LENGTH: 524 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 16 Met Gly Trp Ser Cys Ile Ile
Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 35 40 45 Asn
Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 50 55
60 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser
Arg
65 70 75 80 Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser 85 90 95 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln His Tyr Thr 100 105 110 Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175 Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185
190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly
Ser Gly Gly Ser 225 230 235 240 Gly Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser Gly 245 250 255 Gly Ser Gly Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln 260 265 270 Pro Gly Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile 275 280 285 Lys Asp Thr
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 290 295 300 Glu
Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala 305 310
315 320 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn 325 330 335 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val 340 345 350 Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe
Tyr Ala Met Asp Tyr 355 360 365 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly 370 375 380 Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 385 390 395 400 Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 405 410 415 Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 420 425 430
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 435
440 445 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val 450 455 460 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys 465 470 475 480 Ser Cys Asp Ala Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu 485 490 495 Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr 500 505 510 Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 515 520 <210> SEQ ID NO 17
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 17 Arg Arg Gly Ser Gly Gly Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser 1 5 10 15 Gly Gly Ser Gly Arg Ser Arg Lys
Arg Arg 20 25 <210> SEQ ID NO 18 <211> LENGTH: 26
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide <400> SEQUENCE: 18 Lys
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10
15 Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys 20 25 <210> SEQ ID
NO 19 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
peptide <400> SEQUENCE: 19 Glu Asn Leu Tyr Phe Gln Gly 1 5
<210> SEQ ID NO 20 <211> LENGTH: 9 <212> TYPE:
PRT <213> ORGANISM: Unknown <220> FEATURE: <223>
OTHER INFORMATION: Description of Unknown: Hedgehog peptide
<400> SEQUENCE: 20 Gly Asp Trp Asn Ala Arg Trp Cys Phe 1 5
<210> SEQ ID NO 21 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (3)..(116) <400> SEQUENCE: 21 gg
cgc gcc gga ggt tca gct caa ctt gag aag gag ctg caa gct ctg 47 Arg
Ala Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln Ala Leu 1 5 10 15
gaa aag gag aac gct caa ctg gaa tgg gag ctg caa gct ctg gaa aag 95
Glu Lys Glu Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys 20
25 30 gag ctg gct caa gga gct acc tga 119 Glu Leu Ala Gln Gly Ala
Thr 35 <210> SEQ ID NO 22 <211> LENGTH: 119 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polynucleotide <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (3)..(116) <400>
SEQUENCE: 22 gg cgc gcc gga ggt tcc gct caa ctt aag aag aag ctt caa
gct ctg 47 Arg Ala Gly Gly Ser Ala Gln Leu Lys Lys Lys Leu Gln Ala
Leu 1 5 10 15 aag aag aag aac gct caa ctt aag tgg aag ctg caa gct
ctg aag aag 95 Lys Lys Lys Asn Ala Gln Leu Lys Trp Lys Leu Gln Ala
Leu Lys Lys 20 25 30 aag ctg gct caa gga gct acc tga 119 Lys Leu
Ala Gln Gly Ala Thr 35 <210> SEQ ID NO 23 <211> LENGTH:
900 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <400> SEQUENCE:
23 acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc
tttttctagt 60 agcaactgca actggagtac attcagaagt tcagctggtg
gagtctggcg gtggcctggt 120 gcagccaggg ggctcactcc gtttgtcctg
tgcagcttct ggcttcaaca ttaaagacac 180 ctatatacac tgggtgcgtc
aggccccggg taagggcctg gaatgggttg caaggattta 240 tcctacgaat
ggttatacta gatatgccga tagcgtcaag ggccgtttca ctataagcgc 300
agacacatcc aaaaacacag cctacctgca gatgaacagc ctgcgtgctg aggacactgc
360 cgtctattat tgttctagat ggggagggga cggcttctat gctatggact
actggggtca 420 aggaaccctg gtcaccgtct cctcggcctc caccaagggc
ccatcggtct tccccctggc 480 accctcctcc aagagcacct ctgggggcac
agcggccctg ggctgcctgg tcaaggacta 540 cttccccgaa ccggtgacgg
tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac 600 cttcccggct
gtcctacagt cctcaggact ctactccctc agcagcgtgg tgactgtgcc 660
ctctagcagc ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac
720 caaggtggac aagaaagttg agcccaaatc ttgtgacgca actcacacat
gcccaccgtg 780 cccagcacct gaactcctgg ggggaccgtc agtcttcctc
ttccccccaa aacccaagga 840 caccctcatg atctcccgga cccctgaggt
cacatgcgtg gtggtggacg tgagccacga 900 <210> SEQ ID NO 24
<211> LENGTH: 1600 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (29)..(1600)
<400> SEQUENCE: 24 acctcggttc tatcgattga attccacc atg gga tgg
tca tgt atc atc ctt 52 Met Gly Trp Ser Cys Ile Ile Leu 1 5 ttt cta
gta gca act gca act gga gta cat tca gat atc cag atg acc 100 Phe Leu
Val Ala Thr Ala Thr Gly Val His Ser Asp Ile Gln Met Thr 10 15 20
cag tcc ccg agc tcc ctg tcc gcc tct gtg ggc gat agg gtc acc atc 148
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 25
30 35 40 acc tgc cgt gcc agt cag gat gtg aat act gct gta gcc tgg
tat caa 196 Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp
Tyr Gln 45 50 55 cag aaa cca gga aaa gct ccg aaa cta ctg att tac
tcg gca tcc ttc 244 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
Ser Ala Ser Phe 60 65 70 ctc tac tct gga gtc cct tct cgc ttc tct
ggt tcc aga tct ggg acg 292 Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser Arg Ser Gly Thr 75 80 85 gat ttc act ctg acc atc agc agt
ctg cag ccg gaa gac ttc gca act 340 Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr 90 95 100 tat tac tgt cag caa cat
tat act act cct ccc acg ttc gga cag ggt 388 Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly 105 110 115 120 acc aag gtg
gag atc aaa cga act gtg gct gca cca tct gtc ttc atc 436 Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 125 130 135 ttc
ccg cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg 484 Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 140 145
150 tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag
532 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
155 160 165 gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc
aca gag 580 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu 170 175 180 cag gac agc aag gac agc acc tac agc ctc agc agc
acc ctg acg ctg 628 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu 185 190 195 200 agc aaa gca gac tac gag aaa cac aaa
gtc tac gcc tgc gaa gtc acc 676 Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr 205 210 215 cat cag ggc ctg agc tcg ccc
gtc aca aag agc ttc aac agg gga gag 724 His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu 220 225 230 tgt gga gga ggt tca
gga ggt tct ggt ggt tcg gga gga tcc gga gga 772 Cys Gly Gly Gly Ser
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 235 240 245 tct gga ggt
tca gga ggt tct ggt ggg tca gga gaa gtt cag ctg gtg 820 Ser Gly Gly
Ser Gly Gly Ser Gly Gly Ser Gly Glu Val Gln Leu Val 250 255 260 gag
tct ggc ggt ggc ctg gtg cag cca ggg ggc tca ctc cgt ttg tcc 868 Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 265 270
275 280 tgt gca gct tct ggc ttc aac att aaa gac acc tat ata cac tgg
gtg 916 Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp
Val 285 290 295 cgt cag gcc ccg ggt aag ggc ctg gaa tgg gtt gca agg
att tat cct 964 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg
Ile Tyr Pro 300 305 310 acg aat ggt tat act aga tat gcc gat agc gtc
aag ggc cgt ttc act 1012 Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
Val Lys Gly Arg Phe Thr 315 320 325 ata agc gca gac aca tcc aaa aac
aca gcc tac ctg cag atg aac agc 1060 Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr Leu Gln Met Asn Ser 330 335 340 ctg cgt gct gag gac
act gcc gtc tat tat tgt tct aga tgg gga ggg 1108 Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly 345 350 355 360 gac
ggc ttc tat gct atg gac tac tgg ggt caa gga acc ctg gtc acc 1156
Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 365
370 375 gtc tcc tcg gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca
ccc 1204 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 380 385 390 tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg
ggc tgc ctg gtc 1252 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val 395 400 405 aag gac tac ttc ccc gaa ccg gtg acg
gtg tcg tgg aac tca ggc gcc 1300 Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala 410 415 420 ctg acc agc ggc gtg cac
acc ttc ccg gct gtc cta cag tcc tca gga 1348 Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 425 430 435 440 ctc tac
tcc ctc agc agc gtg gtg act gtg ccc tct agc agc ttg ggc 1396 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 445 450
455 acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag
1444 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys 460 465 470 gtg gac aag aaa gtt gag ccc aaa tct tgt gac gca act
cac aca tgc 1492 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Ala
Thr His Thr Cys 475 480 485 cca ccg tgc cca gca cct gaa ctc ctg ggg
gga ccg tca gtc ttc ctc 1540 Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu 490 495 500 ttc ccc cca aaa ccc aag gac
acc ctc atg atc tcc cgg acc cct gag 1588 Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 505 510 515 520 gtc aca tgc
gtg 1600 Val Thr Cys Val <210> SEQ ID NO 25 <211>
LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic peptide <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Any amino acid <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Any amino acid <400>
SEQUENCE: 25 Arg Xaa Arg Xaa Arg Arg 1 5 <210> SEQ ID NO 26
<400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <400>
SEQUENCE: 27 000 <210> SEQ ID NO 28 <400> SEQUENCE: 28
000 <210> SEQ ID NO 29 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1) <223>
OTHER INFORMATION: Any hydrophobic amino acid residue or Asparagine
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(14) <223> OTHER INFORMATION: This sequence
consists of 2 heptad repeats; see specification as filed for
detailed description of preferred embodiments <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(3)
<223> OTHER INFORMATION: Any amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Any hydrophobic amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Any
charged amino acid residue <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Any amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(8)..(8) <223> OTHER INFORMATION: Any hydrophobic amino acid
residue or Asparagine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(10) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Any charged
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Any amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (14)..(14) <223> OTHER INFORMATION: Any charged
amino acid residue <400> SEQUENCE: 29 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 <210> SEQ ID NO 30
<211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic peptide
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: Any hydrophobic
amino acid residue or Asparagine <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (1)..(14) <223>
OTHER INFORMATION: This sequence consists of 2 heptad repeats; see
specification as filed for detailed description of preferred
embodiments <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (2)..(3) <223> OTHER INFORMATION: Any
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (4)..(4) <223> OTHER
INFORMATION: Any hydrophobic amino acid residue <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(5)..(5) <223> OTHER INFORMATION: Any charged amino acid
residue <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (6)..(6) <223> OTHER INFORMATION: Any
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Any charged amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (8)..(8)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
or Asparagine <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (9)..(10) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Any hydrophobic amino acid residue
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (12)..(12) <223> OTHER INFORMATION: Any charged
amino acid residue <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (13)..(13) <223> OTHER
INFORMATION: Any amino acid residue <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (14)..(14)
<223> OTHER INFORMATION: Any charged amino acid residue
<400> SEQUENCE: 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 10 <210> SEQ ID NO 31 <211> LENGTH:
33 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 31
Arg Gly Arg Cys Arg Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5
10 15 Ser Gly Gly Ser Gly Gly Ser Gly Arg Ser Arg Lys Arg Arg Glu
Val 20 25 30 Gln <210> SEQ ID NO 32 <211> LENGTH: 33
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 32
Arg Gly Glu Cys Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5
10 15 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Glu
Val 20 25 30 Gln <210> SEQ ID NO 33 <211> LENGTH: 36
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polypeptide <400> SEQUENCE: 33
Gly Gly Ser Ala Gln Leu Glu Lys Glu Leu Gln Ala Leu Glu Lys Glu 1 5
10 15 Asn Ala Gln Leu Glu Trp Glu Leu Gln Ala Leu Glu Lys Glu Leu
Ala 20 25 30 Gln Gly Ala Thr 35 <210> SEQ ID NO 34
<211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 34 Gly Gly Ser Ala Gln Leu Lys
Lys Lys Leu Gln Ala Leu Lys Lys Lys 1 5 10 15 Asn Ala Gln Leu Lys
Trp Lys Leu Gln Ala Leu Lys Lys Lys Leu Ala 20 25 30 Gln Gly Ala
Thr 35 <210> SEQ ID NO 35 <211> LENGTH: 28 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic peptide <400> SEQUENCE: 35 Arg Cys Arg
Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly
Ser Gly Gly Ser Gly Arg Ser Arg Lys Arg Arg 20 25 <210> SEQ
ID NO 36 <211> LENGTH: 30 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 36 Arg Lys Arg Lys Arg Arg Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Ser Gly Gly Ser Gly
Gly Ser Gly Arg Ser Arg Lys Arg Arg 20 25 30 <210> SEQ ID NO
37 <211> LENGTH: 33 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polypeptide <400> SEQUENCE: 37 Arg Gly Arg Ala Arg Arg Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Ser Gly Gly Ser Gly
Gly Ser Gly Arg Ser Arg Lys Arg Arg Glu Val 20 25 30 Gln
<210> SEQ ID NO 38 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 38 Arg Gly Glu Cys Arg
Lys Arg Lys Arg Arg Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Arg Ser Arg Lys 20 25 30 Arg
Arg Glu Val Gln 35 <210> SEQ ID NO 39 <211> LENGTH: 10
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic peptide
<400> SEQUENCE: 39 Arg Gly Glu Cys Arg Lys Arg Lys Arg Arg 1
5 10
* * * * *