U.S. patent application number 11/121282 was filed with the patent office on 2006-05-04 for specificity exchangers that redirect antibodies to a pathogen.
Invention is credited to Jan-Ingmar Flock, Matti Sallberg.
Application Number | 20060094862 11/121282 |
Document ID | / |
Family ID | 29588026 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094862 |
Kind Code |
A1 |
Sallberg; Matti ; et
al. |
May 4, 2006 |
Specificity exchangers that redirect antibodies to a pathogen
Abstract
Specificity exchangers and methods of making and using
specificity exchangers are disclosed. Specificity exchangers are
useful for preventing and treating human diseases including cancer
and those resulting from pathogens such as bacteria, yeast,
parasites, fungus, viruses, and the like. More specifically,
specificity exchangers can redirect existing antibodies in a
subject to pathogens and cancer cells.
Inventors: |
Sallberg; Matti; (Alvsjo,
SE) ; Flock; Jan-Ingmar; (Bromma, SE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
29588026 |
Appl. No.: |
11/121282 |
Filed: |
May 3, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10372735 |
Feb 21, 2003 |
6933366 |
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11121282 |
May 3, 2005 |
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10234579 |
Aug 30, 2002 |
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10372735 |
Feb 21, 2003 |
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09839666 |
Apr 19, 2001 |
6469143 |
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10234579 |
Aug 30, 2002 |
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09532106 |
Mar 21, 2000 |
6245895 |
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09839666 |
Apr 19, 2001 |
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09246258 |
Feb 8, 1999 |
6040137 |
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09532106 |
Mar 21, 2000 |
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08737085 |
Dec 27, 1996 |
5869232 |
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PCT/SE95/00468 |
Apr 27, 1995 |
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09246258 |
Feb 8, 1999 |
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09664945 |
Sep 19, 2000 |
6660842 |
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10372735 |
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09664025 |
Sep 19, 2000 |
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10372735 |
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PCT/IB01/02327 |
Sep 19, 2001 |
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10372735 |
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10153271 |
May 21, 2002 |
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10372735 |
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09556605 |
Apr 21, 2000 |
6417324 |
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10153271 |
May 21, 2002 |
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09839447 |
Apr 20, 2001 |
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10372735 |
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Current U.S.
Class: |
530/350 ;
530/395 |
Current CPC
Class: |
Y02A 50/30 20180101;
C07K 16/109 20130101; Y02A 50/466 20180101; C07K 16/082 20130101;
C12N 2740/16222 20130101; C12N 2770/32622 20130101; C07K 16/087
20130101; C07K 16/1009 20130101; C12N 2710/20022 20130101; C12N
2770/24222 20130101; C07K 14/75 20130101; C07K 14/78 20130101; G01N
33/569 20130101; C07K 14/005 20130101; C12N 2710/16622 20130101;
A61K 38/00 20130101; C07K 2317/73 20130101; C12N 2730/10122
20130101; C07K 7/08 20130101; C07K 2317/565 20130101; C07K 14/195
20130101; C07K 16/00 20130101; A61K 2039/505 20130101; C07K 2318/10
20130101; C07K 16/1063 20130101; C07K 7/06 20130101; C07K 2317/50
20130101; G01N 33/531 20130101; C07K 19/00 20130101 |
Class at
Publication: |
530/350 ;
530/395 |
International
Class: |
C07K 14/195 20060101
C07K014/195 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 1994 |
SE |
9401460 |
Claims
1. A ligand/receptor specificity exchanger comprising at least one
specificity domain comprising a fragment of a ligand for a
bacterial receptor joined to an oligo/polysaccharide.
2. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain comprises at least 3 and up to 27
consecutive amino acids of a ligand for a bacterial receptor.
3. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain contains between 3-200 consecutive amino
acids of a ligand for a bacterial receptor.
4. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain contains between 5-100 consecutive amino
acids of a ligand for a bacterial receptor.
5. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain contains between 8-50 consecutive amino
acids of a ligand for a bacterial receptor.
6. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain contains between 10-25 consecutive amino
acids of a ligand for a bacterial receptor.
7. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain contains between 3-20 consecutive amino
acids of a ligand for a bacterial receptor.
8. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain contains between 3-30 consecutive amino
acids of a ligand for a bacterial receptor.
9. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain comprises at least three consecutive amino
acids of fibrinogen.
10. The ligand/receptor specificity exchanger of claim 1, wherein
said specificity domain is selected from the group consisting of
fibrinogen, collagen, vitronectin, laminin, plasminogen,
thrombospondin, and fibronectin.
11. The ligand/receptor specificity exchanger of claim 1, wherein
said bacterial receptor is selected from the group consisting of
extracellular fibrinogen binding protein (Efb), collagen binding
protein, vitronectin binding protein, laminin binding protein,
plasminogen binding protein, thrombospondin binding protein,
clumping factor A (ClfA), clumping factor B (ClfB), fibronectin
binding protein, coagulase, and extracellular adherence
protein.
12. The ligand/receptor specificity exchanger of claim 6,
comprising a sequence selected from the group consisting of SEQ.
ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID. No. 4, SEQ. ID.
No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No. 8, SEQ. ID. No.
9, SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No. 12, SEQ. ID. No.
13, SEQ. ID. No. 14, SEQ. ID. No. 15, SEQ. ID. No. 16, SEQ. ID. No.
17, SEQ. ID. No. 18, SEQ. ID. No. 19, SEQ. ID. No. 20, SEQ. ID. No.
21, SEQ. ID. No. 22, SEQ. ID. No. 23, SEQ. ID. No. 24, SEQ. ID. No.
25, SEQ. ID. No. 26, SEQ. ID. No. 27, SEQ. ID. No. 28, SEQ. ID. No.
29, SEQ. ID. No. 30, SEQ. ID. No. 31, SEQ. ID. No. 32, SEQ. ID. No.
33, SEQ. ID. No. 34, SEQ. ID. No. 35, SEQ. ID. No. 36, SEQ. ID. No.
37, SEQ. ID. No. 38, SEQ. ID. No. 39, SEQ. ID. No. 40, SEQ. ID. No.
41, SEQ. ID. No. 42, SEQ. ID. No. 43, SEQ. ID. No. 44, SEQ. ID. No.
45, SEQ. ID. No. 46, and SEQ. ID. No. 47.
13. A ligand/receptor specificity exchanger comprising at least one
specificity domain comprising a fragment of a ligand for a
bacterial receptor joined to a carbohydrate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of prior U.S.
application Ser. No. 10/372,735, filed Feb. 21, 2003, which is a
Continuation-In-Part of prior U.S. application Ser. No. 10/234,579,
filed Aug. 30, 2002, which is a continuation of prior U.S.
application Ser. No. 09/839,666, filed Apr. 19, 2001 (now U.S. Pat.
No. 6,469,143, issued Oct. 22, 2002), which is a continuation of
prior U.S. application Ser. No. 09/532,106, filed Mar. 21, 2000
(now U.S. Pat. No. 6,245,895, issued Jun. 12, 2001), which is a
continuation of prior U.S. application Ser. No. 09/246,258, filed
Feb. 8, 1999 (now U.S. Pat. No. 6,040,137, issued Mar. 21, 2000),
which is a continuation of prior U.S. application Ser. No.
08/737,085, filed Dec. 27, 1996 (now U.S. Pat. No. 5,869,232,
issued Feb. 9, 1999), which is a National Phase application under
35 U.S.C. .sctn.371 of PCT/SE95/00468.
[0002] The present application is a continuation of prior U.S.
application Ser. No. 10/372,735, filed Feb. 21, 2003, which is also
a Continuation-In-Part of prior U.S. application Ser. No.
09/664,945, filed Sep. 19, 2000 (now U.S. Pat. No. 6,660,842,
issued Dec. 9, 2003).
[0003] The present application is a continuation of prior U.S.
application Ser. No. 10/372,735, filed Feb. 21, 2003, which is also
a Continuation-In-Part of prior U.S. application Ser. No.
09/664,025, filed Sep. 19, 2000.
[0004] The present application is a continuation of prior U.S.
application Ser. No. 10/372,735, filed Feb. 21, 2003, which is also
a Continuation-In-Part of international application number
PCT/IB01/02327, and claims the benefit of priority of international
application number PCT/IB01/02327, having an international filing
date of Sep. 19, 2001, designating the United States of America and
published in English, which claims the benefit of priority of U.S.
application Ser. No. 09/664,025, filed Sep. 19, 2000.
[0005] The present application is a continuation of prior U.S.
application Ser. No. 10/372,735, filed Feb. 21, 2003, which is also
a Continuation-In-Part of prior U.S. application Ser. No.
10/153,271, filed May 21, 2002, which is a divisional of prior U.S.
application Ser. No. 09/556,605, filed Apr. 21, 2000 (now U.S. Pat.
No. 6,417,324, issued Jul. 9, 2002).
[0006] The present application is a continuation of prior U.S.
application Ser. No. 10/372,735, filed Feb. 21, 2003, which is also
a Continuation-In-Part of prior U.S. application Ser. No.
09/839,447, filed Apr. 20, 2001, which is a continuation-in-part of
prior U.S. application Ser. No. 09/556,605, filed Apr. 21, 2000
(now U.S. Pat. No. 6,417,324, issued Jul. 9, 2002).
[0007] The present application claims priority to all of the
above-referenced prior applications and the disclosures of these
prior applications are hereby expressly incorporated by reference
in their entireties.
FIELD OF THE INVENTION
[0008] The present disclosure generally relates to compositions and
methods for preventing and treating human diseases including cancer
and those resulting from pathogens such as bacteria, yeast,
parasites, fungus, viruses, and the like. More specifically,
embodiments described herein concern the manufacture and use of
specificity exchangers, which redirect existing antibodies in a
subject to pathogens and cancer cells.
BACKGROUND OF THE INVENTION
[0009] Infection by pathogens, such as bacteria, yeast, parasites,
fungus, and viruses, and the onset and spread of cancer present
serious health concerns for all animals, including humans, farm
livestock, and household pets. These health threats are exacerbated
by the rise of strains that are resistant to vaccination and/or
treatment. In the past, practitioners of pharmacology have relied
on traditional methods of drug discovery to generate safe and
efficacious compounds for the treatment of these diseases.
Traditional drug discovery methods typically involve blindly
testing potential drug candidate-molecules, often selected at
random, in the hope that one might prove to be an effective
treatment for some disease. With the advent of molecular biology,
however, the focus of drug discovery has shifted to the
identification of molecular targets associated with the etiological
agent and the design of compounds that interact with these
molecular targets.
[0010] One promising class of molecular targets are antigens found
on the surface of bacteria, yeast, parasites, fungus, viruses,
toxins and cancer cells. It has been shown that synthetic peptides
corresponding to antibody regions (e.g., a CDR) can act as a mini
antibody by binding to a particular antigen on a pathogen or cancer
cell and neutralizing the pathogen or cancer cell in vitro.
Although several antigen antagonists have promising therapeutic
potential, there still remains a need for new compositions and
methods to treat and prevent infection by pathogens and other
disease.
[0011] Another promising class of molecular targets are the
receptors found on the surface of bacteria, yeast, parasites,
fungus, viruses, toxins and cancer cells, especially receptors that
allow for attachment to a host cell or host protein (e.g., an
extracellular matrix protein). Research in this area primarily
focuses on the identification of the receptor and its ligand and
the discovery of molecules that interrupt the interaction of the
ligand with the receptor and, thereby, block adhesion to the host
cell or protein. Although several receptor antagonists have
promising therapeutic potential, there still remains a need for new
compositions and methods to treat and prevent infection by
pathogens and other diseases.
SUMMARY OF THE INVENTION
[0012] Embodiments described herein are directed to specificity
exchangers comprising at least one specificity domain and at least
one antigenic domain joined to said specificity domain, wherein
said antigenic domain comprises a peptide or an epitope obtained
from a pathogen or toxin. In some embodiments, the specificity
exchanger is an antigen/antibody specificity exchanger that
comprises a specificity domain having a sequence obtained from an
antibody joined to an antigenic domain that comprises a peptide or
an epitope obtained from a pathogen or toxin, preferably a viral
antigen such as polio virus, TT virus, herpes virus, hepatitis
virus, or human immunodeficiency virus (HIV). In other embodiments,
the specificity exchanger is a ligand/receptor specificity
exchanger that comprises a specificity domain having a ligand for a
receptor joined to an antigenic domain that comprises a peptide or
an epitope obtained from a pathogen or toxin, preferably a viral
antigen such as polio virus, TT virus, herpes virus, hepatitis
virus, or human immunodeficiency virus.
[0013] The length of the specificity domain of the specificity
exchangers is desirably between at least 3-200 amino acids,
preferably between at least 5-100 amino acids, more preferably
between 8-50 amino acids, and still more preferably between 10-25
amino acids. The length of the antigenic domain of the specificity
exchangers is desirably between at least 3-200 amino acids,
preferably between at least 5-100 amino acids, more preferably
between 8-50 amino acids, and still more preferably between 10-25
amino acids.
[0014] The specificity exchangers described herein comprise
specificity domains that interact with antigens or receptors on
pathogens, including, but not limited to, bacteria, yeast,
parasites, fungus, and cancer cells. Some embodiments, for example,
comprise a sequence obtained from an antibody that binds to a
bacteria, hepatitis virus, or HIV. Other embodiments have a
specificity domain that comprises a fragment of an extracellular
matrix protein (e.g., between 3 and 14 amino acids, such as 3 to 5,
8, 9, 10, 12, or 14 consecutive amino acids of fibrinogen), a
ligand for a receptor on a virus, or a ligand for a receptor on a
cancer cell. In preferred embodiments, for example, the specificity
domain comprises a ligand that is a fragment (e.g., between 3 and
20 amino acids, such as 3 to 5, 8, 9, 10, 12, 14, 17, and 20
consecutive amino acids) of an extracellular matrix protein
selected from the group consisting of fibrinogen, collagen,
vitronectin, laminin, plasminogen, thrombospondin, and
fibronectin.
[0015] Several of the specificity exchangers described herein bind
to a receptor found on a pathogen (vis a vis antigen/antibody
interaction or ligand/receptor interaction). In some embodiments,
the receptor is a bacterial adhesion receptor, for example, a
bacterial adhesion receptor selected from the group consisting of
extracellular fibrinogen binding protein (Efb), collagen binding
protein, vitronectin binding protein, laminin binding protein,
plasminogen binding protein, thrombospondin binding protein,
clumping factor A (ClfA), clumping factor B (ClfB), fibronectin
binding protein, coagulase, and extracellular adherence
protein.
[0016] In some embodiments, the specificity exchangers comprise a
specificity domain that comprises at least one of the following
sequences: SEQ. ID. No. 1, SEQ. ID. No. 2, SEQ. ID. No. 3, SEQ. ID.
No. 4, SEQ. ID. No. 5, SEQ. ID. No. 6, SEQ. ID. No. 7, SEQ. ID. No.
8, SEQ. ID. No. 9, SEQ. ID. No. 10, SEQ. ID. No. 11, SEQ. ID. No.
12, SEQ. ID. No. 13, SEQ. ID. No. 14, SEQ. ID. No. 15, SEQ. ID. No.
16, SEQ. ID. No. 17, SEQ. ID. No. 18, SEQ. ID. No. 19, SEQ. ID. No.
20, SEQ. ID. No. 21, SEQ. ID. No. 22, SEQ. ID. No. 23, SEQ. ID. No.
24, SEQ. ID. No. 25, SEQ. ID. No. 26, SEQ. ID. No. 27, SEQ. ID. No.
28, SEQ. ID. No. 29, SEQ. ID. No. 30, SEQ. ID. No. 31, SEQ. ID. No.
32, SEQ. ID. No. 33, SEQ. ID. No. 34, SEQ. ID. No. 35, SEQ. ID. No.
36, SEQ. ID. No. 37, SEQ. ID. No. 38, SEQ. ID. No. 39, SEQ. ID. No.
40, SEQ. ID. No. 41, SEQ. ID. No. 42, SEQ. ID. No. 43, SEQ. ID. No.
44, SEQ. ID. No. 45, SEQ. ID. No. 46, or SEQ. ID. No. 47.
[0017] In other embodiments, the specificity exchangers comprise an
antigenic domain that comprises at least one of the following
sequences: SEQ. ID. No. 48, SEQ. ID. No. 49, SEQ. ID. No. 50, SEQ.
ID. No. 51, SEQ. ID. No. 52, SEQ. ID. No. 53, SEQ. ID. No. 54, SEQ.
ID. No. 55, SEQ. ID. No. 56, SEQ. ID. No. 57, and SEQ. ID. No. 58,
SEQ. ID. No. 59, SEQ. ID. No. 60, SEQ. ID. No. 61, SEQ. ID. No. 62,
SEQ. ID. No. 63, SEQ. ID. No. 64, SEQ. ID. No. 65, SEQ. ID. No. 66,
SEQ. ID. No. 67, SEQ. ID. No. 68, SEQ. ID. No. 69, SEQ. ID. No. 70,
or SEQ. ID. No. 71.
[0018] More embodiments include specificity exchangers that
comprise at least one of the following sequences: SEQ. ID. No. 72,
SEQ. ID. No. 73, SEQ. ID. No. 74, SEQ. ID. No. 75, SEQ. ID. No. 76,
SEQ. ID. No. 77, SEQ. ID. No. 78, SEQ. ID. No. 79, SEQ. ID. No. 80,
SEQ. ID. No. 81, SEQ. ID. No. 82, SEQ. ID. No. 83, SEQ. ID. No. 84,
SEQ. ID. No. 85, SEQ. ID. No. 86, SEQ. ID. No. 87, SEQ. ID. No. 88,
SEQ. ID. No. 89, SEQ. ID. No. 90, SEQ. ID. No. 91, SEQ. ID. No. 92,
SEQ. ID. No. 93, SEQ. ID. No. 94, SEQ. ID. No. 95, SEQ. ID. No. 96,
SEQ. ID. No. 97, SEQ. ID. No. 98, SEQ. ID. No. 99, SEQ. ID. No.
100, SEQ. ID. No. 101, SEQ. ID. No. 102, SEQ. ID. No. 103, SEQ. ID.
No. 104, SEQ. ID. No. 105, SEQ. ID. No. 106, SEQ. ID. No. 107, SEQ.
ID. No. 108, SEQ. ID. No. 109, SEQ. ID. No. 110, SEQ. ID. No. 111,
SEQ. ID. No. 112, SEQ. ID. No. 113, SEQ. ID. No. 114, SEQ. ID. No.
115, SEQ. ID. No. 118, SEQ. ID. No. 119, SEQ. ID. No. 120, SEQ. ID.
No. 121, SEQ. ID. No. 122, SEQ. ID. No. 123, SEQ. ID. No. 124, SEQ.
ID. No. 125, SEQ. ID. No. 126, SEQ. ID. No. 127, SEQ. ID. No. 128,
SEQ. ID. No. 159, SEQ. ID. No. 160, SEQ. ID. No. 161, SEQ. ID. No.
162, SEQ. ID. No. 163, SEQ. ID. No. 164, SEQ. ID. No. 165, SEQ. ID.
No. 166, SEQ. ID. No. 167, SEQ. ID. No. 168, SEQ. ID. No. 169, SEQ.
ID. No. 170, SEQ. ID. No. 171, SEQ. ID. No. 172, SEQ. ID. No. 173,
SEQ. ID. No. 174, SEQ. ID. No. 175, SEQ. ID. No. 176, SEQ. ID. No.
177, SEQ. ID. No. 178, SEQ. ID. No. 179, SEQ. ID. No. 180, SEQ. ID.
No. 181, SEQ. ID. No. 182, SEQ. ID. No. 183, SEQ. ID. No. 184, SEQ.
ID. No. 185, SEQ. ID. No. 186, SEQ. ID. No. 187, SEQ. ID. No. 188,
SEQ. ID. No. 189, SEQ. ID. No. 190, SEQ. ID. No. 191, SEQ. ID. No.
192, SEQ. ID. No. 193, SEQ. ID. No. 194, SEQ. ID. No. 195, or SEQ.
ID. No. 196.
[0019] Several embodiments also concern specificity exchangers that
can be used to treat or prevent infection by a pathogen. One
approach, for example, involves providing a therapeutically
effective amount of a specificity exchanger to a subject, wherein
said specificity exchanger comprises a specificity domain that
interacts with a receptor or antigen on said pathogen, and an
antigenic domain that comprises a peptide or an epitope obtained
from a pathogen or toxin. Many of the specificity exchangers
described herein can be used with these approaches. In some
embodiments, the subject is monitored for a reduction of the
pathogen after providing the specificity exchanger. In other
approaches, the subject is identified as one in need of a molecule
that redirects antibodies present in the subject to the pathogen
prior to providing the specificity exchanger.
[0020] Several embodiments also concern specificity exchangers that
can be used to treat or prevent bacterial infection. By one
approach, a therapeutically effective amount of a specificity
exchanger is provided to a subject, wherein said specificity
exchanger comprises a specificity domain that interacts with a
receptor or antigen on said bacteria, and an antigenic domain that
comprises a peptide or an epitope obtained from a pathogen or
toxin. Several specificity exchangers that interact with bacteria,
for example, Staphylococcus, are described herein and any one of
these can be used with these methods. In some approaches, the
subject is monitored for a reduction of the bacteria after
providing the specificity exchanger. In other approaches, the
subject is identified as one in need of a molecule that redirects
antibodies present in the subject to the bacteria prior to
providing the specificity exchanger.
[0021] Still more embodiments concern specificity exchangers that
can be used to treat or prevent viral infection. By one approach, a
therapeutically effective amount of a specificity exchanger is
provided to a subject, wherein said specificity exchanger comprises
a specificity domain that interacts with a receptor or antigen on a
virus, and an antigenic domain that comprises a peptide or an
epitope obtained from a pathogen or toxin. Several specificity
exchangers that interact with a virus, for example, a hepatitis
virus, are described herein and any one of these can be used with
this method. In some approaches, the subject is monitored for a
reduction of the virus after providing the specificity exchanger.
In other approaches, the subject is identified as one in need of a
molecule that redirects antibodies present in the subject to the
virus prior to providing the specificity exchanger.
[0022] Still more embodiments concern specificity exchangers that
can be used to treat or prevent cancer. By one approach, a
therapeutically effective amount of a specificity exchanger is
provided to a subject, wherein said specificity exchanger comprises
a specificity domain that interacts with a receptor or antigen on a
cancer cell, and an antigenic domain that comprises a peptide or an
epitope obtained from a pathogen or toxin. Several specificity
exchangers that interact with a cancer cell, for example, a myeloma
cell, are described herein and any one of these can be used with
this method. In some approaches, the subject is monitored for a
reduction of the pathogen after providing the specificity
exchanger. In other approaches, the subject is identified as one in
need of a molecule that redirects antibodies present in the subject
to the pathogen prior to providing the specificity exchanger.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] The following sections describe the manufacture,
characterization, and use of specificity exchangers that bind
pathogens and redirect antibodies that are present in a subject to
the pathogen. Specificity exchangers are generally composed of two
domains, a specificity domain and an antigenic domain. There are
two general types of specificity exchangers differentiated by the
nature of their specificity domains. The first type of specificity
exchanger is an antigen/antibody specificity exchanger. Several
antigen/antibody specificity exchangers are known in the art. See
e.g., U.S. Pat. Nos. 5,869,232; 6,040,137; 6,245,895; 6,417,324;
6,469,143; and U.S. application Ser. Nos. 09/839,447 and
09/839,666; and International App. Nos. PCT/SE95/00468 and
PCT/IB01/00844, all of which are hereby expressly incorporated by
reference in their entireties.
[0024] Antigen/antibody specificity exchangers comprise an amino
acid sequence of an antibody that specifically binds to an antigen
(i.e., the specificity domain) joined to an amino acid sequence to
which an antibody binds (i.e., the antigenic domain). Some
specificity domains of antigen/antibody specificity exchangers
comprise an amino acid sequence of a complementarity determining
region (CDR), are at least 5 and less than 35 amino acids in
length, are specific for bacterial antigens, HIV-1 antigens, or are
specific for hepatitis viral antigens. Some antigenic domains of
antigen/antibody specificity exchangers comprise a peptide having
an antibody-binding region (epitope) of viral, bacterial, or fungal
origin, are at least 5 and less than 35 amino acids in length, or
contain antigenic peptides obtained from the polio virus, measles
virus, hepatitis B virus, hepatitis C virus, or HIV-1.
[0025] The second type of specificity exchanger, the
ligand/receptor specificity exchanger, is also composed of a
specificity domain and an antigenic domain, however, the
specificity domain of the ligand/receptor specificity exchanger
comprises a ligand for a receptor that is present on a pathogen, as
opposed to a sequence of an antibody that binds to an antigen. That
is, a ligand/receptor specificity exchanger differs from an
antibody/antigen specificity exchanger in that the ligand/receptor
specificity exchanger does not contain a sequence of an antibody
that binds an antigen but, instead, adheres to the pathogen through
a ligand interaction with a receptor that is present on the
pathogen. Several ligand/receptor specificity exchangers are also
known in the art. See e.g., U.S. application Ser. Nos. 09/664,945
and 09/664,025; and International App. No. PCT/IB01/02327, all of
which are hereby expressly incorporated by reference in their
entireties.
[0026] Some specificity domains of ligand/receptor specificity
exchangers comprise an amino acid sequence that is a ligand for a
bacterial adhesion receptor (e.g., extracellular fibrinogen binding
protein or clumping factor A or B), are at least 3 and less than 27
amino acids in length, or are specific for bacteria, viruses, or
cancer cells. Some antigenic domains of ligand/receptor specificity
exchangers comprise a peptide having an antibody-binding region
(epitope) of a pathogen or toxin, are at least 5 and less than 35
amino acids in length, or contain antigenic peptides obtained from
polio virus, TT virus, hepatitis B virus, and herpes simplex
virus.
[0027] As used herein, the term "specificity exchanger" refers to
both ligand/receptor specificity exchangers and antigen/antibody
specificity exchangers. If a specific type of specificity exchanger
is being described, either the term "ligand/receptor specificity
exchanger" or "antigen/antibody specificity exchanger" is used.
While there are two main types of specificity exchangers, certain
embodiments include specificity exchangers with one or more ligands
and one or more amino acid sequences of an antibody that
specifically binds to an antigen. In some embodiments, the
specificity exchangers described herein can have 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 ligands, which are the same or different molecules,
in their specificity domain. Likewise, specificity exchangers can
have a specificity domain that includes 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10 amino acid sequences of an antibody that specifically bind to
an antigen, which are the same or different molecules. The
following section describes some general features of specificity
exchangers.
[0028] Specificity Exchangers
[0029] Specificity exchangers have a variety of chemical structures
but are typically characterized as having at least one specificity
domain that interacts with a pathogen (vis a vis antigen/antibody
interaction or ligand/receptor interaction), cancer cell, or toxin
(e.g. a receptor or an antigen) and at least one antigenic domain
that interacts with an antibody. Generally, the specificity
exchangers described herein (i.e., antibody/antigen specificity
exchangers and ligand/receptor specificity exchangers) comprise a
specificity domain, which is at least 3 and less than or equal to
200 amino acids in length, joined to an antigenic domain (e.g., a
peptide backbone), which is at least 3 and less than or equal to
200 amino acids in length, and the antigenic domain in conjunction
with the specificity domain or by itself, reacts with high titer
antibodies that are present in a subject (e.g., a human).
[0030] In some embodiments, for example, the specificity exchangers
comprise a specificity domain that is any length between 3 and 200
amino acids. That is, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino
acids in length and said specificity domain is joined to an
antigenic domain, which is any length between 3 and 200 amino
acids; that is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino
acids in length.
[0031] Preferred specificity exchangers are peptides but some
embodiments comprise derivatized or modified peptides, a
peptidomimetic structure or chemicals. For example, a typical
peptide-based specificity exchanger can be modified to have
substituents not normally found on a peptide or to have
substituents that are normally found on a peptide but are
incorporated at regions that are not normal. In this vein, a
peptide-based specificity exchanger can be acetylated, acylated, or
aminated and the substituents that can be included on the peptide
so as to modify it include, but are not limited to, H, alkyl, aryl,
alkenyl, alkynl, aromatic, ether, ester, unsubstituted or
substituted amine, amide, halogen or unsubstituted or substituted
sulfonyl or a 5 or 6 member aliphatic or aromatic ring. Thus, the
term "specificity exchanger" is a broad one that encompasses
modified or unmodified peptide structures, as well as
peptidomimetics and chemical structures.
[0032] There are many ways to make a peptidomimetic that resembles
a peptide-based specificity exchanger. The naturally occurring
amino acids employed in the biological production of peptides all
have the L-configuration. Synthetic peptides can be prepared
employing conventional synthetic methods, utilizing L-amino acids,
D-amino acids, or various combinations of amino acids of the two
different configurations. Synthetic compounds that mimic the
conformation and desirable features of a peptide but that avoid the
undesirable features, e.g., flexibility (loss of conformation) and
bond breakdown are known as a "peptidomimetics". (See, e.g.,
Spatola, A. F. Chemistry and Biochemistry of Amino Acids. Peptides,
and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, Marcel
Dekker, New York (1983), which describes the use of the
methylenethio bioisostere [CH.sub.2 S] as an amide replacement in
enkephalin analogues; and Szelke et al., In peptides: Structure and
Function, Proceedings of the Eighth American Peptide Symposium,
(Hruby and Rich, Eds.); pp. 579-582, Pierce Chemical Co., Rockford,
Ill. (1983), which describes renin inhibitors having both the
methyleneamino [CH.sub.2 NH] and hydroxyethylene [CHOHCH.sub.2]
bioisosteres at the Leu-Val amide bond in the 6-13 octapeptide
derived from angiotensinogen, all of which are expressly
incorporated by reference in their entireties).
[0033] In general, the design and synthesis of a peptidomimetic
that resembles a specificity exchanger involves starting with the
sequence of a specificity exchanger and conformation data (e.g.,
geometry data, such as bond lengths and angles) of a desired
specificity exchanger (e.g., the most probable simulated peptide),
and using such data to determine the geometries that should be
designed into the peptidomimetic. Numerous methods and techniques
are known in the art for performing this step, any of which could
be used. (See, e.g., Farmer, P. S., Drug Design, (Ariens, E. J.
ed.), Vol. 10, pp. 119-143 (Academic Press, New York, London,
Toronto, Sydney and San Francisco) (1980); Farmer, et al., in TIPS,
9/82, pp. 362-365; Verber et al., in TINS, 9/85, pp. 392-396;
Kaltenbronn et al., in J. Med. Chem. 33: 838-845 (1990); and
Spatola, A. F., in Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins, Vol. 7, pp. 267-357, Chapter 5, "Peptide
Backbone Modifications: A Structure-Activity Analysis of Peptides
Containing Amide Bond Surrogates. Conformational Constraints, and
Relations" (B. Weisten, ed.; Marcell Dekker: New York, pub.)
(1983); Kemp, D. S., "Peptidomimetics and the Template Approach to
Nucleation of .beta.-sheets and .alpha.-helices in Peptides,"
Tibech, Vol. 8, pp. 249-255 (1990), all of which are expressly
incorporated by reference in their entireties). Additional
teachings can be found in U.S. Pat. Nos. 5,288,707; 5,552,534;
5,811,515; 5,817,626; 5,817,879; 5,821,231; and 5,874,529, all of
which are expressly incorporated by reference in their entireties.
Once the peptidomimetic is designed, it can be made using
conventional techniques in peptide chemistry and/or organic
chemistry.
[0034] Some embodiments include a plurality of specificity domains
and/or a plurality of antigenic domains. One type of specificity
exchanger that has a plurality of specificity domains and/or
antigenic domains is referred to as a "multimerized specificity
exchanger" because it has multiple specificity domains and/or
antigenic domains that appear (e.g., are fused) in tandem on the
same molecule. For example, a multimerized specificity domain can
have two or more ligands that interact with one type of receptor,
two or more ligands that interact with different types of receptors
on the pathogen, two or more ligands that interact with different
types of receptors on different pathogens, two or more antibody
sequences that interact with one type of antigen on a pathogen, two
or more antibody sequences that interact with different types of
antigens on a pathogen, or two or more antibody sequences that
interact with different types of antigens on different
pathogens.
[0035] Similarly, a multimerized antigenic domain can be
constructed to have multimers of the same epitope of a pathogen or
different epitopes of a pathogen, which can also be multimerized.
That is, some multimerized antigenic domains are multivalent
because the same epitope is repeated. In contrast, some
multimerized antigenic domains have more than one epitope present
on the same molecule in tandem but the epitopes are different. In
this respect, these antigenic domains are multimerized but not
multivalent. Further, some multimerized antigenic domains are
constructed to have different epitopes but the different epitopes
are themselves multivalent because each type of epitope is
repeated.
[0036] Some specificity exchangers or specificity domains or
antigenic domains are disposed on or comprise a support. A
"support" can be a carrier, a protein, a resin, a cell membrane, or
any macromolecular structure used to join or immobilize specificity
domains, antigenic domains, or the specificity exchangers
themselves. For example, the antigenic domain can be thought of as
a support (e.g., a backbone) onto which one or more specificity
domains are joined. Further, a multimeric specificity exchanger can
be made by joining a plurality of specificity domains to support
that may be an antigenic domain in itself or may have a plurality
of antigenic domains joined. Similarly, a specificity domain can be
joined to a support onto which one or more antigenic domains are
joined. Thus, a support can be used to link one or more specificity
domains to one or more antigenic domains or the support can be an
antigenic domain in itself.
[0037] Solid supports include, but are not limited to, the walls of
wells of a reaction tray, test tubes, polystyrene beads, magnetic
beads, nitrocellulose strips, membranes, microparticles such as
latex particles, animal cells, Duracyte.RTM., artificial cells, and
others. A specificity exchanger or parts thereof can also be joined
to inorganic supports, such as silicon oxide material (e.g. silica
gel, zeolite, diatomaceous earth or aminated glass) by, for
example, a covalent linkage through a hydroxy, carboxy, or amino
group and a reactive group on the support.
[0038] In some embodiments, the macromolecular support has a
hydrophobic surface that interacts with a portion of the
specificity exchanger, by a hydrophobic non-covalent interaction.
In some cases, the hydrophobic surface of the support is a polymer
such as plastic or any other polymer in which hydrophobic groups
have been linked such as polystyrene, polyethylene or polyvinyl.
Additionally, a specificity exchanger, specificity domain, or
antigenic domain can be covalently bound to supports including
proteins and oligo/polysaccharides (e.g. cellulose, starch,
glycogen, chitosane or aminated sepharose). In some embodiments, a
reactive group on the molecule, such as a hydroxy or an amino
group, is used to join to a reactive group on the carrier so as to
create the covalent bond. Additional specificity exchangers
comprise a support that has other reactive groups that are
chemically activated so as to attach the specificity exchanger or
parts thereof. For example, cyanogen bromide activated matrices,
epoxy activated matrices, thio and thiopropyl gels, nitrophenyl
chloroformate and N-hydroxy succinimide chlorformate linkages, or
oxirane acrylic supports can be used. (Sigma). Furthermore, in some
embodiments, a liposome or lipid bilayer (natural or synthetic) is
contemplated as a support and a specificity exchanger, specificity
domain, or antigenic domain can be attached to the membrane surface
or are incorporated into the membrane by techniques in liposome
engineering. By one approach, liposome multimeric supports comprise
a specificity exchanger or parts thereof that is exposed on the
surface.
[0039] Some specificity exchangers also comprise other elements in
addition to the specificity domain and antigenic domain such as
sequences that facilitate purification (e.g., poly-histidine tail),
linkers that provide greater flexibility and reduce steric
hindrance, and sequences that either provide greater stability to
the specificity exchanger (e.g., resistance to protease
degradation) or promote degradation (e.g., protease recognition
sites). For example, the specificity exchangers can comprise
cleavable signal sequences that promote cytoplasmic export of the
peptide and/or cleavable sequence tags that facilitate purification
on antibody columns, glutathione columns, and metal columns.
[0040] Specificity exchangers can also comprise elements that
promote flexibility of the molecule, reduce steric hindrance, or
allow the specificity exchanger to be attached to a support or
other molecule. These elements are collectively referred to as
"linkers". One type of linker that can be incorporated with a
specificity exchanger, for example, is avidin or streptavidin (or
their ligand--biotin). Through a biotin-avidin/streptavidin
linkage, multiple specificity exchangers can be joined together
(e.g., through a support, such as a resin, or directly) or
individual specificity domains and antigenic domains can be joined.
Another example of a linker that can be included in a specificity
exchanger is referred to as a ".lamda. linker" because it has a
sequence that is found on .lamda. phage. Preferred .lamda.
sequences are those that correspond to the flexible arms of the
phage. These sequences can be included in a specificity exchanger
(e.g., between the specificity domain and the antigenic domain or
between multimers of the specificity and/or antigenic domains) so
as to provide greater flexibility and reduce steric hindrance.
Additionally, a plurality of alanine residues or other peptide
sequences can be used as linkers.
[0041] Specificity exchangers can also include sequences that
either confer resistance to protease degradation or promote
protease degradation. By incorporating multiple cysteines in a
specificity exchanger, for example, greater resistance to protease
degradation can be obtained. These embodiments of the
ligand/receptor specificity exchanger are expected to remain in the
body for extended periods, which may be beneficial for some
therapeutic applications. In contrast, specificity exchangers can
also include sequences that promote rapid degradation so as to
promote rapid clearance from the body. Many sequences that serve as
recognition sites for serine, cysteine, and aspartic proteases are
known and can be included in a specificity exchanger. The section
below describes the specificity domains of antigen/antibody
specificity exchangers in greater detail.
[0042] Specificity Domains of Antigen/Antibody Specificity
Exchangers
[0043] The specificity domain of antigen/antibody specificity
exchangers can include the amino-acid sequence of any antibody
which specifically binds to a certain antigen, such as a hapten,
for example. Preferred specificity domains of antigen/antibody
specificity exchangers comprise an amino acid sequence of a
complementarity determining region (CDR) or a framework region of a
certain antibody. The CDRs of antibodies are responsible for the
specificity of the antibody. X-ray crystallography has shown that
the three CDRs of the variable (V) region of the heavy chain and
the three CDRs of the V region of the light chain may all have
contact with the epitope in an antigen-antibody complex.
[0044] In certain embodiments, single peptides corresponding to the
CDRs of mAbs to various antigens and that are capable of mimicking
the recognition capabilities of the respective mAb can be included
in the specificity domain of the antigen/antibody specificity
exchangers. Specifically a peptide corresponding to CDRH3 of a mAb
specific for the V3 region of human immuno deficiency virus-1 gp160
can be included in the specificity domain. This peptide was shown
to have neutralizing capacity when assayed in vitro. The CDRH3 can
be derived from mAb F58, and Ab C1-5, and the like. Like CDRH3, the
CDRH1 and/or CDRH2 domain of Ab C1-5 can also be used in the
specificity domains described herein. In other embodiments the
specificity domain can include a peptide corresponding to CDRH2 of
a mAb to hepatitis B virus core antigen (HBcAg). CDRH2 has
demonstrated an ability to capture HBcAg. Several other peptides,
derived from antibodies that bind HBcAg or hepatitis B virus e
antigen (HBeAg) have been identified. See U.S. Pat. No. 6,417,324,
issued Jul. 9, 2002; and U.S. patent application Ser. No.
09/839,447, filed Apr. 20, 2001 and U.S. patent application Ser.
No. 10/153,271, filed May 21, 2002, all of which are hereby
incorporated by reference in their entireties. These peptides
(specificity domains) can be incorporated into antigen/antibody
specificity exchangers so as to redirect antibodies present in a
subject to hepatitis B virus.
[0045] TABLE I provides a non-exclusive list of specificity domains
that can be used in the antigen/antibody specificity exchangers
described herein. The section following TABLE I describes the
specificity domains of ligand/receptor specificity exchangers in
greater detail. TABLE-US-00001 TABLE I SPECIFICITY DOMAINS FOR
ANTIGEN/ANTIBODY SPECIFICITY EXCHANGERS SEQ ID NO: 43:
CDLIYYDYEEDYYF SEQ ID NO: 44: CDLIYYDYEEDYY SEQ ID NO: 45 TYAMN SEQ
ID NO: 46 RVRSKSFNYATYYADSVKG SEQ ID NO: 47 PAQGIYFDYGGFAY
[0046] Specificity Domains for Ligand/Receptor Specficity
Exchangers
[0047] The diversity of ligand/receptor specificity exchangers is
also equally vast because many different ligands that bind many
different receptors on many different pathogens can be incorporated
into a ligand/receptor specificity exchanger. The term "pathogen"
generally refers to any etiological agent of disease in an animal
including, but not limited to, bacteria, parasites, fungus, mold,
viruses, and cancer cells. Similarly, the term "receptor" is used
in a general sense to refer to a molecule (usually a peptide other
than a sequence found in an antibody, but can be a carbohydrate,
lipid, or nucleic acid) that interacts with a "ligand" (usually a
peptide other than a sequence found in an antibody, or a
carbohydrate, lipid, nucleic acid or combination thereof). The
receptors contemplated do not have to undergo signal transduction
and can be involved in a number of molecular interactions
including, but not limited to, adhesion (e.g., integrins) and
molecular signaling (e.g., growth factor receptors).
[0048] In certain embodiments, desired specificity domains include
a ligand that has a peptide sequence that is present in an
extracellular matrix protein (e.g., fibrinogen, collagen,
vitronectin, laminin, plasminogen, thrombospondin, and fibronectin)
and some specificity domains comprise a ligand that interacts with
a bacterial adhesion receptor (e.g., extracellular fibrinogen
binding protein (Efb), collagen binding protein, vitronectin
binding protein, laminin binding protein, plasminogen binding
protein, thrombospondin binding protein, clumping factor A (ClfA),
clumping factor B (ClfB), fibronectin binding protein, coagulase,
and extracellular adherence protein).
[0049] Investigators have mapped the regions of extracellular
matrix proteins that interact with several receptors. (See e.g.,
McDevvit et al., Eur. J. Biochem., 247:416-424 (1997); Flock,
Molecular Med. Today, 5:532 (1999); and Pei et al., Infect. and
Immun. 67:4525 (1999), all of which are herein expressly
incorporated by reference in their entirety). Some receptors bind
to the same region of the extracellular matrix protein, some have
overlapping binding domains, and some bind to different regions
altogether. Preferably, the ligands that make up the specificity
domain have an amino acid sequence that has been identified as
being involved in adhesion to an extracellular matrix protein. It
should be understood, however, that random fragments of known
ligands for any receptor on a pathogen can be used to generate
ligand/receptor specificity exchangers and these candidate
ligand/receptor specificity exchangers can be screened in the
characterization assays described infra to identify the molecules
that interact with the receptors on the pathogen.
[0050] Some specificity domains have a ligand that interacts with a
bacterial adhesion receptor including, but not limited to,
extracellular fibrinogen binding protein (Efb), collagen binding
protein, vitronectin binding protein, laminin binding protein,
plasminogen binding protein, thrombospondin binding protein,
clumping factor A (ClfA), clumping factor B (ClfB), fibronectin
binding protein, coagulase, and extracellular adherence protein.
Ligands that have an amino acid sequence corresponding to the
C-terminal portion of the gamma-chain of fibrinogen have been shown
to competitively inhibit binding of fibrinogen to ClfA, a
Staphylococcus aureus adhesion receptor. (McDevvit et al., Eur. J.
Biochem., 247:416-424 (1997)). Further, Staphylococcus organisms
produce many more adhesion receptors such as Efb, which binds to
the alpha chain fibrinogen, ClfB, which interacts with both the
.alpha. and .beta. chain of fibrinogen, and Fbe, which binds to the
.beta. chain of fibrinogen. (Pei et al., Infect. and Immun. 67:4525
(1999)). Accordingly, preferred specificity domains comprise
between 3 and 30 amino acids, that is, at least 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 consecutive amino acids of a sequence present
in a molecule (e.g., fibrinogen) that can bind to a bacterial
adhesion receptor.
[0051] Specificity domains can also comprise a ligand that
interacts with a viral receptor. Several viral receptors and
corresponding ligands are known and these ligands or fragments
thereof can be incorporated into a ligand/receptor specificity
exchanger. For example, Tong et al., has identified an Hepadnavirus
receptor, a 170 kd cell surface glycoprotein that interacts with
the pre-S domain of the duck hepatitis B virus envelope protein
(U.S. Pat. No. 5,929,220) and Maddon et al., has determined that
the T cell surface protein CD4 (or the soluble form termed T4)
interacts with gp120 of HIV (U.S. Pat. No. 6,093,539); both
references are herein expressly incorporated by reference in their
entireties. Thus, specificity domains that interact with a viral
receptor can comprise regions of the pre-S domain of the duck
hepatitis B virus envelope protein (e.g., amino acid residues
80-102 or 80-104) or regions of the T cell surface protein CD4 (or
the soluble form termed T4) that interacts with gp120 of HIV (e.g.,
the extracellular domain of CD4/T4 or fragments thereof). Many more
ligands for viral receptors exist and these molecules or fragments
thereof can be used as a specificity domain.
[0052] Specificity domains can also comprise a ligand that
interacts with a receptor present on a cancer cell. The
proto-oncogene HER-2/neu (C-erbB2) encodes a surface growth factor
receptor of the tyrosine kinase family, p185HER2. Twenty to thirty
percent of breast cancer patients over express the gene encoding
HER-2/neu (C-erbB2), via gene amplification. Thus, ligand/receptor
specificity exchangers comprising a specificity domain that encodes
a ligand for HER-2/neu (C-erbB2) are desirable embodiments.
[0053] Many types of cancer cells also over express or
differentially express integrin receptors. Many preferred
embodiments comprise a specificity domain that interacts with an
integrin receptor. Although integrins predominantly interact with
extracellular matrix proteins, it is known that these receptors
interact with other ligands such as invasins, RGD-containing
peptides (i.e., Arginine-Glycine-Aspartate), and chemicals. (See
e.g., U.S. Pat. Nos. 6,090,944 and 6,090,388; and Brett et al., Eur
J Immunol, 23:1608 (1993), all of which are hereby expressly
incorporated by reference in their entireties). Ligands for
integrin receptors include, but are not limited to, molecules that
interact with a vitronectin receptor, a laminin receptor, a
fibronectin receptor, a collagen receptor, a fibrinogen receptor,
an .alpha..sub.4.beta..sub.1 receptor, an
.alpha..sub.6.beta..sub.1receptor, an
.alpha..sub.3.beta..sub.1receptor, an .alpha..sub.5.beta..sub.1
receptor, and an .alpha..sub.v.beta..sub.3receptor. Preferably, the
specificity domain of an antigen/antibody specificity exchanger is
between 5-35 amino acids in length. TABLE II lists several
preferred specificity domains for ligand/receptor specificity
exchangers. The section that follows TABLE II describes the
antigenic domains of specificity exchangers in greater detail.
TABLE-US-00002 TABLE II SPECIFICITY DOMAINS FOR LIGAND/RECEPTOR
SPECIFICITY EXCHANGERS YGEGQQHHLGGAKQAGDV (SEQ. ID. No. 1)
MSWSLHPRNLILYFYALLFL (SEQ. ID. No. 2) ILYFYALLFLSTCVAYVAT (SEQ. ID.
No. 3) SSTCVAYVATRDNCCILDER (SEQ. ID. No. 4) RDNCCILDERFGSYCPTTCG
(SEQ. ID. No. 5) FGSYCPTTCGIADFLSTYQT (SEQ. ID. No. 6)
IADFLSTYQTKVDKDLQSLE (SEQ. ID. No. 7) KVDKDLQSLEDILHQVENKT (SEQ.
ID. No. 8) DILHQVENKTSEVKQLIKAI (SEQ. ID. No. 9)
SEVKQLIKAIQLTYNPDESS (SEQ. ID. No. 10) QLTYNPDESSKPNMIDAATL (SEQ.
ID. No. 11) KPNMIDAATLKSRIMLEEIM (SEQ. ID. No. 12)
KSRIMLEEIMKYEASILTHD (SEQ. ID. No. 13) KYEASILTHDSSIRYLQEIY (SEQ.
ID. No. 14) SSIRYLQEIYNSNNQKIVNL (SEQ. ID. No. 15)
NSNNQKIVNLKEKVAQLEAQ (SEQ. ID. No. 16) CQEPCKDTVQIHDITGKDCQ (SEQ.
ID. No. 17) IHDITGKDCQDIANKGAKQS (SEQ. ID. No. 18)
DIANKGAKQSGLYFIKPLKA (SEQ. ID. No. 19) GLYFIKPLKANQQFLVYCEI (SEQ.
ID. No. 20) NQQFLVYCEIDGSGNGWTVF (SEQ. ID. No. 21)
DGSGNGWTVFQKRLDGSVDF (SEQ. ID. No. 22) QKRLDGSVDFKKNWIQYKEG (SEQ.
ID. No. 23) KKNWIQYKEGFGHLSPTGTT (SEQ. ID. No. 24)
FGHLSPTGTTEFWLGNEKIH (SEQ. ID. No. 25) EFWLGNEKIHLISTQSAIPY (SEQ.
ID. No. 26) LISTQSAIPYALRVELEDWN (SEQ. ID. No. 27)
ALRVELEDWNGRTSTADYAM (SEQ. ID. No. 28) GRTSTADYAMFKVGPEADKY (SEQ.
ID. No. 29) FKVGPEADKYRLTYAYFAGG (SEQ. ID. No. 30)
RLTYAYFAGGDAGDAFDGFD (SEQ. ID. No. 31) DAGDAFDGFDFGDDPSDKFF (SEQ.
ID. No. 32) FGDDPSDKFFTSHNGMQFST (SEQ. ID. No. 33)
TSHNGMQFSTWDNDNDKFEG (SEQ. ID. No. 34) WDNDNDKFEGNCAEQDGSGW (SEQ.
ID. No. 35) NCAEQDGSGWWMNKCHAGHL (SEQ. ID. No. 36)
WMNKCHAGHLNGVYYQGGTY (SEQ. ID. No. 37) NGVYYQGGTYSKASTPNGYD (SEQ.
ID. No. 38) SKASTPNGYDNGIIWATWKT (SEQ. ID. No. 39)
NGIIWATWKTRWYSMKKTTM (SEQ. ID. No. 40) RWYSMKKTTMKIIPFNRLTI (SEQ.
ID. No. 41) KIIPFNRLTIGEGQQHHLGGAKQAGDV (SEQ. ID. No. 42)
[0054] Antigenic Domains
[0055] The diversity of antigenic domains that can be used in the
ligand/receptor specificity exchangers and antibody/antigen
specificity exchangers is quite large because a pathogen or toxin
can present many different epitopes. Desirably, the antigenic
domains used with the specificity exchangers are peptides obtained
from surface proteins or exposed proteins from bacteria, fungi,
plants, molds, viruses, cancer cells, and toxins. It is also
desired that the antigenic domains comprise a peptide sequence that
is rapidly recognized as non-self by existing antibodies in a
subject, preferably by virtue of naturally acquired immunity or
vaccination. For example, many people are immunized against
childhood diseases including, but not limited to, small pox,
measles, mumps, rubella, and polio. Thus, antibodies to epitopes on
these pathogens can be produced by an immunized person. Desirable
antigenic domains have a peptide that contains one or more epitopes
that is recognized by antibodies in the subject that are present in
the subject to respond to pathogens such as small pox, measles,
mumps, rubella, herpes, hepatitis, and polio.
[0056] Some embodiments, however, have antigenic domains that
interact with an antibody that has been administered to the
subject. For example, an antibody that interacts with an antigenic
domain on a specificity exchanger can be co-administered with the
specificity exchanger. Further, an antibody that interacts with a
specificity exchanger may not normally exist in a subject but the
subject has acquired the antibody by introduction of a biologic
material or antigen (e.g., serum, blood, or tissue) so as to
generate a high titer of antibodies in the subject. For example,
subjects that undergo blood transfusion acquire numerous
antibodies, some of which can interact with an antigenic domain of
a specificity exchanger. Some preferred antigenic domains for use
in a specificity exchanger also comprise viral epitopes or peptides
obtained from pathogens such as the herpes simplex virus, hepatitis
B virus, TT virus, and the poliovirus.
[0057] Preferably, the antigenic domains comprise an epitope or
peptide obtained from a pathogen or toxin that is recognized by a
"high-titer antibody." The term "high-titer antibody" as used
herein, refers to an antibody that has high affinity for an antigen
(e.g., an epitope on an antigenic domain). For example, in a
solid-phase enzyme linked immunosorbent assay (ELISA), a high titer
antibody corresponds to an antibody present in a serum sample that
remains positive in the assay after a dilution of the serum to
approximately the range of 1:100-1:1000 in an appropriate dilution
buffer. Other dilution ranges include 1:200-1:1000, 1:200-1:900,
1:300-1:900, 1:300-1:800, 1:400-1:800, 1:400-1:700, 1:400-1:600,
and the like. In certain embodiments, the ratio between the serum
and dilution buffer is approximately: 1:100, 1:150, 1:200, 1:250,
1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700,
1:750, 1:800, 1:850, 1:900, 1:950, 1:1000. Approaches to determine
whether the epitope or peptide obtained from a pathogen or toxin is
recognizable by a high titer antibody are also provided infra in
the Examples.
[0058] Epitopes or peptides of a pathogen that can be included in
an antigenic domain of a specificity exchanger include the epitopes
or peptide sequences disclosed in Swedish Pat No. 9901601-6; U.S.
Pat. No. 5,869,232; Mol. Immunol. 28: 719-726 (1991); and J. Med.
Virol. 33:248-252 (1991); all which are herein expressly
incorporated by reference in their entireties. Preferred antigenic
domains, have an epitope or peptide obtained form herpes simplex
virus gG2 protein, hepatitis B virus s antigen (HBsAg), hepatitis B
virus e antigen (HBeAg), hepatitis B virus c antigen (HBcAg), TT
virus, and the poliovirus or combination thereof or comprise a
sequence selected from the group consisting of SEQ. ID. Nos. 48-71.
TABLE III provides the amino acid sequence of several preferred
antigenic domains that can be used with the specificity exchangers
described herein. The section that follows TABLE III describes
several approaches to make specificity exchangers. TABLE-US-00003
TABLE III ANTIGENIC DOMAINS GLYSSIWLSPGRSYFET (SEQ. ID. No. 48)
YTDIKYNPFTDRGEGNM (SEQ. ID. No. 49) DQNIHMNARLLIRSPFT (SEQ. ID. No.
50) LIRSPFTDPQLLVHTDP (SEQ. ID. No. 51) QKESLLFPPVKLLRRVP (SEQ. ID.
No. 52) PALTAVETGAT (SEQ. ID. No. 53) STLVPETT (SEQ. ID. No. 54)
TPPAYRPPNAPIL (SEQ. ID. No. 55) EIPALTAVE (SEQ. ID. No. 56)
LEDPASRDLV (SEQ. ID. No. 57) HRGGPEEF (SEQ. ID. No. 58) HRGGPEE
(SEQ. ID. No. 59) VLICGENTVSRNYATHS (SEQ. ID. No. 60)
KINTMPPFLDTELTAPS (SEQ. ID. No. 61) PDEKSQREILLNKIASY (SEQ. ID. No.
62) TATTTTYAYPGTNRPPV (SEQ. ID. No. 63) STPLPETT (SEQ. ID. No. 64)
PPNAPILS (SEQ. ID. No. 65) RPPNAPILST (SEQ. ID. No. 66)
KEIPALTAVETG (SEQ. ID. No. 67) PAHSKEIPALTA (SEQ. ID. No. 68)
WGCSGKLICT (SEQ. ID. No. 69) CTTAVPWNAS (SEQ. ID. No. 70)
QRKTKRNTNRR (SEQ. ID. No. 71)
[0059] Methods of Making Specificity Exchangers
[0060] Many different specificity exchangers can be made using
conventional techniques in recombinant engineering and/or peptide
chemistry. In some embodiments, the specificity domains and
antigenic domains of the specificity exchangers are made separately
and are subsequently joined together (e.g., through linkers or by
association with a common carrier molecule) and in other
embodiments, the specificity domain and antigenic domain are made
as part of the same molecule. For example, any of the specificity
domains listed in TABLES I and II can be joined to any of the
antigenic domains of TABLE III. Although the specificity and
antigenic domains can be made separately and joined together
through a linker or carrier molecule (e.g., a complex comprising a
biotinylated specificity domain, streptavidin, and a biotinylated
antigenic domain), it is preferred that the specificity exchangers
are made as fusion proteins. Thus, preferred embodiments include
fusion proteins comprising any of the specificity domains listed in
TABLES I and II can be joined to any of the antigenic domains of
TABLE III.
[0061] Specificity exchangers can be generated in accordance with
conventional methods of protein engineering, protein chemistry,
organic chemistry, and molecular biology. Additionally, some
commercial enterprises manufacture made-to-order peptides and a
specificity exchanger can be obtained by providing such a company
with the sequence of a desired specificity exchanger and employing
their service to manufacture the agent according to particular
specifications (e.g., Bachem AG, Switzerland). Preferably, the
specificity exchangers are prepared by chemical synthesis methods
(such as solid phase peptide synthesis) using techniques known in
the art, such as those set forth by Merrifield et al., J. Am. Chem.
Soc. 85:2149 (1964), Houghten et al., Proc. Natl. Acad. Sci. USA,
82:51:32 (1985), Stewart and Young (Solid phase peptide synthesis,
Pierce Chem Co., Rockford, Ill. (1984), and Creighton, 1983,
Proteins: Structures and Molecular Principles, W. H. Freeman &
Co., N.Y.; all references are herein expressly incorporated by
reference in their entireties.
[0062] By another approach, solid phase peptide synthesis is
performed using a peptide synthesizer, such as an Applied
Biosystems 430A peptide synthesizer (Applied Biosystems, Foster
City, Calif.). Each synthesis uses a p-methylbenzylhydrylamine
solid phase support resin (Peptide International, Louisville, Ky.)
yielding a carboxyl terminal amide when the peptides are cleaved
off from the solid support by acid hydrolysis. Prior to use, the
carboxyl terminal amide can be removed and the specificity
exchangers can be purified by high performance liquid
chromatography (e.g., reverse phase high performance liquid
chromatography (RP-HPLC) using a PepS-15 C18 column (Pharmacia,
Uppsala, Sweden)) and sequenced on an Applied Biosystems 473A
peptide sequencer. An alternative synthetic approach uses an
automated peptide synthesizer (Syro, Multisyntech, Tubingen,
Germany) and 9-fluorenylmethoxycarbonyl (fmoc) protected amino
acids (Milligen, Bedford, Mass.).
[0063] In still other embodiments, the specificity exchangers can
be synthesized with a Milligen 9050 peptide synthesizer using
9-fluorenylmethoxy-carbonyl-protected amino acid esters.
Synthesized specificity exchangers can be analysed and/or purified
by reverse phase HPLC using a Pep-S 5 m column (Pharmacia-LKB,
Uppsala, Sweden), run with a gradient from 10% to 60% CH3CN against
water containing 0.1% trifluoro-acetic acid.
[0064] While the specificity exchangers can be chemically
synthesized, it can be more efficient to produce these polypeptides
by recombinant DNA technology using techniques well known in the
art. Such methods can be used to construct expression vectors
containing nucleotide sequences encoding a specificity exchanger
and appropriate transcriptional and translational control signals.
The expression construct can then be transfected to cells. After
the transfected cells express the specificity exchanger, the
specificity exchanger can be purified or isolated from the cells or
cell supernatent. It is important to note that any recombinant
methodology can be used to synthesize the specificity exchangers
described herein, including, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination.
[0065] Alternatively, RNA capable of encoding a specificity
exchanger can be chemically synthesized using, for example,
synthesizers. See, for example, the techniques described in
Oligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0066] A variety of host-expression vector systems can be utilized
to express the specificity exchangers. Where the specificity
exchanger is a soluble molecule it can be recovered from the
culture, i.e., from the host cell in cases where the peptide or
polypeptide is not secreted, and from the culture media in cases
where the peptide or polypeptide is secreted by the cells. However,
the expression systems also encompass engineered host cells that
express membrane bound specificity exchangers. Purification or
enrichment of the specificity exchangers from such expression
systems can be accomplished using appropriate detergents and lipid
micelles and methods well known to those skilled in the art.
[0067] The expression systems that can be used include, but are not
limited to, microorganisms such as bacteria (e.g., E. coli or B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing nucleotide
sequences encoding a specificity exchanger; yeast (e.g.,
Saccharomyces, Pichia) transformed with recombinant yeast
expression vectors containing nucleotide sequences encoding
specificity exchangers; insect cell systems infected with
recombinant virus expression vectors (e.g., Baculovirus) containing
nucleic acids encoding the specificity exchangers; or mammalian
cell systems (e.g., HeLa, COS, CHO, BHK, 293, or 3T3 cells)
harboring recombinant expression constructs containing nucleic
acids encoding specificity exchangers.
[0068] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
specificity exchanger. For example, when a large quantity is
desired (e.g., for the generation of pharmaceutical compositions of
specificity exchangers) vectors that direct the expression of high
levels of fusion protein products that are readily purified can be
desirable. Such vectors include, but are not limited, to the E.
coli expression vector pUR278 (Ruther et al., EMBO J., 2:1791
(1983), in which the specificity exchanger coding sequence can be
ligated individually into the vector in frame with the lacZ coding
region so that a fusion protein is produced; pIN vectors (Inouye
& Inouye, Nucleic Acids Res., 13:3101-3109 (1985); Van Heeke
& Schuster, J. Biol. Chem., 264:5503-5509 (1989)); and the
like. The pGEX vectors can also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can be
purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0069] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The
specificity exchanger gene coding sequence can be cloned
individually into non-essential regions (for example the polyhedrin
gene) of the virus and placed under control of an AcNPV promoter
(for example the polyhedrin promoter). Successful insertion of a
specificity exchanger gene coding sequence will result in
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus, (i.e., virus lacking the proteinaceous coat
coded for by the polyhedrin gene). These recombinant viruses are
then used to infect Spodoptera frugiperda cells in which the
inserted gene is expressed. (E.g., see Smith et al., J. Virol. 46:
584 (1983); and Smith, U.S. Pat. No. 4,215,051).
[0070] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, a nucleic acid sequence encoding a specificity
exchanger can be ligated to an adenovirus transcription/translation
control complex, e.g., the late promoter and tripartite leader
sequence. This chimeric gene can then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a
non-essential region of the viral genome (e.g., region E1 or E3)
will result in a recombinant virus that is viable and capable of
expressing the specificity exchanger gene product in infected
hosts. (See e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA
81:3655-3659 (1984)). Specific initiation signals can also be
required for efficient translation of inserted specificity
exchanger nucleotide sequences (e.g., the ATG initiation codon and
adjacent sequences). In most cases, an exogenous translational
control signal, including, perhaps, the ATG initiation codon,
should be provided. Furthermore, the initiation codon should be in
phase with the reading frame of the desired coding sequence to
ensure translation of the entire insert. These exogenous
translational control signals and initiation codons can be of a
variety of origins, both natural and synthetic. The efficiency of
expression can also be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc.
(See Bittner et al., Methods in Enzymol., 153:516-544 (1987)).
[0071] In addition, a host cell strain can be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for some embodiments.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
foreign protein expressed. To this end, eukaryotic host cells that
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
can be used. Such mammalian host cells include, but are not limited
to, HeLa, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138
cells.
[0072] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the specificity exchangers described above can
be engineered. Rather than using expression vectors that contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells are allowed
to grow for 1-2 days in an enriched media, and then are switched to
a selective media. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci
which in turn are cloned and expanded into cell lines. This method
is advantageously used to engineer cell lines which express a
specificity exchanger.
[0073] A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:2026 (1962)), and adenine
phosphoribosyltransferase (Lowy, et al., Cell 22:817 (1980)) genes
can be employed in tk..sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci.
USA 77:3567 (1980)); O'Hare, et al., Proc. Natl. Acad. Sci. USA
78:1527 (1981)); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981));
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., J. Mol. Biol. 150:1 (1981)); and hygro,
which confers resistance to hygromycin (Santerre, et al., Gene
30:147 (1984)). The following section describes several types of in
vitro and in vivo characterization assays that can be used to
identify specificity exchangers that bind to pathogens and redirect
antibodies present in a subject to the pathogen.
[0074] Specificity Exchanger Characterization Assays
[0075] Preferably, after a specificity exchanger is synthesized it
is analyzed for its ability to interact with a receptor or antigen
and/or the ability to interact with an antibody that is specific
for the antigenic domain. The term "characterization assay" refers
to an assay, experiment, or analysis made on a specificity
exchanger, which evaluates the ability of a specificity exchanger
to interact with a receptor or antigen (e.g., a surface receptor or
protein present in bacteria, virus, mold, or fungi) and/or an
antibody present in a subject or made to be present in a subject
(e.g., an antibody that recognizes an epitope or peptide of a
pathogen that is part of an antigenic domain), or effect the
proliferation of a pathogen. Encompassed by the term
"characterization assay" are binding studies (e.g., enzyme
immunoassays (EIA), enzyme-linked immunoassays (ELISA), competitive
binding assays, computer generated binding assays, support bound
binding studies, and one and two hybrid systems), and infectivity
studies (e.g., reduction of viral infection, propagation, and
attachment to a host cell). For example, some in vitro
characterization assays evaluate the ability of a specificity
exchanger to bind to a support having a receptor or antigen of a
pathogen or fragment thereof disposed thereon or vice versa. Other
in vitro characterization assays assess the ability of a
specificity exchanger to bind to an antibody specific for the
antigenic domain of the specificity exchanger.
[0076] Several of these types of in vitro approaches employ a
multimeric specificity exchanger, specificity domain, or antigenic
domain, as described above. For example, a support-bound
ligand/receptor specificity exchanger can be contacted with "free"
adhesion receptors and an association can be determined directly
(e.g., by using labeled adhesion receptors) or indirectly (e.g., by
using a labeled ligand directed to an adhesion receptor). Thus,
candidate ligand/receptor specificity exchangers are identified as
bona fide ligand/receptor specificity exchangers by virtue of the
association of the receptors with the support-bound candidate
ligand/receptor specificity exchanger. Alternatively, support-bound
adhesion receptors can be contacted with "free" ligand/receptor
specificity exchangers and the amount of associated ligand/receptor
specificity exchanger can be determined directly (e.g., by using
labeled ligand/receptor specificity exchanger) or indirectly (e.g.,
by using a labeled antibody directed to the antigenic domain of the
ligand/receptor specificity exchanger). Similarly, by using an
antibody specific for the antigenic domain of a specificity
exchanger disposed on a support and labeled specificity exchanger
(or a secondary detection reagent, e.g., a labeled receptor or
antibody to the specificity exchanger) the ability of the antibody
to bind to the antigenic domain of the specificity exchanger can be
determined. Additionally, some characterization assays are designed
to determine whether a specificity exchanger can bind to both the
target and the redirected antibody.
[0077] Cellular characterization assays are also employed to
evaluate the ability of the specificity exchanger to bind to a
pathogen or affect infection or proliferation of the pathogen in
cultured cells. In vivo characterization assays are also employed
to evaluate the ability of specificity exchangers to redirect
antibodies to a pathogen or to reduce the proliferation of a
pathogen in diseased animals. In general, the characterization
assays can be classified as: (1) in vitro characterization assays,
(2) cellular characterization assays, and (3) in vivo
characterization assays. A discussion of each type of
characterization assay is provided in the following sections.
[0078] In Vitro Characterization Assays
[0079] There are many types of in vitro assays that can be used to
determine whether a specificity exchanger binds to a particular
receptor or antigen and whether an antibody found in a subject can
bind to the antigenic domain of the specificity exchanger. Most
simply, the receptor or antigen is bound to a support (e.g., a
petri dish) and the association of the specificity exchanger with
the receptor or antigen is monitored directly or indirectly, as
described above. Similarly, a primary antibody directed to the
antigenic domain of a specificity exchanger (e.g., an antibody
found in a subject) can be bound to a support and the association
of the specificity exchanger with the primary antibody can be
determined directly (e.g., using labeled specificity exchanger) or
indirectly (e.g., using labeled receptor, antigen or a labeled
secondary antibody that interacts with an epitope on the
specificity exchanger that does not compete with the epitope
recognized by the primary antibody).
[0080] Another approach involves a sandwich-type assay, wherein the
receptor or antigen is bound to a support, the specificity
exchanger is bound to the receptor or antigen, and the primary
antibody is bound to the specificity exchanger. If labeled primary
antibody is used, the presence of a receptor or antigen/specificity
exchanger/primary antibody complex can be directly determined. The
presence of the receptor or antigen/specificity exchanger/primary
antibody complex can also be determined indirectly by using, for
example, a labeled secondary antibody that reacts with the primary
antibody at an epitope that does not interfere with the binding of
the primary antibody to the specificity exchanger. In some cases,
it may be desired to use a labeled tertiary antibody to react with
an unlabeled secondary antibody, thus, forming a receptor or
antigen/specificity exchanger/primary antibody/secondary
antibody/labeled tertiary antibody complex.
[0081] The following examples (EXAMPLES 1-5) describe the
preparation and characterization of antigen/antibody specificity
exchangers. EXAMPLE 1 describes the preparation of several
antigen/antibody specificity exchangers.
EXAMPLE 1
[0082] The antigen/antibody specificity exchangers provided in
TABLE IV are synthetic peptides synthesized according to a method
for multiple peptide synthesis and by a Milligen 9050 peptide
synthesizer using 9-fluorenylmethoxy-carbonyl-protected amino acid
esters. These peptides were analysed and/or purified by reverse
phase HPLC using a Pep-S 5 m column (Pharmacia-LKB, Uppsala,
Sweden), run with a gradient from 10% to 60% CH3CN against water
containing 0.1% trifluoro-acetic acid. TABLE-US-00004 TABLE IV
Peptide 1: CDLIYYDYEEDYYFPPNAPILS (SEQ ID NO: 118) Peptide 2:
CDLIYYDYEEDYYFRPPNAPILST (SEQ ID NO: 119) Peptide 3:
CDLIYYDYEEDYYFKEIPALTAVETG (SEQ ID NO: 120) Peptide 4:
CDLIYYDYEEDYYFPAHSKEIPALTA (SEQ ID NO: 121) Peptide 5:
CDLIYYDYEEDYYFWGCSGKLICT (SEQ ID NO: 122) Peptide 6:
CDLIYYDYEEDYYFCTTAVPWNAS (SEQ ID NO: 123) Peptide 7:
CDLIYYDYEEDYYFKRPPNAPILSTCDLIYYDYEEDYYF (SEQ ID NO: 124) Peptide 8:
TYAMNPPNAPILS (SEQ ID NO: 125) Peptide 9:
RVRSKSFNYATYYADSVKGPPNAPILS (SEQ ID NO: 126) Peptide 10:
PAQGIYFDYGGFAYPPNAPILS (SEQ ID NO: 127) Peptide 11:
CDLIYYDYEEDYYQRKTKRNTNRR (SEQ ID NO: 128)
[0083] TABLE V illustrates the specific regions of the
antigen/antibody specificity exchangers provided in TABLE IV. These
antigen/antibody specificity exchangers include specificity domains
that comprise peptides containing the CDRH3 domain of mAb F58 or
CDRH1, CDRH2, CDRH3 domain of mAb C1-5. These antigen/antibody
specificity exchangers further comprise antigenic domains obtained
from various viral proteins. TABLE-US-00005 TABLE V Peptide
Antigenic Source of Antigenic No. Specificity Domain link Domain
Domain aas 1. SEQ ID NO 43 peptide bond SEQ ID NO 65 HBc/eAg, aas
134-141 2. SEQ ID NO 43 peptide bond SEQ ID NO 66 HBc/eAg, aas
133-142 3. SEQ ID NO 43 peptide bond SEQ ID NO 67 Polio VP1, aas
39-50 4. SEQ ID NO 43 peptide bond SEQ ID NO 68 Polio VP1, aas
35-46 5. SEQ ID NO 43 peptide bond SEQ ID NO 69 HIV-1 gp41, aas
596-605 6. SEQ ID NO 43 peptide bond SEQ ID NO 70 HIV-1 gp41, aas
603-612 7. 2(SEQ ID NO 43) Lys SEQ ID NO 66 HBc/eAg, aas 133-142 8.
SEQ ID NO 45 peptide bond SEQ ID NO 65 HBc/eAg, aas 134-141 9. SEQ
ID NO 46 peptide bond SEQ ID NO 65 HBc/eAg, aas 134-141 10. SEQ ID
NO 47 peptide bond SEQ ID NO 65 HBc/eAg, aas 134-141 11. SEQ ID NO
44 peptide bond SEQ ID NO 71 HCV core 8-18 Note: aas = amino
acids
[0084] The following example describes an evaluation of the ability
of the specificity exchangers described in EXAMPLE 1 to bind to
antigen.
EXAMPLE 2
[0085] The specificity exchangers prepared in EXAMPLE 1 were then
evaluated using enzyme immuno assays (EIAS). Strain-specific HIV-1
V3 peptides were coated on microtiter wells (Nunc 96F Certificated;
Nunc, Copenhagen, Denmark) in 100 ml portions at concentrations of
from 10 mg/ml to 0.01 mg/ml in 0.05 M sodium carbonate buffer, pH
9.6, overnight at +4.degree. C. Excess peptides were removed by
washing with PBS containing 0.05% Tween 20. The peptide-coated
plates were assayed for binding using the specificity exchangers
prepared in EXAMPLE 1, diluted from 100 mg/ml to 0.01 mg/ml in PBS
containing 1% BSA, 2% goat serum, and 0.05% Tween 20. The dilutions
of these specificity exchangers were added in 100 ml portions and
incubated with the adsorbed V3 peptides for 60 minutes at
+37.degree. C. Excess specificity exchangers were removed by
washing. Bound specificity exchangers were indicated using the
respective mAb or anti-serum and incubating for 60 minutes at
+37.degree. C. The amount of bound antibody was indicated by an
additional incubation of enzyme-labeled secondary antibody, rabbit
anti-mouse Ig (P260, Dako, Copenhagen, Denmark) for mAbs, and goat
anti-human IgG (A-3150; Sigma Chemicals, St. Louis, Mo.) for human
antibodies. The amount of bound conjugate was determined by
addition of substrate and the absorbencies were measured at 492 nm
or 405 nm in a spectrophotometer. When adsorbed to microplates all
specificity exchangers provided in TABLE IV except for Peptide Nos.
4 and 7 were found to be reactive with the respective antibodies.
TABLE VI provides the binding results for Peptides 1-4.
TABLE-US-00006 TABLE VI Peptide Antibody Amount peptide added
(ng/0.1 ml) to solid phase No. used 1,000 100 10 1 0.1 0.01 1 14E11
2.500 1.675 0.030 0.010 0.009 0.008 2 14E11 2.500 1.790 0.008 0.006
0.008 0.006 3 CBV 2.500 1.142 0.036 0.020 0.019 0.036 human A 1.945
1.850 0.486 0.088 0.115 0.116 human B 1.342 0.770 0.130 0.065 0.090
0.095 4 CBV 0.020 0.018 0.015 0.016 0.017 0.018 human A 0.059 0.081
0.108 0.109 0.097 0.100 human B 0.052 0.072 0.091 0.098 0.083 0.100
Note: Regression analysis of the relation between absorbance and
peptide concentration gives p < 0.01.
[0086] The next example demonstrates the ability of the
antigen/antibody specificity exchangers described in EXAMPLE 1 to
simultaneously bind to a particular antigen, HIV-1 V3 peptide,
MN-strain, and antibodies that are specific for the respective
antigenic domains.
EXAMPLE 3
[0087] As indicated by data shown in TABLES VII, VIII, and IX, all
of the HIV-specific antigen/antibody specificity exchangers were
found to directly bind to the HIV-1 V3 peptide. The data provided
in TABLES VII, VIII, and IX also show that the reactivity to the
HIV-1 V3 peptide was found to be dependent on both concentrations
of the specificity exchangers and the V3 peptides, indicating a
specific reactivity.
[0088] TABLE VII illustrates the ability of the antigen/antibody
specificity exchanger to simultaneously bind the HIV-1 V3 peptide
antigen (vis a vis the CDR sequence of the specificity domain) and
the monoclonal antibodies specific for the particular antigenic
domain of the specificity exchangers. Values are given as the
absorbance at 492 nm. TABLE VIII illustrates the ability of the
antigen/antibody specificity exchanger to simultaneously bind the
HIV-1 V3 peptide antigen (vis a vis the CDR sequence of the
specificity domain) and human anti-polio VP1 polyclonal antibodies
specific for the antigenic region on the tested specificity
exchanger. Values are given as the absorbance at 405 nm. TABLE IX
illustrates the ability of the antigen/antibody specificity
exchanger to simultaneously bind the HIV-1 V3 peptide antigen (vis
a vis the CDR sequence of the specificity domain) and the human
anti-HCV core polyclonal anti-bodies specific for the antigenic
region on the tested specificity exchanger. Values are given as the
absorbance at 405 nm.
[0089] The results provided in TABLES VII, VIII, and IX clearly
show that antibodies specific for HIV-1 gp41, HBc/eAg, poliovirus 1
VP1, and HCV core proteins were redirected to the HIV-1 V3 peptide
antigen. It was also found, that pre-incubation of equimolar
concentrations of mAb 14E11 and the corresponding specificity
exchanger did not alter the ability of the specificity exchanger
complex to bind to the V3 peptide. This indicated that antigenic
domains could be joined to a CDR peptide (a specificity domain)
while retaining the antigen binding ability of the specificity
domain. TABLE-US-00007 TABLE VII a: Amount of test Pep- peptide
Amount V3 peptide added tide Antibody (ng/ (ng/0.1 ml) to solid
phase No. used 0.1 ml) 1,000 500 250 125 62.5 31.25 1 14E11 10,000
2.500 2.500 2.500 2.338 1.702 1.198 5,000 2.500 2.500 2.500 2.190
1.622 1.122 2,500 2.500 2.500 2.500 2.039 1.394 0.990 1,250 2.500
2.500 2.500 1.712 0.930 0.771 625 1.936 0.824 0.380 0.152 0.056
0.053 312 0.196 0.085 0.044 0.043 0.030 0.025 b: Amount of test
Pep- peptide Amount V3 peptide added tide Antibody (ng/ (ng/0.1 ml)
No. used 0.1 ml) 1,000 500 250 125 62.5 31.25 4 14E11 10,000 2.500
2.500 2.133 1.560 1.070 0.829 5,000 2.500 2.500 1.963 1.645 1.074
0.981 2,500 2.500 2.500 1.729 1.404 0.962 0.747 1,250 2.500 2.424
1.433 1.327 0.795 0.488 625 0.835 0.359 0.200 0.120 0.088 0.073 312
0.099 0.054 0.042 0.049 0.045 0.025 c: Amount of test Pep- peptide
Amount peptide added tide Antibody (ng/ (ng/0.1 ml) to solid phase
No. used 0.1 ml) 1,000 100 10 1 0.1 0.01 3 CBV 10,000 0.523 0.498
0.162 0.161 0.017 0.017 1,000 0.053 0.054 0.031 0.027 0.010 0.010
100 0.034 0.037 0.025 0.029 0.010 0.010 10 0.023 0.022 0.014 0.014
0.010 0.009 1 0.013 0.044 0.014 0.017 0.027 0.009 0.1 0.011 0.009
0.008 0.032 0.013 0.013 Note: Regression analysis of the relation
between absorbance and CDR peptide concentration, and relation
between absorbance and V3 peptide concentration gives p < 0.01,
respectively.
[0090] TABLE-US-00008 TABLE VIII Amount of test Pep- peptide Amount
V3 peptide added tide Antibody (ng/ (ng/0.1 ml) to solid phase No.
used 0.1 ml) 1,000 500 250 125 62.5 31.25 a: 3 human A 10,000 1.538
1.356 1.448 1.052 0.280 0.123 5,000 1.179 1.050 1.006 0.557 0.136
0.087 2,500 0.684 0.558 0.604 0.216 0.084 0.067 1,250 0.367 0.358
0.332 0.162 0.075 0.062 625 0.228 0.238 0.220 0.121 0.083 0.063 312
0.171 0.154 0.154 0.103 0.072 0.060 b: 3 human B 10,000 0.366 0.352
0.352 0.200 0.074 0.056 5,000 0.206 0.217 0.188 0.131 0.063 0.053
2,500 0.134 0.132 0.126 0.091 0.061 0.055 1,250 0.107 0.114 0.108
0.077 0.060 0.054 625 0.082 0.104 0.087 0.075 0.063 0.056 312 0.083
0.091 0.094 0.077 0.068 0.060 Note: Regression analysis of the
relation between absorbance and CDR peptide concentration, and
relation between absorbance and V3 peptide concentration gives p
< 0.01, respectively.
[0091] TABLE-US-00009 TABLE IX Pep- Anti- Amount of Amount of test
peptide added tide body V3 peptide (ng/0.1 ml) No. used (ng/0.1 ml)
62 31 15 7.5 3.7 1.8 11 human 625 2.500 2.416 2.097 1.473 0.973
0.630 HCV-C 78 2.500 2.335 1.781 1.225 0.825 0.564 39 2.389 2.287
1.626 1.081 0.664 0.389 11 human 625 1.999 1.490 1.184 0.751 0.458
0.428 HCV-D 78 1.758 1.370 1.025 0.612 0.468 0.380 39 1.643 0.993
0.833 0.497 0.343 0.287 11 human 625 2.368 2.165 1.104 0.645 0.462
HCV-E 78 2.156 1.824 1.396 0.733 0.514 0.352 39 1.893 1.683 1.110
0.756 0.310 0.272
[0092] The next example demonstrates the ability of the
antigen/antibody specificity exchangers to simultaneously bind to
another antigen, residues 71-90 of HBc/eAg with an Ile at position
80, and antibodies that are specific for the respective antigenic
domains.
EXAMPLE 4
[0093] The ability of antigen/antibody specificity exchangers to
redirect antibodies was further evaluated in a system where the
CDRH 1, CDRH2 and CDRH3 sequences from mAb C1-5 were added to the
epitope sequence for mAb 14E11 (residues 135-141 of the HBc/eAg
sequence (PNAPILS SEQ ID No. 116). A peptide corresponding to the
epitope sequence for mAb C1-5, residues 71-90 of HBc/eAg with an
Ile at position 80, was adsorbed to microplates. The
antigen/-antibody specificity exchangers, based on the C1-5 CDRs,
were then added, and the amount bound CDR peptide was indicated by
the epitope specific mAb 14E11. The results provided in TABLE X
clearly show that the mAb 14E11 was redirected by the
antigen/antibody specificity exchanger containing the CDRH2
sequence to the PNAPILS (SEQ ID No. 116) sequence. Also, this
reactivity was dependent on the amount specificity exchanger added,
indicating a specific reaction (p<0.01, Regression analysis).
TABLE-US-00010 TABLE X Amount c71-90 Antibody peptide Amount of
test peptide added (ng/0.1 ml) CDR sequence used (ng/0.1 ml) 10.000
5.000 2.500 1.250 625 312 Peptide 8: 14E11 625 0.003 0.002 0.002
0.002 0.002 0.002 CDRH1 312 0.002 0.002 0.004 0.003 0.006 0.004
(SEQ ID NO 78 0.003 0.003 0.005 0.005 0.003 0.003 45) Peptide 9:
14E11 625 2.500 1.303 0.070 0.012 0.003 0.002 CDRH2 312 2.500 1.070
0.058 0.011 0.003 0.002 (SEQ ID NO 78 2.500 0.868 0.039 0.008 0.003
0.003 46) Peptide 10: 14E11 625 0.004 0.003 0.004 0.003 0.003 0.003
CDRH3 312 0.004 0.003 0.004 0.004 0.003 0.003 (SEQ ID NO 78 0.005
0.004 0.005 0.005 0.004 0.004 47)
[0094] The next example provides more evidence that
antigen/antibody specificity exchangers redirect antibodies.
EXAMPLE 5
[0095] This example describes experiments that verified that
antigen/antibody specificity exchangers containing a CDRH3
sequence, a specificity domain directed to an HIV-1 antigen, and an
antigenic domain that contains an HBc/eAg epitope recognized by mAb
14E11 redirected HBc/eAg specific antibody to HIV-1 V3 peptides of
several different subtypes. As shown in TABLE XI, the HBc/eAg
specific antibody efficiently bound to the specificity exchangers
that were also bound to the HIV-1 antigen, which was affixed to
microtiter plates. Thus, antigen/antibody specificity exchangers
effectively redirect antibodies antigens that are present on
pathogens. TABLE-US-00011 TABLE XI HIV-1 V3 peptide attached
Reactivity (absorbance at 405 nm) of specificity to solid-
exchanger peptide added in the indicated amount (ng) phase 500 250
125 62.5 31.25 15.625 Subtype A 0.378 0.126 0.078 0.068 0.062 0.017
Subtype B 2.686 2.536 1.710 1.329 0.360 0.157 Subtype C 1.261 0.514
0.111 0.077 0.051 0.020 Subtype D 0.17 0.079 0.065 0.028 0.029
0.026 Subtype E 0.22 0.090 0.093 0.032 0.063 0.030
[0096] The following examples (EXAMPLES 6-8) describe the
preparation and characterization of ligand/receptor specificity
exchangers. EXAMPLE 6 describes a characterization assay that was
performed to determine whether a specificity domain derived from
the C-terminal domain of fibrinogen inhibits the binding of
clumping factor (Clf) to fibrinogen.
EXAMPLE 6
[0097] In this example, several peptides corresponding to the
C-terminal domain of fibrinogen (Fib) were analyzed for their
ability to block the binding of clumping factor (Clf) to
fibrinogen. (See TABLE XII). These peptides were manufactured using
standard techniques in peptide synthesis using fmoc chemistry
(Syro, MultiSynTech, Germany). Preferably, the peptides are
purified by reverse-phase HPLC. A competition enzyme immunoassay
was then performed to determine whether the peptides were able to
block the interaction between Clf and fibrinogen. The results of
these experiments are shown in TABLE XII. The smallest peptide from
fibrinogen found to inhibit the interaction between Clf and
fibrinogen was HLGGAKQAGD (SEQ. ID No. 117). Substitution of the
first two amino acids of this peptide with alanine and lysine had a
significant effect on the ability of the peptide to block the
interaction between Clf and fibrinogen (e.g., the peptide
ALGCAKQAGD (SEQ. ID No. 129) was unable to block the Clf/fibrinogen
interaction). TABLE-US-00012 TABLE XII Seq. Inhibition of ID No.
(Fib) peptide (Fib/Clf) interaction 130 LTIGEGQQHHLGGAKQAGDV + 131
GEGQQHHLGGAKQAGDV + 132 QQHHLGGAKQAGDV + 133 QHHLGGAKQAGDV + 134
HHLGGAKQAGDV + 135 HLGGAKQAGDV + 136 LGGAKQAGDV - 137 GGAKQAGDV -
138 GAKQAGDV - 139 QHHLGGAKQAGD + 140 QHHLGGAKQAG + 141 QHHLGGAKQA
- 142 QHHLGGAKQ - 143 QHHLGGAK +/- 144 QHHLGGA - 145 HHLGGAKQAGDV +
146 HHLGGAKQAGD + 147 HHLGGAKQAG + 148 HLGGAKQAGDV + 149 HLGGAKQAGD
+ 150 ALGGAKQAG - 151 HAGGAKQAG + 152 HLAGAKQAG + 153 HLGAAKQAG +
154 HLGGGKQAG + 155 HLGGAAQAG +/- 156 HLGGAKAAG + 157 HLGGAKQGG +
158 HLGGAKQAA +
[0098] The next example describes the preparation and
characterization of several ligand/receptor specificity exchangers
that interact with the ClfA receptor found on Staphylococcus.
EXAMPLE 7
[0099] Ligand/receptor specificity exchangers having specificity
domains (approximately 20 amino acids long) corresponding to
various regions of the fibrinogen gamma-chain sequence were
produced using standard techniques in peptide synthesis using fmoc
chemistry (Syro, MultiSynTech, Germany) and these ligand/receptor
specificity exchangers were analyzed for their ability to bind the
ClfA receptor and an antibody specific for their respective
antigenic domains. The sequences of these ligand/receptor
specificity exchangers are listed in TABLE XIII and are provided in
the Sequence listing (SEQ. ID. Nos. 72-115). The ligand/receptor
specificity exchangers used in this analysis have an antigenic
domain that comprises a peptide having an epitope of herpes simplex
virus gG2 protein, which is recognized by a monoclonal antibody for
herpes simplex virus gG2 proteins. Serial dilutions of these
ligand/receptor specificity exchangers were made in phosphate
buffered saline (PBS) containing 2 .mu.g/ml goat serum. (Sigma
Chemicals, St. Louis, Mo.) and 0.5% Tween 20 (PBS-GT). The receptor
ClfA was passively adsorbed at 10 .mu.g/ml to 96 well microtiter
plates in 50 mM sodium carbonate buffer, pH 9.6, overnight at
+4.degree. C.
[0100] The diluted ligand/receptor specificity exchangers were then
incubated on the plates for 60 minutes. The ability of the
ligand/receptor specificity exchanger to interact with the receptor
was determined by applying a primary antibody to the plate and
incubating for 60 minutes (a 1:1000 dilution of mAb for herpes
simplex virus gG2 proteins). The bound primary mAb was then
indicated by a rabbit anti-mouse IgG (Sigma) secondary antibody and
a peroxidase labeled goat anti-rabbit IgG (Sigma) tertiary
antibody. The plates were developed by incubation with
dinitro-phenylene-diamine (Sigma) and the absorbance at 405 nm was
analyzed.
[0101] Every ligand/receptor specificity exchanger provided in
TABLE XIII (SEQ. ID. Nos. 72-115) appreciably bound the immobilized
ClfA and also bound the mAb specific for HSV gG2 protein.
Accordingly, these ligand/receptor specificity exchangers
redirected antibodies specific for HSV to a receptor found on a
pathogen. Preferred ligand/receptor specificity exchangers are also
provided in TABLE XIV. TABLE-US-00013 TABLE XIII LIGAND/RECEPTOR
SPECIFICITY EXCHANGERS YGEGQQHHLGGAKQAGDV HRGGPEEF (SEQ. ID. No.
72) YGEGQQHHLGGAKQAGDVHRGGPEE (SEQ. ID. No. 73)
YGEGQQHHLGGAKQAGDVSTPLPETT (SEQ. ID. No. 74)
MSWSLHPRNLILYFYALLFLHRGGPEE (SEQ. ID. No. 75)
ILYFYALLFLSTCVAYVATHRGGPEE (SEQ. ID. No. 76)
SSTCVAYVATRDNCCILDERHRGGPEE (SEQ. ID. No. 77)
RDNCCILDERFGSYCPTTCGHRGGPEE (SEQ. ID. No. 78)
FGSYCPTTCGIADFLSTYQTHRGGPEE (SEQ. ID. No. 79)
IADFLSTYQTKVDKDLQSLEHRGGPEE (SEQ. ID. No. 80)
KVDKDLQSLEDILHQVENKTHRGGPEE (SEQ. ID. No. 81)
DILHQVENKTSEVKQLIKAIHRGGPEE (SEQ. ID. No. 82)
SEVKQLIKAIQLTYNPDESSHRGGPEE (SEQ. ID. No. 83)
QLTYNPDESSKPNMIDAATLHRGGPEE (SEQ. ID. No. 84)
KPNMIDAATLKSRIMLEEIMHRGGPEE (SEQ. ID. No. 85)
KSRIMLEEIMKYEASILTHDHRGGPEE (SEQ. ID. No. 86)
KYEASILTHDSSIRYLQEIYHRGGPEE (SEQ. ID. No. 87)
SSIRYLQEIYNSNNQKIVNLHRGGPEE (SEQ. ID. No. 88)
NSNNQKIVNLKEKVAQLEAQHRGGPEE (SEQ. ID. No. 89)
CQEPCKDTVQIHDITGKDCQHRGGPEE (SEQ. ID. No. 90)
IHDITGKDCQDIANKGAKQSHRGGPEE (SEQ. ID. No. 91)
DIANKGAKQSGLYFIKPLKAHRGGPEE (SEQ. ID. No. 92)
GLYFIKPLKANQQFLVYCEIHRGGPEE (SEQ. ID. No. 93)
NQQFLVYCEIDGSGNGWTVFHRGGPEE (SEQ. ID. No. 94)
DGSGNGWTVFQKRLDGSVDFHRGGPEE (SEQ. ID. No. 95)
QKRLDGSVDFKKNWIQYKEGHRGGPEE (SEQ. ID. No. 96)
KKNWIQYKEGFGHLSPTGTTHRGGPEE (SEQ. ID. No. 97)
FGHLSPTGTTEFWLGNEKIHHRGGPEE (SEQ. ID. No. 98)
EFWLGNEKIHLISTQSAIPYHRGGPEE (SEQ. ID. No. 99)
LISTQSAIPYALRVELEDWNHRGGPEE (SEQ. ID. No. 100)
ALRVELEDWNGRTSTADYAMHRGGPEE (SEQ. ID. No. 101)
GRTSTADYAMFKVGPEADKYHRGGPEE (SEQ. ID. No. 102)
FKVGPEADKYRLTYAYFAGGHRGGPEE (SEQ. ID. No. 103)
RLTYAYFAGGDAGDAFDGFDHRGGPEE (SEQ. ID. No. 104)
DAGDAFDGFDFGDDPSDKFFHRGGPEE (SEQ. ID. No. 105)
FGDDPSDKFFTSHNGMQFSTHRGGPEE (SEQ. ID. No. 106)
TSHNGMQFSTWDNDNDKFEGHRGGPEE (SEQ. ID. No. 107)
WDNDNDKFEGNCAEQDGSGWHRGGPEE (SEQ. ID. No. 108)
NCAEQDGSGWWMNKCHAGHLHRGGPEE (SEQ. ID. No. 109)
WMNKCHAGHLNGVYYQGGTYHRGGPEE (SEQ. ID. No. 110)
NGVYYQGGTYSKASTPNGYDHRGGPEE (SEQ. ID. No. 111)
SKASTPNGYDNGIIWATWKTHRGGPEE (SEQ. ID. No. 112)
NGIIWATWKTRWYSMKKTTMHRGGPEE (SEQ. ID. No. 113)
RWYSMKKTTMKIIPFNRLTIHRGGPEE (SEQ. ID. No. 114)
KIIPFNRLTIGEGQQHHLGGAKQAGDVHRGG (SEQ. ID. No. 115) PEE
[0102] TABLE-US-00014 TABLE XIV LIGAND/RECEPTOR SPECIFICITY
EXCHANGERS Ligand/receptor Specificity Exchanger Seq. ID No.
HRGGPEEF-HHLGGAKQAGD 159 HRGGPEEF-HHLGGAKRAGR 160
HRGGPEEF-HHLGGARRAGR 161 HRGGPEEF-HHLGHAKQAGR 162
HRGGPEEF-HHLGHARQAGR 163 HRGGPEEF-HHLGHAKRAGL 164
HRGGPEEF-HHLGHAKRAGR 165 HHLGGAKQAGD-HRGGPEEF 166
HHLGGAKRAGR-HRGGPEEF 167 HHLGGARRAGR-HRGGPEEF 168
HHLGHAKQAGR-HRGGPEEF 169 HHLGHARQAGR-HRGGPEEF 170
HHLGHAKRAGL-HRGGPEEF 171 HHLGHAKRAGR-HRGGPEEF 172
PALTAVETGATNPL-HHLGGAKQAGD 173 PALTAVETGATNPL-HHLGGAKRAGR 174
PALTAVETGATNPL-HHLGGARRAGR 175 PALTAVETGATNPL-HHLGHAKQAGR 176
PALTAVETGATNPL-HHLGHARQAGR 177 PALTAVETGATNPL-HHLGHAKRAGL 178
PALTAVETGATNPL-HHLGHAKRAGR 179 HHLGGAKQAGD-PALTAVETGATNPL 180
HHLGGAKRAGR-PALTAVETGATNPL 181 HHLGGARRAGR-PALTAVETGATNPL 182
HHLGHAKQAGR-PALTAVETGATNPL 183 HHLGHARQAGR-PALTAVETGATNPL 184
HHLGHAKRAGL-PALTAVETGATNPL 185 HHLGHAKRAGR-PALTAVETGATNPL 186
[0103] The next example describes another characterization assay
that was performed to determine whether ligand/receptor specificity
exchangers bind to a receptor that is present on a bacteria and
thereby redirect an antibody specific for the antigenic domain of
the specificity exchanger to the bacterial receptor.
EXAMPLE 8
[0104] Ligand/receptor specificity exchangers having specificity
domains that bind to clumping factor (Clf) and antigenic domains
that correspond to an epitope derived from the polio virus were
produced using standard techniques in peptide synthesis using fmoc
chemistry (Syro, MultiSynTech, Germany). See TABLE XV. These
ligand/receptor specificity exchangers were analyzed for their
ability to inhibit the interaction between CLF and fibrinogen. In
these experiments, the ligand/specificity exchangers described in
TABLE XV were manufactured and various concentrations of these
molecules were added to an enzyme competition immunoassay
containing Clf and fibrinogen. The lowest inhibiting concentration,
which is the lowest peptide concentration needed to inhibit the
Clf/Fib interaction, was ascertained. Accordingly, the lower the
concentration needed to inhibit the Fib/Clf interaction, the more
effective the inhibitor. Additionally, the lowest solid-phase bound
peptide concentration, which is the lowest tested concentration of
peptide recognized by anti-poliovirus antibodies in the
immunoassay, was determined. Some of the peptides used (e.g.,
CPALTAVETGCTNPLAAHHLGGAKQAG (SEQ ID No. 187),
HHLGGAKQAG-AA-CPALTAVETGCTNPL (SEQ ID No. 188),
CPALTAVETGC-TNPLHHLGGAKQAG (SEQ ID No. 189), and
HHLGGAKQAG-CPALTAVETGCTNPL (SEQ ID No. 190)), designated by
asterisks in TABLE XV, were cyclized between the two artificially
introduced cystiene residues. These experiments revealed that
HHLGGAKQAG-AA-CPALTAVETGCTNPL* (SEQ ID No. 191) and
HHLGGAKQAG-CPALTAVETGCTNPL (SEQ ID No. 190) effectively inhibited
the interaction of Clf with fibrinogen and retained functional
poliovirus epitopes. TABLE-US-00015 TABLE XV Lowest Lowest epitope
inhibiting on solid- Conc. phase SEQ ID Peptide sequence (.mu.g/ml)
(.mu.g/ml) 192 CPALTAVETGCTNPL-AA-HHLGGAKQAG* >625 1.6 187
CPALTAVETGCTNPL-AA-HHLGGAKQAG 625 1.6 191
HHLGGAKQAG-AA-CPALTAVETGCTNPL* 69 8 188
HHLGGAKQAG-AA-CPALTAVETGCTNPL 625 >200 193
CPALTAVETGC-TNPLHHLGGAKQAG* 625 1.6 189 CPALTAVETGC-TNPLHHLGGAKQAG
208 1.6 194 HHLGGAKQAG-CPALTAVETGCTNPL* 208 >200 190
HHLGGAKQAG-CPALTAVETGCTNPL 23 1.6 195 PALTAVETGATNPL-HHLGGAKQAG
>625 1.6 196 HHLGGAKQAG-PALTAVETGATNPL >625 >200
[0105] The next section describes several cellular-based
characterization assays that can be performed to determine whether
an antigen/antibody specificity exchanger or a ligand/receptor
specificity exchanger binds to a pathogen or inhibits the
proliferation of a pathogen.
[0106] Cell-Based Characterization Assays
[0107] In another type of characterization assay, a cell-based
approach is used to evaluate the ability of a specificity exchanger
to bind to a pathogen and redirect an antibody specific for the
antigenic domain of the ligand/receptor specificity exchanger to
the pathogen. This analysis also reveals the ability of the
specificity exchanger to inhibit proliferation of a pathogen
because, in the body of a subject, the interaction of the
ligand/receptor specificity exchanger with a pathogen and an
antibody directed to the antigenic domain of the ligand/receptor
specificity exchanger is followed by humoral and cellular responses
that purge the pathogen from the subject (e.g., complement fixation
and macrophage degradation).
[0108] In general, the cell-based characterization assays involve
providing antigen/antibody specificity exchangers or
ligand/receptor specificity exchangers to cultured pathogens and
monitoring the association of the ligand/receptor specificity
exchanger with the pathogen. Several types of cell-based
characterization assays can be used and the example below describes
some of the preferred characterization assays in greater
detail.
EXAMPLE 9
[0109] One type of cell-based characterization assay involves
binding of a specificity exchanger to bacteria disposed on a
support. Accordingly, bacteria (e.g., Staphylococcus aureus, or
Escherichia coli.) are grown in culture or on an agar plate in a
suitable growth media (e.g., LB broth, blood broth, LB agar or
blood agar). The cells are then transferred to a membrane (e.g.,
nitrocellulose or nylon) by either placing the culture on the
membrane under vacuum (e.g., using a dot-blot manifold apparatus)
or by placing the membrane on the colonies for a time sufficient to
permit transfer. The cells that are bound to the membrane are then
provided a serial dilution of a specificity exchanger (e.g., 500
ng, 1 .mu.g, 5 .mu.g, 10 .mu.g, 25 .mu.g, and 50 .mu.g of the
specificity exchanger in a total volume of 200 .mu.l of PBS).
Antigen/antibody specificity exchangers that comprise a specificity
domain that binds to a protein present on the bacteria (e.g., Clf)
can be evaluated in this manner, for example. Ligand/receptor
specificity exchangers having a specificity domain comprising a
ligand for a receptor present on the bacteria (e.g., Clf) can also
be evaluated using this approach.
[0110] In one experiment, for example, the ligand/receptor
specificity exchangers listed in TABLES XIII or XIV are used. The
diluted ligand/receptor specificity exchangers are then incubated
on the membranes for 60 minutes. Subsequently, the non-bound
ligand/receptor specificity exchangers are removed and the membrane
is washed with PBS (e.g., 3 washes with 2 ml of PBS per wash).
Next, a 1:100-1:1000 dilution of a primary antibody that interacts
with the antigenic domain of the particular ligand/receptor
specificity exchanger (e.g., mAb for herpes simplex virus gG2
protein for some of the specificity exchangers) is provided and the
binding reaction is allowed to occur for 60 minutes. Again, the
membrane is washed with PBS (e.g., 3 washes with 2 ml of PBS per
wash) to remove unbound primary antibody. Appropriate controls
include the membrane itself, bacteria on the membrane without a
ligand/receptor specificity exchanger, and bacteria on the membrane
with ligand/receptor specificity exchanger but no primary
antibody.
[0111] To detect the amount of ligand/receptor specificity
exchanger bound to the bacteria on the membrane, a secondary
antibody (e.g., rabbit anti-mouse IgG (Sigma)) and a tertiary
antibody (e.g., a peroxidase labeled goat anti-rabbit IgG (Sigma))
are used. Of course, a labeled secondary antibody that interacts
with the primary antibody can be used as well. As above, the
secondary antibody is contacted with the membrane for 60 minutes
and the non-bound secondary antibody is washed from the membrane
with PBS (e.g., 3 washes with 2 ml of PBS per wash). Then, the
tertiary antibody is contacted with the membrane for 60 minutes and
the non-bound tertiary antibody is washed from the membrane with
PBS (e.g., 3 washes with 2 ml of PBS per wash). The bound tertiary
antibody can be detected by incubating the membrane with
dinitro-phenylene-diamine (Sigma).
[0112] Another approach involves the use of an immobilized
ligand/receptor specificity exchanger. Accordingly, primary
antibody (e.g., mAb for herpes simplex virus gG2 protein for some
of the specificity exchangers) is bound to a petri dish. Once the
primary antibody is bound, various dilutions of a ligand/receptor
specificity exchanger (e.g., a ligand/receptor specificity
exchanger provided in TABLES XIII or XIV) are added to the coated
dish. The ligand/receptor specificity exchanger is allowed to
associate with the primary antibody for 60 minutes and the
non-bound ligand/receptor specificity exchanger is washed away
(e.g., three washes with 2 ml of PBS). Appropriate controls include
petri dishes without primary antibody or ligand/receptor
specificity exchanger.
[0113] Subsequently, a turbid solution of bacteria (e.g.,
Staphylococcus) are added to the petri dishes and the bacteria are
allowed to interact with the immobilized ligand/receptor
specificity exchanger for 60 minutes. The non-bound bacteria are
then removed by washing with PBS (e.g., 3 washes with 2 ml of PBS).
Next, growth media (e.g., LB broth) is added to the petri dish and
the culture is incubated overnight. Alternatively, LB agar is added
to the petri dish and the culture is incubated overnight. An
interaction between the ligand/receptor specificity exchanger and
the bacteria can be observed visually (e.g., turbid growth media,
which can be quantified using spectrophotometry or an analysis of
the appearance of colonies on the agar).
[0114] By modifying the approaches described above, one of skill in
the art can evaluate the ability of a specificity exchanger to
interact with a virus. For example, soluble fragments of T4
glycoprotein have been shown to interact with a human
immunodeficiency virus (HIV) envelope glycoprotein. (See e.g., U.S.
Pat. No. 6,093,539, herein expressly incorporated by reference in
its entirety). Ligand/receptor specificity exchangers having a
specificity domain comprising a fragment of T4 glycoprotein that
interacts with HIV envelope glycoprotein (e.g., amino acids 1-419
of the T4 glycoprotein sequence provided in U.S. Pat. No. 6,093,539
or a portion thereof) can be made by synthesizing a fusion protein
having the specificity domain joined to an antigenic domain.
Although peptide chemistry can be used to make the ligand/receptor
specificity exchanger, it is preferred that an expression construct
having the fragment of T4 glycoprotein joined to an antigenic
domain is made and transfected into a suitable cell. The expression
and purification strategies described in U.S. Pat. No. 6,093,539
and above can also be employed.
[0115] Once the ligand/receptor specificity exchanger has been
constructed a filter binding assay is performed. Accordingly,
serial ten-fold dilutions of HIV inoculum are applied to a membrane
(e.g. nitrocellulose or nylon) in a dot blot apparatus under
constant vacuum. Then serial ten fold dilutions of the
ligand/receptor specificity exchanger are applied to the bound HIV
particles. The ligand/receptor specificity exchanger is contacted
with the particles for 60 minutes before applying vacuum and
washing with PBS (e.g., 3 washes with 2 ml of PBS per wash)). Once
the non-bound ligand/receptor specificity exchanger is removed, ten
fold serial dilutions of the primary antibody, which binds to the
antigenic domain, are added to the samples and the binding reaction
is allowed to occur for 60 minutes. Then a vacuum is applied and
the non-bound primary antibody is washed with PBS (e.g., 3 washes
with 2 ml of PBS per wash)). The detection of the bound primary
antibody can be accomplished, as described above.
[0116] The ability of a specificity exchanger to interact with a
virus can also be evaluated in a sandwich-type assay. Accordingly,
a primary antibody that interacts with the antigenic domain of the
specificity exchanger is immobilized in micro titer wells and
serial dilutions of specificity exchanger are added to the primary
antibody so as to create a primary antibody/specificity exchanger
complex, as described above. Next, ten fold serial dilutions of HIV
inoculum are added and the binding reaction is allowed to occur for
60 minutes. Non-bound HIV particles are removed by successive
washes in PBS. Detection of the bound HIV particles can be
accomplished using a radiolabeled anti-HIV antibody (e.g., antibody
obtained from sera from a person suffering with HIV infection).
[0117] While the examples above describe cell-based assays using
bacteria and a virus, modifications of these approaches can be made
to study the interaction of specificity exchangers with mammalian
cells. For example, the ability of a ligand/receptor specificity
exchanger to interact with an integrin receptor present on a cancer
cell can be determined as follows. Melanoma cells that express an
.alpha..sub.v.beta..sub.3 receptor (e.g., M21 human melanoma cells)
bind fibrinogen and this interaction can be blocked by
administering an RGD containing peptide (See e.g., Katada et al.,
J. Biol. Chem. 272: 7720 (1997) and Felding-Habermann et al., J.
Biol. Chem. 271:5892-5900 (1996); both references herein expressly
incorporated by reference in their entireties). Similarly, many
other types of cancer cells express integrins that interact with
RGD peptides. By one approach, cancer cells that expresses an
RGD-responsive integrin (e.g., M21 human melanoma cells) are
cultured to confluency. M21 cells can be grown in DMEM media with
10% fetal bovine serum, 20 mM Hepes, and 1 mM pyruvate.
[0118] Preferably, the cells are stained with hydroethidine
(Polysciences, Inc., Warrington, Pa.) at 20 .mu.g/ml final
concentration (2.times.10.sup.6 cells/ml) for 30 min at 37.degree.
C. and then washed twice to remove excess dye. Hydroethidine
intercalates into the DNA resulting in a red fluorescent labeling
of the cells and does not impair the cell's adhesive functions. The
staining provides a way to quantify the binding of a
ligand/receptor specificity exchanger to the cells. That is, the
total number of hydroethidine stained cells can be compared to the
number of cells bound to a fluorescently labeled primary
antibody/specificity exchanger complex so as to determine the
binding efficiency.
[0119] Accordingly, the stained cells are incubated with various
dilutions of a ligand/receptor specificity exchanger comprising a
RGD sequence (e.g., GRGDSPHRGGPEE (SEQ. ID No. 197) or
WSRGDWHRGGPEE (SEQ. ID No. 198)). After a 60-minute incubation, the
non-bound ligand/receptor specificity exchanger is removed by
several washes in DMEM media with 10% fetal bovine serum, 20 mM
Hepes, and 1 mM pyruvate (e.g., 3 washes of 5 ml of media). Next, a
1:100-1:1000 dilution of a primary antibody that interacts with the
antigenic domain of the ligand/receptor specificity exchanger
(e.g., mAb for herpes simplex virus gG2 protein) is provided and
the binding reaction is allowed to occur for 60 minutes.
Subsequently, several washes in media are performed to remove any
non-bound primary antibody. Appropriate controls include stained
cells without ligand/receptor specificity exchanger or stained
cells without primary antibody.
[0120] Following binding of the primary antibody, a goat anti-mouse
FITC labeled antibody (1:100 dilution) (Sigma) is added and binding
is allowed to occur for 60 minutes. Again, several media washes are
made to remove any non-bound secondary antibody. Analysis is made
by flow cytometry with filter settings at 543/590 nm for
hydroethidine and 495/525 nm for fluorescin. One will observe an
appreciable binding of primary antibody to the ligand/receptor
specificity exchanger/cell complex, which will demonstrate that the
ligand/receptor specificity exchanger will have an effect on the
cell. It should be emphasized that modifications of the approach
described above can be easily made to accommodate the evaluation of
an antigen/antibody specificity exchanger.
[0121] The next example describes experiments that verified that
ligand/receptor specificity exchangers efficiently bind to
pathogens in culture and redirect antibodies that are specific for
the antigenic domains of the ligand/receptor specificity domains to
the pathogen.
EXAMPLE 10
[0122] A ligand/receptor specificity exchanger comprising a
fragment of fibrinogen (specificity domain) joined to a peptide
obtained from the hepatitis B virus (antigenic domain) was found to
bind to adhesion receptors present on a pathogen in culture (Murine
myeloma cells (SP2/0 cells)). A ligand/receptor specificity
exchanger having the sequence RGDSAATPPAYR (SEQ ID No. 199) was
manufactured using standard techniques in peptide synthesis using
fmoc chemistry (Syro, MultiSynTech, Germany). This peptide has a
specificity domain that binds adhesion receptors on a pathogen, a
spacer (the AA), and an antigenic domain that has an epitope
recognized by the monoclonal antibody 57/8, an epitope present on
the hepatitis B virus e antigen (HBeAg).
[0123] Murine myeloma cells (SP2/0 cells) were washed in serum free
media and were incubated with the ligand/receptor specificity
exchanger or a control peptide derived from hepatitis C virus (HCV)
NS3 domain at a concentration of 50 .mu.g/ml. The cells were then
washed and the amount of surface bound peptide was detected by
labeling the cells with the the anti-HBV (57/8) antibody. Surface
bound antibody was indicated by an FITC labelled anti-mouse IgG
conjugate diluted 1/500 and the level of surface staining was
determined by fluorescent microscopy.
[0124] Microscopy revealed that cells incubated with the control
peptide did not show significant staining, whereas, cells incubated
with the ligand/receptor specificity exchanger showed significant
surface staining consistent with the location of surface expressed
adhesion receptors. These experiments verified that ligand/receptor
specificity exchangers comprising fragments of fibrinogen
effectively bound adhesion recpetors on a pathogen (a myeloma cell)
and redirected anti-HBV antibodies to the tumor cells. It should be
emphasized that modifications of the approach described above can
be easily made to accommodate the evaluation of an antigen/antibody
specificity exchanger. The next section describes characterization
assays that are performed in animals.
[0125] In Vivo Characterization Assays
[0126] Characterization assays also include experiments that
evaluate specificity exchangers in vivo. There are many animal
models that are suitable for evaluating the ability of a a
specificity exchanger to inhibit pathogenic infection. Mice are
preferred because they are easy to maintain and are susceptible to
bacterial infection, viral infection, and cancer. Chimpanzees are
also preferred because of their close genetic relationship to
humans. The next example provides one in vivo approach to evaluate
the ability of a ligand specificity exchanger to bind to a
pathogen, redirect antibodies specific for the antigenic domain of
the ligand/receptor specificity exchanger, and thereby inhibit the
proliferation of the pathogen. It should be emphasized that
modifications of the approach described below can be easily made to
accommodate the evaluation of an antigen/antibody specificity
exchanger.
EXAMPLE 11
[0127] To test the ability of a ligand/receptor specificity
exchanger to treat a bacterial infection in mice, the following
characterization assay can be performed. Several female CF-1
outbred mice (Charles Rivers Laboratories) of approximately 8 weeks
of age and 25 gram body mass are inoculated intraperitoneally with
overnight cultures of Staphylococcus aureus. Blood samples are
drawn from the mice and tests are conducted to verify that
Staphylococcus aureus is present in the subjects.
[0128] The infected mice are injected with a suitable amount of a
ligand specificity exchanger that interacts with the Clf receptor
(e.g., a ligand/receptor specificity exchanger comprising a
fragment of fibrinogen). A small sample (e.g. 0.5 mL) of human
serum that contains antibodies specific for the antigenic domain is
also injected into the infected mice. For various time points after
the injection of the human serum for up to two weeks, the mice are
monitored for the presence and prevalence of Staphylococcus aureus.
The progress or decline in Staphylococcus aureus infection is
plotted. The data will show that the ligand/receptor specificity
exchanger efficiently inhibited the proliferation of Staphylococcus
aureus.
[0129] Another approach to evaluate the efficacy of a
ligand/receptor specificity exchanger in mice is provided in the
next example. It should be emphasized that modifications of the
approach described below can be easily made to accommodate the
evaluation of an antigen/antibody specificity exchanger.
EXAMPLE 12
[0130] To test the ability of a ligand/receptor specificity
exchanger to treat a bacterial infection the following
characterization assay can be performed. Several female CF-1
outbred mice (Charles Rivers Laboratories) of approximately 8 weeks
of age and 25 gram body mass are vaccinated with the antigenic
domains of the ligand/receptor specificity exchangers to be tested.
Preferably, the antigenic domains are coupled to a carrier and are
administered with an adjuvant. For example, the antigenic domains
can be fused to keyhole limpet hemocyanin or bovine serum albumin,
which act as both a carrier and adjuvant or an adjuvant such as
Freund's adjuvant, aluminum hydroxide, or lysolecithin can be used.
Once a high titer of antibody to the antigenic domains can be
verified by, for example, immunodiffusion or EIA, the immunized
mice are inoculated intraperitoneally with overnight cultures of
Staphylococcus aureus NTCC 10649. The inoculums are adjusted to
yield approximately 100.times.LD.sub.50 or log 6.6 for S.
aureus.
[0131] Serial dilutions of ligand/receptor specificity exchangers
(e.g., the ligand/receptor specificity exchangers provide in Table
IV) are formulated in sterile water for injection and are
administered by the subcutaneous (SC) or oral (PO) route at one and
five hours post infection. Concurrently with each trial, the
challenge LD.sub.50 is validated by inoculation of untreated mice
with log dilutions of the bacterial inoculum. Preferably, a five
log dilution range of the bacterial challenges is inoculated into
five groups of ten mice each (ten mice per log dilution). A
mortality rate of 100% will be produced in all groups of untreated
mice at the 100.times.LD.sub.50 challenge inoculum. Mice are
monitored daily for mortality for seven days. The mean effective
dose to protect 50% of the mice (ED.sub.50) can be calculated from
cumulative mortality by logarithmic-probit analysis of a plotted
curve of survival versus dosage as described in Antimicrob. Agents
Chemother. 31: 1768-1774 and Proc. Soc. Exp. Biol. Med. 1994, 57,
261-264, each of which are hereby expressly incorporated by
reference in their entireties. As one of skill in the art will
appreciate, similar approaches can be used to test the ability of
ligand/receptor specificity exchangers to inhibit viral infection
and cancer.
[0132] The specificity exchangers described herein can be
formulated in pharmaceuticals and administered to subjects in need
of an agent that inhibits the proliferation of a pathogen. The
section below describes several pharmaceuticals comprising
specificity exchangers that interact with a receptor on a pathogen.
The following section describes the preparation of pharmaceuticals
comprising a specificity exchanger.
[0133] Pharmaceuticals Comprising a Specificity Exchanger
[0134] The specificity exchangers described herein are suitable for
incorporation into pharmaceuticals for administration to subjects
in need of a compound that treats or prevents infection by a
pathogen. In preferred embodiments, specificity exchangers are
incorporated into pharmaceuticals, as active ingredients for
administration to subjects in need of compounds that treat or
prevent infection by a pathogen or cancer in animals, including
humans. These pharmacologically active compounds can be processed
in accordance with conventional methods of galenic pharmacy to
produce medicinal agents for administration to mammals including
humans. In some embodiments, these pharmaceuticals can contain
excipients, binders, emulsifiers, carriers, and other auxiliary
agents in addition to the specificity exchanger. In other
embodiments, the active ingredients can be incorporated into a
pharmaceutical product with and without modification.
[0135] Further, the manufacture of pharmaceuticals or therapeutic
agents that deliver the pharmacologically active compounds of this
invention by several routes are aspects of the present invention.
For example, and not by way of limitation, DNA, RNA, and viral
vectors having sequences encoding a specificity exchanger that
interacts with a receptor or other antigen on a pathogen are used
with embodiments of the invention. Nucleic acids encoding a
specificity exchanger can be administered alone or in combination
with other active ingredients.
[0136] The specificity exchangers described herein can be employed
in admixture with conventional excipients, i.e., pharmaceutically
acceptable organic or inorganic carrier substances suitable for
parenteral, enteral (e.g., oral) or topical application that do not
deleteriously react with the pharmacologically active ingredients
described herein. Suitable pharmaceutically acceptable carriers
include, but are not limited to, water, salt solutions, alcohols,
gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols,
gelatine, carbohydrates such as lactose, amylose or starch,
magnesium stearate, talc, silicic acid, viscous paraffin, perfume
oil, fatty acid monoglycerides and diglycerides, pentaerythritol
fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone,
etc. Many more vehicles that can be used are described in
Remmington's Pharmaceutical Sciences, 15th Edition, Easton: Mack
Publishing Company, pages 1405-1412 and 1461-1487(1975) and The
National Formulary XIV, 14th Edition, Washington, American
Pharmaceutical Association (1975), herein incorporated by
reference. The pharmaceutical preparations can be sterilized and if
desired mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like so long as the auxiliary agents
does not deleteriously react with the specificity exchangers.
[0137] The effective dosage and method of administration of a
specificity exchanger provided in a pharmaceutical, therapeutic
protocol, or applied to a medical device varies depending on the
intended use, the patient, and the frequency of administration.
Therapeutic efficacy and toxicity of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED.sub.50 (the dose therapeutically
effective in 50% of the population). For example, the effective
dose of a specificity exchanger can be evaluated using the
characterization assays described above. The data obtained from
these assays is then used in formulating a range of dosage for use
with other organisms, including humans. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with no toxicity. The
dosage varies within this range depending upon type of specificity
exchanger, the dosage form employed, sensitivity of the organism,
and the route of administration.
[0138] Normal dosage amounts of a specificity exchanger can vary
from approximately 1 to 100,000 micrograms, up to a total dose of
about 10 grams, depending upon the route of administration.
Desirable dosages include about 250 mg-1 mg, about 50 mg-200 mg,
and about 250 mg-500 mg.
[0139] In some embodiments, the dose of a specificity exchanger
preferably produces a tissue or blood concentration or both from
approximately 0.1 .mu.M to 500 mM. Desirable doses produce a tissue
or blood concentration or both of about 1 to 800 .mu.M. Preferable
doses produce a tissue or blood concentration of greater than about
10 .mu.M to about 500 .mu.M. Although doses that produce a tissue
concentration of greater than 800 .mu.M are not preferred, they can
be used. A constant infusion of a specificity exchanger can also be
provided so as to maintain a stable concentration in the tissues as
measured by blood levels.
[0140] The exact dosage is chosen by the individual physician in
view of the patient to be treated. Dosage and administration are
adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors that can be taken
into account include the severity of the disease, age of the
organism being treated, and weight or size of the organism; diet,
time and frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. Short acting
pharmaceutical compositions are administered daily or more
frequently whereas long acting pharmaceutical compositions are
administered every 2 or more days, once a week, or once every two
weeks or even less frequently.
[0141] Routes of administration of the pharmaceuticals include, but
are not limited to, topical, transdermal, parenteral,
gastrointestinal, transbronchial, and transalveolar. Transdermal
administration is accomplished by application of a cream, rinse,
gel, etc. capable of allowing the specificity exchangers to
penetrate the skin. Parenteral routes of administration include,
but are not limited to, electrical or direct injection such as
direct injection into a central venous line, intravenous,
intramuscular, intraperitoneal, intradermal, or subcutaneous
injection. Gastrointestinal routes of administration include, but
are not limited to, ingestion and rectal. Transbronchial and
transalveolar routes of administration include, but are not limited
to, inhalation, either via the mouth or intranasally.
[0142] Compositions having the specificity exchangers that are
suitable for transdermal or topical administration include, but are
not limited to, pharmaceutically acceptable suspensions, oils,
creams, and ointments applied directly to the skin or incorporated
into a protective carrier such as a transdermal device
("transdermal patch"). Examples of suitable creams, ointments, etc.
can be found, for instance, in the Physician's Desk Reference.
Examples of suitable transdermal devices are described, for
instance, in U.S. Pat. No. 4,818,540 issued Apr. 4, 1989 to Chinen,
et al., herein expressly incorporated by reference in its
entirety.
[0143] Compositions having the specificity exchangers that are
suitable for parenteral administration include, but are not limited
to, pharmaceutically acceptable sterile isotonic solutions. Such
solutions include, but are not limited to, saline and phosphate
buffered saline for injection into a central venous line,
intravenous, intramuscular, intraperitoneal, intradermal, or
subcutaneous injection.
[0144] Compositions having the specificity exchangers that are
suitable for transbronchial and transalveolar administration
include, but are not limited to, various types of aerosols for
inhalation. Devices suitable for transbronchial and transalveolar
administration of these are also embodiments. Such devices include,
but are not limited to, atomizers and vaporizers. Many forms of
currently available atomizers and vaporizers can be readily adapted
to deliver compositions having the specificity exchangers described
herein.
[0145] Compositions having the specificity exchangers that are
suitable for gastrointestinal administration include, but not
limited to, pharmaceutically acceptable powders, pills or liquids
for ingestion and suppositories for rectal administration. Due to
the ease of use, gastrointestinal administration, particularly
oral, is a preferred embodiment. Once the pharmaceutical comprising
the specificity exchanger has been obtained, it can be administered
to an organism in need to treat or prevent pathogenic
infection.
[0146] Aspects of the invention also include a coating for medical
equipment to prevent infection or the spread of disease. The term
"medical equipment" is to be construed broadly and includes for
example prosthetics, implants, and instruments. Coatings suitable
for use on medical devices can be provided by a gel or powder
containing the specificity exchanger or by a polymeric coating into
which a specificity exchanger is suspended. Suitable polymeric
materials for coatings of devices are those that are
physiologically acceptable and through which a therapeutically
effective amount of the specificity exchanger can diffuse. Suitable
polymers include, but are not limited to, polyurethane,
polymethacrylate, polyamide, polyester, polyethylene,
polypropylene, polystyrene, polytetrafluoroethylene,
polyvinyl-chloride, cellulose acetate, silicone elastomers,
collagen, silk, etc. Such coatings are described, for instance, in
U.S. Pat. No. 4,612,337, herein expressly incorporated by reference
in its entirety. The section below describes methods of treating
and preventing disease using the specificity exchangers described
herein.
[0147] Methods of Treatment and Prevention of Disease Using
Specificity Exchangers
[0148] Several embodiments also concern approaches to use the
specificity exchangers to treat or prevent proliferation of a
pathogen. Some methods involve providing a specificity exchanger to
a subject in need of treatment and/or prevention of bacterial
infection, fungal infection, viral infection, and cancer. For
example, pharmaceuticals comprising a specificity exchanger can be
provided to a subject in need to treat and/or prevent infection by
a pathogen that has a receptor or an antigen. Such subjects in need
can include individuals at risk of contacting a pathogen or
individuals who are already infected by a pathogen. These
individuals can be identified by standard clinical or diagnostic
techniques.
[0149] By one approach, for example, a subject suffering from a
bacterial infection is identified as a subject in need of an agent
that inhibits proliferation of a pathogen. This subject is then
provided a therapeutically effective amount of a specificity
exchanger. The specificity exchanger used in this method comprises
a specificity domain that interacts with a receptor (e.g.,
extracellular fibrinogen binding protein (Efb), collagen binding
protein, vitronectin binding protein, laminin binding protein,
plasminogen binding protein, thrombospondin binding protein,
clumping factor A (ClfA), clumping factor B (ClfB), fibronectin
binding protein, coagulase, and extracellular adherence protein) or
another antigen present on the bacteria. The specificity exchanger
also comprises an antigenic domain that has peptide or an epitope
obtained from a pathogen or toxin, preferably, a peptide or an
epitope that is recognized by high titer antibodies that are
already present in the subject in need. It may also be desired to
screen the subject in need for the presence of high titer
antibodies that recognize the antigenic domain prior to providing
the subject with the specificity exchanger. This screening can be
accomplished by EIA or ELISA using immobilized antigenic domain or
specificity exchanger, as described above.
[0150] Similarly, a subject in need of an agent that inhibits viral
infection can be provided a specificity exchanger that recognizes a
receptor or antigen present on the particular etiologic agent.
Accordingly, a subject in need of an agent that inhibits viral
infection is identified by standard clinical or diagnostic
procedures. Next, the subject in need is provided a therapeutically
effective amount of a specificity exchanger that interacts with the
receptor or another antigen present on the type of virus infecting
the individual. As above, it may be desired to determine whether
the subject has a sufficient titer of antibody to interact with the
antigenic domain of the specificity exchanger prior to providing
the specificity exchanger.
[0151] In the same vein, a subject in need of an agent that
inhibits the proliferation of cancer can be provided a specificity
exchanger that interacts with a receptor or antigen present on the
cancer cell. For example, a subject in need of an agent that
inhibits proliferation of cancer is identified by standard clinical
or diagnostic procedures; then the subject in need is provided a
therapeutically effective amount of a specificity exchanger that
interacts with a receptor present on the cancer cells infecting the
subject. As noted above, it may be desired to determine whether the
subject has a sufficient titer of antibody to interact with the
antigenic domain of the specificity exchanger prior to providing
the specificity exchanger.
[0152] Other embodiments include methods of treating a disease or
disorder associated with a known antigen or receptor in an
individual in need of an increased number of antigen-specific
antibodies. Methods can include providing to said individual, a
sufficient amount of a tailor-made specificity exchanger that binds
to the known antigen or receptor and certain antibodies known to
exist in the individual. An individual in need of an increased
number of antigen-specific antibodies against a known antigen or
receptor, which causes a disease or disorder in said individual,
may be one who will benefit from getting a rapid increase in the
number of such antigen-specific antibodies, or who even lacks or
has insufficient ability to elicit antibodies against said known
antigen. The individual may be a human or non-human mammal.
[0153] In certain embodiments, tailor-made specificity exchangers
are designed so that certain antibodies existing in the patient in
question, (e.g. antibodies against viral proteins, such as
antibodies against poliovirus, antibodies against virus causing
measles, antibodies against hepatitis B virus, antibodies against
hepatitis C virus, antibodies against HIV-1, whether induced by
natural infection or vaccination) bind to the amino-acid sequence
of the antigenic domain and the amino-acid sequence of the
specificity domain binds to a known antigen or receptor of a
pathogen causing a disease or disorder in said patient (e.g. HIV).
Thus, existing antibodies in-said patent are redirected to said
known antigen or receptor (against which said patient e.g. lacks or
has insufficient amount of desired antibodies). A specific example
of an specificity exchanger is a peptide which binds to antibodies
against poliovirus and also binds specifically to HIV virus. Thus,
already high titres in a patient of antibodies against poliovirus
may thus be used to fight HIV infection in said patient.
[0154] Specificity exchangers described herein can also be provided
to subjects as a prophylactic to prevent the onset of disease.
Virtually anyone can be provided a specificity exchanger described
herein for prophylactic purposes, (e.g., to prevent a bacterial
infection, viral infection, or cancer). It is desired, however,
that subjects at a high risk of contracting a particular disease
are identified and provided a specificity exchanger. Subjects at
high risk of contracting a disease include individuals with a
family history of disease, the elderly or the young, or individuals
that come in frequent contact with a pathogen (e.g., health care
practitioners). Accordingly, subjects at risk of becoming infected
by a pathogen are identified and then are provided a
prophylactically effective amount of specificity exchanger.
[0155] One prophylactic application for the specificity exchangers
described herein concerns coating or cross-linking the specificity
exchanger to a medical device or implant. Implantable medical
devices tend to serve as foci for infection by a number of
bacterial species. Such device-associated infections are promoted
by the tendency of these organisms to adhere to and colonize the
surface of the device. Consequently, there is a considerable need
to develop surfaces that are less prone to promote the adverse
biological reactions that typically accompany the implantation of a
medical device.
[0156] By one approach, the medical device is coated in a solution
of containing a specificity exchanger. Prior to implantation,
medical devices (e.g., a prosthetic valve) can be stored in a
solution of specificity exchangers, for example. Medical devices
can also be coated in a powder or gel having a specificity
exchanger. For example, gloves, condoms, and intrauterine devices
can be coated in a powder or gel that contains a specificity
exchanger that interacts with a bacterial or viral receptor. Once
implanted in the body, these specificity exchangers provide a
prophylactic barrier to infection by a pathogen.
[0157] In some embodiments, the specificity exchanger is
immobilized to the medical device. As described above, the medical
device is a support to which a specificity exchanger can be
attached. Immobilization may occur by hydrophobic interaction
between the specificity exchanger and the medical device but a
preferable way to immobilize a specificity exchanger to a medical
device involves covalent attachment. For example, medical devices
can be manufactured with a reactive group that interacts with a
reactive group present on the specificity exchanger.
[0158] By one approach, a periodate is combined with a specificity
exchanger comprising a 2-aminoalcohol moiety to form an
aldehyde-functional exchanger in an aqueous solution having a pH
between about 4 and about 9 and a temperature between about 0 and
about 50 degrees Celsius. Next, the aldehyde-functional exchanger
is combined with the biomaterial surface of a medical device that
comprises a primary amine moiety to immobilize the specificity
exchanger on the support surface through an imine moiety. Then, the
imine moiety is reacted with a reducing agent to form an
immobilized specificity exchanger on the biomaterial surface
through a secondary amine linkage. Other approaches for
cross-linking molecules to medical devices, (such as described in
U.S. Pat. No. 6,017,741, herein expressly incorporated by reference
in its entirety); can be modified to immobilize the specificity
exchanger described herein. The next section describes the use of
specificity exchangers as diagnostic reagents.
[0159] Specificity Exchangers as Diagnostic Reagents
[0160] Other embodiments concern the use of specificity exchangers
as diagnostic reagents. In this context, specificity exchangers can
be used to detect the presence or absence of specific antigens or
receptors in biological samples (e.g. body fluid or tissue
samples). Accordingly, in certain embodiments, these diagnostic
specificity exchangers can be used instead of antisera or
monoclonal antibodies in in vitro testing systems, such as
immunological tests, e.g. Enzyme-Linked Immunosorbent Assay
(ELISA), Enzyme Immunoassay (EIA), Western Blot, Radioimmunoassay
(RIA) etc. Furthermore, the diagnostic specificity exchangers can
be used to investigate the biological properties of biological
systems.
[0161] Although the invention has been described with reference to
embodiments and examples, it should be understood that various
modifications can be made without departing from the spirit of the
invention. It is important to note that skilled artisans will
understand that the protocols and characterization assays described
for ligand/receptor specificity exchangers can be similarly
performed on antigen/antibody specificity exchangers by
substituting known receptors for known antigens. Likewise, skilled
artisans will also understand that the protocols and
characterization assays described for antigen/antibody specificity
exchangers can be similarly performed on ligand/receptor
specificity exchangers by substituting known antigens for known
receptors. Accordingly, the invention is limited only by the
following claims. All references cited herein are hereby expressly
incorporated by reference.
Sequence CWU 1
1
199 1 18 PRT Artificial Sequence Artificially Synthesized Peptides
1 Tyr Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly 1
5 10 15 Asp Val 2 20 PRT Artificial Sequence Artificially
Synthesized Peptides 2 Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile
Leu Tyr Phe Tyr Ala 1 5 10 15 Leu Leu Phe Leu 20 3 19 PRT
Artificial Sequence Artificially Synthesized Peptides 3 Ile Leu Tyr
Phe Tyr Ala Leu Leu Phe Leu Ser Thr Cys Val Ala Tyr 1 5 10 15 Val
Ala Thr 4 20 PRT Artificial Sequence Artificially Synthesized
Peptides 4 Ser Ser Thr Cys Val Ala Tyr Val Ala Thr Arg Asp Asn Cys
Cys Ile 1 5 10 15 Leu Asp Glu Arg 20 5 20 PRT Artificial Sequence
Artificially Synthesized Peptides 5 Arg Asp Asn Cys Cys Ile Leu Asp
Glu Arg Phe Gly Ser Tyr Cys Pro 1 5 10 15 Thr Thr Cys Gly 20 6 20
PRT Artificial Sequence Artificially Synthesized Peptides 6 Phe Gly
Ser Tyr Cys Pro Thr Thr Cys Gly Ile Ala Asp Phe Leu Ser 1 5 10 15
Thr Tyr Gln Thr 20 7 20 PRT Artificial Sequence Artificially
Synthesized Peptides 7 Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys
Val Asp Lys Asp Leu 1 5 10 15 Gln Ser Leu Glu 20 8 20 PRT
Artificial Sequence Artificially Synthesized Peptides 8 Lys Val Asp
Lys Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val 1 5 10 15 Glu
Asn Lys Thr 20 9 20 PRT Artificial Sequence Artificially
Synthesized Peptides 9 Asp Ile Leu His Gln Val Glu Asn Lys Thr Ser
Glu Val Lys Gln Leu 1 5 10 15 Ile Lys Ala Ile 20 10 20 PRT
Artificial Sequence Artificially Synthesized Peptides 10 Ser Glu
Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro 1 5 10 15
Asp Glu Ser Ser 20 11 20 PRT Artificial Sequence Artificially
Synthesized Peptides 11 Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser Lys
Pro Asn Met Ile Asp 1 5 10 15 Ala Ala Thr Leu 20 12 20 PRT
Artificial Sequence Artificially Synthesized Peptides 12 Lys Pro
Asn Met Ile Asp Ala Ala Thr Leu Lys Ser Arg Ile Met Leu 1 5 10 15
Glu Glu Ile Met 20 13 20 PRT Artificial Sequence Artificially
Synthesized Peptides 13 Lys Ser Arg Ile Met Leu Glu Glu Ile Met Lys
Tyr Glu Ala Ser Ile 1 5 10 15 Leu Thr His Asp 20 14 20 PRT
Artificial Sequence Artificially Synthesized Peptides 14 Lys Tyr
Glu Ala Ser Ile Leu Thr His Asp Ser Ser Ile Arg Tyr Leu 1 5 10 15
Gln Glu Ile Tyr 20 15 20 PRT Artificial Sequence Artificially
Synthesized Peptides 15 Ser Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn
Ser Asn Asn Gln Lys 1 5 10 15 Ile Val Asn Leu 20 16 20 PRT
Artificial Sequence Artificially Synthesized Peptides 16 Asn Ser
Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln 1 5 10 15
Leu Glu Ala Gln 20 17 20 PRT Artificial Sequence Artificially
Synthesized Peptides 17 Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile
His Asp Ile Thr Gly 1 5 10 15 Lys Asp Cys Gln 20 18 20 PRT
Artificial Sequence Artificially Synthesized Peptides 18 Ile His
Asp Ile Thr Gly Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly 1 5 10 15
Ala Lys Gln Ser 20 19 20 PRT Artificial Sequence Artificially
Synthesized Peptides 19 Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly
Leu Tyr Phe Ile Lys 1 5 10 15 Pro Leu Lys Ala 20 20 20 PRT
Artificial Sequence Artificially Synthesized Peptides 20 Gly Leu
Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val 1 5 10 15
Tyr Cys Glu Ile 20 21 20 PRT Artificial Sequence Artificially
Synthesized Peptides 21 Asn Gln Gln Phe Leu Val Tyr Cys Glu Ile Asp
Gly Ser Gly Asn Gly 1 5 10 15 Trp Thr Val Phe 20 22 20 PRT
Artificial Sequence Artificially Synthesized Peptides 22 Asp Gly
Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu Asp Gly 1 5 10 15
Ser Val Asp Phe 20 23 20 PRT Artificial Sequence Artificially
Synthesized Peptides 23 Gln Lys Arg Leu Asp Gly Ser Val Asp Phe Lys
Lys Asn Trp Ile Gln 1 5 10 15 Tyr Lys Glu Gly 20 24 20 PRT
Artificial Sequence Artificially Synthesized Peptides 24 Lys Lys
Asn Trp Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Ser Pro 1 5 10 15
Thr Gly Thr Thr 20 25 20 PRT Artificial Sequence Artificially
Synthesized Peptides 25 Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu
Phe Trp Leu Gly Asn 1 5 10 15 Glu Lys Ile His 20 26 20 PRT
Artificial Sequence Artificially Synthesized Peptides 26 Glu Phe
Trp Leu Gly Asn Glu Lys Ile His Leu Ile Ser Thr Gln Ser 1 5 10 15
Ala Ile Pro Tyr 20 27 20 PRT Artificial Sequence Artificially
Synthesized Peptides 27 Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala
Leu Arg Val Glu Leu 1 5 10 15 Glu Asp Trp Asn 20 28 20 PRT
Artificial Sequence Artificially Synthesized Peptides 28 Ala Leu
Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala 1 5 10 15
Asp Tyr Ala Met 20 29 20 PRT Artificial Sequence Artificially
Synthesized Peptides 29 Gly Arg Thr Ser Thr Ala Asp Tyr Ala Met Phe
Lys Val Gly Pro Glu 1 5 10 15 Ala Asp Lys Tyr 20 30 20 PRT
Artificial Sequence Artificially Synthesized Peptides 30 Phe Lys
Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr Ala Tyr 1 5 10 15
Phe Ala Gly Gly 20 31 20 PRT Artificial Sequence Artificially
Synthesized Peptides 31 Arg Leu Thr Tyr Ala Tyr Phe Ala Gly Gly Asp
Ala Gly Asp Ala Phe 1 5 10 15 Asp Gly Phe Asp 20 32 20 PRT
Artificial Sequence Artificially Synthesized Peptides 32 Asp Ala
Gly Asp Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser 1 5 10 15
Asp Lys Phe Phe 20 33 20 PRT Artificial Sequence Artificially
Synthesized Peptides 33 Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe Thr
Ser His Asn Gly Met 1 5 10 15 Gln Phe Ser Thr 20 34 20 PRT
Artificial Sequence Artificially Synthesized Peptides 34 Thr Ser
His Asn Gly Met Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp 1 5 10 15
Lys Phe Glu Gly 20 35 20 PRT Artificial Sequence Artificially
Synthesized Peptides 35 Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn
Cys Ala Glu Gln Asp 1 5 10 15 Gly Ser Gly Trp 20 36 20 PRT
Artificial Sequence Artificially Synthesized Peptides 36 Asn Cys
Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His 1 5 10 15
Ala Gly His Leu 20 37 20 PRT Artificial Sequence Artificially
Synthesized Peptides 37 Trp Met Asn Lys Cys His Ala Gly His Leu Asn
Gly Val Tyr Tyr Gln 1 5 10 15 Gly Gly Thr Tyr 20 38 20 PRT
Artificial Sequence Artificially Synthesized Peptides 38 Asn Gly
Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser Thr Pro 1 5 10 15
Asn Gly Tyr Asp 20 39 20 PRT Artificial Sequence Artificially
Synthesized Peptides 39 Ser Lys Ala Ser Thr Pro Asn Gly Tyr Asp Asn
Gly Ile Ile Trp Ala 1 5 10 15 Thr Trp Lys Thr 20 40 20 PRT
Artificial Sequence Artificially Synthesized Peptides 40 Asn Gly
Ile Ile Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Met Lys 1 5 10 15
Lys Thr Thr Met 20 41 20 PRT Artificial Sequence Artificially
Synthesized Peptides 41 Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys
Ile Ile Pro Phe Asn 1 5 10 15 Arg Leu Thr Ile 20 42 27 PRT
Artificial Sequence Artificially Synthesized Peptides 42 Lys Ile
Ile Pro Phe Asn Arg Leu Thr Ile Gly Glu Gly Gln Gln His 1 5 10 15
His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 20 25 43 14 PRT
Artificial Sequence Artificially Synthesized Peptides 43 Cys Asp
Leu Ile Tyr Tyr Asp Tyr Glu Glu Asp Tyr Tyr Phe 1 5 10 44 13 PRT
Artificial Sequence Artificially Synthesized Peptides 44 Cys Asp
Leu Ile Tyr Tyr Asp Tyr Glu Glu Asp Tyr Tyr 1 5 10 45 5 PRT
Artificial Sequence Artificially Synthesized Peptides 45 Thr Tyr
Ala Met Asn 1 5 46 19 PRT Artificial Sequence Artificially
Synthesized Peptides 46 Arg Val Arg Ser Lys Ser Phe Asn Tyr Ala Thr
Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Gly 47 14 PRT Artificial
Sequence Artificially Synthesized Peptides 47 Pro Ala Gln Gly Ile
Tyr Phe Asp Tyr Gly Gly Phe Ala Tyr 1 5 10 48 17 PRT Artificial
Sequence Artificially Synthesized Peptides 48 Gly Leu Tyr Ser Ser
Ile Trp Leu Ser Pro Gly Arg Ser Tyr Phe Glu 1 5 10 15 Thr 49 17 PRT
Artificial Sequence Artificially Synthesized Peptides 49 Tyr Thr
Asp Ile Lys Tyr Asn Pro Phe Thr Asp Arg Gly Glu Gly Asn 1 5 10 15
Met 50 17 PRT Artificial Sequence Artificially Synthesized Peptides
50 Asp Gln Asn Ile His Met Asn Ala Arg Leu Leu Ile Arg Ser Pro Phe
1 5 10 15 Thr 51 17 PRT Artificial Sequence Artificially
Synthesized Peptides 51 Leu Ile Arg Ser Pro Phe Thr Asp Pro Gln Leu
Leu Val His Thr Asp 1 5 10 15 Pro 52 17 PRT Artificial Sequence
Artificially Synthesized Peptides 52 Gln Lys Glu Ser Leu Leu Phe
Pro Pro Val Lys Leu Leu Arg Arg Val 1 5 10 15 Pro 53 11 PRT
Artificial Sequence Artificially Synthesized Peptides 53 Pro Ala
Leu Thr Ala Val Glu Thr Gly Ala Thr 1 5 10 54 8 PRT Artificial
Sequence Artificially Synthesized Peptides 54 Ser Thr Leu Val Pro
Glu Thr Thr 1 5 55 13 PRT Artificial Sequence Artificially
Synthesized Peptides 55 Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro
Ile Leu 1 5 10 56 9 PRT Artificial Sequence Artificially
Synthesized Peptides 56 Glu Ile Pro Ala Leu Thr Ala Val Glu 1 5 57
10 PRT Artificial Sequence Artificially Synthesized Peptides 57 Leu
Glu Asp Pro Ala Ser Arg Asp Leu Val 1 5 10 58 8 PRT Artificial
Sequence Artificially Synthesized Peptides 58 His Arg Gly Gly Pro
Glu Glu Phe 1 5 59 7 PRT Artificial Sequence Artificially
Synthesized Peptides 59 His Arg Gly Gly Pro Glu Glu 1 5 60 17 PRT
Artificial Sequence Artificially Synthesized Peptides 60 Val Leu
Ile Cys Gly Glu Asn Thr Val Ser Arg Asn Tyr Ala Thr His 1 5 10 15
Ser 61 17 PRT Artificial Sequence Artificially Synthesized Peptides
61 Lys Ile Asn Thr Met Pro Pro Phe Leu Asp Thr Glu Leu Thr Ala Pro
1 5 10 15 Ser 62 17 PRT Artificial Sequence Artificially
Synthesized Peptides 62 Pro Asp Glu Lys Ser Gln Arg Glu Ile Leu Leu
Asn Lys Ile Ala Ser 1 5 10 15 Tyr 63 17 PRT Artificial Sequence
Artificially Synthesized Peptides 63 Thr Ala Thr Thr Thr Thr Tyr
Ala Tyr Pro Gly Thr Asn Arg Pro Pro 1 5 10 15 Val 64 8 PRT
Artificial Sequence Artificially Synthesized Peptides 64 Ser Thr
Pro Leu Pro Glu Thr Thr 1 5 65 8 PRT Artificial Sequence
Artificially Synthesized Peptides 65 Pro Pro Asn Ala Pro Ile Leu
Ser 1 5 66 10 PRT Artificial Sequence Artificially Synthesized
Peptides 66 Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr 1 5 10 67 12
PRT Artificial Sequence Artificially Synthesized Peptides 67 Lys
Glu Ile Pro Ala Leu Thr Ala Val Glu Thr Gly 1 5 10 68 12 PRT
Artificial Sequence Artificially Synthesized Peptides 68 Pro Ala
His Ser Lys Glu Ile Pro Ala Leu Thr Ala 1 5 10 69 10 PRT Artificial
Sequence Artificially Synthesized Peptides 69 Trp Gly Cys Ser Gly
Lys Leu Ile Cys Thr 1 5 10 70 10 PRT Artificial Sequence
Artificially Synthesized Peptides 70 Cys Thr Thr Ala Val Pro Trp
Asn Ala Ser 1 5 10 71 11 PRT Artificial Sequence Artificially
Synthesized Peptides 71 Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg
1 5 10 72 26 PRT Artificial Sequence Artificially Synthesized
Peptides 72 Tyr Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln
Ala Gly 1 5 10 15 Asp Val His Arg Gly Gly Pro Glu Glu Phe 20 25 73
25 PRT Artificial Sequence Artificially Synthesized Peptides 73 Tyr
Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Ala Gly 1 5 10
15 Asp Val His Arg Gly Gly Pro Glu Glu 20 25 74 26 PRT Artificial
Sequence Artificially Synthesized Peptides 74 Tyr Gly Glu Gly Gln
Gln His His Leu Gly Gly Ala Lys Gln Ala Gly 1 5 10 15 Asp Val Ser
Thr Pro Leu Pro Glu Thr Thr 20 25 75 27 PRT Artificial Sequence
Artificially Synthesized Peptides 75 Met Ser Trp Ser Leu His Pro
Arg Asn Leu Ile Leu Tyr Phe Tyr Ala 1 5 10 15 Leu Leu Phe Leu His
Arg Gly Gly Pro Glu Glu 20 25 76 26 PRT Artificial Sequence
Artificially Synthesized Peptides 76 Ile Leu Tyr Phe Tyr Ala Leu
Leu Phe Leu Ser Thr Cys Val Ala Tyr 1 5 10 15 Val Ala Thr His Arg
Gly Gly Pro Glu Glu 20 25 77 27 PRT Artificial Sequence
Artificially Synthesized Peptides 77 Ser Ser Thr Cys Val Ala Tyr
Val Ala Thr Arg Asp Asn Cys Cys Ile 1 5 10 15 Leu Asp Glu Arg His
Arg Gly Gly Pro Glu Glu 20 25 78 27 PRT Artificial Sequence
Artificially Synthesized Peptides 78 Arg Asp Asn Cys Cys Ile Leu
Asp Glu Arg Phe Gly Ser Tyr Cys Pro 1 5 10 15 Thr Thr Cys Gly His
Arg Gly Gly Pro Glu Glu 20 25 79 27 PRT Artificial Sequence
Artificially Synthesized Peptides 79 Phe Gly Ser Tyr Cys Pro Thr
Thr Cys Gly Ile Ala Asp Phe Leu Ser 1 5 10 15 Thr Tyr Gln Thr His
Arg Gly Gly Pro Glu Glu 20 25 80 27 PRT Artificial Sequence
Artificially Synthesized Peptides 80 Ile Ala Asp Phe Leu Ser Thr
Tyr Gln Thr Lys Val Asp Lys Asp Leu 1 5 10 15 Gln Ser Leu Glu His
Arg Gly Gly Pro Glu Glu 20 25 81 27 PRT Artificial Sequence
Artificially Synthesized Peptides 81 Lys Val Asp Lys Asp Leu Gln
Ser Leu Glu Asp Ile Leu His Gln Val 1 5 10 15 Glu Asn Lys Thr His
Arg Gly Gly Pro Glu Glu 20 25 82 27 PRT Artificial Sequence
Artificially Synthesized Peptides 82 Asp Ile Leu His Gln Val Glu
Asn Lys Thr Ser Glu Val Lys Gln Leu 1 5 10 15 Ile Lys Ala Ile His
Arg Gly Gly Pro Glu Glu 20 25 83 27 PRT Artificial Sequence
Artificially Synthesized Peptides 83 Ser Glu Val Lys Gln Leu Ile
Lys Ala Ile Gln Leu Thr Tyr Asn Pro 1 5 10 15 Asp Glu Ser Ser His
Arg Gly Gly Pro Glu Glu 20 25 84 27 PRT Artificial Sequence
Artificially Synthesized Peptides 84 Gln Leu Thr Tyr Asn Pro Asp
Glu Ser Ser Lys Pro Asn Met Ile Asp 1 5 10 15 Ala Ala Thr Leu His
Arg Gly Gly Pro Glu Glu 20 25 85 27 PRT Artificial Sequence
Artificially Synthesized Peptides 85 Lys Pro Asn Met Ile Asp Ala
Ala Thr Leu Lys Ser Arg Ile Met Leu 1 5 10 15 Glu Glu Ile Met His
Arg Gly Gly Pro Glu Glu 20 25 86 27 PRT Artificial Sequence
Artificially Synthesized Peptides 86 Lys Ser Arg Ile Met Leu Glu
Glu Ile Met Lys Tyr Glu Ala Ser Ile 1 5 10 15 Leu Thr His Asp His
Arg Gly Gly Pro Glu Glu 20 25 87 27 PRT Artificial Sequence
Artificially Synthesized Peptides 87 Lys Tyr Glu Ala Ser Ile Leu
Thr His Asp Ser Ser Ile Arg Tyr Leu 1 5 10 15 Gln Glu Ile Tyr His
Arg Gly Gly Pro Glu Glu 20 25 88 27 PRT Artificial Sequence
Artificially Synthesized Peptides 88 Ser Ser Ile Arg Tyr Leu Gln
Glu Ile Tyr Asn Ser
Asn Asn Gln Lys 1 5 10 15 Ile Val Asn Leu His Arg Gly Gly Pro Glu
Glu 20 25 89 27 PRT Artificial Sequence Artificially Synthesized
Peptides 89 Asn Ser Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Lys Val
Ala Gln 1 5 10 15 Leu Glu Ala Gln His Arg Gly Gly Pro Glu Glu 20 25
90 27 PRT Artificial Sequence Artificially Synthesized Peptides 90
Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly 1 5
10 15 Lys Asp Cys Gln His Arg Gly Gly Pro Glu Glu 20 25 91 27 PRT
Artificial Sequence Artificially Synthesized Peptides 91 Ile His
Asp Ile Thr Gly Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly 1 5 10 15
Ala Lys Gln Ser His Arg Gly Gly Pro Glu Glu 20 25 92 27 PRT
Artificial Sequence Artificially Synthesized Peptides 92 Asp Ile
Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu Tyr Phe Ile Lys 1 5 10 15
Pro Leu Lys Ala His Arg Gly Gly Pro Glu Glu 20 25 93 27 PRT
Artificial Sequence Artificially Synthesized Peptides 93 Gly Leu
Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val 1 5 10 15
Tyr Cys Glu Ile His Arg Gly Gly Pro Glu Glu 20 25 94 27 PRT
Artificial Sequence Artificially Synthesized Peptides 94 Asn Gln
Gln Phe Leu Val Tyr Cys Glu Ile Asp Gly Ser Gly Asn Gly 1 5 10 15
Trp Thr Val Phe His Arg Gly Gly Pro Glu Glu 20 25 95 27 PRT
Artificial Sequence Artificially Synthesized Peptides 95 Asp Gly
Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu Asp Gly 1 5 10 15
Ser Val Asp Phe His Arg Gly Gly Pro Glu Glu 20 25 96 27 PRT
Artificial Sequence Artificially Synthesized Peptides 96 Gln Lys
Arg Leu Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln 1 5 10 15
Tyr Lys Glu Gly His Arg Gly Gly Pro Glu Glu 20 25 97 27 PRT
Artificial Sequence Artificially Synthesized Peptides 97 Lys Lys
Asn Trp Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Ser Pro 1 5 10 15
Thr Gly Thr Thr His Arg Gly Gly Pro Glu Glu 20 25 98 27 PRT
Artificial Sequence Artificially Synthesized Peptides 98 Phe Gly
His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly Asn 1 5 10 15
Glu Lys Ile His His Arg Gly Gly Pro Glu Glu 20 25 99 27 PRT
Artificial Sequence Artificially Synthesized Peptides 99 Glu Phe
Trp Leu Gly Asn Glu Lys Ile His Leu Ile Ser Thr Gln Ser 1 5 10 15
Ala Ile Pro Tyr His Arg Gly Gly Pro Glu Glu 20 25 100 27 PRT
Artificial Sequence Artificially Synthesized Peptides 100 Leu Ile
Ser Thr Gln Ser Ala Ile Pro Tyr Ala Leu Arg Val Glu Leu 1 5 10 15
Glu Asp Trp Asn His Arg Gly Gly Pro Glu Glu 20 25 101 27 PRT
Artificial Sequence Artificially Synthesized Peptides 101 Ala Leu
Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala 1 5 10 15
Asp Tyr Ala Met His Arg Gly Gly Pro Glu Glu 20 25 102 27 PRT
Artificial Sequence Artificially Synthesized Peptides 102 Gly Arg
Thr Ser Thr Ala Asp Tyr Ala Met Phe Lys Val Gly Pro Glu 1 5 10 15
Ala Asp Lys Tyr His Arg Gly Gly Pro Glu Glu 20 25 103 27 PRT
Artificial Sequence Artificially Synthesized Peptides 103 Phe Lys
Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr Ala Tyr 1 5 10 15
Phe Ala Gly Gly His Arg Gly Gly Pro Glu Glu 20 25 104 27 PRT
Artificial Sequence Artificially Synthesized Peptides 104 Arg Leu
Thr Tyr Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp Ala Phe 1 5 10 15
Asp Gly Phe Asp His Arg Gly Gly Pro Glu Glu 20 25 105 27 PRT
Artificial Sequence Artificially Synthesized Peptides 105 Asp Ala
Gly Asp Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser 1 5 10 15
Asp Lys Phe Phe His Arg Gly Gly Pro Glu Glu 20 25 106 27 PRT
Artificial Sequence Artificially Synthesized Peptides 106 Phe Gly
Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly Met 1 5 10 15
Gln Phe Ser Thr His Arg Gly Gly Pro Glu Glu 20 25 107 27 PRT
Artificial Sequence Artificially Synthesized Peptides 107 Thr Ser
His Asn Gly Met Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp 1 5 10 15
Lys Phe Glu Gly His Arg Gly Gly Pro Glu Glu 20 25 108 27 PRT
Artificial Sequence Artificially Synthesized Peptides 108 Trp Asp
Asn Asp Asn Asp Lys Phe Glu Gly Asn Cys Ala Glu Gln Asp 1 5 10 15
Gly Ser Gly Trp His Arg Gly Gly Pro Glu Glu 20 25 109 27 PRT
Artificial Sequence Artificially Synthesized Peptides 109 Asn Cys
Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His 1 5 10 15
Ala Gly His Leu His Arg Gly Gly Pro Glu Glu 20 25 110 27 PRT
Artificial Sequence Artificially Synthesized Peptides 110 Trp Met
Asn Lys Cys His Ala Gly His Leu Asn Gly Val Tyr Tyr Gln 1 5 10 15
Gly Gly Thr Tyr His Arg Gly Gly Pro Glu Glu 20 25 111 27 PRT
Artificial Sequence Artificially Synthesized Peptides 111 Asn Gly
Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser Thr Pro 1 5 10 15
Asn Gly Tyr Asp His Arg Gly Gly Pro Glu Glu 20 25 112 27 PRT
Artificial Sequence Artificially Synthesized Peptides 112 Ser Lys
Ala Ser Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala 1 5 10 15
Thr Trp Lys Thr His Arg Gly Gly Pro Glu Glu 20 25 113 27 PRT
Artificial Sequence Artificially Synthesized Peptides 113 Asn Gly
Ile Ile Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Met Lys 1 5 10 15
Lys Thr Thr Met His Arg Gly Gly Pro Glu Glu 20 25 114 27 PRT
Artificial Sequence Artificially Synthesized Peptides 114 Arg Trp
Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn 1 5 10 15
Arg Leu Thr Ile His Arg Gly Gly Pro Glu Glu 20 25 115 34 PRT
Artificial Sequence Artificially Synthesized Peptides 115 Lys Ile
Ile Pro Phe Asn Arg Leu Thr Ile Gly Glu Gly Gln Gln His 1 5 10 15
His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val His Arg Gly Gly Pro 20
25 30 Glu Glu 116 7 PRT Artificial Sequence Artificially
Synthesized Peptides 116 Pro Asn Ala Pro Ile Leu Ser 1 5 117 10 PRT
Artificial Sequence Artificially Synthesized Peptides 117 His Leu
Gly Gly Ala Lys Gln Ala Gly Asp 1 5 10 118 22 PRT Artificial
Sequence Artificially Synthesized Peptides 118 Cys Asp Leu Ile Tyr
Tyr Asp Tyr Glu Glu Asp Tyr Tyr Phe Pro Pro 1 5 10 15 Asn Ala Pro
Ile Leu Ser 20 119 24 PRT Artificial Sequence Artificially
Synthesized Peptides 119 Cys Asp Leu Ile Tyr Tyr Asp Tyr Glu Glu
Asp Tyr Tyr Phe Arg Pro 1 5 10 15 Pro Asn Ala Pro Ile Leu Ser Thr
20 120 26 PRT Artificial Sequence Artificially Synthesized Peptides
120 Cys Asp Leu Ile Tyr Tyr Asp Tyr Glu Glu Asp Tyr Tyr Phe Lys Glu
1 5 10 15 Ile Pro Ala Leu Thr Ala Val Glu Thr Gly 20 25 121 26 PRT
Artificial Sequence Artificially Synthesized Peptides 121 Cys Asp
Leu Ile Tyr Tyr Asp Tyr Glu Glu Asp Tyr Tyr Phe Pro Ala 1 5 10 15
His Ser Lys Glu Ile Pro Ala Leu Thr Ala 20 25 122 24 PRT Artificial
Sequence Artificially Synthesized Peptides 122 Cys Asp Leu Ile Tyr
Tyr Asp Tyr Glu Glu Asp Tyr Tyr Phe Trp Gly 1 5 10 15 Cys Ser Gly
Lys Leu Ile Cys Thr 20 123 24 PRT Artificial Sequence Artificially
Synthesized Peptides 123 Cys Asp Leu Ile Tyr Tyr Asp Tyr Glu Glu
Asp Tyr Tyr Phe Cys Thr 1 5 10 15 Thr Ala Val Pro Trp Asn Ala Ser
20 124 39 PRT Artificial Sequence Artificially Synthesized Peptides
124 Cys Asp Leu Ile Tyr Tyr Asp Tyr Glu Glu Asp Tyr Tyr Phe Lys Arg
1 5 10 15 Pro Pro Asn Ala Pro Ile Leu Ser Thr Cys Asp Leu Ile Tyr
Tyr Asp 20 25 30 Tyr Glu Glu Asp Tyr Tyr Phe 35 125 13 PRT
Artificial Sequence Artificially Synthesized Peptides 125 Thr Tyr
Ala Met Asn Pro Pro Asn Ala Pro Ile Leu Ser 1 5 10 126 27 PRT
Artificial Sequence Artificially Synthesized Peptides 126 Arg Val
Arg Ser Lys Ser Phe Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15
Val Lys Gly Pro Pro Asn Ala Pro Ile Leu Ser 20 25 127 22 PRT
Artificial Sequence Artificially Synthesized Peptides 127 Pro Ala
Gln Gly Ile Tyr Phe Asp Tyr Gly Gly Phe Ala Tyr Pro Pro 1 5 10 15
Asn Ala Pro Ile Leu Ser 20 128 24 PRT Artificial Sequence
Artificially Synthesized Peptides 128 Cys Asp Leu Ile Tyr Tyr Asp
Tyr Glu Glu Asp Tyr Tyr Gln Arg Lys 1 5 10 15 Thr Lys Arg Asn Thr
Asn Arg Arg 20 129 10 PRT Artificial Sequence Artificially
Synthesized Peptides 129 Ala Leu Gly Gly Ala Lys Gln Ala Gly Asp 1
5 10 130 20 PRT Artificial Sequence Artificially Synthesized
Peptides 130 Leu Thr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly
Ala Lys Gln 1 5 10 15 Ala Gly Asp Val 20 131 17 PRT Artificial
Sequence Artificially Synthesized Peptides 131 Gly Glu Gly Gln Gln
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp 1 5 10 15 Val 132 14
PRT Artificial Sequence Artificially Synthesized Peptides 132 Gln
Gln His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 133 13
PRT Artificial Sequence Artificially Synthesized Peptides 133 Gln
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 134 12 PRT
Artificial Sequence Artificially Synthesized Peptides 134 His His
Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 135 11 PRT
Artificial Sequence Artificially Synthesized Peptides 135 His Leu
Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 136 10 PRT Artificial
Sequence Artificially Synthesized Peptides 136 Leu Gly Gly Ala Lys
Gln Ala Gly Asp Val 1 5 10 137 9 PRT Artificial Sequence
Artificially Synthesized Peptides 137 Gly Gly Ala Lys Gln Ala Gly
Asp Val 1 5 138 8 PRT Artificial Sequence Artificially Synthesized
Peptides 138 Gly Ala Lys Gln Ala Gly Asp Val 1 5 139 12 PRT
Artificial Sequence Artificially Synthesized Peptides 139 Gln His
His Leu Gly Gly Ala Lys Gln Ala Gly Asp 1 5 10 140 11 PRT
Artificial Sequence Artificially Synthesized Peptides 140 Gln His
His Leu Gly Gly Ala Lys Gln Ala Gly 1 5 10 141 10 PRT Artificial
Sequence Artificially Synthesized Peptides 141 Gln His His Leu Gly
Gly Ala Lys Gln Ala 1 5 10 142 9 PRT Artificial Sequence
Artificially Synthesized Peptides 142 Gln His His Leu Gly Gly Ala
Lys Gln 1 5 143 8 PRT Artificial Sequence Artificially Synthesized
Peptides 143 Gln His His Leu Gly Gly Ala Lys 1 5 144 7 PRT
Artificial Sequence Artificially Synthesized Peptides 144 Gln His
His Leu Gly Gly Ala 1 5 145 12 PRT Artificial Sequence Artificially
Synthesized Peptides 145 His His Leu Gly Gly Ala Lys Gln Ala Gly
Asp Val 1 5 10 146 11 PRT Artificial Sequence Artificially
Synthesized Peptides 146 His His Leu Gly Gly Ala Lys Gln Ala Gly
Asp 1 5 10 147 10 PRT Artificial Sequence Artificially Synthesized
Peptides 147 His His Leu Gly Gly Ala Lys Gln Ala Gly 1 5 10 148 11
PRT Artificial Sequence Artificially Synthesized Peptides 148 His
Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 149 10 PRT
Artificial Sequence Artificially Synthesized Peptides 149 His Leu
Gly Gly Ala Lys Gln Ala Gly Asp 1 5 10 150 9 PRT Artificial
Sequence Artificially Synthesized Peptides 150 Ala Leu Gly Gly Ala
Lys Gln Ala Gly 1 5 151 9 PRT Artificial Sequence Artificially
Synthesized Peptides 151 His Ala Gly Gly Ala Lys Gln Ala Gly 1 5
152 9 PRT Artificial Sequence Artificially Synthesized Peptides 152
His Leu Ala Gly Ala Lys Gln Ala Gly 1 5 153 9 PRT Artificial
Sequence Artificially Synthesized Peptides 153 His Leu Gly Ala Ala
Lys Gln Ala Gly 1 5 154 9 PRT Artificial Sequence Artificially
Synthesized Peptides 154 His Leu Gly Gly Gly Lys Gln Ala Gly 1 5
155 9 PRT Artificial Sequence Artificially Synthesized Peptides 155
His Leu Gly Gly Ala Ala Gln Ala Gly 1 5 156 9 PRT Artificial
Sequence Artificially Synthesized Peptides 156 His Leu Gly Gly Ala
Lys Ala Ala Gly 1 5 157 9 PRT Artificial Sequence Artificially
Synthesized Peptides 157 His Leu Gly Gly Ala Lys Gln Gly Gly 1 5
158 9 PRT Artificial Sequence Artificially Synthesized Peptides 158
His Leu Gly Gly Ala Lys Gln Ala Ala 1 5 159 19 PRT Artificial
Sequence Artificially Synthesized Peptides 159 His Arg Gly Gly Pro
Glu Glu Phe His His Leu Gly Gly Ala Lys Gln 1 5 10 15 Ala Gly Asp
160 19 PRT Artificial Sequence Artificially Synthesized Peptides
160 His Arg Gly Gly Pro Glu Glu Phe His His Leu Gly Gly Ala Lys Arg
1 5 10 15 Ala Gly Arg 161 19 PRT Artificial Sequence Artificially
Synthesized Peptides 161 His Arg Gly Gly Pro Glu Glu Phe His His
Leu Gly Gly Ala Arg Arg 1 5 10 15 Ala Gly Arg 162 19 PRT Artificial
Sequence Artificially Synthesized Peptides 162 His Arg Gly Gly Pro
Glu Glu Phe His His Leu Gly His Ala Lys Gln 1 5 10 15 Ala Gly Arg
163 19 PRT Artificial Sequence Artificially Synthesized Peptides
163 His Arg Gly Gly Pro Glu Glu Phe His His Leu Gly His Ala Arg Gln
1 5 10 15 Ala Gly Arg 164 19 PRT Artificial Sequence Artificially
Synthesized Peptides 164 His Arg Gly Gly Pro Glu Glu Phe His His
Leu Gly His Ala Lys Arg 1 5 10 15 Ala Gly Leu 165 19 PRT Artificial
Sequence Artificially Synthesized Peptides 165 His Arg Gly Gly Pro
Glu Glu Phe His His Leu Gly His Ala Lys Arg 1 5 10 15 Ala Gly Arg
166 19 PRT Artificial Sequence Artificially Synthesized Peptides
166 His His Leu Gly Gly Ala Lys Gln Ala Gly Asp His Arg Gly Gly Pro
1 5 10 15 Glu Glu Phe 167 19 PRT Artificial Sequence Artificially
Synthesized Peptides 167 His His Leu Gly Gly Ala Lys Arg Ala Gly
Arg His Arg Gly Gly Pro 1 5 10 15 Glu Glu Phe 168 19 PRT Artificial
Sequence Artificially Synthesized Peptides 168 His His Leu Gly Gly
Ala Arg Arg Ala Gly Arg His Arg Gly Gly Pro 1 5 10 15 Glu Glu Phe
169 19 PRT Artificial Sequence Artificially Synthesized Peptides
169 His His Leu Gly His Ala Lys Gln Ala Gly Arg His Arg Gly Gly Pro
1 5 10 15 Glu Glu Phe 170 19 PRT Artificial Sequence Artificially
Synthesized Peptides 170 His His Leu Gly His Ala Arg Gln Ala Gly
Arg His Arg Gly Gly Pro 1 5 10 15 Glu Glu Phe 171 19 PRT Artificial
Sequence Artificially Synthesized Peptides 171 His His Leu Gly His
Ala Lys Arg Ala Gly Leu His Arg Gly Gly Pro 1 5 10 15 Glu Glu Phe
172 19 PRT Artificial Sequence Artificially Synthesized Peptides
172 His His Leu Gly His Ala Lys Arg Ala Gly Arg His Arg Gly Gly Pro
1 5 10 15 Glu Glu Phe 173 25 PRT Artificial Sequence Artificially
Synthesized Peptides 173 Pro Ala Leu Thr Ala Val Glu Thr Gly Ala
Thr Asn Pro Leu His His 1 5 10 15 Leu Gly Gly Ala Lys Gln Ala Gly
Asp 20 25 174 25 PRT Artificial Sequence Artificially Synthesized
Peptides 174 Pro Ala Leu Thr Ala Val Glu Thr Gly Ala Thr Asn Pro
Leu His His 1 5
10 15 Leu Gly Gly Ala Lys Arg Ala Gly Arg 20 25 175 25 PRT
Artificial Sequence Artificially Synthesized Peptides 175 Pro Ala
Leu Thr Ala Val Glu Thr Gly Ala Thr Asn Pro Leu His His 1 5 10 15
Leu Gly Gly Ala Arg Arg Ala Gly Arg 20 25 176 25 PRT Artificial
Sequence Artificially Synthesized Peptides 176 Pro Ala Leu Thr Ala
Val Glu Thr Gly Ala Thr Asn Pro Leu His His 1 5 10 15 Leu Gly His
Ala Lys Gln Ala Gly Arg 20 25 177 25 PRT Artificial Sequence
Artificially Synthesized Peptides 177 Pro Ala Leu Thr Ala Val Glu
Thr Gly Ala Thr Asn Pro Leu His His 1 5 10 15 Leu Gly His Ala Arg
Gln Ala Gly Arg 20 25 178 25 PRT Artificial Sequence Artificially
Synthesized Peptides 178 Pro Ala Leu Thr Ala Val Glu Thr Gly Ala
Thr Asn Pro Leu His His 1 5 10 15 Leu Gly His Ala Lys Arg Ala Gly
Leu 20 25 179 25 PRT Artificial Sequence Artificially Synthesized
Peptides 179 Pro Ala Leu Thr Ala Val Glu Thr Gly Ala Thr Asn Pro
Leu His His 1 5 10 15 Leu Gly His Ala Lys Arg Ala Gly Arg 20 25 180
25 PRT Artificial Sequence Artificially Synthesized Peptides 180
His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Pro Ala Leu Thr Ala 1 5
10 15 Val Glu Thr Gly Ala Thr Asn Pro Leu 20 25 181 25 PRT
Artificial Sequence Artificially Synthesized Peptides 181 His His
Leu Gly Gly Ala Lys Arg Ala Gly Arg Pro Ala Leu Thr Ala 1 5 10 15
Val Glu Thr Gly Ala Thr Asn Pro Leu 20 25 182 25 PRT Artificial
Sequence Artificially Synthesized Peptides 182 His His Leu Gly Gly
Ala Arg Arg Ala Gly Arg Pro Ala Leu Thr Ala 1 5 10 15 Val Glu Thr
Gly Ala Thr Asn Pro Leu 20 25 183 25 PRT Artificial Sequence
Artificially Synthesized Peptides 183 His His Leu Gly His Ala Lys
Gln Ala Gly Arg Pro Ala Leu Thr Ala 1 5 10 15 Val Glu Thr Gly Ala
Thr Asn Pro Leu 20 25 184 25 PRT Artificial Sequence Artificially
Synthesized Peptides 184 His His Leu Gly His Ala Arg Gln Ala Gly
Arg Pro Ala Leu Thr Ala 1 5 10 15 Val Glu Thr Gly Ala Thr Asn Pro
Leu 20 25 185 25 PRT Artificial Sequence Artificially Synthesized
Peptides 185 His His Leu Gly His Ala Lys Arg Ala Gly Leu Pro Ala
Leu Thr Ala 1 5 10 15 Val Glu Thr Gly Ala Thr Asn Pro Leu 20 25 186
25 PRT Artificial Sequence Artificially Synthesized Peptides 186
His His Leu Gly His Ala Lys Arg Ala Gly Arg Pro Ala Leu Thr Ala 1 5
10 15 Val Glu Thr Gly Ala Thr Asn Pro Leu 20 25 187 27 PRT
Artificial Sequence Artificially Synthesized Peptides 187 Cys Pro
Ala Leu Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu Ala 1 5 10 15
Ala His His Leu Gly Gly Ala Lys Gln Ala Gly 20 25 188 27 PRT
Artificial Sequence Artificially Synthesized Peptides 188 His His
Leu Gly Gly Ala Lys Gln Ala Gly Ala Ala Cys Pro Ala Leu 1 5 10 15
Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu 20 25 189 25 PRT
Artificial Sequence Artificially Synthesized Peptides 189 Cys Pro
Ala Leu Thr Ala Val Glu Thr Gly Cys Thr Asn Pro Leu His 1 5 10 15
His Leu Gly Gly Ala Lys Gln Ala Gly 20 25 190 25 PRT Artificial
Sequence Artificially Synthesized Peptides 190 His His Leu Gly Gly
Ala Lys Gln Ala Gly Cys Pro Ala Leu Thr Ala 1 5 10 15 Val Glu Thr
Gly Cys Thr Asn Pro Leu 20 25 191 27 PRT Artificial Sequence
Artificially Synthesized Peptides 191 His His Leu Gly Gly Ala Lys
Gln Ala Gly Ala Ala Cys Pro Ala Leu 1 5 10 15 Thr Ala Val Glu Thr
Gly Cys Thr Asn Pro Leu 20 25 192 27 PRT Artificial Sequence
Artificially Synthesized Peptides 192 Cys Pro Ala Leu Thr Ala Val
Glu Thr Gly Cys Thr Asn Pro Leu Ala 1 5 10 15 Ala His His Leu Gly
Gly Ala Lys Gln Ala Gly 20 25 193 25 PRT Artificial Sequence
Artificially Synthesized Peptides 193 Cys Pro Ala Leu Thr Ala Val
Glu Thr Gly Cys Thr Asn Pro Leu His 1 5 10 15 His Leu Gly Gly Ala
Lys Gln Ala Gly 20 25 194 25 PRT Artificial Sequence Artificially
Synthesized Peptides 194 His His Leu Gly Gly Ala Lys Gln Ala Gly
Cys Pro Ala Leu Thr Ala 1 5 10 15 Val Glu Thr Gly Cys Thr Asn Pro
Leu 20 25 195 24 PRT Artificial Sequence Artificially Synthesized
Peptides 195 Pro Ala Leu Thr Ala Val Glu Thr Gly Ala Thr Asn Pro
Leu His His 1 5 10 15 Leu Gly Gly Ala Lys Gln Ala Gly 20 196 24 PRT
Artificial Sequence Artificially Synthesized Peptides 196 His His
Leu Gly Gly Ala Lys Gln Ala Gly Pro Ala Leu Thr Ala Val 1 5 10 15
Glu Thr Gly Ala Thr Asn Pro Leu 20 197 13 PRT Artificial Sequence
Artificially Synthesized Peptides 197 Gly Arg Gly Asp Ser Pro His
Arg Gly Gly Pro Glu Glu 1 5 10 198 13 PRT Artificial Sequence
Artificially Synthesized Peptides 198 Trp Ser Arg Gly Asp Trp His
Arg Gly Gly Pro Glu Glu 1 5 10 199 12 PRT Artificial Sequence
Artificially Synthesized Peptides 199 Arg Gly Asp Ser Ala Ala Thr
Pro Pro Ala Tyr Arg 1 5 10
* * * * *