U.S. patent application number 11/188187 was filed with the patent office on 2006-01-26 for synthetic peptides that bind to the hepatitis b virus core and e antigens.
Invention is credited to Matti Sallberg.
Application Number | 20060020110 11/188187 |
Document ID | / |
Family ID | 24222054 |
Filed Date | 2006-01-26 |
United States Patent
Application |
20060020110 |
Kind Code |
A1 |
Sallberg; Matti |
January 26, 2006 |
Synthetic peptides that bind to the hepatitis B virus core and E
antigens
Abstract
The present invention relates generally to the field of
virology. More particularly, the invention relates to the discovery
that peptides, which bind to the Hepatitis B virus (HBV) core and e
antigens, can be used to inhibit HBV infection. Embodiments concern
"binding partners", which include peptides, peptidomimetics, and
chemicals that resemble these molecules that interact with HBV core
and e antigens, biological complexes having HBV core and e antigens
joined to said binding partners, methods of identifying such
binding partners, pharmaceuticals having binding partners, and
methods of treatments and prevention of HBV infection.
Inventors: |
Sallberg; Matti; (Alvsjo,
SE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
24222054 |
Appl. No.: |
11/188187 |
Filed: |
July 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10369060 |
Feb 14, 2003 |
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11188187 |
Jul 22, 2005 |
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09839447 |
Apr 20, 2001 |
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10369060 |
Feb 14, 2003 |
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09556605 |
Apr 21, 2000 |
6417324 |
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09839447 |
Apr 20, 2001 |
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Current U.S.
Class: |
530/322 ;
435/5 |
Current CPC
Class: |
C07K 14/005 20130101;
C07K 2317/50 20130101; C12N 2730/10122 20130101; A61K 38/00
20130101; C07K 16/082 20130101; Y02A 50/30 20180101; Y02A 50/466
20180101; A61K 2039/505 20130101; C07K 2317/56 20130101; C07K 7/06
20130101; C07K 7/08 20130101 |
Class at
Publication: |
530/322 ;
514/008; 435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 9/00 20060101 C07K009/00; A61K 38/14 20060101
A61K038/14 |
Claims
1. A peptide joined to a carbohydrate, wherein said peptide
comprises a domain of the formula: X.sup.1.sub.nCZASX.sup.2.sub.n,
wherein: "X.sup.1" is any amino acid or absent "C" is cysteine; "Z"
is lysine or arginine; "A" is alanine; "S" is serine; "X.sup.2" is
any amino acid or absent; and "n" is an integer, and wherein said
peptide is less than 50 amino acids in length.
2. The peptide of claim 1, wherein "Z" is lysine.
3. The peptide of claim 1, wherein said "Z" is arginine.
4. The peptide of claim 1, wherein said carbohydrate is an
oligosaccaride.
5. The peptide of claim 1, wherein said domain comprises a sequence
selected from the group consisting of SEQ. ID. Nos.: 5, 6, 17, 28,
29, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 53, 54, 66, 67, 70, 71,
72, 73, 74, 75, 76, 77, and 78.
6. A method of making the peptide of claim 1 comprising: providing
a peptide comprising a domain of the formula:
X.sup.1.sub.nCZASX.sup.2.sub.n, wherein: "X.sup.1" is any amino
acid or absent "C" is cysteine; "Z" is lysine or arginine; "A" is
alanine; "S" is serine; "X.sup.2" is any amino acid or absent; and
"n" is an integer, wherein said peptide is less than 50 amino acids
in length; providing a carbohydrate; joining said carbohydrate to
said peptide so as to make the peptide of claim 1.
7. The method of claim 6, wherein "Z" is lysine.
8. The method of claim 6, wherein said "Z" is arginine.
9. The method of claim 6, wherein said carbohydrate is an
oligosaccaride.
10. The method of claim 6, wherein said domain comprises a sequence
selected from the group consisting of SEQ. ID. Nos.: 5, 6, 17, 28,
29, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 53, 54, 66, 67, 70, 71,
72, 73, 74, 75, 76, 77, and 78.
11. A method of using the peptide of claim 1 to redirect antibodies
present in a subject to hepatitis B virus comprising: identifying a
subject that has antibodies specific for the carbohydrate that is
joined to the preptide of claim 1; and providing to said subject
the peptide of claim 1.
12. The method of claim 11, wherein "Z" is lysine.
13. The method of claim 11, wherein said "Z" is arginine.
14. The method of claim 11, wherein said carbohydrate is an
oligosaccaride.
15. The method of claim 11, wherein said domain comprises a
sequence selected from the group consisting of SEQ. ID. Nos.: 5, 6,
17, 28, 29, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 53, 54, 66, 67,
70, 71, 72, 73, 74, 75, 76, 77, and 78.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/369,060, filed Feb. 14, 2003, which is a continuation of
U.S. application Ser. No. 09/839,447, filed Apr. 20, 2001, now
abandon, which is a continuation-in-part of U.S. Pat. No.
6,417,324, issued Jul. 9, 2002. This application claims the benefit
of priority to U.S. application Ser. Nos. 10/369,060 and 09/839,447
and U.S. Pat. No. 6,417,324, all of which are hereby expressly
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
virology. More particularly, the invention relates to the discovery
that peptides that bind to the hepatitis B virus (HBV) core and e
antigens can be used to inhibit HBV infection.
BACKGROUND OF THE INVENTION
[0003] Of the many viral causes of human hepatitis, few are of
greater global importance than hepatitis B virus (HBV).
Approximately 300 million people worldwide are chronically infected
and some of these chronically infected individuals develop severe
pathologic consequences including chronic hepatic insufficiency,
cirrhosis, and hepatocellular carcinoma (HCC). (See Fields
Virology, third ed., edited by Fields et al., Lipponcott-Raven
Publishers, Philidelphia 1996 pp. 2703 and Lee et al., Cancer,
72:2564-7 (1993)). Primary infection may be asymptomatic (e.g., in
chronically infected individuals) or may result in varying degrees
of acute liver injury. (Milich et al., Springer Seminars in
Immunopathology, 17:149-66 (1995)).
[0004] HBV is unusual among animal viruses in that infected cells
produce multiple types of virus-related particles. (See Fields
Virology, third ed., edited by Fields et al., Lipponcott-Raven
Publishers, Philidelphia 1996 pp. 2704). Electron microscopy of
partially purified preparations of HBV shows three types of
particles, a 42-47 nm infectious particle (referred to as "Dane
particles"), non-infectious 20 nm spheres, and non-infectious 20 nm
diameter filaments of variable length. Id. at 2705-2705. The HBV
genome encodes at least five structural proteins: the envelope or
surface proteins preS1, preS2, and S (HBsAg); the polymerase; and
the core or capsid antigen (HBcAg). All three forms of HBV
particles have HBsAg, which serves as an epitope for neutralizing
antibodies and is the basis for state of the art HBV diagnostics.
In contrast, only the Dane particles have HBcAg, a 21 kD
phosphoprotein that is believed to be phosphorylated in vivo. Id.
at 2705. The HBV genome also encodes the non-structural proteins
HBeAg and X. The HBcAg and the HBeAg are translated from two
different mRNAs that are transcribed from the same open reading
frame. The longer of the two mRNAs encodes HBeAg. HBcAg and the
HBeAg share an amino acid sequence of approximately 150
residues.
[0005] HBcAg is highly immunogenic in humans and mice.
Investigators have observed that HBcAg induces B-cells to produce
IgM and, thus, is currently classified as a partially T cell
independent antigen. (Milich and McLachlan, Science, 234:1398-401
(1986)). HBcAg can also crosslink B-cell surface receptors and
membrane bound IgM on naive B-cells and, in turn, HBcAg can be
taken up, processed, and presented to HBcAg-specific CD4.sup.+ T
cells. (Milich et al., Proc Natl Acad Sci USA, 94:14648-14653
(1997)). Quite surprisingly, B-cells that are able to bind and
present HBcAg exist in great numbers in naive non-immunized mice.
The identification of molecules that inhibit HBV infection by
interacting with HBcAg and/or HBeAg remains a largely unrealized
goal.
BRIEF SUMMARY OF THE INVENTION
[0006] The invention described herein concerns the identification
and manufacture of molecules that interact with HBcAg and/or HBeAg
and thereby inhibit HBV infection or modulate a host immune system
response or both. Molecules that interact with HBcAg and/or HBeAg,
also referred to as "binding partners", are designed from fragments
of antibodies and other proteins that interact with HBcAg and/or
HBeAg. Accordingly, an amino acid sequence corresponding to the
binding domains of monoclonal or polyclonal antibodies or proteins
that bind HBcAg and/or HBeAg is used as a template for the design
of synthetic molecules, including but not limited to, peptides,
derivative or modified peptides, peptidomimetics, and chemicals. A
preferred binding partner, for example, is a molecule called a
"specificity exchanger", which comprises a first domain that
interacts with HBcAg and/or HBeAg and a second domain that has an
epitope for a high titer antibody, preferably an epitope on a
pathogen or a toxin. The binding partners described herein can be
manufactured by conventional techniques in peptide chemistry and/or
organic chemistry.
[0007] Methods to characterize binding partners are also
embodiments. The term "characterization assay" is used to refer to
an experiment or evaluation of the ability of a candidate binding
partner and/or binding partner to interact with HBcAg and/or HBeAg,
inhibit HBV infection, or modulate a host immune response. Some
characterization assays, for example, evaluate the ability of a
binding partner to bind to a multimeric agent having HBcAg and/or
HBeAg disposed thereon or vice versa. Other characterization assays
access the ability of a binding partner to fix complement and/or
bind to a high titer antibody. Additional characterization assays
determine whether a binding partner can effect viral infection in
cultured cell lines or infected animals. Still further, some
embodiments evaluate the ability of a binding partner to modulate a
host immune system response, as measured by cytokine production
and/or T cell proliferation.
[0008] Binding partners can be used as immunochemicals for the
detection of HBcAg and/or HBeAg and can be incorporated into
diagnostic methods and kits. Binding partners, preferably
specificity exchangers can also be incorporated into
pharmaceuticals and used to treat or prevent HBV infection. A
preferred embodiment concerns a method of treating or preventing
HBV infection by identifying a subject in need and administering
said subject a therapeutically effective amount of binding
partner.
[0009] As described herein, embodiments include a peptide that
binds HBcAg or HBeAg having about 3-50 amino acids residues.
Preferably, the sequence of said peptide is selected from the group
consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74. Other
embodiments include a peptide comprising the sequence of at least
one of SEQ. ID. Nos. 1-3, a peptide comprising the sequence of SEQ.
ID. No. 45, a peptide comprising the sequence of SEQ. ID. No. 54, a
peptide comprising the sequence of SEQ. ID. No. 74, and a peptide
having a specificity domain, which binds HBcAg or HBeAg and an
antigenic domain joined to the specificity domain, wherein said
antigenic domain binds a high titer antibody, preferably an epitope
for a pathogen or toxin.
[0010] Related embodiments concern a peptidomimetic that
corresponds to a peptide selected from the group consisting of SEQ.
ID. No. 1, 2, 3, 45, 54, and 74 and an isolated or purified peptide
that is less than 50 amino acids in length having the formula:
X.sup.1.sub.nCKASX.sup.2.sub.n, wherein "X.sup.1" and "X.sup.2" are
any amino acid and "n" is any integer, and wherein the molecule
specifically binds HBcAg and/or HBeAg. Another way of describing
the molecules of this class is by the formula:
"X.sup.1.sub.nCZASX.sup.2.sub.n", wherein: "X.sup.1" and "X.sup.2"
are any amino acid and "n" is any integer, "C" is cysteine, "Z" is
lysine or arginine", "A" is alanine, and "S" is serine. In some
embodiments, the "X.sup.1.sub.n" or "X.sup.2.sub.n" encodes an
epitope that binds a high titer antibody (e.g., an epitope on a
pathogen or a toxin). Other embodiments include an isolated or
purified peptide that is less than 50 amino acids in length having
the formula: X.sup.1.sub.nCRASX.sup.2.sub.n, wherein "X.sup.1" and
"X.sup.2" are any amino acid and "n" is any integer, and wherein
the molecule specifically binds HBcAg and/or HBeAg. As above,
another way of describing the molecules of this class is by the
formula: "X.sup.1.sub.nCZASX.sup.2.sub.n", wherein: "X.sup.1" and
"X.sup.2", are any amino acid and "n" is any integer, "C" is
cysteine, "Z" is lysine or arginine", "A" is alanine, and "S" is
serine. In some embodiments, "X.sup.1.sub.n" or "X.sup.2.sub.n"
encodes an epitope that binds a high titer antibody. Additional
embodiments include a nucleic acid encoding a peptide selected from
the group consisting of SEQ. ID. Nos. 1, 2, 3, 45, 54, and 74.
[0011] Some embodiments include a method of making a binding
partner that interacts with HBcAg or HBeAg. By one approach, a
region of a polypeptide that interacts with HBcAg or HBeAg is
identified, the sequence of said region of the polypeptide is
determined, and a synthetic or recombinant binding partner that
corresponds to the sequence of said region of the polypeptide is
produced. In some aspects of this embodiment, the polypeptide is an
antibody and, in other aspects, the binding partner is a
specificity exchanger. The specificity exchangers described herein
are bi-functional binding partners that have one domain
("specificity domain"), which binds to HBeAg, HBcAg, an HBV viral
capsid, or HBV itself, joined to a second domain ("antigenic
domain") that is recognized by an antibody present in a subject
(e.g., an antigenic domain comprising at least one of the sequences
of SEQ. ID. Nos. 79-95). Preferred specificity exchangers include
SEQ. ID. Nos: 5, 6, 13, 16, 17, 28, 29, 33, 36, 37, and 74.
[0012] Embodiments also include biological complexes comprising the
binding partners described herein, including specificity
exchangers, joined to HBeAg, HBcAg, an HBV viral capsid, HBV
itself, or fragments thereof. Preferred embodiments include
complexes comprising the binding partners described herein joined
to fragments of HBcAG including peptides comprising, consisting
essentially of, or consisting of the peptides of SEQ. ID. Nos: 103
and 104 or nucleic acids encoding these molecules. These peptides
and nucleic acids can be used as research tools or components for
vaccines.
[0013] More embodiments include methods of making a pharmaceutical.
By one approach, a binding partner that interacts with HBcAg or
HBeAg is identified and a therapeutically effective amount of said
binding partner is incorporated into a pharmaceutical. In preferred
aspects of this method, the binding partner has a sequence selected
from the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71,
and 74. Another method described herein concerns an approach to
treat or prevent HBV infection. Accordingly, a subject in need of a
molecule that inhibits HBV infection is identified and said subject
is provided a binding partner that interacts with HBcAg or HBeAg,
or both. Preferred aspects of this method involve a binding partner
that has a sequence selected from the group consisting of SEQ. ID.
Nos. 4-45, 53, 54, 66-69, 71, and 74.
[0014] Methods of identifying a binding partner that interacts with
HBcAg or HBeAg are also embodiments. By one approach, a support
comprising HBcAg or HBeAg is provided, the support is contacted
with a candidate binding partner, and a biological complex
comprising HBcAg or HBeAg and the candidate binding partner is
detected, wherein detection of such complex indicates that said
candidate binding partner is a binding partner interacts with HBcAg
or HBeAg. In preferred aspects of this embodiment, the candidate
binding partner has an amino acid sequence selected from the group
consisting of SEQ. ID. Nos. 1-78. Another method of identifying a
binding partner that inhibits HBV infection involves providing a
cell that is infected with HBV, contacting said cell with a
candidate binding partner, and identifying said binding partner
when the presence of said candidate binding partner with said cell
is associated with a decrease in HBV infection.
[0015] Furthermore, methods are provided that identify a binding
partner that modulates an immune system response. Accordingly, one
method is practiced by providing a naive antigen presenting cell,
contacting said naive antigen presenting cell with a binding
partner and a T cell that reacts to HBcAg or HBeAg, and detecting
an inhibition or enhancement of T cell stimulation. In some
embodiments, the detection step is performed by evaluating a change
in cytokine production or T cell proliferation.
[0016] In another embodiment, a computerized system for identifying
a binding partner that interacts with HBcAg or HBeAg is provided.
This system includes a first data base comprising protein models of
HBcAg or HBeAg; a second data base comprising the composition of a
plurality of candidate binding partners; a search program that
compares the protein model of the first data base with the
compositions of the candidate binding partners of the second
database; and a retrieval program that identifies a binding partner
that interacts with the protein model of the first database. In
some aspects of this embodiment, the candidate binding partners
have an amino acid sequence selected from the group consisting of
SEQ. ID. Nos. 1-78.
[0017] Additionally, a computer-based system for identifying a
candidate binding partner having homology to a binding partner is
provided. This system has a database with at least one of the
sequences of SEQ ID NOS: 1-78 or a representative fragment thereof,
a search program that compares a sequence of a candidate binding
partner to sequences in the database to identify homologous
sequence(s), and a retrieval program that obtains said homologous
sequence(s).
[0018] A method of determining the presence of HBV in a biological
sample is also an embodiment. This method is practiced by providing
a biological sample, providing a binding partner that binds to
HBcAg and/or HBeAg, wherein said binding partner has a sequence
selected from the group consisting of SEQ. ID. Nos. 4-45, 53, 54,
66-69, 71, and 74, and determining the presence of HBV in the
biological sample by monitoring whether said binding partner binds
to HBcAg and/or HBeAg. Diagnostic kits for the detection of HBV
infection are embodiments, as well. One such kit has a binding
partner, wherein said binding partner has a sequence selected from
the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and
74.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The disclosure herein describes the manufacture,
characterization, and use of molecules that bind hepatitis B virus
(HBV) core (HBcAg) and e (HBeAg) antigens and thereby inhibit HBV
infection and/or modulate a host immune system response. The
molecules that bind to HBcAg and/or HBeAg, such as peptides,
modified or derivatized peptides, peptidomimetics, and chemicals,
are collectively referred to as "binding partners". Binding
partners can be obtained by synthesizing the heavy (VH) and light
(VL) chains of antibodies (e.g., polyclonal, monoclonal, or
fragments thereof), synthesizing the domains of proteins that
interact with HBcAg and/or HBeAg, and by employing techniques in
rational drug design and combinatorial chemistry.
[0020] Several synthetic peptides, derived from the variable
domains of monoclonal antibodies (mAbs) specific for the hepatitis
B virus HBcAg and/or HBeAg, were obtained as follows. The mRNAs
encoding the VH and VL chains of HBcAg and/or HBeAg monoclonal
antibodies (mAbs) were sequenced and the protein sequences
corresponding to these mRNAs were determined. Several synthetic
peptides corresponding to these sequences were then synthesized
using conventional protein chemistry. These "candidate binding
partners", which have the potential to bind HBcAg and/or HBeAg,
were tested for the ability to interact with HBcAg and HBeAg. Five
peptides, in particular, were discovered to bind HBcAg and/or HBeAg
with high affinity and these "high affinity" binding partners had
either the conserved motif "CKAS" (SEQ. ID. No. 77) or "CRAS" (SEQ.
ID. No. 78). Thus, preferred embodiments include peptides,
derivative or modified peptides, or peptidomimetics having the
formula "X.sup.1.sub.nCKASX.sup.2.sub.n" or
"X.sup.1.sub.nCRASX.sup.2.sub.n", wherein "X.sup.1" and "X.sup.2"
are any amino acid and "n" is an integer, that bind HBcAg and/or
HBeAg. Another way of describing the molecules of this class is by
the formula: "X.sup.1.sub.nCZASX.sup.2.sub.n", wherein: "X.sup.1"
and "X.sup.2" are any amino acid and "n" is any integer, "C" is
cysteine, "Z" is lysine or arginine", "A" is alanine, and "S" is
serine.
[0021] By a similar approach, synthetic peptides corresponding to
the binding domains of polyclonal antibodies specific for HBcAg
and/or HBeAg can be manufactured. Polyclonal antibodies specific
for HBcAg and/or HBeAg are generated by inoculating animals with
HBcAg and/or HBeAg. The mRNAs encoding the polyclonal antibodies
are isolated, sequenced, and the protein sequences corresponding to
these mRNAs are determined. Synthetic peptides corresponding to
these protein sequences are then made using conventional techniques
in protein chemistry. Several strategies for obtaining the mRNAs
that encode polyclonal antibodies that bind HBcAg and/or HBeAg are
contemplated including, but not limited to, yeast one-hybrid
screens, yeast two-hybrid screens, and phage display techniques.
Ideally, cDNA expression libraries corresponding to mRNAs encoding
polyclonal antibodies that bind HBcAg and/or HBeAg are created.
Embodiments that employ such libraries can express recombinant
binding partners, which can be isolated or purified, characterized,
and used in lieu of or in addition to synthetic binding
partners.
[0022] The term "isolated" requires that the material be removed
from its original environment (e.g., the natural environment if it
is naturally occurring). For example, a naturally-occurring
polypeptide present in a living animal is not isolated, but the
same polypeptide, separated from some or all of the coexisting
materials in the natural system, is isolated. It is also
advantageous that the sequences be in purified form. The term
"purified" does not require absolute purity; rather, it is intended
as a relative definition. Isolated proteins have been
conventionally purified to electrophoretic homogeneity by Coomassie
staining, for example. Purification of starting material or natural
material to at least one order of magnitude, preferably two or
three orders, and more preferably four or five orders of magnitude
is expressly contemplated.
[0023] In addition to antibodies and peptides derived from
antibodies, other molecules that bind HBcAg and/or HBeAg can be
identified by the methods described herein. That is, techniques in
high throughput screening, combinatorial chemistry, and rational
drug design can be employed to identify more binding partners. By
one approach, for example, a high throughput screen based on a
yeast two hybrid system is employed.
[0024] Accordingly, cDNA expression libraries are generated from
any organism (e.g., plant, bacteria, virus, insect, amphibian,
reptile, bird, or mammal) or a plurality of such organisms or from
random or directed oligonucleotide synthesis. In some embodiments,
the organism will have been immunized for HBcAg and/or HBeAg prior
to creation of the cDNA expression library. To create the cDNA
library for the yeast two-hybrid screen, isolated cDNA made from
total mRNA obtained from the organism or generated by
oligonucleotide synthesis is cloned into a first expression
construct having a nucleic acid encoding a transcriptional
activation domain (e.g., GAL4). As one of skill will appreciate,
the cloning of the first expression construct is conducted such
that cells having the construct will express a fusion protein
comprising the cDNA of interest and the transcriptional activation
domain when induced.
[0025] Next, a second expression construct (referred to as the
"bait") is made. This construct has nucleic acid encoding HBcAg
and/or HBeAg or fragments thereof joined to a transcriptional
binding domain (e.g., GAL4). When a cell having the second
expression construct is properly induced, a second fusion protein
comprising the HBcAg and/or HBeAg or fragments thereof joined to
the transcriptional binding domain (the "bait") is expressed. The
two expression constructs are transferred into yeast, which harbor
a DNA template having at least one DNA binding domain specific for
the transcriptional binding domain encoded by the bait construct, a
minimal promoter, and a downstream reporter gene (e.g., Lac Z or
Green Fluorescent Protein (GFP)). When a peptide from the cDNA
library (i.e., the fusion protein expressed from the first
construct) binds to the bait (i.e., the fusion protein from the
second construct) a detectable signal is generated from the
reporter gene. The yeast clones can be presented in addressable
arrays, which allows for the precise determination of the clone
containing an insert that encodes a protein that binds HBcAg and/or
HBeAg. In this manner, protein/protein interactions between HBcAg
and/or HBeAg and the proteins expressed from the cDNA library can
be rapidly identified.
[0026] Clones that display a signal after induction of the first
and second constructs but fail to produce a signal without
induction are isolated, amplified, and the cDNA inserts are
sequenced. The nucleic acid sequence information can be converted
to an amino acid sequence and peptides corresponding to these
sequences can be synthesized by conventional protein chemistry.
Alternatively, recombinant peptides expressed from the positive
clones can be isolated and/or purified. These candidate binding
partners can then be screened for the ability to interact with
HBcAg and/or HBeAg. The binding partners identified by the
approaches above can also be used as templates for the design of
modified or derivative peptides, peptidomimetics, and for rational
drug design. For example, computer modeling and combinatorial
chemistry can be employed to design and manufacture derivative
binding partners.
[0027] The term "binding partner" also refers to a bi-functional
binding partner or "specificity exchanger" comprising a
"specificity domain", which binds HBcAg and/or HBeAg, and an
"antigenic domain", which binds an antibody or other molecule that
can be unrelated to a molecule that binds HBcAg and/or HBeAg.
Desirable antigenic domains have an epitope found on a pathogen
such as a bacteria, virus, fungi, or mold or a toxin. Preferred
antigenic domains have epitopes that bind high titer antibodies
(e.g., the antigenic domains described in SEQ. ID. Nos. 79-85).
These bi-functional binding partners can redirect antibodies that
already exist in an organism to a desired antigen. Such specificty
exchangers can be manufactured by joining a molecule that binds
HBcAg and/or HBeAg, such as a binding partner identified by a
method described above, to an epitope for any antibody using
conventional techniques in molecular biology. In one embodiment,
for example, a specificity domain comprising the CKAS (SEQ. ID. No.
77) motif was joined to an antigenic domain comprising the epitope
for a monoclonal antibody specific for the herpes simplex virus
type 1 gG2 (HSVgG2) protein.
[0028] Desirably, the binding partners are evaluated in a
"characterization assay", which determines the ability of the
molecule to interact with HBcAg and/or HBeAg, inhibit HBV
infectivity, or modulate (inhibit or enhance) a host immune system
response. Several characterization assays described herein involve
binding assays that analyze whether a binding partner can interact
with HBcAg and/or HBeAg and to what extent a binding partner can
compete with other ligands for HBcAg and/or HBeAg (e.g., multimeric
support-based assays and computer generated binding assays).
Additionally, some characterization assays determine the efficacy
of binding partners as inhibitors of HBV infection in vitro and in
vivo. Further, characterization assays are designed to analyze
whether a binding partner can modulate a host immune system
response, as indicated by the activation of an antigen presenting
cell (e.g., a B cell or dendritic cell), production of a cytokine,
or T cell proliferation.
[0029] The binding partners can be used as biotechnological tools,
diagnostic reagents, and the active ingredients in pharmaceuticals.
In some embodiments, for example, the binding partners are used as
detection reagents in conventional immunohistochemical techniques.
In other embodiments, the binding partners are expressed in a cell
in vitro or in vivo. Still in other embodiments, the binding
partners are used as diagnostic reagents to detect the presence or
absence of HBV in a biological sample obtained from a subject.
According to this later aspect, the binding partners can also be
used to determine the efficacy of an HBV treatment protocol by
monitoring the levels of HBcAg and/or HBeAg before, during, and
after treatment.
[0030] Further, binding partners can be incorporated into
pharmaceuticals that can be administered to subjects in need of an
agent that interacts with HBcAg and/or HBeAg, such as a human in
need of treatment and/or prevention of HBV infection. Preferably,
these pharmaceuticals comprise formulations having a specificity
exchanger that promotes rapid clearance of HBV particles.
Additionally, the pharmaceuticals can include nucleic acid
constructs manufactured such that binding partners (preferably
specificity exchangers) are expressed in a variety of cells of the
body. The pharmaceuticals can be administered to individuals in
need of treatment and/or prevention of HBV infection. The section
below describes several approaches to identify and manufacture
binding partners specific for HBcAg and/or HBeAg.
[0031] Identification and Manufacture of Binding Partners Specific
for HBcAg and/or HBeAg
[0032] In general, the approach to make the binding partners
described herein involves: (1) obtaining molecules that bind to
HBcAg and/or HBeAg; (2) determining the molecular structure or
sequence of said molecules; and (3) synthesizing peptides that have
said molecular structure or sequence. In one aspect, for example,
antibodies or other peptides that bind to HBcAg and/or HBeAg are
generated and/or identified; the mRNA sequence encoding the binding
partner is obtained, converted to cDNA, and sequenced; and, from
this sequence, peptides are synthesized. The HBcAg and
HBeAg-specific peptides can be modified, derivatized, and can also
be used as templates for the design of peptidomimetics and rational
drug discovery. Through techniques in combinatorial chemistry and
rational drug design, many more binding partners can be identified.
The term "binding partner" refers to a molecule that binds HBcAg
and/or HBeAg, and should be distinguished from the term "candidate
binding partner", which refers to a molecule that potentially binds
to HBcAg and/or HBeAg. Desirably, binding partners inhibit viral
infectivity and/or modulate (inhibit or enhance) a host immune
system response (e.g., antigen presenting cell activation, cytokine
production, and/or T cell proliferation).
[0033] By one approach, the design and manufacture of peptides that
bind HBcAg and HBeAg involves the manufacture of mAbs directed to
HBcAg and HBeAg. Depending on the context, the term "antibodies"
can encompass polyclonal, monoclonal, chimeric, single chain, Fab
fragments and fragments produced by a Fab expression library.
Furthermore, the terms "low titer antibody" and "high titer
antibody" are also used to refer to an antibody having a low
avidity to an antigen and a high avidity to an antigen,
respectively. That is, whether a particular antibody is a "low
titer antibody" or a "high titer antibody" depends on the dilution
of antibody containing sera at which an antigen is no longer
detectable in an enzyme immunoassay (e.g., an enzyme immunoassay
(EIA) or ELISA assay); 200 ng of target antigen is typically used
with a 1:1000 dilution of secondary antibody. Thus, a "low titer
antibody" generally no longer detects an antigen at a dilution that
is less than 1:10000 under the conditions for ELISA described above
and a "high titer antibody" is characterized by the ability to
detect an antigen at a dilution that is greater than or equal to
1:10000.
[0034] For the production of antibodies, whether monoclonal or
polyclonal, various hosts including goats, rabbits, rats, mice,
etc. can be immunized by injection with HBcAg and/or HBeAg or any
portion, fragment or oligopeptide that retains immunogenic
properties. Depending on the host species, various adjuvants can be
used to increase immunological response. Such adjuvants include,
but are not limited to, Freund's, mineral gels such as aluminum
hydroxide, and surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin, and dinitrophenol. BCG (Bacillus
Calmette-Guerin) and Corynebacterium parvum are also potentially
useful adjuvants.
[0035] Peptides used to induce specific antibodies can have an
amino acid sequence consisting of at least three amino acids, and
preferably at least 10 to 15 amino acids. Short stretches of amino
acids encoding fragments of HBcAg and/or HBeAg can be fused with
those of another protein such as keyhole limpet hemocyanin such
that an antibody is produced against the chimeric molecule. While
antibodies capable of specifically recognizing HBcAg and/or HBeAg
can be generated by injecting synthetic 3-mer, 10-mer, and 15-mer
peptides that correspond to a protein sequence of a binding partner
into an appropriate organism, a more diverse set of antibodies are
generated by using recombinant HBcAg and/or HBeAg.
[0036] Monoclonal antibodies directed to HBcAg and/or HBeAg can be
prepared using any technique that provides for the production of
antibody molecules by continuous cell lines in culture. These
include, but are not limited to, the hybridoma technique originally
described by Koehler and Milstein (Nature 256:495-497 (1975)), the
human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72
(1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the
EBV-hybridoma technique Cole et al. Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985)).
In addition, techniques developed for the production of "chimeric
antibodies", the splicing of mouse antibody genes to human antibody
genes to obtain a molecule with appropriate antigen specificity and
biological activity can be used. (Morrison et al. Proc Natl Acad
Sci 81:6851-6855 (1984); Neuberger et al. Nature 312:604-608(1984);
Takeda et al. Nature 314:452-454(1985)). Alternatively, techniques
described for the production of single chain antibodies (U.S. Pat.
No. 4,946,778) can be adapted to produce HBcAg and/or
HBeAg-specific single chain antibodies. Antibodies can also be
produced by inducing in vivo production in the lymphocyte
population or by screening recombinant immunoglobulin libraries or
panels of highly specific binding reagents as disclosed in Orlandi
et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and
Milstein C; Nature 349:293-299 (1991).
[0037] Antibody fragments that contain specific binding sites for
HBcAg and/or HBeAg can also be generated. For example, such
fragments include, but are not limited to, the F(ab').sub.2
fragments that can be produced by pepsin digestion of the antibody
molecule and the Fab fragments that can be generated by reducing
the disulfide bridges of the F(ab').sub.2 fragments. Alternatively,
Fab expression libraries can be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
[0038] By one approach, monoclonal antibodies to HBcAg and/or HBeAg
or fragments thereof are made as follows. Briefly, a mouse is
repetitively inoculated with a few micrograms of the selected
protein or peptides derived therefrom over a period of a few weeks.
The mouse is then sacrificed, and the antibody producing cells of
the spleen isolated. The spleen cells are fused in the presence of
polyethylene glycol with mouse myeloma cells, and the excess
unfused cells destroyed by growth of the system on selective media
comprising aminopterin (HAT media). The successfully fused cells
are diluted and aliquots of the dilution are placed in wells of a
microtiter plate where growth of the culture is continued.
Antibody-producing clones are identified by detection of antibody
in the supernatant fluid of the wells by immunoassay procedures,
such as ELISA, as originally described by Engvall, E., Meth.
Enzymol. 70:419 (1980), and derivative methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Davis, L. et al. Basic Methods
in Molecular Biology Elsevier, New York. Section 21-2. By using the
approach described in Example 1, several monoclonal antibodies
specific for HBcAg and/or HBeAg were made.
EXAMPLE 1
[0039] The mAbs that were used to create the binding partners
specific for HBcAg and/or HBeAg were made by conventional
techniques, as described above, using recombinant peptides. Full
length recombinant HBcAg (rHBcAg) encompassing residues 1-183 was
produced in Escherichia coli, as previously described. (Schodel et
al., J Biol Chem, 268:1332-7 (1993), herein expressly incorporated
by reference in its entirety). A truncated recombinant form of
HBeAg containing nine residues of pre-core and the 150 first
residues of HBcAg was also made. Further, a non-structural 3
protein (NS3) of the hepatitis C virus (HCV) was also made to serve
as a control. (Jin and Peterson, Arch. Biochem. Biophys., 323:47-53
(1995), herein expressly incorporated by reference in its
entirety). Another control peptide, an analogue of HBcAg
(.DELTA.HBcAg), wherein region 76-85 was replaced by an irrelevant
sequence, was also made.
[0040] Balb/c and CBA mice (purchased from BK Universal,
Sollentuna, Sweden) were immunized (Freunds complete adjuvant; CFA)
and boosted (Freunds incomplete adjuvant; IFA) by 10 .mu.g of the
recombinant peptides. The mice were immunized one to three times,
with two weeks between each immunization. Three days after the last
injection, spleen cells were harvested and fused with the SP2/0
myeloma cells by standard procedures. The SP2/0 cell lines
expressing mAbs were maintained in RPMI-1640 medium supplemented
with 10% FCS, 2 mM L-Glutamine, 100 U/ml Penicilin and 100 .mu.g/ml
Streptomycin (GIBCO-BRL, Gaithesburgh, Md.). All cells were
incubated at 37.degree. C. with 7% CO.sub.2. Following three cycles
of cloning and screening by enzyme immunoassay (EIA) using the
indicated antigens, stable hybridomas were selected for antibody
analysis and extraction of mRNA. The antibodies were purified on
immobilized protein A/G (Pierce, Rockford, Ill.). By using the
approach described in this example, several mAbs that bind HBcAg
and/or HBeAg were obtained.
[0041] Four of the mAbs obtained by the approach described in
Example 1 were characterized for their reactivity to HBcAg,
.DELTA.HBcAg, denaturated HBcAg, and HBeAg. The example below
describes the approach that was used to characterize the reactivity
of mAbs directed to HBcAg and/or HBeAg.
EXAMPLE 2
[0042] To determine the reactivity and specificity of a mAb,
recombinant proteins or fragments thereof (e.g., HBcAg, HBeAg,
.DELTA.HBcAg, denatured HBcAg, or NS3 proteins) were 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. Serial
dilutions of mAbs were made in phosphate buffered saline (PBS)
containing 2% goat serum (Sigma Chemicals, St Louis, Mo.), and
0.05% Tween 20 (PBS-GT). The various dilutions were then incubated
on the plates for 60 minutes. Bound mAbs were detected either by
rabbit anti-mouse IgG (Sigma), or rabbit anti-mouse IgG1, IgG2a,
IgG2b or IgG3 (Sigma) followed by a peroxidase labeled goat
anti-rabbit IgG (Sigma). The plates were developed by incubation
with dinitro-phenylene-diamine (Sigma) and the absorbance at 490 nm
was determined. Additionally, the reactivity in Abbott anti-HBc and
HBe IMX assays was determined according to the manufacturer's
instructions. The results of these studies are provided in TABLE 1.
Further analysis revealed that none of the mAbs were reactive in
the Sorin anti-HBe assay (Sorin Biomedica, Sluggia, Italy) but the
4-2 mAb was reactive in the Abbott IMX anti-Hbe assay, indicating
an unusual specificity. TABLE-US-00001 TABLE 1 MAb reactivity to
HBcAg, mutant HBcAg (.DELTA.HBcAg), denaturated HBcAg (dHBcAg), and
HBeAg*. Reactivity in Abbott IMX assay for anti-HBc or Endpoint
titre to indicated antigen Dominating anti-HBe MAb HBcAg
.DELTA.HBcAg dHBcAg HBeAg specificity in EIA HBc HBe 3-4 3.125 1 1
15.625 HBeAg - - 4-2 3.125 625 15.625 125 dHBcAg - + 5H7 625 0 1 1
HBcAg - - 9C8 78.125 25 25 25 HBcAg - - *Values are given as the
endpoint titres (the highest dilution giving an OD at 490 nm of
three times the negative control).
[0043] Once the specificity of binding of the mAbs was determined,
the mRNAs encoding the VH and/or VL domains of three mAbs (i.e.,
mAbs 4-2, 5H7, and 9C8) were sequenced and these mRNA sequences
were converted to protein sequences. The next example describes the
method that was used to determine the protein sequence of a binding
domain of an antibody that interacts with HBcAg and/or HBeAg.
EXAMPLE 3
[0044] To determine the protein sequence of an antibody binding
domain, total cellular mRNA was extracted using magnetic beads
coated with oligo-dT25 (Dynal A. S, Oslo, Norway). The variable
domains of the heavy (VH) and light (VL) chains of mAbs were
amplified from cDNA by the Polymerase Chain Reaction (PCR) using
the recombinant phage antibody system (Pharmacia Biotech, Uppsala,
Sweden). The amplified cDNA fragments were directly ligated to the
TA cloning vector pCR 2.1 (Invitrogen, San Diego, USA) as
described. (Zhang et al., Clin. Diagn. Lab. Immunol., 7:58-63
(2000), herein expressly incorporated by reference in its
entirety). The DNA sequences were determined by an automated
sequencer (ALF express, Pharmacia, Uppsala, Sweden) as described.
(Zhang et al., Clin. Diagn. Lab. Immunol., 7:58-63 (2000), herein
expressly incorporated by reference in its entirety). From the cDNA
sequence, a corresponding protein sequence was deduced. The protein
sequences deduced from VH cDNA clones of mAbs 4-2 and 9C8 and VL
cDNA clone 5H7 are provided in TABLE 2 and in the Sequence Listing
(SEQ. ID. Nos. 1-3). The approach described above can be used to
determine the protein sequence of the binding domain of either a
monoclonal or polyclonal antibody. TABLE-US-00002 TABLE 2 The
deduced VH and/or VL sequences of mAbs 4-2, 5H7 and 9C8. 4-2 VH
VKLQQSGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQTPEQGLEWIGWIFPGE (SEQ. ID.
No. 1) GSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSEDSAVYFCARGDYDYYRRYF
DLWGQGTTVTVS 5H7 VL
DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYA (SEQ. ID.
No. 2) SNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEI
KRADAAPAVSIFPPSSKLG 9C8 VH
IQLQQSGAELVKPGASVKISCKASGYSFTGYNMNWVKQSHGKSLEWIGNINPY (SEQ. ID. No.
3) YGSTSYNQKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARGKGTGFAY
WGQGTLVTVSAAKTTPPSVYPLVPV
[0045] Synthetic peptides corresponding to the VH and/or VL
sequences of mAbs 4-2, 5H7, and 9C8 or fragments thereof were then
synthesized by conventional techniques in protein chemistry. These
synthetic peptides are referred to as "candidate binding partners"
because they are molecules that potentially bind HBcAg and/or
HBeAg. The example below describes an approach that was used to
synthesize a peptide that corresponds to the binding domain of a
mAb specific for HBcAg and/or HBeAg.
EXAMPLE 4
[0046] Peptides that correspond to regions of a binding domain of a
mAb specific for HBcAg and/or HBeAg were manufactured as follows.
Overlapping peptides (20 amino acids long with a 10 amino acid
residue overlap) corresponding to the VH and/or VL of the mAbs 4-2,
5H7, and 9C8 were produced by standard techniques (Sallberg et al.,
Immunol Lett, 30:59-68 (1991), herein expressly incorporated by
reference in its entirety) using a multiple peptide synthesizer and
standard Fmoc chemistry (Syro, MultiSynTech, Germany). Additional
deletion and alanine substitution analogues of reactive peptides
were synthesized by the same technique. In some cases the peptides
were purified by high performance liquid chromatography using
standard protocols. (Sallberg et al., Immunol Lett, 30:59-68
(1991), herein expressly incorporated by reference in its
entirety). There are many ways to synthesize or produce the
peptides described herein (e.g., through recombinant technology)
and the approach described above is one such method.
[0047] In addition to using monoclonal antibodies, the design and
manufacture of peptides that bind HBcAg and HBeAg can involve the
manufacture of polyclonal antibodies directed to HBcAg and HBeAg.
Accordingly, animals are repetitively inoculated with HBcAg and/or
HBeAg so as to raise a population of high titer polyclonal
antibodies specific for HBcAg and/or HBeAg. The mRNAs that encode
the polyclonal antibodies are isolated, converted to cDNA, and the
protein sequences corresponding to these cDNAs are deduced. (See
Examples 3 and 4 for an approach to manufacture peptides that
correspond to a binding domain of an antibody). Synthetic peptides
having these protein sequences are then made using conventional
techniques in peptide chemistry.
[0048] Polyclonal antiserum containing antibodies to heterogenous
epitopes of a single protein can be prepared by immunizing suitable
animals with HBcAg and/or HBeAg or fragments thereof, which can be
unmodified or modified to enhance immunogenicity. Effective
polyclonal antibody production is affected by many factors related
both to the antigen and the host species. For example, small
molecules tend to be less immunogenic than others and can require
the use of carriers and adjuvant. Also, host animals vary in
response to site of inoculations and dose, with both inadequate or
excessive doses of antigen resulting in low titer antisera. Small
doses (ng level) of antigen administered at multiple intradermal
sites appears to be most reliable. An effective immunization
protocol for rabbits can be found in Vaitukaitis, J. et al. J.
Clin. Endocrinol. Metab. 33:988-991 (1971), herein expressly
incorporated by reference in its entirety.
[0049] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. (See
e.g., Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental
Immunology D. Wier (ed) Blackwell (1973), herein expressly
incorporated by reference in its entirety). Plateau concentration
of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum
(about 12 .mu.M). Affinity of the antisera for the antigen is
determined by preparing competitive binding curves, as described,
for example, by Fisher, D., Chap. 42 in: Manual of Clinical
Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For
Microbiol., Washington, D.C. (1980), herein expressly incoprporated
by reference in its entirety.
[0050] Once it can be verified that polyclonal antibodies directed
to HBcAg and/or HBeAg have been generated, the mRNA obtained from
the spleens of animals inoculated with HBcAg and/or HBeAg can be
obtained and used as a template for the synthesis of cDNA. This
cDNA, which represents (among other things) RNA encoding regions of
polyclonal antibodies, can be inserted into phage that are
engineered such that the cDNA insert is expressed and displayed on
the surface of the phage. That is, a "phage display" cDNA library
can be created from the cDNA corresponding to the mRNA encoding
polyclonal antibodies that bind HBcAg and/or HBeAg. Many phage
display kits that are suitable for this testing are commercially
available.
[0051] Once the phage display library is obtained, a technique
called "panning" is employed to isolate phage having an insert that
encodes a peptide that binds HBcAg and/or HBeAg. Accordingly, HBcAg
and/or HBeAg or fragments thereof are disposed on a support (e.g.,
a plate) and are brought in contact with the phage display library.
After a sufficient time for binding has occurred, unbound phage are
removed by successive washes with a isotonic buffer. Next, a plate
having a bacterial lawn is brought in contact with the phage that
remain bound to the plate having immobilized HBcAg and/or HBeAg.
The two plates are held in position for sufficient time for
infection of the bacteria and, after inoculation, the plate is
incubated overnight at 37.degree. C. The appearance of clear zones
on the bacterial lawn, indicative of phage proliferation, provides
evidence that the phage within the zone contain a cDNA that encodes
a peptide that binds HBcAg and/or HBeAg. The DNA from such phage
can be isolated, sequenced, and the protein sequence of the binding
peptides can be deduced, as described above. Synthetic peptides can
then be manufactured based on these sequences. Further, the cDNA
inserts from positive binding phage can be subcloned into cDNA
expression libraries for the production of recombinant binding
partners.
[0052] Another approach to isolate molecules that bind HBcAg and/or
HBeAg takes advantage of techniques developed to analyze
protein/protein interactions. Conventional one and two hybrid
systems, for example, can be readily adapted to identify binding
partners. Such approaches include: [0053] (1) the two-hybrid
systems (Field & Song, Nature 340:245-246 (1989); Chien et al.,
Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991); and Young K H,
Biol. Reprod. 58:302-311 (1998), all of which are expressly
incorporated by reference in their entirety); [0054] (2) reverse
two-hybrid system (Leanna & Hannink, Nucl. Acid Res.
24:3341-3347 (1996), herein incorporated by reference in their
entirety); [0055] (3) repressed transactivator system (Sadowski et
al., U.S. Pat. No. 5,885,779), herein incorporated by reference in
their entirety); [0056] (4) phage display (Lowman H B, Annu. Rev.
Biophys. Biomol. Struct. 26:401-424 (1997), herein incorporated by
reference in their entirety); and [0057] (5) GST/HIS pull down
assays, mutant operators (Granger et al., WO 98/01879) and the like
(See also Mathis G., Clin. Chem. 41:139-147 (1995); Lam K. S.
Anticancer Drug Res., 12:145-167 (1997); and Phizicky et al.,
Microbiol. Rev. 59:94-123 (1995), all of which are expressly
incorporated by reference in their entirety).
[0058] An adaptation of the system described by Chien et al., 1991,
Proc. Natl. Acad. Sci. USA, 88:9578-9582, herein incorporated by
reference in its entirety, which is commercially available from
Clontech (Palo Alto, Calif.) is as follows. Plasmids are
constructed that encode two hybrid proteins: one plasmid consists
of nucleotides encoding the DNA-binding domain of a transcription
activator protein fused to a nucleotide sequence encoding HBcAg
and/or HBeAg and the other plasmid consists of nucleotides encoding
the transcription activator protein's activation domain fused to a
cDNA encoding a candidate binding partner. The DNA-binding
domain/HBcAg and/or HBeAg fusion plasmid and the candidate binding
partner cDNA are transformed into a strain of the yeast
Saccharomyces cerevisiae that contains a reporter gene (e.g., GFP
or lacZ) whose regulatory region contains the transcription
activator's binding site. Either hybrid protein alone cannot
activate transcription of the reporter gene: the DNA-binding domain
hybrid cannot because it does not provide activation function and
the activation domain hybrid cannot because it cannot localize to
the activator's binding sites. Interaction of the two hybrid
proteins reconstitutes the functional activator protein and results
in expression of the reporter gene, which is detected by an assay
for the reporter gene product.
[0059] The two-hybrid system or related methodology can also be
used to rapidly screen candidate binding partner libraries
generated from animals for proteins that interact with the "bait"
gene product (HBcAg and/or HBeAg/DNA binding domain). For example,
total cDNA (representing total mRNA) generated from animals that
may or may not have polyclonal antibodies specific for HBcAg and/or
HBeAg can be fused to the DNA encoding an activation domain. This
library and a plasmid encoding a hybrid of a bait gene encoding the
HBcAg and/or HBeAg product fused to the DNA-binding domain are
co-transformed into a yeast reporter strain, and the resulting
transformants are screened for those that express the reporter
gene.
[0060] For example, and not by way of limitation, a bait gene
sequence encoding HBcAg and/or HBeAg can be cloned into a vector
such that it is translationally fused to the DNA encoding the
DNA-binding domain of the GAL4 protein. These colonies are purified
and the library plasmids responsible for reporter gene expression
are isolated. DNA sequencing is then used to identify the proteins
encoded by the library plasmids.
[0061] A cDNA/activation domain library representative of the total
mRNA from an organism that may or may not have polyclonal
antibodies directed to HBcAg and/or HBeAg and an activation domain
that interacts with the HBcAg and/or HBeAg/DNA binding domain
fusion protein can be made using methods routinely practiced in the
art. According to the particular system described herein, for
example, the cDNA fragments can be inserted into a vector such that
they are translationally fused to the transcriptional activation
domain of GAL4. This library can be co-transformed along with the
bait HBcAg and/or HBeAg gene-GAL4 fusion plasmid into a yeast
strain that contains a LacZ gene driven by a promoter which
contains GAL4 activation sequence. A cDNA encoded protein, fused to
GAL4 transcriptional activation domain, that interacts with bait
HBcAg and/or HBeAg gene product will reconstitute an active GAL4
protein and thereby drive expression of the LacZ gene. Colonies
that express lacZ can be detected and the cDNA can then be purified
from these strains, sequenced, and synthetic peptides corresponding
to these sequences can be generated, derivatized modified, or used
as templates for rational drug design. The section below describes
binding partners in greater detail.
[0062] Binding Partners Specific for HBcAg and/or HBeAg
[0063] While the most preferred peptide embodiments are at least 13
amino acids in length, many candidate binding partners and binding
partners are between about 3 amino acids and about 100 amino acids
or more in length. That is, desirable candidate binding partners
and binding partners are about 3-125 amino acids in length, more
desirable candidate binding partners and binding partners are
between about 3-100 amino acids in length, preferred candidate
binding partners and binding partners are between about 3-75 amino
acids in length, more preferred candidate binding partners and
binding partners are between about 3-50 amino acids in length, and
most preferred candidate binding partners and binding partners are
between about 13-25 amino acids in length. Some embodiments, for
example have the formula "X.sup.1.sub.nCKASX.sup.2.sub.n" or
"X.sup.1.sub.nCRASX.sup.2.sub.n", wherein "X.sup.1" and "X.sup.2"
are any amino acid and "n" is any integer, and the molecule
specifically binds HBcAg and/or HBeAg. Another way of describing
the molecules of this class is by the formula:
"X.sup.1.sub.nCZASX.sup.2.sub.n", wherein: "X.sup.1" and "X.sup.2"
are any amino acid and "n" is any integer, "C" is cysteine, "Z" is
lysine or arginine", "A" is alanine, and "S" is serine.
[0064] The peptides not only include those molecules containing as
a primary amino acid sequence all or part of the amino acid
sequence of SEQ. ID. Nos.1-78, for example, but also altered
sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a silent
change. Accordingly, one or more amino acid residues within the
sequence of SEQ. ID. Nos. 1-78 can be substituted by another amino
acid of a similar polarity that acts as a functional equivalent,
resulting in a silent alteration. Substitutes for an amino acid
within the sequence can be selected from other members of the class
to which the amino acid belongs. For example, the non-polar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan, and methionine. The
uncharged polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine and glutamine. The
positively charged (basic) amino acids include arginine, lysine,
and histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. The aromatic amino acids include
phenylalanine, tryptophan, and tyrosine.
[0065] Additional embodiments are nucleic acids that encode the
binding partners and candidate binding partners described herein.
The nucleic acid embodiments can be DNA or RNA and these molecules
can be provided in constructs, plasmids, vectors, chromosomes and
can be transferred to plants, virus, bacteria, insects, amphibians,
reptiles, birds, animals, and mammals, including humans. Most
preferably, the nucleic acid embodiments are between about 9 and
about 300 nucleotides in length, although it is recognized that a
fusion protein of almost any length can incorporate a nucleic acid
embodiment. That is, the nucleic acid embodiments desirably have
about 9-1000 nucleotides, preferably about 9-700 nucleotides, more
preferably about 9-500 nucleotides, and most preferably about 9-300
nucleotides. Some nucleic acid embodiments, for example encode a
peptide having the formula "X.sup.1.sub.nCKASX.sup.2.sub.n" or
"X.sup.1.sub.nCRASX.sup.2.sub.n", wherein "X.sup.1" and "X.sup.2"
are any amino acid and "n" is any integer and wherein the peptide
encoded by the nucleic acid specifically binds to HBcAg and/or
HBeAg. The section below describes the manufacture and use of
modified and derivatized binding partners that resemble peptides
(e.g., peptidomimetics) that bind HbcAg and/or HBeAg.
[0066] Modified and Derivatized Binding Partners Specific for HBcAg
and/or HBeAg
[0067] The peptides described herein can be modified (e.g., the
binding partners can have substituents not normally found on a
peptide or the binding partners can have substituents that are
normally found on the peptide but are incorporated at regions of
the peptide that are not normal). The peptides can be acetylated,
acylated, or aminated, for example. 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 "binding partner" can refer to a
modified or unmodified peptide and a chemical or a peptidomimetic
that structurally (three-dimensionally or two-dimensionally)
resembles a peptide that binds HBcAg and/or HBeAg.
[0068] There are many ways to make a peptidomimetic that resembles
the peptides described herein. 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 binding partner 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).
[0069] In general, the design and synthesis of a peptidomimetic
involves starting with the sequence of the peptide and the
conformation data (e.g., geometry data, such as bond lengths and
angles) of a desired peptide (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.
[0070] Preferred peptidomimetics have structures that resemble a
peptide whose sequence is provided in SEQ. ID. No. 1-78. While the
most preferred peptidomimetics have structures that resemble
peptides that are at least 13 amino acids in length, many
peptidomimetics resemble peptides that are between about 3 amino
acids and about 100 amino acids or more in length. That is,
desirable peptidomimetics resemble peptides that are about 3-125
amino acids in length, more desirable peptidomimetics resemble
peptides that are between about 3-100 amino acids in length,
preferred peptidomimetics resemble peptides that are between about
3-75 amino acids in length, more preferred peptidomimetics resemble
peptides that are between about 3-50 amino acids in length, and
most preferred peptidomimetics resemble peptides that are between
about 13-25 amino acids in length. Some embodiments, for example,
are peptidomimetics that resemble a peptide having the formula
"X.sup.1.sub.nCKASX.sup.2.sub.n" or
"X.sup.1.sub.nCRASX.sup.2.sub.n", wherein "X.sup.1" and "X.sup.2"
are any amino acid and "n" is any integer, and the molecule
specifically binds HBcAg and/or HBeAg. In the discussion that
follows, several methods of using candidate binding partners and
binding partners as templates for molecular modeling and rational
drug design are described. These techniques can be applied to
identify additional molecules that bind to HBcAg and/or HBeAg and
thereby inhibit viral infectivity and/or modulate a host immune
response.
[0071] Rational Drug Design Approaches to Identify Binding Partners
Specific for HBcAg and/or HBeAg
[0072] Several methods of molecular modeling and rational drug
design can be used to identify more molecules that bind to HBcAg
and/or HBeAg. Rational drug design involving polypeptides requires
identifying and defining a first peptide and using this first
target peptide to define the requirements for a second peptide.
With such requirements defined, one can find or prepare an
appropriate peptide or non-peptide molecule that meets all or
substantially all of the defined requirements. Thus, one goal of
rational drug design is to produce structural or functional analogs
of biologically active polypeptides of interest in order to fashion
drugs that are, for example, more or less potent forms of a
particular binding partner. (See, e.g., Hodgson, Bio. Technology
9:19-21 (1991)).
[0073] Combinatorial chemistry can also be used to rapidly make and
test the materials constructed by rational drug design.
Combinatorial chemistry is the science of synthesizing and testing
compounds for bioactivity en masse, instead of one by one, the aim
being to discover drugs and materials more quickly and
inexpensively than was formerly possible. Many high throughput
systems for rapidly testing whether a target molecule can be bound
by a candidate compound are known in the art. These systems can be
adapted to determine whether a candidate binding partner can
interact with HBcAg and/or HBeAg or a fragment thereof.
[0074] Rational drug design and combinatorial chemistry have become
more intimately related in recent years due to the development of
approaches in computer-aided protein modeling and drug discovery.
(See e.g., U.S. Pat. Nos. 4,908,773; 5,884,230; 5,873,052;
5,331,573; and 5,888,738). Not only is it possible to view
molecules on computer screens in three dimensions but it is also
possible to examine the interactions of macromolecules such as
enzymes and receptors and rationally design derivative molecules to
test. (See Boorman, Chem. Eng. News 70:18-26 (1992)). A vast amount
of user-friendly software and hardware is now available and
virtually all pharmaceutical companies have computer modeling
groups devoted to rational drug design. Molecular Simulations Inc.,
for example, sells several sophisticated programs that allow a user
to start from an amino acid sequence, build a two or
three-dimensional model of the protein or polypeptide, compare it
to other two and three-dimensional models, and analyze the
interactions of compounds, drugs, and peptides with a three
dimensional model in real time.
[0075] Accordingly, in some embodiments, software is used to
compare regions of binding partners with other molecules, such as
peptides, peptidomimetics, and chemicals, so that therapeutic
interactions can be predicted and designed. (See Schneider, Genetic
Engineering News December: page 20 (1998), Tempczyk et al.,
Molecular Simulations Inc. Solutions April (1997) and Butenhof,
Molecular Simulations Inc. Case Notes (August 1998) for a
discussion of molecular modeling). For example, the protein or
nucleic acid sequence of a candidate binding partner, binding
partner, or a domain of these molecules can be entered onto a
computer readable medium for recording and manipulation. It will be
appreciated by those skilled in the art that a computer readable
medium having these sequences can interface with software that
converts or manipulates the sequences to obtain structural and
functional information, such as protein models. That is, the
functionality of a software program that converts or manipulates
these sequences includes the ability to compare these sequences to
other sequences or structures of molecules that are present on
publicly and commercially available databases so as to conduct
rational drug design.
[0076] The candidate binding partner or binding partner polypeptide
or nucleic acid sequence or both can be stored, recorded, and
manipulated on any medium that can be read and accessed by a
computer. As used herein, the words "recorded" and "stored" refer
to a process for storing information on computer readable medium. A
skilled artisan can readily adopt any of the presently known
methods for recording information on a computer readable medium to
generate manufactures comprising the desired nucleotide or
polypeptide sequence information. A variety of data storage
structures are available to a skilled artisan for creating a
computer readable medium having recorded thereon a nucleotide or
polypeptide sequence. The choice of the data storage structure will
generally be based on the component chosen to access the stored
information. Computer readable media include magnetically readable
media, optically readable media, or electronically readable media.
For example, the computer readable media can be a hard disc, a
floppy disc, a magnetic tape, zip disk, CD-ROM, DVD-ROM, RAM, or
ROM as well as other types of other media known to those skilled in
the art. The computer readable media on which the sequence
information is stored can be in a personal computer, a network, a
server or other computer systems known to those skilled in the
art.
[0077] Embodiments utilize computer-based systems that contain the
sequence information described herein and convert this information
into other types of usable information (e.g., protein models for
rational drug design). The term "a computer-based system" refers to
the hardware, software, and any database used to analyze a
candidate binding partner or a binding partner nucleic acid or
polypeptide sequence or both, or fragments of these biomolecules so
as to construct models or to conduct rational drug design. The
computer-based system preferably includes the storage media
described above, and a processor for accessing and manipulating the
sequence data. The hardware of the computer-based systems of this
embodiment comprise a central processing unit (CPU) and a database.
A skilled artisan can readily appreciate that any one of the
currently available computer-based systems are suitable.
[0078] In one particular embodiment, the computer system includes a
processor connected to a bus that is connected to a main memory
(preferably implemented as RAM) and a variety of secondary storage
devices, such as a hard drive and removable medium storage device.
The removable medium storage device can represent, for example, a
floppy disk drive, a DVD drive, an optical disk drive, a compact
disk drive, a magnetic tape drive, etc. A removable storage medium,
such as a floppy disk, a compact disk, a magnetic tape, etc.
containing control logic and/or data recorded therein can be
inserted into the removable storage device. The computer system
includes appropriate software for reading the control logic and/or
the data from the removable medium storage device once inserted in
the removable medium storage device. The candidate binding partner
or binding partner nucleic acid or polypeptide sequence or both can
be stored in a well known manner in the main memory, any of the
secondary storage devices, and/or a removable storage medium.
Software for accessing and processing these sequences (such as
search tools, compare tools, and modeling tools etc.) reside in
main memory during execution.
[0079] As used herein, "a database" refers to memory that can store
a candidate binding partner or binding partner nucleotide or
polypeptide sequence information, protein model information,
information on other peptides, chemicals, peptidomimetics, and
other agents that interact with HbcAg and/or HBeAg, and values or
results from characterization assays. Additionally, a "database"
refers to a memory access component that can access manufactures
having recorded thereon candidate binding partner or binding
partner nucleotide or polypeptide sequence information, protein
model information, information on other peptides, chemicals,
peptidomimetics, and other agents that interact with HbcAg and/or
HBeAg, and values or results from characterization assays. The
sequence data and values or results from characterization assays
can be stored and manipulated in a variety of data processor
programs in a variety of formats. For example, the sequence data
can be stored as text in a word processing file, such as Microsoft
WORD or WORDPERFECT, an ASCII file, a html file, or a pdf file in a
variety of database programs familiar to those of skill in the art,
such as DB2, SYBASE, or ORACLE.
[0080] A "search program" refers to one or more programs that are
implemented on the computer-based system to compare a candidate
binding partner or binding partner nucleotide or polypeptide
sequence with other nucleotide or polypeptide sequences and other
agents including but not limited to peptides, peptidomimetics, and
chemicals stored within a database. A search program also refers to
one or more programs that compare one or more protein models to
several protein models that exist in a database and one or more
protein models to several peptides, peptidomimetics, and chemicals
that exist in a database. Still further, a search program can be
used to compare values or results from characterization assays and
agents that modulate binding partner-mediated effects on viral
infectivity and/or host immune system response.
[0081] A "retrieval program" refers to one or more programs that
can be implemented on the computer-based system to identify a
homologous nucleic acid sequence, a homologous protein sequence, or
a homologous protein model. A retrieval program can also used to
identify peptides, peptidomimetics, and chemicals that interact
with HBcAg and/or HBeAg or a protein model of HBcAg and/or HBeAg
stored in a database. A retrieval program can also be used to
obtain "a binding partner profile" that is composed of a chemical
structure, nucleic acid sequence, or polypeptide sequence or model
of an molecule that interacts with HBcAg and/or HBeAg and, thereby
inhibits viral infectivity or modulates a host immune response to
HBV.
[0082] As a starting point to rational drug design, a two or three
dimensional model of a polypeptide of interest is created (e.g., a
binding partner or candidate binding partner whose sequence is
provided in SEQ. ID. Nos. 1-78). In the past, the three-dimensional
structure of proteins has been determined in a number of ways.
Perhaps the best known way of determining protein structure
involves the use of x-ray crystallography. A general review of this
technique can be found in Van Holde, K. E. Physical Biochemistry,
Prentice-Hall, N.J. pp. 221-239 (1971), herein expressly
incorporated by reference in its entirety. Using this technique, it
is possible to elucidate three-dimensional structure with good
precision. Additionally, protein structure can be determined
through the use of techniques of neutron diffraction, or by nuclear
magnetic resonance (NMR). (See, e.g., Moore, W. J., Physical
Chemistry, 4.sup.th Edition, Prentice-Hall, N.J. (1972), herein
expressly incorporated by reference in its entirety).
[0083] Alternatively, protein models of a polypeptide of interest
can be constructed using computer-based protein modeling
techniques. By one approach, the protein folding problem is solved
by finding target sequences that are most compatible with profiles
representing the structural environments of the residues in known
three-dimensional protein structures. (See, e.g., U.S. Pat. No.
5,436,850, herein expressly incorporated by reference in its
entirety). In another technique, the known three-dimensional
structures of proteins in a given family are superimposed to define
the structurally conserved regions in that family. This protein
modeling technique also uses the known three-dimensional structure
of a homologous protein to approximate the structure of a
polypeptide of interest. (See e.g., U.S. Pat. Nos. 5,557,535;
5,884,230; and 5,873,052, all of which are expressly incorporated
by reference in their entireties). Conventional homology modeling
techniques have been used routinely to build models of proteases
and antibodies. (Sowdhamini et al., Protein Engineering 10:207, 215
(1997), herein expressly incorporated by reference in its
entirety). Comparative approaches can also be used to develop
three-dimensional protein models when the protein of interest has
poor sequence identity to template proteins. In some cases,
proteins fold into similar three-dimensional structures despite
having very weak sequence identities. For example, the
three-dimensional structures of a number of helical cytokines fold
in similar three-dimensional topology in spite of weak sequence
homology.
[0084] The recent development of threading methods and "fuzzy"
approaches now enables the identification of likely folding
patterns and functional protein domains in a number of situations
where the structural relatedness between target and template(s) is
not detectable at the sequence level. By one method, fold
recognition is performed using Multiple Sequence Threading (MST)
and structural equivalences are deduced from the threading output
using the distance geometry program DRAGON that constructs a low
resolution model. A full-atom representation is then constructed
using a molecular modeling package such as QUANTA.
[0085] According to this 3-step approach, candidate templates are
first identified by using the novel fold recognition algorithm MST,
which is capable of performing simultaneous threading of multiple
aligned sequences onto one or more 3-D structures. In a second
step, the structural equivalences obtained from the MST output are
converted into interresidue distance restraints and fed into the
distance geometry program DRAGON, together with auxiliary
information obtained from secondary structure predictions. The
program combines the restraints in an unbiased manner and rapidly
generates a large number of low resolution model confirmations. In
a third step, these low resolution model confirmations are
converted into full-atom models and organized to energy
minimization using the molecular modeling package QUANTA. (See
e.g., Aszodi et al., Proteins: Structure, Function, and Genetics,
Supplement 1:38-42 (1997), herein expressly incorporated by
reference in its entirety).
[0086] In a preferred approach, the commercially available "Insight
II 98" program (Molecular Simulations Inc.) and accompanying
modules are used to create a two and/or three dimensional model of
a polypeptide of interest from an amino acid sequence. Insight II
is a three-dimensional graphics program that can interface with
several modules that perform numerous structural analysis and
enable real-time rational drug design and combinatorial chemistry.
Modules such as Builder, Biopolymer, Consensus, and Converter, for
example, allow one to rapidly create a two dimensional or three
dimensional model of a polypeptide, carbohydrate, nucleic acid,
chemical or combinations of the foregoing from their sequence or
structure. The modeling tools associated with Insight II support
many different data file formats including Brookhaven and Cambridge
databases; AMPAC/MOPAC and QCPE programs; Molecular Design Limited
Molfile and SD files, Sybel Mol2 files, VRML, and Pict files.
[0087] Additionally, the techniques described above can be
supplemented with techniques in molecular biology to design models
of the protein of interest. For example, a known binding partner
can be analyzed by an alanine scan (Wells, Methods in Enzymol.
202:390-411 (1991), herein expressly incorporated by reference in
its entirety) or other types of site-directed mutagenesis analysis.
In alanine scan, each amino acid residue of the binding partner is
sequentially replaced by alanine in a step-wise fashion (i.e., only
one alanine point mutation is incorporated per, molecule starting
at position #1 and proceeding through the entire molecule), and the
effect of the mutation on the peptide's activity in a
characterization assay is determined. Each of the amino acid
residues of the peptide is analyzed in this manner and the regions
important for the binding to HBcAg and/or HBeAg are determined.
These functionally important regions can be recorded on a computer
readable medium, stored in a database in a computer system, and a
search program can be employed to generate a protein model of the
functionally important regions. The example below describes a
rational drug design approach that was used to identify fragments
of the binding domains of mAbs that specifically bind HBcAg and/or
HBeAg.
EXAMPLE 5
[0088] One approach to rational drug design involves sequential
amino acid deletion of a known binding partner starting from either
the amino or carboxy termini. Amino-terminal deletions of the
binding partners of SEQ. ID. Nos. 5 and 17 were made and these
peptide fragments were joined to a support and analyzed for the
ability to bind HBcAg. By using this technique, a fine map of the
peptide sequence involved in binding to HBcAg was obtained. As
shown in TABLE 3, the HBcAg binding sequences for the peptides of
SEQ. ID. Nos. 5 and 17 included KLSCKASGYIFTS (SEQ. ID. No. 45) and
CRASQSVSTSSYSYMHWY (SEQ. ID. No. 54), respectively. The
amino-terminal deletions of the peptide of SEQ. ID. Nos. 5 were
also evaluated for the ability to inhibit binding of mAb 4-2 to
HBcAg. (See TABLE 4). The amino-terminal deletion products of the
peptide of SEQ. ID. No 5 that were most effective at inhibiting
binding of mAb 4-2 were found to have at least the sequence
VKLSCKASGYIFTS (SEQ. ID. No. 44), which provided evidence that the
valine residue in SEQ. ID. No. 44 was intimately involved in
binding of mAb4-2 to HBcAg. TABLE-US-00003 TABLE 3 Mapping of the
HBcAg binding sequence using support-bound amino terminal deletion
peptides* OD Amino terminal deletion peptides at 490 nm
VKPGASVKLSCKASGYIFTS (SEQ. ID. No. 5) 3.257 KPGASVKLSCKASGYIFTS
(SEQ. ID. No. 39) 1.337 PGASVKLSCKASGYIFTS (SEQ. ID. No. 40) 1.722
GASVKLSCKASGYIFTS (SEQ. ID. No. 41) 2.863 ASVKLSCKASGYIFTS (SEQ.
ID. No. 42) 3.219 SVKLSCKASGYIFTS (SEQ. ID. No. 43) 3.364
VKLSCKASGYIFTS (SEQ. ID. No. 44) 3.703 KLSCKASGYIFTS (SEQ. ID. No.
45) 3.694 LSCKASGYIFTS (SEQ. ID. No. 46) 0.565 SCKASGYIFTS (SEQ.
ID. No. 47) 0.297 CKASGYIFTS (SEQ. ID. No. 48) 0.255 KASGYIFTS
(SEQ. ID. No. 49) 0.237 ASGYIFTS (SEQ. ID. No. 50) 0.407 SGYIFTS
(SEQ. ID. No. 51) 0.389 GYIFTS (SEQ. ID. No. 52) 0.414
ISCRASQSVSTSSYSYMHWY (SEQ. ID. NO.17) 1.939 SCRASQSVSTSSYSYMHWY
(SEQ. ID. No. 53) 1.452 CRASQSVSTSSYSYMHWY (SEQ. ID. No. 54) 1.415
RASQSVSTSSYSYMHWY (SEQ. ID. No. 55) 0.429 ASQSVSTSSYSYMHWY (SEQ.
ID. No. 56) 0.324 SQSVSTSSYSYMHWY (SEQ. ID. No. 57) 0.310
QSVSTSSYSYMHWY (SEQ. ID. No. 58) 0.282 SVSTSSYSYMHWY (SEQ. ID. No.
59) 0.305 VSTSSYSYMHWY (SEQ. ID. No. 60) 0.369 STSSYSYMHWY (SEQ.
ID. No. 61) 0.372 TSSYSYMHWY (SEQ. ID. No. 62) 0.317 SSYSYMHWY
(SEQ. ID. No. 63) 0.311 SYSYMHWY (SEQ. ID. No. 63) 0.283 YSYMHWY
(SEQ. ID. No. 64) 0.245 SYMHWY (SEQ. ID. No. 65) 0.218 *HBcAg was
added at 5 .mu.g/ml. Values are given as the OD at 490 nm. The
original starting peptide has been written in bold face.
[0089] TABLE-US-00004 TABLE 4 Inhibition of mAb 4-2 binding to
HBcAg by the amino terminal deletion peptides added prior to
addition of mAb*. OD Amino terminal deletion peptides at 490 nm
VKPGASVKLSCKASGYIFTS (SEQ. ID. No. 5) 0.453 KPGASVKLSCKASGYIFTS
(SEQ. ID. No. 39) 0.202 PGASVKLSCKASGYIFTS (SEQ. ID. No. 40) 0.182
GASVKLSCKASGYIFTS (SEQ. ID. No. 41) 0.205 ASVKLSCKASGYIFTS (SEQ.
ID. No. 42) 0.207 SVKLSCKASGYIFTS (SEQ. ID. No. 43) 0.175
VKLSCKASGYIFTS (SEQ. ID. No. 44) 0.152 KLSCKASGYIFTS (SEQ. ID. No.
45) 0.808 LSCKASGYIFTS (SEQ. ID. No. 46) 0.777 SCKASGYIFTS (SEQ.
ID. No. 47) 0.784 CKASGYIFTS (SEQ. ID. No. 48) 0.851 KASGYIETS
(SEQ. ID. No. 49) 0.866 ASGYIFTS (SEQ. ID. No. 50) 0.920 SGYIFTS
(SEQ. ID. No. 51) 0.887 GYIFTS (SEQ. ID. No. 52) 0.903 *The
uninhibited control gave a mean OD at 490 nm of 0.871. The original
starting peptide has been written in bold face.
[0090] Once a model or map of a binding partner is created, it can
be compared to other models or maps so as to identify new members
of a particular binding partner family. By starting with the amino
acid sequence or protein model of a binding partner, for example,
molecules having two-dimensional and/or three-dimensional homology
can be rapidly identified. In one approach, a percent sequence
identity can be determined by standard methods that are commonly
used to compare the similarity and position of the amino acid of
two polypeptides. Using a computer program such as BLAST or FASTA,
two polypeptides can be aligned for optimal matching of their
respective amino acids (either along the full length of one or both
sequences, or along a predetermined portion of one or both
sequences). Such programs provide "default" opening penalty and a
"default" gap penalty, and a scoring matrix such as PAM 250 (a
standard scoring matrix; see Dayhoff et al., in: Atlas of Protein
Sequence and Structure, Vol. 5, Supp. 3 (1978)) can be used in
conjunction with the computer program. The percent identity can
then be calculated as: total .times. .times. number .times. .times.
of .times. .times. identical .times. .times. matches [ length
.times. .times. of .times. .times. the .times. .times. longer
.times. .times. sequence .times. .times. within .times. .times. the
.times. .times. matched .times. .times. span + number .times.
.times. of .times. .times. gaps .times. .times. introduced .times.
.times. into .times. .times. the .times. .times. longer .times.
.times. sequence .times. .times. in order .times. .times. to
.times. .times. align .times. .times. the .times. .times. two
.times. .times. sequences ] .times. 100 ##EQU1##
[0091] Accordingly, the protein sequence corresponding to a binding
partner or a binding partner or a fragment or derivative of these
molecules can be compared to known sequences on a protein basis.
Protein sequences corresponding to a binding partner, or a binding
partner or a fragment or derivative of these molecules are
compared, for example, to known amino acid sequences found in
Swissprot release 35, PIR release 53 and Genpept release 108 public
databases using BLASTP with the parameter W=8 and allowing a
maximum of 10 matches. In addition, the protein sequences are
compared to publicly known amino acid sequences of Swissprot using
BLASTXi with the parameter E=0.001. The example below describes
database searches that were performed on the identified binding
partners so as to find homologous molecules that are expected to
bind HBcAg and/or HBeAg.
EXAMPLE 6
[0092] To identify new candidate binding partners, the sequences of
identified binding partners were used to search publicly available
databases. The sequences KLSCKASGYIFTS (SEQ. ID. No. 45) and
CRASQSVSTSSYSYMHWY (SEQ. ID. No. 54), obtained from mus musculins,
were used to search for homologous molecules in Genebank, for
example. Many sequences with a high degree of homology were found.
Noticeably, the sequences uncovered in the search were mAb
sequences from various species including, Homo sapiens, Carassius
auratus, Canis familiaris, and Caiman crocodilus. Fragments of
these sequences are provided in TABLE 5 and the Sequence Listing
(SEQ. ID. Nos. 66, 67, 68, and 69). Not only did these findings
demonstrate that homology-based methods of rational drug design can
yield new candidate binding partners but the data also provided
evidence that HBcAg can bind naive B cells in a plurality of
different species. TABLE-US-00005 TABLE 5 Alignment of an HBcAg and
HBeAg binding with sequences obtained from a Genbank and Swissprot
search* Genebank/ swissprot Sequence identity Sequence accession
Pept. #2 (mus musc.) V K P G A S V K L S C K A S G Y F T S (SEQ.
ID. No. 5) Homo sapiens P G A S V R I S C K A S G Y P80421 A F
(SEQ. ID. No. 66) Carassius auratus K P G D S L R L S C K A S G Y
P19180 T F S (SEQ. ID. No. 67) Canis familiaris V K P G G S L R L S
C V A S G F P01785 F S S (SEQ. ID. No. 68) Caiman crocodilus K P G
D S L R L S C K G S G F P03981 F S N (SEQ. ID. No. 69) *The
homology search was made prior to Dec. 11, 1999.
[0093] In another embodiment, computer modeling and the
sequence-to-structure-to-function paradigm is exploited to identify
more binding partners and candidate binding partners. By this
approach, first the structure of a binding partner or a candidate
binding partner having a known response in a characterization assay
is determined from its sequence using a threading algorithm, which
aligns the sequence to the best matching structure in a structural
database. Next, the peptide's active site (i.e., the site important
for a desired response in the characterization assay) is identified
and a "fuzzy functional form" (FFF)--a three-dimensional descriptor
of the active site of a protein--is created. (See e.g., Fetrow et
al., J. Mol. Biol. 282:703-711 (1998) and Fetrow and Skolnick, J.
Mol. Biol. 281: 949-968 (1998), herein expressly incorporated by
reference in its entirety). The mapping techniques described above
can be used to facilitate description of the active site of the
peptide.
[0094] The FFFs are built by iteratively superimposing the protein
geometries from a series of functionally related proteins with
known structures. The FFFs are not overly specific, however, and
the degree to which the descriptors can be relaxed is explored. In
essence, conserved and functionally important residues for a
desired response are identified and a set of geometric and
conformational constraints for a specific function are defined in
the form of a computer algorithm. The program then searches
experimentally determined protein structures from a protein
structural database for sets of residues that satisfy the specified
constraints. In this manner, homologous three-dimensional
structures can be compared and degrees (e.g., percentages of
three-dimensional homology) can be ascertained. The ability to
search three-dimensional structure databases for structural
similarity to a protein of interest can also be accomplished by
employing the Insight II using modules such as Biopolymer, Binding
Site Analysis, and Profiles-3D.
[0095] By using this computational protocol, genome sequence data
bases such as maintained by various organizations can be rapidly
screened for specific protein active sites and for identification
of the residues at those active sites that resemble a desired
molecule. Several other groups have developed databases of short
sequence patterns or motifs designed to identify a given function
or activity of a protein. Many of these databases, notably Prosite,
Blocks, Prints, the Molecular Modelling Database (MMDB), and the
Protein Data Bank can use short stretches of sequence information
to identify sequence patterns that are specific for a given
function; thus they avoid the problems arising from the necessity
of matching entire sequences.
[0096] By a similar approach, a binding partner can be identified
and manufactured as follows. First, a molecular model of one or
more binding partners are created using one of the techniques
discussed above or as known in the art. Next, chemical libraries
and databases are searched for molecules similar in structure to
the known molecule. That is, a search can be made of a three
dimensional data base for non-peptide (organic) structures (e.g.,
non-peptide analogs) having three dimensional similarity to the
known structure of the target compound. See, e.g., the Cambridge
Crystal Structure Data Base, Crystallographic Data Center,
Lensfield Road, Cambridge, CB2 1EW, England; and Allen, F. H., et
al., Acta Crystallogr., B35: 2331-2339 (1979), all of which are
expressly incorporated by reference in their entireties. One
program that allows for such analysis is Insight II having the Ludi
module. Further, the Ludi/ACD module allows a user access to over
65,000 commercially available drug candidates (MDL's Available
Chemicals Directory) and provides the ability to screen these
compounds for interactions with HBcAg and/or HBeAg on the computer.
The identified candidate binding partners can then be analyzed in a
characterization assay and new molecules can be modeled after the
candidate binding partners that produce a desirable response. By
cycling in this fashion, libraries of molecules that interact HBcAg
and/or HBeAg and produce a desirable or optimal response in a
characterization assay can be selected.
[0097] It is noted that search algorithms for three dimensional
data base comparisons are available in the literature. See, e.g.,
Cooper, et al., J. Comput.-Aided Mol. Design, 3: 253-259 (1989) and
references cited therein; Brent, et al., J. Comput.-Aided Mol.
Design, 2: 311-310 (1988) and references cited therein, all of
which are expressly incorporated by reference in their entireties.
Commercial software for such searches is also available from
vendors such as Day Light Information Systems, Inc., Irvine, Calif.
92714, and Molecular Design Limited, 2132 Faralton Drive, San
Leandro, Calif. 94577. The searching is done in a systematic
fashion by simulating or synthesizing analogs having a substitute
moiety at every residue level. Preferably, care is taken that
replacement of portions of the backbone does not disturb the
tertiary structure and that the side chain substitutions are
compatible to retain the protein:protein interactions.
[0098] Alternatively, these methods can be used to identify
improved binding partners from an already known binding partner.
The composition of the known binding partner can be modified and
the structural effects of modification can be determined using the
experimental and computer modeling methods described above. The
altered structure can be compared to the active site structure of
HBcAg and/or HBeAg to determine if an improved fit or interaction
results. In this manner systematic variations in composition, such
as by varying side groups, can be quickly evaluated to obtain
modified binding partners of improved specificity or activity.
[0099] Additionally, a computer model of HBcAg and/or HBeAg can be
obtained using the approaches described above, and this model can
be compared with libraries of candidate binding partners in real
time. For example, a search program can locate several structures
within the database that have a given set of molecular properties,
which correspond to the constraints provided by the HBcAg and/or
HBeAg model. With the aid of computer graphics and a retrieval
program, candidate binding partners can be obtained from the
database, modeled, and evaluated for the ability to interact with
HBcAg and/or HBeAg. This approach is referred to as a "computer
generated binding assay". Such assays can be performed in the
presence or absence of competing molecules.
[0100] A number of articles review computer modelling of drugs
interactive with specific-proteins, such as Rotivinen, et al.,
1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist
54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev.
Pharmacol. Toxiciol. 29:111-122; Perry and Davies, OSAR:
Quantitative Structure-Activity Relationships in Drug Design pp.
189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R.
Soc. Lond. 236:125-140 and 141-162; and, with respect to a model
receptor for nucleic acid components, Askew, et al., 1989, J. Am.
Chem. Soc. 111:1082-1090, all of which are expressly incorporated
by reference in their entireties. Other computer programs that
screen and graphically depict chemicals are available from
companies such as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc.
(Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge,
Ontario).
[0101] Many more computer programs and databases can be used with
embodiments to identify candidate binding partners and binding
partners that inhibit viral infectivity and/or modulate a host
immune system response. The following list is intended not to limit
the invention but to provide guidance to programs and databases
that are useful with the approaches discussed above. The programs
and databases that can be used include, but are not limited to:
MacPattern (EMBL), DiscoveryBase (Molecular Applications Group),
GeneMine (Molecular Applications Group), Look (Molecular
Applications Group), MacLook (Molecular Applications Group), BLAST
and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol.
215: 403 (1990), herein incorporated by reference), FASTA (Pearson
and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), herein
incorporated by reference), Catalyst (Molecular Simulations Inc.),
Catalyst/SHAPE (Molecular Simulations Inc.), Cerius.sup.2.DBAccess
(Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.),
Insight II, (Molecular Simulations Inc.), Discover (Molecular
Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix
(Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.),
QuanteMM, (Molecular Simulations Inc.), Homology (Molecular
Simulations Inc.), Modeler (Molecular Simulations Inc.), Modeller 4
(Sali and Blundell J. Mol. Biol. 234:217-241 (1997)), ISIS
(Molecular Simulations Inc.), Quanta/Protein Design (Molecular
Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab
Diversity Explorer (Molecular Simulations Inc.), Gene Explorer
(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.),
Biopendium (Inpharmatica), SBdBase (Structural Bioinformatics), the
EMBL/Swissprotein database, the MDL Available Chemicals Directory
database, the MDL Drug Data Report data base, the Comprehensive
Medicinal Chemistry database, Derwents's World Drug Index database,
and the BioByteMasterFile database. Many other programs and data
bases would be apparent to one of skill in the art given the
present disclosure.
[0102] Although the peptides described above can effectively
modulate an immune response to HBV in a subject, binding
partner-fusion proteins can be created to take advantage of already
existing highly potent immune responses in a subject. That is,
peptides that bind HBcAg and/or HBeAg can be fused with molecules,
which are known to elicit a potent immune response in a subject. In
this manner, high titer antibodies present in the subject are
redirected to HBcAg and/or HBeAg and, thus, HBV can more
effectively be cleared from the subject. These binding
partner-fusion proteins are referred to as "specificity exchangers"
and the section below describes their manufacture and use in
detail.
[0103] Binding Partner Specificity Exchangers
[0104] Antibodies can be redirected to new antigens using
bi-functional synthetic peptides called binding partner-fusion
proteins or specificity exchangers. (See Sallberg et al.,
Biochemical & Biophysical Research Communications, 205:1386-90
(1994) and U.S. Pat. No. 5,869,232, both disclosures are herein
incorporated by reference in their entireties). One portion of the
specificity exchanger, referred to as the "specificity domain",
comprises a molecule that binds a desired molecule (e.g., a peptide
that binds HBcAg and/or HbeAg, such as provided in SEQ. ID. Nos. 5,
16, 17, 28, 29, and 44), which may resemble an antibody binding
domain or fragment thereof and another portion of the specificity
exchanger, called the "antigenic domain", which serves as an
antigen for antibody recognition (preferably recognition by a high
titer antibody). The specificity domain can be a binding partner
itself (e.g., peptide, peptidomimetic, or chemical that binds to
HbcAg and/or HBeAg), whereas, the antigenic domain can comprise
molecules including, but not limited to, carbohydrates, lipids,
proteins, and nucleic acids that have epitopes, which are
recognized by antibodies present in an animal.
[0105] Desirably, the antigenic domain comprises an epitope found
on a pathogen (e.g., bacteria, mold, fungus, or virus) or a toxin
(e.g., pertussis toxin or cholera toxin) or a non-self antigen.
Many antigenic domains are between 3 and 40 amino acids in length,
desirably between 5 and 35 amino acids in length, more desirably
between 5 and 30 amino acids in length, preferably between 5 and 25
amino acids in length, and most preferably between 5 and 20 amino
acids in length. The diversity of antigenic domains that can be
used in the specificity exchangers is quite large because a
pathogen or toxin can present many different epitopes. Antigenic
domains that comprise an epitope of a pathogen that already exists
in a subject by virtue of naturally acquired immunity or
vaccination are highly desirable. Epitopes of pathogens that cause
childhood diseases, for example, can be used as antigenic domains.
Epitopes of a pathogen that can be included in an antigenic domain
of a specificity exchanger can include, for example, epitopes on
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 references are herein expressly incorporated
by reference in their entireties.
[0106] Some embodiments 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 (e.g.,
serum, blood, or tissue). For example, subjects that undergo blood
transfusion acquire numerous antibodies, some of which can interact
with an antigenic domain of a specificity exchanger. Preferred
antigenic domains for use in a specificity exchanger embodiment
comprise viral epitopes including, but not limited to, the herpes
simplex virus, hepatitis B virus, TT virus, and the poliovirus.
Highly preferred antigenic domains are provided in Table 6 and the
sequence listing (SEQ. ID. Nos. 79-95). TABLE-US-00006 TABLE 6
Antigenic Domains GLYSSIWLSPGRSYFET (SEQ. ID. No. 79)
YTDIKYNPFTDRGEGNM (SEQ. ID. No. 80) DQNIHMNARLLIRSPFT (SEQ. ID. No.
81) LIRSPFTDPQLLVHTDP (SEQ. ID. No. 82) QKESLLFPPVKLLRRVP (SEQ. ID.
No. 83) PALTAVETGAT (SEQ. ID. No. 84) STLVPETT (SEQ. ID. No. 85)
TPPAYRPPNAPIL (SEQ. ID. No. 86) EIPALTAVE (SEQ. ID. No. 87)
LEDPASRDLV (SEQ. ID. No. 88) HRGGPEEF (SEQ. ID. No. 89) HRGGPEE
(SEQ. ID. No. 90) VLICGENTVSRNYATHS (SEQ. ID. No. 91)
KINTMPPFLDTELTAPS (SEQ. ID. No. 92) PDEKSQREILLNKIASY (SEQ. ID. No.
93) TATTTTYAYPGTNRPPV (SEQ. ID. No. 94) STPLPETT (SEQ. ID. No.
95)
[0107] In some embodiments, the specificity and antigenic domains
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 binding partners, peptides or specificity domains disclosed
herein can be joined to any of the antigenic domains of TABLE 6
(SEQ. ID. Nos: 79-95). Although the specificity and antigenic
domains could 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 exchanger
is made as a fusion protein. Thus, preferred embodiments include
fusion proteins comprising any of the peptides, binding partners,
or specificity domains described herein joined to any of the
antigenic domains of TABLE 6 (SEQ. ID. Nos:79-95). Thus, preferred
specificity exchangers include, but are not limited to, peptides
comprising, consisting, or consisting essentially of a sequence
selected from the group consisting of SEQ. ID. Nos. 5, 6, 13, 16,
17, 28, 29, 33, 36, and 37.
[0108] Any of the approaches used to identify and characterize
binding partners described herein can be used to identify and
characterize the specificity domains of a specificity exchanger
specific for HBV. The specificity exchangers themselves can also
can be analyzed in the characterization assays described above or
in modified characterization assays, as will be apparent to one of
skill in the art provided the description herein. The example below
describes the manufacture of specificity exchangers having a
specificity domain directed to HBcAg and/or HBeAg and an antigenic
domain directed to an anti-HSV mAb.
EXAMPLE 7
[0109] An approach to manufacture specificity exchangers that can
redirect high titer antibodies to HBV is provided in this example.
A first set of specificity exchangers having a specificity domain
containing the HBcAg binding sequence KLSCKASGYIFTS (SEQ. ID. No.
45) and a C terminal antigenic domain containing the epitope for a
monoclonal antibody directed to the herpes simplex virus type 1 gG2
(HSV gG2) protein was created. A second set of specificity
exchangers having a specificity domain containing the HBcAg binding
sequence VKLSCKASGYIFTS (SEQ. ID. No. 44) and a C-terminal
antigenic domain containing the epitope for a monoclonal antibody
directed to the HSV gG2 protein was also constructed. The sequences
of these binding partner fusion proteins are provided in the
Sequence Listing (SEQ. ID. Nos. 70-76). These molecules were made
by conventional peptide synthesis.
[0110] Once candidate binding partners have been identified,
desirably, they are analyzed in a characterization assay. Further
cycles of modeling and characterization assays can be employed to
more narrowly define the parameters needed in a binding partner.
Each binding partner and its response in a characterization assay
can be recorded on a computer readable media and a database or
library of binding partners and respective responses in a
characterization assay can be generated. These databases or
libraries can be used by researchers to identify important
differences between active and inactive molecules so that compound
libraries are enriched for binding partners that have favorable
characteristics. The section below describes several binding
partner characterization assays that can be used to evaluate
candidate binding partners.
[0111] Binding Partner and Candidate Binding Partner
Characterization Assays
[0112] The evaluation of candidate binding partners and, thus, the
determination whether a candidate binding partner is, in fact, a
binding partner can be accomplished by using a "characterization
assay". The term "characterization assay" refers to an assay,
experiment, or analysis made on a candidate binding partner or
binding partner, which evaluates the ability of said candidate
binding partner or binding partner to interact with HBcAg and/or
HBeAg or fragments thereof, effect viral infection, and a host
immune system response. 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), infectivity studies (e.g.,
reduction of viral infection, propagation, attachment to a host
cell), and analysis of host immune system response e.g., (clearance
of viral particles, reduction in viral lode, activation of antigen
presenting cells, and effect on B and T cell presentation). In
general, the characterization assays can be described in three
general catagories: (1) assays that determine whether a candidate
binding partner binds to HBcAg and/or HBeAg; (2) assays that
determine whether a binding partner reduces viral infectivity; and
(3) assays that determine whether a binding partner modulates a
host immune system response.
[0113] Preferred HBcAg and/or HBeAg binding assays use multimeric
agents. One form of multimeric agent concerns a manufacture
comprising a candidate binding partner or binding partner, or
fragments thereof disposed on a support. Another form of multimeric
agent involves a manufacture comprising HBcAg and or HBeAg or
fragments thereof disposed on a support. These multimeric agents
provide the attached molecule in such a form or in such a way that
a sufficient affinity is achieved. A "support" can be a termed a
carrier, a protein, a resin, a cell membrane, or any macromolecular
structure used to join or immobilize such molecules. 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 candidate
binding partner or binding partner 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.
[0114] In some multimeric agents, the macromolecular support has a
hydrophobic surface that interacts with a portion of the candidate
binding partner, binding partner, or viral antigen (e.g., HBcAg
and/or HBeAg) 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, candidate binding partner, binding partner, or viral
antigen can be covalently bound to supports including proteins and
oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane
or aminated sepharose). In these later multimeric agents, 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 multimeric agents comprise a
support that has other reactive groups that are chemically
activated so as to attach the candidate binding partner or binding
partner or fragments 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).
[0115] Furthermore, in some embodiments, a liposome or lipid
bilayer (natural or synthetic) is contemplated as a support and a
candidate binding partner, binding partner, or viral antigen 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 candidate binding partner,
binding partner, or viral antigen that is exposed on the surface. A
hydrophobic domain can be joined to the candidate binding partner
or binding partner so as to facilitate the interaction with the
membrane.
[0116] Supports for use in the body, (i.e. for prophylactic or
therapeutic applications) are desirably physiological, non-toxic
and preferably, non-immunoresponsive. Suitable carriers for use in
the body include poly-L-lysine, poly-D, L-alanine, liposomes, and
Chromosorb.RTM. (Johns-Manville Products, Denver Co.). Ligand
conjugated Chromosorb.RTM. (Synsorb-Pk) has been tested in humans
for the prevention of hemolytic-uremic syndrome and was reported as
not presenting adverse reactions. (Armstrong et al. J. Infectious
Diseases 171:1042-1045 (1995)). For some embodiments, a "naked"
carrier (i.e., lacking an attached binding partner) that has the
capacity to attach a binding partner in the body of a organism is
administered. By this approach, a "prodrug-type" therapy is
envisioned in which the naked carrier is administered separately
from the binding partner and, once both are in the body of the
organism, the carrier and the binding partner are assembled into a
multimeric complex.
[0117] The insertion of linkers, such as linkers (e.g., ".lamda.
linkers" engineered to resemble the flexible regions of .lamda.
phage) of an appropriate length between the candidate binding
partner, binding partner, or viral antigen and the support are also
contemplated so as to encourage greater flexibility of the
candidate binding partner, binding partner, or viral antigen and
thereby overcome any steric hindrance that can be presented by the
support. The determination of an appropriate length of linker that
allows for optimal binding to HBcAg and/or HBeAg, inhibition of
viral infectivity, and modulation of host immune response can be
determined by screening the attached molecule with varying linkers
in the characterization assays detailed in the present
disclosure.
[0118] A composite support comprising more than one type of binding
partner is also envisioned. A "composite support" can be a carrier,
a resin, or any macromolecular structure used to attach or
immobilize two or more different binding partners. In some
embodiments, a liposome or lipid bilayer (natural or synthetic) is
contemplated for use in constructing a composite support or binding
partners are attached to the membrane surface or are incorporated
into the membrane using techniques in liposome engineering.
[0119] As above, the insertion of linkers, such as .lamda. linkers,
of an appropriate length between the binding partner and the
support is also contemplated so as to encourage greater flexibility
in the molecule and thereby overcome any steric hindrance that can
occur. The determination of an appropriate length of linker that
allows for optimal binding to HBcAg and/or HBeAg, inhibition of
viral infectivity, and modulation of host immune response or lack
thereof, can be determined by screening the binding partners with
varying linkers in the characterization assays detailed in the
present disclosure.
[0120] Several approaches to identify agents that interact with
HBcAg and/or HBeAg, employ a multimeric support having candidate
binding partner, binding partner, or HBcAg and/or HBeAg or a
fragment thereof. For example, support-bound candidate binding
partner can be contacted with "free" HBcAg and/or HBeAg and an
association can be determined directly (e.g., by using labeled
HBcAg and/or HBeAg) or indirectly (e.g., by using a labeled
antibody directed to the HBcAg and/or HBeAg). Thus, candidate
binding partners are identified as binding partners by virtue of
the association of HBcAg and/or HBeAg with the support-bound
candidate binding partner. Alternatively, support-bound HBcAg
and/or HBeAg can be contacted with "free" candidate binding partner
and the amount of associated candidate binding partner can be
determined directly (e.g., by using labeled binding partner) or
indirectly (e.g., by using a labeled antibody directed to the
binding partner).
[0121] Assays that determine whether a candidate binding partner
binds to HBcAg and/or HBeAg can be conducted in a variety of
formats. Most simply, techniques in immunology can be employed or
readily adapted to ascertain whether a candidate binding partner
has bound HBcAg and/or HBeAg. In one set of characterization
assays, the interaction of 20 amino acid long candidate binding
partners with HBcAg was determined by enzyme immunoassays (EIA), as
described in the example below.
EXAMPLE 8
[0122] To determine whether a candidate binding partner bound to
HBcAg and/or HbeAg, a multimeric support-based binding assay was
performed. Accordingly, synthetic peptides corresponding to the VH
and VL domains of the sequenced mAbs were passively adsorbed to 96
well microplates in serial dilutions starting at 200 .mu.g/ml. The
peptide coated plates were then incubated with HBcAg and HBeAg
serially diluted in PBS-GT. The amount of HBcAg and HBeAg bound by
the peptides was then detected using a mAb directed to an epitope
common to HBcAg and HBeAg (See Sallberg et al., J General Virology
74:1335-1340 (1993), herein expressly incorportaed by reference in
its entirety) diluted 1:3000 in PBS-GT. The quantity of bound mAb
was determined by addition by peroxidase labeled rabbit anti-mouse
IgG (P260, Dako A S, Denmark). The plates were developed by
incubation with ortho-phenylene-diamine (Sigma) and the absorbance
at 492 nm was evaluated.
[0123] As shown in TABLE 7, many peptides were found to bind to
HBcAg in this characterization assay. Two conserved domains were
found in five of the binding partners (SEQ. ID. Nos. 5, 16, 17, 28,
and 29), which bound HBcAg with high affinity. These conserved
domains were Cys-Lys-Ala-Ser (SEQ. ID. No. 77) and Cys-Arg-Ala-Ser
(SEQ. ID. No. 78). Taken together, the levels of affinity of the
five binding partners and the conservation of the two domains
provided evidence that these domains are intimately involved in
binding to HBcAg. TABLE-US-00007 TABLE 7 Binding of support-bound
peptides to HBcAg and NS3 OD at 405 nm to indicated antigen Peptide
HBcAg HBcAg HCV # Sequence of peptide 2 .mu.g/well 0.2 .mu.g/well
NS3 MAb 4-2 1 VKLQQSGTEVVKPGASVKLS (SEQ. ID. No. 4) 0.323 0.124
0.021 2 VKPGASVKLSCKASGYIFTS (SEQ. ID. No. 5) 3.692 1.146 0.269 3
CKASGYIFTSYDIDWVRQTP (SEQ. ID. No. 6) 0.525 0.187 0.032 4
YDIDWVRQTPEQGLEWIGWI (SEQ. ID. No. 7) 0.551 0.202 0.089 5
EQGLEWIGWIFPGEGSTEYN (SEQ. ID. No. 8) 0.706 0.256 0.182 6
FPGEGSTEYNEKFKGRATLS (SEQ. ID. No. 9) 0.325 0.121 0.109 7
EKFKGRATLSVDKSSSTAYM (SEQ. ID. No. 10) 0.883 0.194 0.035 8
VDKSSSTAYMELTRLTSEDS (SEQ. ID. No. 11) 0.363 0.134 0.041 9
ELTRLTSEDSAVYFCARGDY (SEQ. ID. No. 12) 0.574 0.195 0.073 10
AVYFCARGDYDYYRRYFDLW (SEQ. ID. No. 13) 0.981 0.304 0.038 11
DYYRRYFDLWGQGTTVTVS (SEQ. ID. No. 14) 0.356 0.133 0.022 MAb 5H7 12
DIVLTQSPASLAVSLGQRAT (SEQ. ID. No. 15) 0.53 0.156 0.025 13
LAVSLGQRATISCRASQSVS (SEQ. ID. No. 16) 3.113 0.807 0.128 14
ISCRASQSVSTSSYSYMHWY (SEQ. ID. No. 17) 2.475 0.449 0.156 15
TSSYSYMHWYQQKPGQPPKL (SEQ. ID. No. 18) 1.442 0.574 0.028 16
QQKPGQPPKLLIKYASNLES (SEQ. ID. No. 19) 0.299 0.064 0.016 17
LIKYASNLESGVPARFSGSG (SEQ. ID. No. 20) 0.357 0.112 0.020 18
GVPARFSGSGSGTDFTLNIH (SEQ. ID. No. 21) 0.409 0.141 0.027 19
SGTDFTLNIHPVEEEDTATY (SEQ. ID. No. 22) 0.649 0.205 0.206 20
PVEEEDTATYYCQHSWEIPY (SEQ. ID. No. 23) 0.625 0.207 0.124 21
YCQHSWEIPYTFGGGTKLEI (SEQ. ID. No. 24) 0.498 0.173 0.052 22
TFGGGTKLEIKRADAAPAV (SEQ. ID. No. 25) 0.273 0.084 0.024 23
KRADAAPAVSIFPPSSKLG (SEQ. ID. No. 26) 0.465 0.162 0.021 MAb 9C8 24
IQLQQSGAELVKPGASVKIS (SEQ. ID. No. 27) 0.369 0.123 0.021 25
VKPGASVKISCKASGYSFTG (SEQ. ID. No. 28) 3.129 0.845 0.097 26
CKASGYSFTGYNMNWVKQSH (SEQ. ID. No. 29) 2.29 0.356 0.053 27
YNMNWVKQSHGKSLEWIGNI (SEQ. ID. No. 30) 0.157 0.114 0.021 28
GKSLEWIGNINIPYYGSTSYN (SEQ. ID. No. 31) 0.289 0.114 0.028 29
NPYYGSTSYNQKFKGKATLT (SEQ. ID. No. 32) 0.783 0.213 0.021 30
QKFKGKATLTVDKSSSTAYM (SEQ. ID. No. 33) 1.115 0.207 0.035 31
VDKSSSTAYMQLNSLTSEDS (SEQ. ID. No. 34) 0.338 0.114 0.106 32
QLNSLTSEDSAVYYCARGKG (SEQ. ID. No. 35) 0.528 0.121 0.035 33
AVYYCARGKGTGFAYWGQGT (SEQ. ID. No. 36) 1.203 0.227 0.035 34
TGFAYWGQGTLVTVSAAKTT (SEQ. ID. No. 37) 0.898 0.192 0.035 35
LVTVSAAKTTPPSVYPLVPV (SEQ. ID. No. 38) 0.59 0.198 0.032
[0124] The five "high affinity" binding partners (SEQ. ID. Nos. 5,
16, 17, 28, and 29) were also bound to a support and were analyzed
for the ability to bind various dilutions of "free" HBcAg and
HBeAg. As shown in TABLE 8, the five peptides exhibited appreciable
binding to HBcAg and HBeAg at concentrations as low as 0.67
.mu.g/ml. The converse of an aspect of this experiment was also
performed. That is, various dilutions of support-bound binding
partners (SEQ. ID. Nos. 5, 16, 17, 28, and 29) were contacted with
10 .mu.g/ml of HBcAg and binding was evaluated. As shown in TABLE
9, the peptides of SEQ. ID. Nos. 5, 16, 17, and 28 showed
appreciable binding at low concentrations of HBcAg and/or HBeAg. In
each of these experiments, the amount of peptide binding was
directly proportional to the amount of HBcAg or HBeAg added or the
amount of peptide disposed on the support. TABLE-US-00008 TABLE 8
Binding of dilutions of HBcAg and HBeAg to support-bound peptides
Amount HBcAg added (.mu.g/ml) Amount HBeAg added (.mu.g/ml) Peptide
# 20 10 5 2.5 1.25 0.67 20 10 5 2.5 1.25 0.67 2 3.453 1.692 0.989
0.374 0.155 0.034 0.751 0.222 0.096 0.063 0.043 0.081 13 2.635
0.872 0.501 0.195 0.079 0.028 0.393 0.060 0.038 0.030 0.023 0.024
14 2.660 1.150 0.633 0.252 0.117 0.039 0.687 0.246 0.132 0.102
0.056 0.046 25 2.652 0.897 0.359 0.159 0.058 0.036 0.319 0.096
0.050 0.115 0.034 0.022 26 1.479 0.601 0.245 0.101 0.038 0.028
0.241 0.091 0.091 0.063 0.026 0.027 27 0.305 0.115 0.059 0.042
0.026 0.020 0.040 0.037 0.029 0.032 0.021 0.018
[0125] TABLE-US-00009 TABLE 9 Binding of 10 .mu.g/ml HBcAg to
dilutions of support-bound peptides Amount peptide coated
(.mu.g/ml) Peptide # 100 50 25 12.5 6.25 3.125 2 1.453 1.218 1.039
0.913 0.597 0.333 13 0.900 0.434 0.276 0.240 0.388 0.355 14 0.641
0.569 0.636 0.514 0.630 0.413 25 1.011 0.766 0.569 0.423 0.298
0.217 26 0.422 0.198 0.181 0.179 0.156 0.171 27 0.151 0.170 0.162
0.181 0.175 0.168
[0126] Variations of the characterization assays described above
include competitive binding assays. For example, the five high
affinity peptides (SEQ. ID. Nos. 5, 16, 17, 28, and 29) were
analyzed for the ability to prevent binding of mAb 4-2 to HBcAg.
Initial experiments were conducted by contacting binding partner to
HBcAg prior to introducing the antibody. As shown in TABLE 10, all
five high affinity binding partners were able to inhibit binding of
mAb 4-2 when the concentration of peptide was raised to 200
.mu.g/ml. The peptides of SEQ. ID. Nos. 5, 28, and 29 effectively
reduced binding of the antibody at concentrations as low as 100
.mu.g/ml and the peptides of SEQ. ID. No. 5 and 29 appreciably
reduced binding of mAb 4-2 at concentrations as low as 50 .mu.g/ml.
The ability of the five high affinity binding partners to compete
for binding of the mAb 4-2 to HBcAg was also analyzed. When the
peptides of SEQ. ID. Nos. 5, 16, 28, and 29 were provided at 200
.mu.g/ml in the presence of mAb 4-2, the binding of the antibody to
HBcAg was significantly inhibited. (See TABLE 11). Similarly, the
peptides of SEQ. ID. Nos. 5, 16, 28, and 29 effectively competed
with mAb 4-2 at 100 .mu.g/ml. The peptide of SEQ. ID. No. 28
prevented binding of mAb 4-2 at a concentration as low as 12.5
.mu.g/ml, whereas, the peptides of SEQ. ID. Nos. 5 and 16 prevented
binding of mAb 4-2 at 6.25 .mu.g/ml. TABLE-US-00010 TABLE 10
Inhibition of mAb 4-2 binding to HBcAg coated at 1 .mu.g/ml by
prior incubation with synthetic peptides*. Amount peptide
(.mu.g/ml) added prior to mAb 4-2 Peptide # 200 100 50 25 12.5 6.25
2 0.078 0.083 0.188 0.341 0.331 0.336 13 0.299 0.345 0.364 0.429
0.365 0.350 14 0.305 0.370 0.402 0.439 0.380 0.370 25 0.173 0.274
0.312 0.359 0.339 0.329 26 0.201 0.280 0.337 0.383 0.357 0.351 16
0.368 0.394 0.383 0.406 0.391 0.336 *Values have been given as the
OD at 490 nm. 100% binding of mAb without addition of peptide
corresponds to an mean OD of 0.379
[0127] TABLE-US-00011 TABLE 11 Inhibition of mAb 4-2 binding to
HBcAg coated at 5 .mu.g/ml by simultaneous addition of mAb and
synthetic peptides*. Amount peptide (.mu.g/ml) added simultaneous
as mAb 4-2 Peptide # 200 100 50 25 12.5 6.25 2 0.139 0.194 0.395
0.698 0.824 0.867 13 0.859 0.891 0.998 1.027 1.037 0.979 14 1.468
1.458 1.557 1.390 1.442 1.264 25 0.302 0.910 1.217 1.200 1.207
1.178 26 0.341 0.947 1.250 1.347 1.275 1.196 16 0.925 1.115 1.232
1.239 1.235 1.065 *Values have been given as the OD at 490 nm. 100%
binding of mAb without addition of peptide corresponds to an mean
OD of 1.229
[0128] Preferably, the specificity exchangers are also evaluated in
characterization assays. Some of these characterization assays
evaluate the ability of the specificity exchanger to interact with
the target molecule and the redirecting antibody. Other
characterization assays evaluate the ability of the specificity
exchanger to fix complement. Still more characterization assays are
designed to determine whether a specificity exchanger can bind to
the target molecule, bind to the redirecting antibody, and fix
complement. The example below describes several characterization
assays that were conducted on the specificity exchangers.
EXAMPLE 9
[0129] To evaluate the efficacy of the specificity exchangers
described above, several binding assays using a mAb specific for
HSV gG2 were conducted. In one assay, the various specificity
exchangers were coated onto microtiterplates and were bound with
the mAb specific for HSV gG2. As shown in TABLE 12, the specificity
exchangers provided in SEQ. ID. Nos. 71, 74, and 75 appreciably
bound the mAb. In another assay, HBcAg was coated onto microtiter
plates, specificity exchanger was added (20 .mu.g/well), and the
binding of the mAb specific for HSV gG2 was determined. The
specificity exchangers provided in SEQ. ID. Nos. 71 and 74
appreciably bound the immobilized HBcAg and allowed for the binding
of the mAb specific for HSV gG2. In another binding assay, HBeAg
was coated onto microtiter plates, specificity exchanger was added
(20 .mu.g/well), and the binding of the mAb specific for HSV gG2
was determined. Similarly, the specificity exchangers provided in
SEQ. ID. Nos. 71 and 74 appreciably bound the immobilized HBeAg and
allowed for the binding of the mAb specific for HSV gG2. These
results demonstrate that specificity exchangers specific for HBcAg
and/or HBeAg can be manufactured and used to redirect HBV to high
titer antibodies. The characterization assays used to evaluate
binding partners can also be used and/or modified for the analysis
of the specificity exchangers. TABLE-US-00012 TABLE 12 Redirection
of antibodies specific for an epitope within HSV gG2 gG2 mAb
binding to indicated antigen on solid phase fusion peptide HBcAg
HBeAg Addition of fusion peptide in solution Peptide (20
.mu.g/well) # Fusion peptide sequence no yes yes 13
KLSCKASGYIFTSEHRGGPEE (SEQ. ID. No. 70) 0.040 0.020 0.022 14
KLSCKASGYIFTSHRGGPEEF (SEQ. ID. No. 71) 1.622 0.550 0.638 15
KLSCKASGYIFTSRGGPEEFE (SEQ. ID. No. 72) 0.062 0.016 0.040 16
VKLSCKASGYIFTSEHRGGPE (SEQ. ID. No. 73) 0.060 0.038 0.029 17
VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) 0.884 1.462 1.727 18
VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 75) 0.213 0.037 0.032 19
VKLSCKASGYIFTSGGPEEFE (SEQ. ID. No. 76) 0.018 0.025 0.024 Neg ctrl
Peptide 16 Not tested 0.038 0.022
[0130] In another experiment, it was found that binding of the
peptide VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) to HBcAg can be
inhibited by a peptide derived from HBcAg. That is, the precise
domain on HbcAg that binds to the peptide VKLSCKASGYIFTSHRGGPEE
(SEQ. ID. No. 74) was discovered. In this experiment, microplates
were coated overnight with 1 .mu.g/ml HBcAg and, subsequently, a
100 .mu.l mixture containing 5 .mu.g of the peptide
VKLSCKASGYIFTSHRGGPEE (SEQ. ID No. 74) and 5 .mu.g of an HBcAg
peptide was added to the coated plate. The HBcAg peptides were
approximately 20 amino acids in length and corresponded to regions
of HBcAg spanning amino acid residues 11-183. The experiment was
performed in triplicate. The amount of bound peptide (SEQ. ID. No.
74) was then determined by adding a 1:1000 dilution of an HSV gG2
mAb followed by the addition of enzyme conjugated anti-mouse Ig,
which interacts with the HSV gG2 mAb. The amount of enzyme present,
which directly correlates to the amount of peptide (SEQ. ID. No.
74) that bound to the coated plates, was then determined by adding
the substrate ortho-phenylene diamine and analyzing the absorbance
at 490 nm. As shown in TABLE 13, the HBcAg peptide
SRDLVVSYVNTNMGLKFRQL (SEQ. ID. NO. 103) and TNMGLKFRQLLWFHISCLTF
(SEQ. ID. No.104), which correspond to amino acid residues 81-100
and 91-110 of HbcAg, respectively consistently reduced the binding
of the peptide VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74). This result
indicated that the HBcAg-binding domain of the peptide
VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) involves amino acid
residues 91-110 of HBcAg.
[0131] Thus, embodiments also include complexes of binding partners
with HBcAg, HBeAg, HBV capsids, and HBV itself or fragments
thereof. Preferred embodiments include complexes of binding partner
and peptides comprising, consiting essentially of or consisting of
either SEQ. ID. No. 103 or SEQ. ID. No. 104. Highly preferred
embodiments include complexes comprising a peptide comprising,
consiting essentially of or consisting of the peptide of SEQ. ID.
No. 74 joined to either SEQ. ID. Nos. 103 or 104. Additional
embodiments include peptides comprising, consiting essentially of
or consisting of the sequence of SEQ. ID. Nos. 103 or 104 or
nucleic acids encoding these molecules. These embodiments are
valuable research tools and can be used as active ingredients in
vaccines or therapeutics. TABLE-US-00013 TABLE 13 Std.
Corresponding Sequence Deviation Sequence of inhibiting residues
within ID OD at from the HBcAg peptide HBcAg No. 490 nm mean none
-- -- 1.454 0.831 ATVELLSFLPSDFFPSVRDL 11-30 96 1.387 0.696
SDFFPSVRDLLDTASALYRE 21-40 97 1.576 1.077 LDTASALYREALESPEHCSP
31-50 98 1.589 1.103 ALESPEHCSPHHTALRQAIL 41-60 99 1.372 0.665
HHTALRQAILCWGELMTLAT 51-70 100 0.646 -0.798 CWGELMTLATWVGVNLEDPA
61-80 101 1.395 0.712 WVGVNLEDPASRDLVVSYVN 71-90 102 1.411 0.744
SRDLVVSYVNTNMGLKFRQL 81-100 103 0.386 -1.323 TNMGLKFRQLLWFHISCLTF
91-110 104 0.058 -1.984 LWFHISCLTFGRETVIEYLV 101-120 105 1.129 .175
GRETVIEYLVSFGVWIRTPP 111-131 106 0.832 -0.423 SFGVWIRTPPAYRPPNAPIL
121-140 107 0.701 -0.688 AYRPPNAPILSTLPETTVVR 131-150 108 1.564
1.052 STLPETTVVRRRGRSPRRRT 141-160 109 0.811 -0.466
RRGRSPRRRTPSPRRRRSQS 151-170 110 0.732 -0.625
PSPRRRRSQSPRRRRSQSRESQC 161-183 111 0.678 -0.734 Mean OD .+-. SD --
-- 1.042 .+-. 0,469 --
[0132] To demonstrate that the biological activities encompassed by
the Fc domain of the HSV mAb was introduced to the specificity
exchanger (SEQ. ID. No. 74), a complement binding assay was
performed. Accordingly, the assay was conducted by mixing the
antibody to be tested with the appropriate antigen and allowing
binding to occur overnight. Subsequently, rabbit complement, which
is consumed if the antibody binds the antigen and the complement,
was added. To determine the amount of residual complement in the
mixture, sheep erythrocytes and an antibody to sheep erythrocytes
was added. If the complement has been exhausted (i.e. a complement
binding antibody has bound to the antigen) lysis of red blood cells
will not occur. If no antibody-antigen complex has formed then the
remaining complement will lyse the red blood cells bound by the
specific antibody.
[0133] The complement binding of the HBcAg-specific mAb 9C8 was
evaluated together with the ability of the specificity exchanger
peptide (SEQ. ID. No. 74) to inhibit the binding of mAb 9C8 to
HBcAg. Serial dilutions of mAb 9C8 were mixed with HBcAg in the
presence and absence of the specificity exchanger peptide (SEQ. ID.
No. 74). As shown in TABLE 14, the mAb 9C8 bound to HBcAg
appreciably bound complement and the specificity exchanger peptide
(SEQ. ID. No. 74) was unable to compete away bound mAb 9C8.
TABLE-US-00014 TABLE 14 Complement binding activity of mAb 9C8 in
the presence of a specificity exchanger Amout peptide Amount Amout
mAb 9C8 SEQ.ID. No. 74 HBcAg (pmol) (pmol) (pmol) 6.7 3.3 1.7 0.8
none none - - - - none 24 + + - - 4000 24 + + + - 400 24 + + - ? 40
24 + + - - 4 24 + + - - 0.4 24 + + - -
[0134] The complement binding of mAb 4-2 was also evaluated in
conjunction with the ability of the specificity exchanger peptide
(SEQ. ID. No. 74) to inhibit binding of mAb 4-2 to HBcAg.
Accordingly, serial dilutions of mAb 4-2 were mixed with HBcAg in
the presence and absence of the specificity exchanger peptide (SEQ.
ID. No. 74). As shown in TABLE 15, the mAb 4-2 bound to HBcAg also
bound complement. Further, the specificity exchanger (SEQ. ID. No.
74) appreciably inhibited the binding of mAb 4-2 to HBcAg.
TABLE-US-00015 TABLE 15 Complement binding activity of mAb 4-2 in
the presence of a specificity exchanger Amout peptide Amount Amount
4-2 SEQ.ID. No. 74 HBcAg mAb (pmol) (pmol) (pmol) 6.7 3.3 1.7 0.8
none none - - - - none 24 +/- + - - 4000 24 - - - - 400 24 - - - -
40 24 - - - - 4 24 +/- - - +/- 0.4 24 +/- +/- - -
[0135] The ability of the complex of HSV mAb, specificity exchanger
(SEQ. ID. No. 74), and HBcAg to bind complement was also evaluated.
Accordingly, serial dilutions of the HSV mAb were mixed with HBcAg
in the presence and absence of the specificity exchanger (SEQ. ID.
No. 74). As shown in TABLE 16, the HSV mAb did not activate
complement in the absence of the specificity exchanger (SEQ. ID.
No. 74). However, when the HSV mAb and the specificity exchanger
(SEQ. ID. No. 74) were present at equimolar ratios, the HSV mAb
bound HBcAg through the specificity exchanger (SEQ. ID. No. 74) and
the mab-specificity exchanger-HBcAg complex was able to bind
complement. These data confirm that an antibody bound to the
specificity exchanger (SEQ. ID. No. 74) can impart the biological
activity of the antibody to the specificity exchanger peptide.
TABLE-US-00016 TABLE 16 Complement binding activity of a
specificity exchanger bound to HBcAg and a HSV mAb Amout peptide
Amount Amout HSV mAb (pmol) SEQ. ID. No. 74 HBcAg Excess Excess
Excess Excess (pmol) (pmol) 6.7 peptide 3.3 peptide 1.7 peptide 0.8
peptide none none - - - - None 24 - - - - 4000 24 +/- 597 - 1212 -
2352 - 5000 400 24 - 59.7 - 121.2 - 235.2 + 500 40 24 - 5.97 -
12.12 - 23.52 - 50 4 24 - 0.597 +/- 1.212 + 2.352 - 5 0.4 24 -
0.0597 - 0.1212 +/- 0.2352 - 0.5
[0136] In another type of characterization assay, the ability of a
binding partner to inhibit viral infectivity is analyzed. Several
types of HBV viral infectivity assays can determine the efficacy of
anti-HBV materials by detecting a reduction in HBV nucleic acid
synthesis and/or the presence of HBV antigens. These assays can be
readily adapted to determine whether a binding partner inhibits HBV
propagation. By one approach, a human hepatoblastoma cell culture
assay is used to evaluate the ability of binding partners to
inhibit HBV replication. (See Korba and Gerin, Antiviral Res. 19:
55-70 (1992) and Korba and Milman, Antiviral Res. 15:217-228
(1991), both references herein incorporated by reference in their
entirety). By another approach, persistently infected HepG2 cells
can be used to evaluate the ability of binding partners to inhibit
HBV replication. The toxicity of binding partners can also be
assessed under the same culture and treatment conditions. The
example below describes several characterization assays that can be
used to determine the ability of a binding partner to inhibit HBV
infectivity.
EXAMPLE 10
[0137] A human hepatoblastoma cell culture assay can be used to
evaluate the ability of a binding partner to inhibit HBV
replication. The detection of HBV propagation is determined by
analyzing the presence of HBV nucleic acid in the media and cells.
Human hepatoblastoma 2.2.15 cells are grown to confluency and are
provided a daily dose of binding partner in RPMI1640 medium with 2%
fetal bovine serum. Ideally, a titration of binding partner (e.g.,
500 .mu.M to 1 .mu.M) is provided to aliquots of cells. As a
control, scrambled peptides having a similar amino acid content are
added at the same concentrations. Medium is analyzed for HBV virion
DNA before treatment and daily during treatment. HBV DNA can be
extracted from medium and analyzed by slot blot analysis, for
example. Intracellular HBV DNA is also analyzed after 10 days of
treatment. Preferably, the cellular DNA is prepared and analyzed by
Southern blot analysis using a .sup.32P-labelled 3.2 kb EcoRI HBV
DNA fragment as a probe. Quantitation can be accomplished by
comparison to HBV standards loaded on each gel. Desirable binding
partners will inhibit HBV propagation by greater than 50%, as
demonstrated by a reduction of HBV virion DNA in the medium and 3.2
Kb DNA syntheses.
[0138] Additionally, the effect of a binding partner on HBV
propagation in HEP-G2 cells can be monitored by detecting the
presence of HBV antigens (e.g., HBsAg and HBeAg) in the media. Kits
that detect these hepatitis markers are commercially available.
(Abbott Laboratories). As above, human hepatoblastoma 2.2.15 cells
are grown to confluency and are provided a daily dose (200
.mu.g/ml) of binding partner in RPMI1640 medium with 2% fetal
bovine serum. Medium is assayed for HBV virion DNA before treatment
and periodically during treatment for the presence of a viral
antigen by using a commercially available kit (Abbott
Laboratories). Desirable binding partners will inhibit HBV
propagation by greater than 50%, as demonstrated by a reduction of
viral antigen present in the medium.
[0139] By another approach, HEP-G2 cells, persistently infected
with HBV, are treated daily with fresh D-MEM containing 20% FBS and
a titration of binding partner, as above. After 9 days incubation
and treatment, the cells and overlay medium are harvested
separately to assay the quantity of nucleic acids. Extracellular
virion DNA in untreated cells will range from from 50 to 150 pg/ml
in the overlay medium. Intracellular HBV DNA replication
intermediates (RI) in untreated cells will range from 50 to 100
pg/.mu.g cell DNA. Hybridization analysis will show that
approximately 1 pg intracellular HBV DNA/.mu.g cellular DNA to 2-3
genomic copies per cell and 1.0 pg of extracellular HBV DNA/ml
overlay medium to 3.times.10.sup.3 viral particles/ml. HBV RNA can
also be analyzed by Northern blot hybridization analysis using a
.sup.32P-labeled 3.2 Kb gel-purified cloned genomic probe.
Quarititive analysis of intracellular HBV DNA and 14BV RNA can be
performed using an AMBIS beta scanner. Desirable binding partners
will inhibit the level of HBV propagation demonstrated by untreated
cells by a value of greater than 50%, as demonstrated by a
reduction of virion replication intermediates (RI) and 3.2 Kb DNA
syntheses.
[0140] Additionally, the effect of a binding partner on HBV
propagation in a human hepatoblastoma cell culture assay is
monitored by detecting the presence of HBV antigens (e.g., HBsAg
and HBeAg) in the media. Kits that detect these hepatitis markers
are commercially available. (Abbott Laboratories). HEP-G2 cells are
persistently infected with HBV and are treated daily with fresh
D-MEM containing 20% FBS and 200 .mu.g/ml binding partner. After 9
days of incubation and treatment, the overlay medium is harvested
to assay the quantity of HBsAg and HBeAg by EIA (Abbott
Laboratories). The overlay medium is diluted to levels of antigen
in the linear range of the assay. Standard curves using dilutions
of positive HBsAg and HBeAg controls are included in each assay.
Desirable binding partners will inhibit HBV propagation by greater
than 50%, as demonstrated by a reduction of viral antigen present
in the medium.
[0141] Toxicity of a binding partner can also be determined by the
exclusion of neutral red dye uptake in cells grown in 96-well
plates and treated as described above. One day after the final
addition of binding partner, medium is removed and 0.2 ml of DPBS
containing 0.01% neutral red dye (Sigma, Inc.) is added to each
well. Cells are allowed to recover for two hours. Dye is removed,
cells are washed with DPBS and then 0.2 ml of 50% EtOH/1% glacial
acetic acid is added to each well. After 30 minutes of gentle
mixing, absorbance at 510 nm is measured and compared to untreated
control cultures. Desirable binding partners will demonstrate a
toxicity of less than 5% at concentrations ten fold greater than
that shown to be effective at inhibiting HBV propagation. The in
vitro assays described in this example can be used to rapidly
determine whether a binding partner can inhibit HBV infection.
[0142] Characterization assays also include experiments designed to
test binding partners in vivo. There are many animal models that
are suitable for evaluating the ability of a binding partner to
inhibit HBV infection. The woodchuck model has been used in
hepatitis research for over a decade. (See e.g., Gerin, J. L. 1984.
In Advances in Hepatitis Research. F. Chisari, ed. Masson
Publishing USA, Inc. New York, pp. 40-48; Gerin et al. 1986 In
Vaccines 86: New approaches to Immunization. F. Brown et al., eds.
Cold Spring Harbor Laboratory Press, N.Y., pg 383-386, herein
expressly incorporated by reference in its entirety). The woodchuck
hepatitis virus (WHV) is closely related to HBV, both
immunologically and in terms of sequence homology. Woodchucks are
now bred and reared for experimental hepatitis research. Infection
of young animals with defined WHV inocula yields chronic carriers
for drug testing and research. At least one commercial testing
facility is devoted to testing of compounds in woodchucks. Tennant,
B. C. and J. L. Gerin. 1994. In The Liver: Biology and
Pathobiology, Third Edition. I. M. Arias et al., eds. Raven Press,
Ltd., N.Y. pp 1455-1466, herein expressly incorporated by reference
in its entirety. Because of the sequence homology between HBV and
WHV, the efficacy of the binding partners can be evaluated in the
woodchuck model. Furthermore, demonstration of binding partner
efficacy in this model is a clear demonstration of a specific
pharmacologic effect to those of skill in the art.
[0143] A more recently developed animal model for HBV uses
transgenic rats that express human hepatitis B virus genes. (See
e.g., Takahashi et al., Proc. Natl. Acad. Sci. U.S.A. 92, 1470-1474
(1995), herein expressly incorporated by reference in its
entirety). These animals develop acute hepatitis and viral
particles and HBeAg are seen in the blood between three and seven
days after transfection. HBV is expressed in the liver and liver
cell death results. These effects and the subsequent clearing of
virions from the blood mimic the effects of acute HBV infection in
humans. Therefore activity of binding partners in this model is
indicative of therapeutic activity in humans to those of skill in
the art.
[0144] Chimpanzees are hosts for HBV, and therefore constitute
another animal model for HBV induced disease. The serological
events following infection in chimpanzees are identical to that
observed in humans. Both acute and chronic infections result from
exposure of chimpanzees to HBV. However, chimpanzees do not have
recognizable clinical symptoms of hepatitis. Cornelius, C. E.,
1988, in The Liver: Biology and Pathobiology, Second Ed. I. M.
Arias et al., eds. Raven Press, Ltd., N.Y., pp. 1315-1336, herein
expressly incorporated by reference in its entirety. Demonstration
of activity in this model, in which the animal is infected with the
same virus that infects humans, is also indicative of therapeutic
effect in humans to those skilled in the art. The example below
describes in vivo assays that can be performed in woodchucks to
determine whether a binding partner can inhibit HBV infection.
EXAMPLE 11
[0145] Binding partners can be evaluated for their ability to
inhibit HBV infection at a commercial facility, which routinely
screens anti-HBV and anti-hepatocellular carcinoma drug candidates
in the woodchuck hepatitis model. (Marmotech, Inc. of Ithaca,
N.Y.). Two doses of binding partner are tested, 20 mg/kg and 2
mg/kg, with three animals receiving each dose. Binding partners are
administered intravenously in 0.1 ml of PBS every other day for 30
days, for a total of 15 doses. The primary end point of the assay
is level of circulating virus. Blood samples are collected on day
0, prior to drug treatment, and at days 1, 2, 4, 8, 15, 22 and 30
of treatment. Virus is quantitated by dot blot or Southern blot
analysis using the methods described above or by monitoring the
presence of viral antigens in the blood using a commercially
available kit (Abbott Laboratories). Alternatively, binding
partners can be evaluated in rats or mice made transgenic for HBV
genes. (See Takahashi et al., Proc. Natl. Acad. Sci. U.S.A.,
92:1470-1474 (1995), herein incorporated by reference in its
entirety). The next example describes an approach that was used to
evaluate the efficacy of a binding partner (e.g., a specificity
exchanger) for use as the active ingredient in a pharmaceutical
that is administered to treat or prevent HBV infection.
EXAMPLE 12
[0146] By using an animal model of HBV infection, the therapeutic
efficacy of a specificity exchanger directed to HBV was determined.
In a first group of experiments, two HBeAg-transgenic mice
(obtained from Dr. David R Milich, The Scripps Research Institute,
La Jolla, Calif.) were injected ip. with 500 .mu.l of undiluted
hybridoma supernatants. The mice were then bled on day zero, two,
six and nine post-injection. Sera were then tested for the change
in serum HBeAg levels as determined by EIA (Sorin Biomedica,
Saluggia, Italy). As shown in TABLE 17, neither PBS or mAb 3-4 had
an effect on the HBeAg levels in serum. In contrast, mAb 4-2
complexed an appreciable amount of serum HBeAg (i.e., over a two
fold increase in serum HBeAg was detected by EIA). This data
confirms that mAb 4-2 binds HBeAg in vivo at an epitope that does
not compete with the antibodies present in the commercial EIA.
TABLE-US-00017 TABLE 17 In vivo aggregation of serum HBeAg in
transgenic mice by mAbs 3-4 and 4-2* Fold change in serum HBeAg
levels as determined by EIA in Mab given HBeAg-Tg mice at indicated
day from injection in vivo 0 2 6 9 PBS 1 0.8 1.1 1 3-4 1 0.8 1.1
1.1 4-2 1 2.7 2.2 1.7 *Mabs were injected i.p. as 0.5 ml culture
supernatants containing around 10 .mu.g/ml of mAb (.sup..about.30
pmole/mouse).
[0147] In a second group of experiments, two HBeAg-transgenic mice
were injected i.p. with either culture media (RPMI), the HSV mAb,
the HBcAg-specific mAb 9C8, or the specificity exchanger peptide
(SEQ. ID. No. 74). As shown in TABLE 18, the negative controls RPMI
and the HSV mAb alone show only a 1.2 to 1.9 fold changes in
aggregation of serum HBeAg. By day nine, the mice that received the
specificity exchanger peptide (SEQ. ID. No. 74) had a 3.3 fold
increase in aggregation of serum HBeAg levels. Similar to the 4-2
mAb, the specificity exchanger peptide (SEQ. ID. No. 74) retained
the ability to complex serum HBeAg in vivo. These results verify
that a specificity exchanger directed to an HBV antigen can be
created and that such an agent can appreciably aggregate HBcAg
and/or HBeAg in vivo. TABLE-US-00018 TABLE 18 In vivo aggregation
of serum HBeAg in transgenic mice* Fold change in serum HBeAg
levels mAb given as determined by EIA in HBeAg-Tg in vivo mice at
indicated day from injection Day 0 1 3 6 9 RPMI 1 1.2 1.7 1.4 1.7
HSV mAb 1 1.2 1.9 1.5 1.7 9C8 1 1.4 2.0 1.7 1.8 Peptide of 1 1.5
2.3 2.3 3.3 SEQ.ID. No. 74 *Mabs were injected i.p. as 0.2 to 0.5
ml culture supernatants containing approx. 10 .mu.g/ml of mAb
(.sup..about.30 pmole/mouse). The final volume injected per mouse
was 0.5 ml.
[0148] Another particularly desirable characterization assay
evaluates specificity exchangers in chimpanzees infected with HBV.
The next example describes this characterization assay in
detail.
EXAMPLE 13
[0149] An approach to evaluate the efficacy of a specificity
exchanger in chimpanzees is provided below. Accordingly,
chimpanzees are repeatedly inoculated with an antigen that is known
to promote a high titer antibody response (e.g., HSVgG2). After the
course of immunization, the presence of polyclonal antibodies to
the antigen is verified. Subsequently, the chimpanzee is infected
with HBV and after a stable infection is verified, the infected
animal is provided a therapeutically effective dose of the
specificity exchanger (SEQ. ID. No. 74). The dosages used in
various animals include 100 .mu.g/kg body weight to 500 .mu.g/kg
body weight. Aggregation of HBcAg and/or HBeAg is verified and the
clearance of viral particles and long term monitoring of infection
is conducted by analyzing blood samples for the presence of HBV
nucleic acid or protein by using the techniques described in
Example 10. Over a period of several treatments with the
specificity exchanger, a reduction in viral lode will be
observed.
[0150] Characterization assays also include experiments that
evaluate the ability of a binding partner to modulate a host immune
response to HBV. It is contemplated that HBV reduces the CTL
response of an infected host by targeting HBcAg to a high number of
B cells, which then process the antigen. Through antigen leakage,
HBcAg-peptides are present on the class I molecules of B cells
whereby the CTL response against HBcAg and HBV is inhibited. To
prevent this molecular cascade and, thereby, modulate a host immune
response to HBV, binding partners that inhibit the binding of HBcAg
to B cells can be provided so as to prevent tolerization of the
HBcAg-specific CTL response of an infected host. Subjects infected
with HBV have antigen presenting cells (e.g., dendritic cells and B
cells) that display HBV viral antigens and effect T cell
proliferation. By analyzing the ability of binding partners to
modulate B cell presentation of HBV antigens in vitro, for example,
one can accurately determine whether a binding partner will
modulate a host immune response to HBV. Accordingly, this type of
characterization assay is performed by obtaining "naive" B cells
(i.e., B cells from animals that have not come in contact with HBV
or an HBV antigen) and contacting the naive B cells with
"experienced" T cells (i.e., T cells from animals that have been
immunized with a HBV viral antigen or infected with HBV) in the
presence of a binding agent. The ability of the binding agent to
modulate a host immune response to HBV is then determined by
monitoring the production of cytokines and/or T cell proliferation.
The example below describes an assay that was used to determine the
ability of a binding partner to modulate an immune response in a
subject.
EXAMPLE 14
[0151] A characterization assay that was used to evaluate the
ability of a binding partner to modulate a host immune system
response to HBV was conducted as follows. Groups of Balb/c mice
were immunized subcutaneously with 20 .mu.g HBcAg. Ten days later,
draining lymph nodes were harvested and experienced CD4.sup.+ T
cells were purified using anti-CD4 coated magnetic beads (Dynal A
S, Oslo, Norway) according to the manufacturers instructions. Naive
B cells were obtained from syngeneic mice, which had not been
immunized. These B cells were used as antigen presenting cells in
the characterization assay.
[0152] The ability of the different mAbs and peptides to block B
cell uptake and antigen presentation of HBcAg-peptides to
HBcAg-specific CD4+ T cells was then tested in vitro. The cell
populations were mixed at a B/T cell ratio of 1:5 and added at a
final concentration of approximately 2.5.times.10.sup.5 to
5.0.times.10.sup.5 cells per well in a 96 well microplate.
Approximately, 20 .mu.g of HBcAg was preincubated with the binding
partner. The binding partner mixtures were then added to the cells
and the plates were incubated for 26 to 96 hours at 37.degree. C.
The effect of the binding partners on T cell proliferation was
determined by monitoring [.sup.3H] thymidine incorporation.
[0153] As shown in TABLE 19, mAb 9C8 and mAb 4-2 and the HBcAg and
HBeAg binding peptide (SEQ.ID. No. 74) inhibited B cell mediated
antigen presentation of HBcAg to specific CD4+ T cells. In
contrast, the control peptide (SEQ.ID. No. 19) showed no
inhibition. Thus, the specificity exchanger (SEQ.ID. No. 74) not
only bound HBcAg and HBeAg but also possessed the ability to block
uptake and antigen presentation of HBcAg by B cells. TABLE-US-00019
TABLE 19 Inhibition of B cell - mediated antigen presentation of
HBcAg to experienced T cells* % inhibition* Cell population Antigen
Inhibitor cpm SD of APC T cells 20 .mu.g HBcAg none 82 30 Negative
control T + B cells none none 110 46 background proliferation T + B
cells 20 .mu.g HBcAg none 573 28 Positive control T + B cells 20
.mu.g HBcAg Mab 9C8 20 1 100 T + B cells 20 .mu.g HBcAg MAb 4-2 245
123 71 T + B cells 20 .mu.g HBcAg 4-2 peptide 345 8 49 (SEQ.ID. No.
74) T + B cells 20 .mu.g HBcAg Control peptide 572 183 0 (SEQ.ID.
No. 19) *Calculated as 1 minus [(cpm of cells with inhibitor minus
cpm of background proliferation) divided by (positive control cpm
minus cpm of background proliferation)].
[0154] It is contemplated that some binding partners will be highly
efficient B cell stimulatory molecules in that they effect a rapid
and potent T cell response. Other binding partners are contemplated
to weakly activate antigen presenting cells and, thus, stimulate a
weak T cell response, if any at all. Classes of such weak and
strong binding partners can be created based on similarities in
structure and function. These classes and profiles can be entered
onto a computer readable media, placed in a database, and accessed
for comparison so as to develop more effective weak and strong
binding partners. The section below describes the use of binding
partners as biotechnological tools and diagnostic reagents.
[0155] Biotechnological Tools and Diagnostic Reagents
[0156] In one aspect, binding partners are used as biotechnological
tools that detect the presence or absence, as well as the
concentration of HBcAg or HBeAg in a biological sample. The
peptides can be used in many different immunohistochemical
techniques including but not limited to, immunoprecipitation,
Western blot, affinity purification, and in situ analysis.
Advantageously, some embodiments can be used as high affinity
probes that detect HBV in tissues that are difficult to label using
conventional antibodies.
[0157] Desirably, the binding partners are used as diagnostic
reagents to determine the presence of HBV infection in a subject or
to monitor the treatment of HBV infection in a subject. Further,
the manufacture of kits that incorporate the binding partners are
contemplated. The detection component of these kits will typically
be supplied in combination with one or more of the following
reagents. A support capable of absorbing or otherwise binding
protein will often be supplied. Available supports include
membranes of nitrocellulose, nylon or derivatized nylon that can be
characterized by bearing an array of positively charged
substituents. One or more control reagents, buffers, enzymes, and
detection material (e.g., radioisotope, enzyme conjugate and
substrate, magnetic particle, gold particle, or secondary antibody
with or without conjugate) can be supplied in these kits.
[0158] The presence of HBV in a protein sample can be detected by
using conventional assays and a binding partner or specificity
exchanger. In some embodiments, a binding partner or specificity
exchanger is used to immunoprecipitate HBV viral antigens from
solution or are used to react with HBV viral antigens on Western or
Immunoblots. Favored diagnostic embodiments also include
enzyme-linked immunosorbant assays (ELISA), radioimmunoassays
(RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays
(IEMA), including sandwich assays using monoclonal and/or
polyclonal antibodies. Exemplary sandwich assays are described by
David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby
incorporated by reference. Other embodiments employ aspects of the
immune-strip technology disclosed in U.S. Pat. Nos. 5,290,678;
5,604,105; 5,710,008; 5,744,358; and 5,747,274, herein incorporated
by reference.
[0159] In another preferred protein-based diagnostic, binding
partners or specificity exchangers are attached to a support in an
ordered array, wherein a plurality of binding partners or
specificity exchangers are attached to distinct regions of the
support that do not overlap with each other. These arrays are
designed to be "addressable" such that the distinct locations are
recorded and can be accessed as part of an assay procedure. The
binding partners or specificity exchangers (collectively referred
to as "probes" in this context) are joined to the support in
different known locations. The knowledge of the precise location of
each probe makes these "addressable" arrays particularly useful in
binding assays. For example, an addressable array can comprise a
support having several regions to which are joined a plurality of
probes that specifically recognize the presence of HBcAg and/or
HBeAg in a biological sample obtained from subjects suspected of
having contact with HBV.
[0160] Accordingly, proteins are recovered from biological samples
from subjects suspected of contracting HBV and are labeled by
conventional approaches (e.g., radioactivity, calorimetrically, or
fluorescently). The labeled protein samples are then applied to the
array under conditions that permit binding to the probes. If a
protein in the sample binds to a probe on the array, then a signal
will be detected at a position on the support that corresponds to
the location of the probe-protein complex. Since the identity of
each labeled sample is known and the region of the support on which
the labeled sample was applied is known, an identification of the
presence of HBV infection can be rapidly determined. These
approaches are easily automated using technology known to those of
skill in the art of high throughput diagnostic analysis.
[0161] In another embodiment, an opposite approach to that
presented above can be employed. Proteins present in biological
samples can be disposed on a support so as to create an addressable
array. Preferably, the protein samples are disposed on the support
at known positions that do not overlap. The presence of a viral
antigen in each sample is then determined by applying labeled
probes that recognize HBcAg and/or HBeAg. Because the identity of
the biological sample and its position on the array is known, an
identification of the presence of HBV infection can be rapidly
determined. As detailed above, any addressable array technology
known in the art can be employed with this aspect and display the
protein arrays on the chips in an attempt to maximize antibody
binding patterns and diagnostic information.
[0162] Although many embodiments were chemically synthesized using
conventional techniques in peptide chemistry, nucleic acids
encoding the peptides can be introduced into cells in vitro or in
vivo and the recipient cells can be made to express a binding
partner, preferably a specificity exchanger. A description of
several approaches to make cells that express a binding partner is
given in the section below.
[0163] Cells Made to Express Binding Partners
[0164] Cells made to express a binding partner, whether in vivo or
in vitro, are embodiments of the invention. The concentration of a
binding partner, preferably a specificity exchanger, can be raised
in a cell in vitro by transfecting expression constructs encoding
these molecules. In vivo expression constructs can also be used to
deliver a nucleic acid encoding a binding partner to liver cells in
an animal. Liposome mediated transfer can also be used to transfer
a nucleic acid encoding a binding partner to a cell in vivo or in
vitro.
[0165] The following is provided as one possible method to express
a binding partner or specificity exchanger in a cell in vitro.
First, the methionine initiation codon for a binding partner or
specificity exchanger and the poly A signal of the gene are
identified. If the nucleic acid encoding the polypeptide to be
expressed lacks a methionine to serve as the initiation site, an
initiating methionine can be introduced next to the first codon of
the nucleic acid using conventional techniques. Similarly, if the
nucleic acid lacks a poly A signal, this sequence can be added to
the construct by, for example, splicing out the Poly A signal from
pSG5 (Stratagene) using BglI and SalI restriction endonuclease
enzymes and incorporating it into the mammalian expression vector
pXT1 (Stratagene). The vector pXT1 contains the LTRs and a portion
of the gag gene from Moloney Murine Leukemia Virus. The position of
the LTRs in the construct allow efficient stable transfection. The
vector includes the Herpes Simplex Thymidine Kinase promoter and
the selectable neomycin gene.
[0166] The nucleic acid encoding the polypeptide to be expressed
can be obtained by PCR from a bacterial vector having the binding
partner using oligonucleotide primers complementary to the nucleic
acid and containing restriction endonuclease sequences for Pst I
incorporated into the 5'primer and BglII at the 5' end of the
corresponding cDNA 3' primer, taking care to ensure that the
nucleic acid is positioned in frame with the poly A signal. The
purified fragment obtained from the resulting PCR reaction is
digested with PstI, blunt ended with an exonuclease, digested with
Bgl II, purified and ligated to pXT1, now containing a poly A
signal and digested with BglII. The ligated product is transfected
into a suitable cell line using Lipofectin (Life Technologies,
Inc., Grand Island, N.Y.) under conditions outlined in the product
specification. Positive transfectants are selected after growing
the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.).
Preferably the expressed protein is released into the culture
medium, thereby facilitating purification.
[0167] Another approach utilizes the "Xpress system for expression
and purification" (Invitrogen, San Diego, Calif.). The Xpress
system is designed for high-level production and purification of
recombinant proteins from bacterial, mammalian, and insect cells.
The Xpress vectors produce recombinant proteins fused to a short
N-terminal leader peptide that has a high affinity for divalent
cations. Using a nickel-chelating resin (Invitrogen), the
recombinant protein can be purified in one step and the leader can
be subsequently removed by cleavage with enterokinase.
[0168] One preferred vector for the expression of binding partners
and fragments of binding partner is the pBlueBacHis2 Xpress. The
pBlueBacHis2 Xpress vector is a Baculovirus expression vector
containing a multiple cloning site, an ampicillin resistance gene,
and a Lac Z gene. By one approach, the binding partner or
specificity exchanger nucleic acid is cloned into the pBlueBacHis2
Xpress vector and SF9 cells are infected. The expression protein is
then isolated or purified according to the manufacturer's
instructions. Several other cultured cell lines having recombinant
constructs or vectors comprising a binding partner or specificity
exchanger are embodiments and their manufacture would be routine
given the present disclosure.
[0169] By similar approaches, a nucleic acid encoding a binding
partner can be incorporated into a vector that expresses the
binding partner or specificity exchanger in liver cells in vivo.
(Huber et al. Proc. Natl. Acad. Sci. USA 88:8039-8043(1991), herein
expressly incorporated by reference in its entirety. Many such
organ specific vectors have been described in the literature and
nucleic acids encoding a binding partner or specificity exchanger
can be incorporated into these vectors by conventional techniques
in molecular biology. (See U.S. Pat. Nos. 5,981,274; 5,998,205; and
6,025,195, all of which are herein incorporated by reference in
their entirety.) In the disclosure below, several pharmaceutical
embodiments are described.
[0170] Pharmaceutical Preparations and Methods of
Administration
[0171] Binding partners, preferably a specificity exchanger, are
suitable for incorporation into pharmaceuticals for administration
to subjects in need of a compound that treats or prevents HBV
infection. 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. The active ingredients can be incorporated into a
pharmaceutical product with and without modification. Further, the
manufacture of pharmaceuticals or therapeutic agents that deliver
the pharmacologically active compounds of this invention by several
routes are aspects. For example, and not by way of limitation, DNA,
RNA, and viral vectors having sequence encoding a binding partner
or specificity exchanger are used with embodiments. Nucleic acids
encoding a binding partner or specificity exchanger can be
administered alone or in combination with other active
ingredients.
[0172] The compounds 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, polyetylene 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 suitable
vehicles 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 that do
not deleteriously react with the active compounds.
[0173] The effective dose and method of administration of a
particular pharmaceutical formulation having a binding partner or
specificity exchanger can vary based on the individual needs of the
patient and the treatment or preventative measure sought.
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, a binding partner
or 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 ED50 with no toxicity. The dosage
varies within this range depending upon type of binding partner or
specificity exchanger, the dosage form employed, sensitivity of the
organism, and the route of administration.
[0174] Normal dosage amounts of a binding partner or 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 .mu.g-1 mg,
about 50 mg-200 mg, and about 250 mg-500 mg.
[0175] In some embodiments, the dose of a binding partner or
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 with some
embodiments. A constant infusion of a binding partner or
specificity exchanger can also be provided so as to maintain a
stable concentration in the tissues as measured by blood
levels.
[0176] 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, 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.
[0177] 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 pharmacologically active
compounds 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.
[0178] Compositions having pharmacologically active compounds
described herein 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 incorporated by
reference.
[0179] Compositions having pharmacologically active compounds 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.
[0180] Compositions having pharmacologically active compounds that
are suitable for transbronchial and transalveolar administration
include, but 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 pharmacologically active
compounds.
[0181] Compositions having pharmacologically active compounds 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 binding partner or specificity exchanger has been obtained, it
can be administered to a organism in need to treat or prevent HBV
infection.
[0182] Aspects of the invention also include a coating for medical
equipment such as prosthetics, implants, and instruments. Coatings
suitable for use in medical devices can be provided by a gel or
powder containing the binding partners or by polymeric coating into
which a binding partner is suspended. Suitable polymeric materials
for coatings or devices are those that are physiologically
acceptable and through which a therapeutically effective amount of
the binding partner 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, issued Sep.
16, 1986 to Fox et al. that is incorporated herein by reference in
its entirety.
[0183] In several aspects, a pharmaceutical having a binding
partner, preferably a specificity exchanger, is provided to a
subject in need of an agent that treats or prevents HBV infection.
These pharmaceuticals can be formulated with or without a carrier
or other agent in addition to the active ingredient, as described
above. Methods to formulate such pharmaceuticals that inhibit HBV
infection and/or modulate a host immune response to HBV are
embodiments.
[0184] Other embodiments involve methods to treat or prevent HBV
infection. Accordingly, a subject in need of a binding partner or
specificity exchanger that inhibits HBV infection and/or modulates
a host immune response to HBV is provided a therapeutically
effective amount of a pharmaceutical having said binding partner or
specificity exchanger. Such subjects in need can include
individuals at risk of contracting HBV or are already afflicted
with HBV. These individuals can be identified by clinical or
biochemical techniques.
[0185] 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.
Accordingly, the invention is limited only by the following claims.
All references cited herein are hereby expressly incorporated by
reference.
Sequence CWU 1
1
111 1 119 PRT Artificial Sequence Synthetically prepared amino acid
sequence 1 Val Lys Leu Gln Gln Ser Gly Thr Glu Val Val Lys Pro Gly
Ala Ser 1 5 10 15 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe
Thr Ser Tyr Asp 20 25 30 Ile Asp Trp Val Arg Gln Thr Pro Glu Gln
Gly Leu Glu Trp Ile Gly 35 40 45 Trp Ile Phe Pro Gly Glu Gly Ser
Thr Glu Tyr Asn Glu Lys Phe Lys 50 55 60 Gly Arg Ala Thr Leu Ser
Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Thr Arg
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 85 90 95 Arg Gly
Asp Tyr Asp Tyr Tyr Arg Arg Tyr Phe Asp Leu Trp Gly Gln 100 105 110
Gly Thr Thr Val Thr Val Ser 115 2 129 PRT Artificial Sequence
Synthetically prepared amino acid sequence 2 Asp Ile Val Leu Thr
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala
Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser
Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn
Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys
Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala Pro Ala Val Ser
Ile Phe Pro Pro Ser Ser Lys Leu 115 120 125 Gly 3 130 PRT
Artificial Sequence Synthetically prepared amino acid sequence 3
Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 1 5
10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
Asn 20 25 30 Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu
Trp Ile Gly 35 40 45 Asn Ile Asn Pro Tyr Tyr Gly Ser Thr Ser Tyr
Asn Gln Lys Phe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Gln Leu Asn Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Lys Gly Thr
Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Val 115 120 125 Pro
Val 130 4 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 4 Val Lys Leu Gln Gln Ser Gly Thr Glu Val Val Lys Pro
Gly Ala Ser 1 5 10 15 Val Lys Leu Ser 20 5 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 5 Val Lys Pro
Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr 1 5 10 15 Ile
Phe Thr Ser 20 6 20 PRT Artificial Sequence Synthetically prepared
amino acid sequence 6 Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr
Asp Ile Asp Trp Val 1 5 10 15 Arg Gln Thr Pro 20 7 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 7
Tyr Asp Ile Asp Trp Val Arg Gln Thr Pro Glu Gln Gly Leu Glu Trp 1 5
10 15 Ile Gly Trp Ile 20 8 20 PRT Artificial Sequence Synthetically
prepared amino acid sequence 8 Glu Gln Gly Leu Glu Trp Ile Gly Trp
Ile Phe Pro Gly Glu Gly Ser 1 5 10 15 Thr Glu Tyr Asn 20 9 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 9
Phe Pro Gly Glu Gly Ser Thr Glu Tyr Asn Glu Lys Phe Lys Gly Arg 1 5
10 15 Ala Thr Leu Ser 20 10 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 10 Glu Lys Phe Lys Gly
Arg Ala Thr Leu Ser Val Asp Lys Ser Ser Ser 1 5 10 15 Thr Ala Tyr
Met 20 11 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 11 Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu
Thr Arg Leu Thr 1 5 10 15 Ser Glu Asp Ser 20 12 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 12 Glu Leu Thr
Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 1 5 10 15 Arg
Gly Asp Tyr 20 13 20 PRT Artificial Sequence Synthetically prepared
amino acid sequence 13 Ala Val Tyr Phe Cys Ala Arg Gly Asp Tyr Asp
Tyr Tyr Arg Arg Tyr 1 5 10 15 Phe Asp Leu Trp 20 14 19 PRT
Artificial Sequence Synthetically prepared amino acid sequence 14
Asp Tyr Tyr Arg Arg Tyr Phe Asp Leu Trp Gly Gln Gly Thr Thr Val 1 5
10 15 Thr Val Ser 15 20 PRT Artificial Sequence Synthetically
prepared amino acid sequence 15 Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr 20 16 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 16
Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 1 5
10 15 Gln Ser Val Ser 20 17 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 17 Ile Ser Cys Arg Ala
Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr 1 5 10 15 Met His Trp
Tyr 20 18 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 18 Thr Ser Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln
Lys Pro Gly Gln 1 5 10 15 Pro Pro Lys Leu 20 19 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 19 Gln Gln Lys
Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr Ala Ser 1 5 10 15 Asn
Leu Glu Ser 20 20 20 PRT Artificial Sequence Synthetically prepared
amino acid sequence 20 Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly
Val Pro Ala Arg Phe 1 5 10 15 Ser Gly Ser Gly 20 21 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 21
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5
10 15 Leu Asn Ile His 20 22 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 22 Ser Gly Thr Asp Phe
Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 1 5 10 15 Thr Ala Thr
Tyr 20 23 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 23 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys
Gln His Ser Trp 1 5 10 15 Glu Ile Pro Tyr 20 24 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 24 Tyr Cys Gln
His Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr 1 5 10 15 Lys
Leu Glu Ile 20 25 19 PRT Artificial Sequence Synthetically prepared
amino acid sequence 25 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Ala Asp Ala Ala 1 5 10 15 Pro Ala Val 26 19 PRT Artificial
Sequence Synthetically prepared amino acid sequence 26 Lys Arg Ala
Asp Ala Ala Pro Ala Val Ser Ile Phe Pro Pro Ser Ser 1 5 10 15 Lys
Leu Gly 27 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 27 Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys
Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser 20 28 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 28 Val Lys Pro
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 1 5 10 15 Ser
Phe Thr Gly 20 29 20 PRT Artificial Sequence Synthetically prepared
amino acid sequence 29 Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
Asn Met Asn Trp Val 1 5 10 15 Lys Gln Ser His 20 30 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 30
Tyr Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp 1 5
10 15 Ile Gly Asn Ile 20 31 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 31 Gly Lys Ser Leu Glu
Trp Ile Gly Asn Ile Asn Pro Tyr Tyr Gly Ser 1 5 10 15 Thr Ser Tyr
Asn 20 32 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 32 Asn Pro Tyr Tyr Gly Ser Thr Ser Tyr Asn Gln Lys
Phe Lys Gly Lys 1 5 10 15 Ala Thr Leu Thr 20 33 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 33 Gln Lys Phe
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 1 5 10 15 Thr
Ala Tyr Met 20 34 20 PRT Artificial Sequence Synthetically prepared
amino acid sequence 34 Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln
Leu Asn Ser Leu Thr 1 5 10 15 Ser Glu Asp Ser 20 35 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 35
Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 1 5
10 15 Arg Gly Lys Gly 20 36 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 36 Ala Val Tyr Tyr Cys
Ala Arg Gly Lys Gly Thr Gly Phe Ala Tyr Trp 1 5 10 15 Gly Gln Gly
Thr 20 37 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 37 Thr Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ala 1 5 10 15 Ala Lys Thr Thr 20 38 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 38 Leu Val Thr
Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 1 5 10 15 Leu
Val Pro Val 20 39 19 PRT Artificial Sequence Synthetically prepared
amino acid sequence 39 Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Ile 1 5 10 15 Phe Thr Ser 40 18 PRT Artificial
Sequence Synthetically prepared amino acid sequence 40 Pro Gly Ala
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe 1 5 10 15 Thr
Ser 41 17 PRT Artificial Sequence Synthetically prepared amino acid
sequence 41 Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile
Phe Thr 1 5 10 15 Ser 42 16 PRT Artificial Sequence Synthetically
prepared amino acid sequence 42 Ala Ser Val Lys Leu Ser Cys Lys Ala
Ser Gly Tyr Ile Phe Thr Ser 1 5 10 15 43 15 PRT Artificial Sequence
Synthetically prepared amino acid sequence 43 Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10 15 44 14 PRT
Artificial Sequence Synthetically prepared amino acid sequence 44
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10 45
13 PRT Artificial Sequence Synthetically prepared amino acid
sequence 45 Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5
10 46 12 PRT Artificial Sequence Synthetically prepared amino acid
sequence 46 Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10
47 11 PRT Artificial Sequence Synthetically prepared amino acid
sequence 47 Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10 48
10 PRT Artificial Sequence Synthetically prepared amino acid
sequence 48 Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10 49 9 PRT
Artificial Sequence Synthetically prepared amino acid sequence 49
Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 50 8 PRT Artificial
Sequence Synthetically prepared amino acid sequence 50 Ala Ser Gly
Tyr Ile Phe Thr Ser 1 5 51 7 PRT Artificial Sequence Synthetically
prepared amino acid sequence 51 Ser Gly Tyr Ile Phe Thr Ser 1 5 52
6 PRT Artificial Sequence Synthetically prepared amino acid
sequence 52 Gly Tyr Ile Phe Thr Ser 1 5 53 19 PRT Artificial
Sequence Synthetically prepared amino acid sequence 53 Ser Cys Arg
Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met 1 5 10 15 His
Trp Tyr 54 18 PRT Artificial Sequence Synthetically prepared amino
acid sequence 54 Cys Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr
Ser Tyr Met His 1 5 10 15 Trp Tyr 55 17 PRT Artificial Sequence
Synthetically prepared amino acid sequence 55 Arg Ala Ser Gln Ser
Val Ser Thr Ser Ser Tyr Ser Tyr Met His Trp 1 5 10 15 Tyr 56 16 PRT
Artificial Sequence Synthetically prepared amino acid sequence 56
Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met His Trp Tyr 1 5
10 15 57 15 PRT Artificial Sequence Synthetically prepared amino
acid sequence 57 Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met
His Trp Tyr 1 5 10 15 58 14 PRT Artificial Sequence Synthetically
prepared amino acid sequence 58 Gln Ser Val Ser Thr Ser Ser Tyr Ser
Tyr Met His Trp Tyr 1 5 10 59 13 PRT Artificial Sequence
Synthetically prepared amino acid sequence 59 Ser Val Ser Thr Ser
Ser Tyr Ser Tyr Met His Trp Tyr 1 5 10 60 12 PRT Artificial
Sequence Synthetically prepared amino acid sequence 60 Val Ser Thr
Ser Ser Tyr Ser Tyr Met His Trp Tyr 1 5 10 61 11 PRT Artificial
Sequence Synthetically prepared amino acid sequence 61 Ser Thr Ser
Ser Tyr Ser Tyr Met His Trp Tyr 1 5 10 62 10 PRT Artificial
Sequence Synthetically prepared amino acid sequence 62 Thr Ser Ser
Tyr Ser Tyr Met His Trp Tyr 1 5 10 63 9 PRT Artificial Sequence
Synthetically prepared amino acid sequence 63 Ser Ser Tyr Ser Tyr
Met His Trp Tyr 1 5 64 7 PRT Artificial Sequence Synthetically
prepared amino acid sequence 64 Tyr Ser Tyr Met His Trp Tyr 1 5 65
6 PRT Artificial Sequence Synthetically prepared amino acid
sequence 65 Ser Tyr Met His Trp Tyr 1 5 66 16 PRT Artificial
Sequence Synthetically prepared amino acid sequence 66 Pro Gly Ala
Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe 1 5 10 15 67 18
PRT Artificial Sequence Synthetically prepared amino acid sequence
67 Lys Pro Gly Asp Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr
1 5 10 15 Phe Ser 68 20 PRT Artificial Sequence Synthetically
prepared amino acid sequence 68 Val Lys Pro Gly Gly Ser Leu Arg Leu
Ser Cys Val Ala Ser Gly Phe 1 5 10 15 Thr Phe Ser Ser 20 69 19 PRT
Artificial Sequence Synthetically prepared amino acid sequence 69
Lys Pro Gly Asp Ser Leu Arg Leu Ser Cys Lys Gly Ser Gly Phe Thr 1 5
10 15 Phe Ser Asn 70 21 PRT Artificial Sequence Synthetically
prepared amino acid sequence 70 Lys Leu Ser Cys Lys Ala Ser Gly Tyr
Ile Phe Thr Ser Glu His Arg 1 5 10 15 Gly Gly Pro Glu Glu 20 71 21
PRT Artificial Sequence Synthetically prepared amino acid sequence
71 Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser His Arg Gly
1 5 10 15 Gly Pro Glu Glu Phe 20 72 21 PRT Artificial Sequence
Synthetically prepared amino acid sequence 72 Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Ile Phe Thr Ser Arg Gly Gly 1 5 10 15 Pro Glu Glu
Phe Glu 20 73 21 PRT Artificial Sequence Synthetically prepared
amino acid sequence 73 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile
Phe Thr Ser Glu His 1 5 10 15 Arg Gly Gly Pro Glu 20 74 21 PRT
Artificial Sequence Synthetically prepared amino acid sequence 74
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser His Arg 1 5
10 15 Gly Gly Pro Glu Glu 20 75 21 PRT Artificial Sequence
Synthetically prepared amino acid sequence 75 Val Lys Leu Ser Cys
Lys Ala Ser Gly Tyr Ile Phe Thr Ser His Arg 1 5 10 15 Gly Gly Pro
Glu Glu 20 76 21 PRT Artificial Sequence Synthetically prepared
amino acid sequence 76 Val Lys Leu Ser Cys Lys
Ala Ser Gly Tyr Ile Phe Thr Ser Gly Gly 1 5 10 15 Pro Glu Glu Phe
Glu 20 77 4 PRT Artificial Sequence Synthetically prepared amino
acid sequence 77 Cys Lys Ala Ser 1 78 4 PRT Artificial Sequence
Synthetically prepared amino acid sequence 78 Cys Arg Ala Ser 1 79
17 PRT Artificial Sequence Synthetically prepared amino acid
sequence 79 Gly Leu Tyr Ser Ser Ile Trp Leu Ser Pro Gly Arg Ser Tyr
Phe Glu 1 5 10 15 Thr 80 17 PRT Artificial Sequence Synthetically
prepared amino acid sequence 80 Tyr Thr Asp Ile Lys Tyr Asn Pro Phe
Thr Asp Arg Gly Glu Gly Asn 1 5 10 15 Met 81 17 PRT Artificial
Sequence Synthetically prepared amino acid sequence 81 Asp Gln Asn
Ile His Met Asn Ala Arg Leu Leu Ile Arg Ser Pro Phe 1 5 10 15 Thr
82 17 PRT Artificial Sequence Synthetically prepared amino acid
sequence 82 Leu Ile Arg Ser Pro Phe Thr Asp Pro Gln Leu Leu Val His
Thr Asp 1 5 10 15 Pro 83 17 PRT Artificial Sequence Synthetically
prepared amino acid sequence 83 Gln Lys Glu Ser Leu Leu Phe Pro Pro
Val Lys Leu Leu Arg Arg Val 1 5 10 15 Pro 84 11 PRT Artificial
Sequence Synthetically prepared amino acid sequence 84 Pro Ala Leu
Thr Ala Val Glu Thr Gly Ala Thr 1 5 10 85 8 PRT Artificial Sequence
Synthetically prepared amino acid sequence 85 Ser Thr Leu Val Pro
Glu Thr Thr 1 5 86 13 PRT Artificial Sequence Synthetically
prepared amino acid sequence 86 Thr Pro Pro Ala Tyr Arg Pro Pro Asn
Ala Pro Ile Leu 1 5 10 87 9 PRT Artificial Sequence Synthetically
prepared amino acid sequence 87 Glu Ile Pro Ala Leu Thr Ala Val Glu
1 5 88 10 PRT Artificial Sequence Synthetically prepared amino acid
sequence 88 Leu Glu Asp Pro Ala Ser Arg Asp Leu Val 1 5 10 89 8 PRT
Artificial Sequence Synthetically prepared amino acid sequence 89
His Arg Gly Gly Pro Glu Glu Phe 1 5 90 7 PRT Artificial Sequence
Synthetically prepared amino acid sequence 90 His Arg Gly Gly Pro
Glu Glu 1 5 91 17 PRT Artificial Sequence Synthetically prepared
amino acid sequence 91 Val Leu Ile Cys Gly Glu Asn Thr Val Ser Arg
Asn Tyr Ala Thr His 1 5 10 15 Ser 92 17 PRT Artificial Sequence
Synthetically prepared amino acid sequence 92 Lys Ile Asn Thr Met
Pro Pro Phe Leu Asp Thr Glu Leu Thr Ala Pro 1 5 10 15 Ser 93 17 PRT
Artificial Sequence Synthetically prepared amino acid sequence 93
Pro Asp Glu Lys Ser Gln Arg Glu Ile Leu Leu Asn Lys Ile Ala Ser 1 5
10 15 Tyr 94 17 PRT Artificial Sequence Synthetically prepared
amino acid sequence 94 Thr Ala Thr Thr Thr Thr Tyr Ala Tyr Pro Gly
Thr Asn Arg Pro Pro 1 5 10 15 Val 95 8 PRT Artificial Sequence
Synthetically prepared amino acid sequence 95 Ser Thr Pro Leu Pro
Glu Thr Thr 1 5 96 20 PRT Artificial Sequence Synthetically
prepared amino acid sequence 96 Ala Thr Val Glu Leu Leu Ser Phe Leu
Pro Ser Asp Phe Phe Pro Ser 1 5 10 15 Val Arg Asp Leu 20 97 20 PRT
Artificial Sequence Synthetically prepared amino acid sequence 97
Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp Thr Ala Ser Ala 1 5
10 15 Leu Tyr Arg Glu 20 98 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 98 Leu Asp Thr Ala Ser
Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu 1 5 10 15 His Cys Ser
Pro 20 99 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 99 Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His
Thr Ala Leu Arg 1 5 10 15 Gln Ala Ile Leu 20 100 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 100 His His Thr
Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met 1 5 10 15 Thr
Leu Ala Thr 20 101 20 PRT Artificial Sequence Synthetically
prepared amino acid sequence 101 Cys Trp Gly Glu Leu Met Thr Leu
Ala Thr Trp Val Gly Val Asn Leu 1 5 10 15 Glu Asp Pro Ala 20 102 20
PRT Artificial Sequence Synthetically prepared amino acid sequence
102 Trp Val Gly Val Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu Val Val
1 5 10 15 Ser Tyr Val Asn 20 103 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 103 Ser Arg Asp Leu Val
Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 1 5 10 15 Phe Arg Gln
Leu 20 104 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 104 Thr Asn Met Gly Leu Lys Phe Arg Gln Leu Leu Trp
Phe His Ile Ser 1 5 10 15 Cys Leu Thr Phe 20 105 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 105 Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile 1 5 10 15 Glu
Tyr Leu Val 20 106 20 PRT Artificial Sequence Synthetically
prepared amino acid sequence 106 Gly Arg Glu Thr Val Ile Glu Tyr
Leu Val Ser Phe Gly Val Trp Ile 1 5 10 15 Arg Thr Pro Pro 20 107 20
PRT Artificial Sequence Synthetically prepared amino acid sequence
107 Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn
1 5 10 15 Ala Pro Ile Leu 20 108 20 PRT Artificial Sequence
Synthetically prepared amino acid sequence 108 Ala Tyr Arg Pro Pro
Asn Ala Pro Ile Leu Ser Thr Leu Pro Glu Thr 1 5 10 15 Thr Val Val
Arg 20 109 20 PRT Artificial Sequence Synthetically prepared amino
acid sequence 109 Ser Thr Leu Pro Glu Thr Thr Val Val Arg Arg Arg
Gly Arg Ser Pro 1 5 10 15 Arg Arg Arg Thr 20 110 20 PRT Artificial
Sequence Synthetically prepared amino acid sequence 110 Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg 1 5 10 15 Arg
Ser Gln Ser 20 111 23 PRT Artificial Sequence Synthetically
prepared amino acid sequence 111 Pro Ser Pro Arg Arg Arg Arg Ser
Gln Ser Pro Arg Arg Arg Arg Ser 1 5 10 15 Gln Ser Arg Glu Ser Gln
Cys 20
* * * * *