Synthetic peptides that bind to the hepatitis B virus core...

Details Synthetic peptides that bind to the hepatitis B virus core... Synthetic peptides that bind to the hepatitis B virus core...

2000-04-21

2002-07-09

Housel, James (Department: 1648)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C435S005000

Reexamination Certificate

active

06417324

ABSTRACT:

FIELD OF THE INVENTION
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
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)).
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 preS 1, 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.
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
−
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
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.
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.
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.
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.
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
1
n
CKASX
2
n
, wherein “X
1
” and “X
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
1
n
CZASX
2
n
”, wherein: “X
1
” and “X
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
1
n
” or “X
2
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
1
n
CRASX
2
n
, wherein “X
1
” and “X
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
1
n
CZASX
2
n
”, wherein: “X
1
” and “X
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
1
n
” or “X
2
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.
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

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