U.S. patent application number 14/035172 was filed with the patent office on 2014-05-15 for human antibodies against hepatitis c virus (hcv) and uses thereof.
The applicant listed for this patent is University of Massachusetts. Invention is credited to Donna M. AMBROSINO, Gregory J. BABCOCK, Teresa BROERING, Susan SLOAN, William D. THOMAS, JR..
Application Number | 20140134175 14/035172 |
Document ID | / |
Family ID | 39537518 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134175 |
Kind Code |
A1 |
AMBROSINO; Donna M. ; et
al. |
May 15, 2014 |
HUMAN ANTIBODIES AGAINST HEPATITIS C VIRUS (HCV) AND USES
THEREOF
Abstract
Isolated human monoclonal antibodies which bind to hepatitis C
virus (HCV), and related antibody-based compositions and molecules,
are disclosed. The human antibodies can be produced in a
transfectoma or in a non-human transgenic animal, e.g., a
transgenic mouse, capable of producing multiple isotypes of human
monoclonal antibodies by undergoing V-D-J recombination and isotype
switching. Also disclosed are pharmaceutical compositions
comprising the human antibodies, non-human transgenic animals, and
hybridomas which produce the human antibodies, and therapeutic and
diagnostic methods for using the human antibodies.
Inventors: |
AMBROSINO; Donna M.;
(Jamaica Plain, MA) ; THOMAS, JR.; William D.;
(Dedham, MA) ; BABCOCK; Gregory J.; (Marlborough,
MA) ; BROERING; Teresa; (Brookline, MA) ;
SLOAN; Susan; (Watertown, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Massachusetts |
Boston |
MA |
US |
|
|
Family ID: |
39537518 |
Appl. No.: |
14/035172 |
Filed: |
September 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12527663 |
Mar 4, 2010 |
8551484 |
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PCT/US08/01922 |
Feb 13, 2008 |
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14035172 |
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60902432 |
Feb 21, 2007 |
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Current U.S.
Class: |
424/139.1 ;
530/387.9 |
Current CPC
Class: |
C07K 2317/21 20130101;
C12N 2770/24222 20130101; A61K 39/12 20130101; C07K 14/005
20130101; A61P 31/14 20180101; A61K 2039/5258 20130101; A61K 39/00
20130101; A61K 2039/55566 20130101; C12N 2770/24234 20130101; A61P
31/12 20180101; C07K 16/109 20130101; C07K 2317/34 20130101; A61P
31/00 20180101; A61K 2039/505 20130101; A61K 39/29 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9 |
International
Class: |
C07K 16/10 20060101
C07K016/10 |
Claims
1.-66. (canceled)
67. An isolated human antibody, or antigen binding portion thereof
that competes for binding to an E2 protein of hepatitis C virus
(HCV) with a second antibody, or antigen binding portion thereof,
said second antibody or antigen binding portion thereof comprising:
(a) heavy and light chain variable regions comprising the sequences
of SEQ ID NOs:1 and 2, SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6,
respectively; (b) a heavy chain variable region CDR3 sequence
comprising the sequence of SEQ ID NO:9, a light chain variable
region CDR3 sequence comprising the sequence of SEQ ID NO:18, a
heavy chain variable region CDR2 sequence comprising the sequence
of SEQ ID NO:8, a light chain variable region CDR2 sequence
comprising the sequence of SEQ ID NO:17, a heavy chain variable
region CDRI sequence comprising the sequence of SEQ ID NO:7, and a
light chain variable region CDRI sequence comprising the sequence
of SEQ ID NO:16; (c) a heavy chain variable region CDR3 sequence
comprising the sequence of SEQ ID NO:12, a light chain variable
region CDR3 sequence comprising the sequence of SEQ ID NO:21, a
heavy chain variable region CDR2 sequence comprising the sequence
of SEQ ID NO:11, a light chain variable region CDR2 sequence
comprising the sequence of SEQ ID NO:20, a heavy chain variable
region CDRI sequence comprising the sequence of SEQ ID NO:10, and a
light chain variable region CDRI sequence comprising the sequence
of SEQ ID NO:19; or (d) a heavy chain variable region CDR3 sequence
comprising the sequence of SEQ ID NO:15, a light chain variable
region CDR3 sequence comprising the sequence of SEQ ID NO:24, a
heavy chain variable region CDR2 sequence comprising the sequence
of SEQ ID NO:14, a light chain variable region CDR2 sequence
comprising the sequence of SEQ ID NO:23, a heavy chain variable
region CDRI sequence comprising the sequence of SEQ ID NO:13, and a
light chain variable region CDRI sequence comprising the sequence
of SEQ ID NO:22.
68. The isolated human antibody or antigen binding portion of claim
67, wherein the second antibody or antigen binding portion
comprises heavy and light chain variable regions comprising the
sequences of SEQ ID NOs:1 and 2, SEQ ID NOs:3 and 4, or SEQ ID
NOs:5 and 6, respectively.
69. The isolated human antibody, or antigen binding portion thereof
of claim 67, wherein the second antibody, or antigen binding
portion thereof comprises heavy and light chain variable regions
comprising the sequences of SEQ ID NOs:1 and 2.
70. The isolated human antibody of claim 67, wherein the antibody
is a full-length antibody.
71. The isolated human antibody, or antigen binding portion thereof
of claim 67, wherein the antibody or antigen binding portion is
selected from the group consisting of an antibody comprising an Fc
domain, a single-chain antibody, and a Fab fragment.
72. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody or antigen binding portion inhibits
binding of hepatitis C virus (HCV) to mammalian cells.
73. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody or antigen binding portion inhibits
binding of an HCV E2 protein to mammalian cells.
74. The isolated human antibody or antigen binding portion of claim
67, which binds to a non-conformational (linear) epitope on the HCV
E2 protein and inhibits the ability of the virus to infect
cells.
75. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody or antigen binding portion neutralizes HCV
in an HCV pseudovirus neutralization assay.
76. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody inhibits HCV infection in vivo in a
subject.
77. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody or antigen binding portion thereof,
inhibits HCV-mediated hepatocyte pathology in a subject.
78. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody binds to two or more HCV genotypes of an
HCV E2 protein or fragment thereof.
79. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody binds an HCV E2 protein from a HCV virus
having a genotype selected from the group consisting of 1a, 1b, 2b,
3a, 4a, 5, 5a, 6a, 6g, 6k, and any combination thereof.
80. The isolated human antibody or antigen binding portion of claim
67, wherein the epitope is between amino acid residues 412-464,
412-423, or 413-420 of the E2 protein (SEQ ID NO:93).
81. The isolated human antibody or antigen binding portion of claim
67, wherein the antibody or antigen binding portion thereof
specifically binds to an HCV E2 protein, or fragment thereof, with
a KD of at least about 1.times.10.sup.-7 M, 1.times.10.sup.-8 M,
1.times.10.sup.-9 M, 1.times.10.sup.-10 M, 1.times.10.sup.-11 M,
1.times.10.sup.-12 M or better.
82. The isolated human antibody or antigen binding portion of claim
67, further comprising a label or a toxin.
83. A composition comprising the isolated human antibody or antigen
binding portion of claim 67 in a pharmaceutically acceptable
carrier.
84. A composition suitable for treating an HCV-mediated disease or
disorder in a mammal, said composition comprising the isolated
human antibody or antigen binding portion of claim 67 in a
pharmaceutically acceptable carrier.
85. A kit comprising the isolated human antibody or antigen binding
portion of claim 67 and instruction for use of said antibody of
antigen binding portion in treating an HCV-mediated disease.
Description
RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
Provisional Patent Application No. 60/902,432, filed Feb. 21, 2007,
for all subject matter common to said application. The disclosure
of the above-mentioned application is hereby incorporated by
reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Hepatitis C virus (HCV), originally named "non-A non-B
hepatitis" (NANBH), is a positive, single-stranded RNA virus of the
Flaviviridae family. Its genome contains a single long open reading
frame which encodes a polyprotein of about three-thousand amino
acid residues. (Choo et al. (1989) Science 244:359-362). The
polyprotein is processed by the host cell and viral proteases into
three major structural proteins and several non-structural proteins
necessary for viral replication. The nucleotide sequence of HCV is
highly variable, the most divergent isolates sharing only 60%
nucleotide sequence homology. Several different genotypes of HCV
with slightly different genomic sequences have been identified.
[0003] Isolates from all over the world have now been grouped into
6 main types, each with several subtypes, based on sequence data
(Simmonds et al. (1995) Hepatology 21: 570-83). Types 1-3 account
for almost all infections in Europe, type 4 is prevalent in Egypt
and Zaire, type 5 in South Africa and type 6 in Hong Kong.
[0004] The virus is transmitted primarily by blood and blood
products. The majority of infected individuals have either received
blood transfusions prior to 1990 (when screening of the blood
supply for HCV was implemented) or have used intravenous drugs.
Approximately 200 to 400 million people worldwide are chronically
infected with HCV, about 2 to 5 million of these are in the United
States. The chronic infection of HCV, which effects about 80% of
those infected, lasts lifelong and often leads to the development
of cirrhosis and liver cancer.
[0005] The development of vaccines against HCV has been slowed by
the extreme antigenic variability of the virus. Even when
available, vaccines will not alleviate the problems faced by
millions of chronic HCV carriers worldwide.
[0006] Most antibodies to HCV do not play a major role in clearance
of infection, although neutralizing antibodies do exist. However,
these tend to be strain-specific and are ineffective against
emerging strains.
[0007] Presently, the only therapy with any demonstrated efficacy
against HCV-induced liver disease involves the use of alpha-MN, but
this approach has achieved only limited success. Even the best
clinical trials with alpha-IFN treatment report only 40-50% of
chronic sufferers respond and 50% of these individuals relapse when
treatment is stopped. There is also evidence that alpha-IFN
treatment is much less effective against some HCV genotypes than
others.
[0008] Currently, no conventional drugs against HCV infection
(other than alpha-IFN) have yet been developed. Accordingly,
improved immunotherapies for treating and preventing HCV infection
are needed.
SUMMARY OF THE INVENTION
[0009] The present invention solves the foregoing problems by
providing a recombinant fully human anti-HCV monoclonal antibody
that specifically binds a broad variety of HCV isolates and
inhibits the ability of the virus to infect cells.
[0010] In one embodiment, this is demonstrated by the antibodies
ability to neutralize (i.e., inhibit or block) HCV in vitro (e.g.,
in a HCV pseudovirus neutralization assay). In another embodiment,
this is demonstrated by the antibodies ability to inhibit HCV
infectivity in vivo in a subject, such as an animal or a human.
[0011] Human monoclonal antibodies of the invention can be made
efficiently, in virtually unlimited amounts, in highly purified
form. Accordingly, the antibodies are suitable for prognosing,
diagnosing, and/or treating an individual exposed or suspected of
having been exposed to HCV. The antibodies can be produced using a
variety of techniques for making human antibodies known in the art.
For example, as exemplified herein, the antibodies can be generated
in transgenic animals expressing human immunoglobulin gene
segments, e.g., transgenic mice comprising a human Ig locus.
Moreover, the antibodies can be administered alone or in
combination, e.g., with an anti-HCV vaccine or other antibodies, to
increase survival rates of subjects (e.g., animals and humans)
infected with HCV.
[0012] Accordingly, the invention provides several advantages that
include, but are not limited to, the following: [0013] a fully
human recombinant anti-HCV antibody for prognosing, diagnosing,
and/or treating HCV in a subject, e.g., protect from or inhibit
HCV-mediated morbidity or mortality in a subject; [0014] a
composition (e.g., pharmaceutical) and/or a kit comprising one or
more fully human recombinant anti-HCV antibodies that can be used
alone or in combination with commercially available vaccines to
treat HCV infection and; [0015] an improved method of passive
immunotherapy for treating a subject infected with HCV which can be
used alone or in combination with active immunotherapy (HCV
vaccine).
[0016] In one embodiment, the human monoclonal antibodies or
antigen binding portions thereof of the invention specifically bind
to a non-conformational epitope of the HCV E2 protein. Particular
antibodies or antigen binding portions thereof specifically bind to
an epitope within the HCV E2 protein. Such epitopes can reside, for
example, within amino acids 1-50, 50-100, 100-150, 150-200,
200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550,
550-600, 600-650, 650-700, 700-746 of the HCV E2 protein, or any
interval, portion or range thereof. In one embodiment, the
antibodies or antigen binding portions thereof specifically bind to
an epitope between about amino acid residues 412-464, 412-423, or
413-420. In another embodiment, the epitope of the HCV E2 protein
comprises amino acid residues 412-423. In another embodiment, the
epitope of the HCV E2 protein consists of amino acid residues
412-423. In one embodiment, the HCV E2 protein comprises amino acid
residues 413, 418, and/or 420.
[0017] In other embodiments, the human monoclonal antibodies or
antigen binding portions thereof can be characterized as
specifically binding to an HCV E2 protein with a K.sub.D of less
than about 10.times.10.sup.-6 M. In a particular embodiment, the
antibody or antigen binding portion thereof specifically binds to
an HCV E2 protein (or fragment thereof) with a K.sub.D of at least
about 10.times.10.sup.-7 M, at least about 10.times.10.sup.-8 M, at
least about 10.times.10.sup.-9 M, at least about
10.times.10.sup.-10 M, at least about 10.times.10.sup.-11 M, or at
least about 10.times.10.sup.-12 M or a K.sub.D even more
favorable.
[0018] In various other embodiments, the antibodies or antigen
binding portions thereof include a variable heavy chain region
comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 98%,
99% or more identical to a variable heavy chain region amino acid
sequence of the antibody produced by clone 83-128 (SEQ ID NO: 1),
95-2 (SEQ ID NO: 3) or 073-1 (SEQ ID NO: 5).
[0019] In other embodiments, the antibodies or antigen binding
portions thereof include a variable light chain region comprising
an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% or
more identical to a variable light chain region amino acid
sequence' of the antibody produced by clone 83-128 (SEQ ID NO: 2),
95-2 (SEQ ID NO: 4) or 073-1 (SEQ ID NO: 6).
[0020] In still other embodiments, the antibodies or antigen
binding portions thereof include both a variable heavy chain region
comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 98%,
99% or more identical to a variable heavy chain region amino acid
sequence of the antibody produced by clone 83-128 (SEQ ID NO: 1),
95-2 (SEQ ID NO: 3) or 073-1 (SEQ ID NO: 5) and a variable light
chain region comprising an amino acid sequence at least 80%, 85%,
90%, 95%, 98%, 99%, or more identical to a variable light chain
amino acid sequence of clone 83-128 (SEQ ID NO: 2), 95-2 (SEQ ID
NO: 4) or 073-1 (SEQ ID NO: 5).
[0021] In certain other embodiments, the antibodies or antigen
binding portions thereof specifically bind to an epitope that
overlaps with an epitope bound by an antibody produced by clone
83-128, 95-2 or 073-1 and/or competes for binding to a HCV, or
portion thereof with an antibody produced by clone 83-128, 95-2 or
073-1.
[0022] The variable heavy and light chain regions of the antibodies
or antigen binding portions thereof typically include one or more
complementarity determining regions (CDRs). These include the CDR1,
CDR2, and CDR3 regions. In particular embodiments, the variable
heavy chain CDRs are at least 80%, 85%, 90%, 95%, or 99%, or more
identical to a CDR of the antibody produced by clone 83-128 (SEQ ID
NOs: 7-9), 95-2 (SEQ ID NOs: 10-12) or 073-1 (SEQ ID NO: 13-15). In
other particular embodiments, variable light chain CDRs are at
least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a
variable light chain region of the antibody produced by clone
83-128 (SEQ ID NOs: 16-18), 95-2 (SEQ ID NOs: 19-21) or 073-1 (SEQ
ID NO: 22-24).
[0023] Accordingly, particular antibodies or fragments of the
invention comprise a variable heavy chain region that includes one
or more complementarity determining regions (CDRs) that are at
least 80%, 85%, 90%, 95%, or 99%, or more identical to a CDR of a
variable heavy chain region of the antibody produced by clone
83-128 (SEQ ID NOs: 7-9), 95-2 (SEQ ID NOs: 10-12) or 073-1 (SEQ ID
NO: 13-15) and a variable light chain region that includes one or
more CDRs that are at least 80%, 85%, 90%, 95%, 99%, or more
identical to a CDR of a variable light chain region of the antibody
produced by clone 83-128 (SEQ ID NOs: 16-18), 95-2 (SEQ ID NOs:
19-21) or 073-1 (SEQ ID NO: 22-24).
[0024] The variable heavy chain region of the antibodies or antigen
binding portions thereof can also include all three CDRs that are
at least 80%, 85%, 90%, 95%, or 99%, or more identical to the CDRs
of the variable heavy chain region of the antibody produced by
clone 83-128 (SEQ ID NOs: 7-9), 95-2 (SEQ ID NOs: 10-12) or 073-1
(SEQ ID NO: 13-15) and/or all three CDRs that are at least 80%,
85%, 90%, 95%, 99%, or more identical to the CDRs of the variable
light chain region of the antibody produced by clone 83-128 (SEQ ID
NOs: 16-18), 95-2 (SEQ ID NOs: 19-21) or 073-1 (SEQ ID NO:
22-24).
[0025] In another embodiment, the variable heavy chain region of
the antibodies or antigen binding portions thereof can also include
one or more heavy chain consensus sequence CDRs as set forth in SEQ
ID NOs: 87-89 and/or one or more light chain consensus CDRs asset
forth in SEQ ID NOs: 90-92.
[0026] In another embodiment of the invention, the human antibodies
or antigen binding portions thereof (a) include a heavy chain
variable region that is encoded by or derived from (i.e., is the
product of) a human VH 3-33 gene; and/or (b) include a light chain
variable region that is encoded by or derived from a human V.kappa.
L6 gene.
[0027] Human monoclonal antibodies of the present invention include
full-length antibodies, for example, that include an effector
domain, (e.g., an Fc domain), as well as antibody portions or
fragments, such as single-chain antibodies and Fab fragments. The
antibodies can also be linked to a variety of therapeutic agents
(e.g., antiviral agents or toxins) and/or a label.
[0028] In another aspect, the invention features isolated
polypeptides that include an antigen binding portion of an antibody
produced by hybridoma clone of 83-128, 073-1, 95-2, 95-14, 95-15,
95-18, 95-20, 95-21, 95-25, 95-26, 95-30, 95-38, 95-39, 95-42,
95-43, 95-48, 95-49, 95-52, 95-54, 95-58, 95-62, and combinations
thereof.
[0029] In another aspect, the invention features isolated nucleic
acids including a sequence encoding a antibody heavy chain variable
region which is at least 75%, 80%, 85%, 90%, 95%, 99%, or more
identical to SEQ ID NO: 25, 27 or 29. The invention also features
isolated nucleic acids that include a sequence encoding an antibody
light chain variable region which is at least 75%, 80%, 85%, 90%,
95%, 99%, or more identical to SEQ ID NO: 26, 28 or 30. The
invention also features expression vectors including any of the
foregoing nucleic acids either alone or in combination (e.g.,
expressed from one or more vectors), as well as host cells
comprising such expression vectors.
[0030] Suitable host cells for expressing antibodies of the
invention include a variety of eukaryotic cells, e.g., yeast cells,
mammalian cells, e.g., Chinese hamster ovary (CHO) cells, NS0
cells, myeloma cells, or plant cells.
[0031] In another aspect, the invention features compositions and
kits that include one or more isolated human monoclonal antibodies
or antigen binding portions thereof as described herein that
specifically bind to HCV and inhibit the ability of the virus to
infect mammalian cells. The composition or kit can further include
one or more antibodies (e.g., human monoclonal or polyclonal
antibodies) or antigen-binding portions thereof that specifically
bind to HCV. In one embodiment, the polyclonal antibody or antigen
binding portion thereof specifically binds to HCV E2 protein. In a
particular embodiment, the composition or kit includes both (a) an
isolated human monoclonal antibody that specifically binds to a
first HCV isolate; and (b) an isolated human monoclonal antibody
that specifically binds to a second HCV isolate.
[0032] The invention also features methods of treating HCV disease
in a subject by administering to the subject an isolated human
monoclonal antibody or antigen binding portion thereof as described
herein (i.e., that specifically binds to HCV) in an amount
effective to inhibit HCV disease, e.g., HCV-mediated symptoms or
morbidity.
[0033] Human monoclonal antibodies or portions thereof (and
compositions comprising the antibodies or portions thereof) of the
invention can be administered in a variety of suitable fashions,
e.g., intravenously (IV), subcutaneously (SC), or, intramuscularly
(IM) to the subject. The antibody or antigen-binding portion
thereof can be administered alone or in combination with another
therapeutic agent, e.g., a second human monoclonal antibody or
antigen binding portion thereof. In one example, the second human
monoclonal antibody or antigen binding portion thereof specifically
binds to a second HCV isolate that differs from the isolate bound
to the first antibody. In another example, the antibody is
administered together with another agent, for example, an antiviral
agent. In another example, the antibody is administered together
with a polyclonal gamma-globulin (e.g., human gamma-globulin). In
another example, the antibody is administered before, after, or
contemporaneously with a HCV vaccine.
[0034] In another aspect, the invention features methods for making
an antibody or antigen binding portion thereof that specifically
binds to a HCV. In one embodiment, the method involves immunizing a
transgenic non-human animal having a genome comprising a human
heavy chain transgene and a human light chain transgene with a
composition that includes a HCV, e.g., live or inactivated virus
and isolating an antibody, antibody producing cell, or antibody
encoding nucleic acid from the animal. The HCV can be inactivated,
for example, by chemical treatment and/or lyophilization. The
method can further include evaluating binding of the antibody to
the HCV or HCV E2 protein.
[0035] The invention also features methods for making the
antibodies or antigen binding portions thereof by expressing
nucleic acids encoding human antibodies in a host cell (e.g.,
nucleic acids encoding the antigen binding region portion of an
antibody). In yet another aspect, the invention features a
hybridoma or transfectoma including the aforementioned nucleic
acids.
[0036] The invention also features a method for making a hybridoma
that expresses an antibody that specifically binds to a HCV by
immunizing a transgenic non-human animal having a genome that
includes a human heavy chain transgene and a human light chain
transgene, with a composition that includes the HCV or HCV E2
protein; isolating splenocytes from the animal; generating
hybridomas from the splenocytes; and selecting a hybridoma that
produces an antibody that specifically binds to HCV or HCV E2
protein thereof.
[0037] In another embodiment, the invention provides an antigen
comprising a non-conformational epitope of the HCV E2 protein,
amino acids 412-464, 412-423, or 413-420. The antigen can be used
for raising, screening, or detecting the presence of an anti-HCV E2
antibody or can be used as an agent in active immunotherapy, i.e.
as a vaccine. As a vaccine, the antigen can be used alone or in
combination with an appropriate adjuvant or hapten, e.g., mixed or
conjugated either chemically or genetically. Conjugates include but
are not limited to toxoids, virus-like particles (VLPs), and
protein carriers all of which are designed to enhance the immune
response to the vaccine antigen. The antigen when used for active
immunotherapy can be also used in combination with passive
immunotherapy, for example, with any of the anti-HCV E2 antibodies
disclosed herein.
[0038] Treatment of humans with human monoclonal antibodies offers
several advantages. For example, the antibodies are likely to be
less immunogenic in humans than non-human antibodies. The therapy
is also rapid because HCV inactivation can occur as soon as the
antibody reaches sites of infection and directly neutralizes the
disease-causing HCV. Human antibodies also localize to appropriate
sites in humans more efficiently than non-human antibodies.
Furthermore, the treatment is specific for HCV, and is recombinant
and highly purified and, unlike traditional therapies, avoids the
potential of being contaminated with adventitious agents.
[0039] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIGS. 1A and 1B are graphs showing binding of human antibody
95-2 (FIG. 1A) and human antibody 83-128 (FIG. 1B) to soluble HCV
genotype 1a E2-660 (triangles) and soluble HCV genotype 1b E2-661
(squares). ELISA plates were coated with 2 .mu.g/ml of antigen and
probed with antibody in two-fold dilutions. Bound antibody was
detected with goat-anti-human secondary antibody conjugated to
alkaline phosphatase and PNPP substrate.
[0041] FIGS. 2A and 2B are graphs showing neutralization of
infection of Hep3B cells with various HCV genotype pseudovirus in
the presence of human antibodies 95-2, 83-128, and 17C7 (irrelevant
human mAb). Five-fold dilutions of antibody were incubated with
each HCV pseudovirus for one hour at room temperature. The
virus-antibody mixture was added to Hep3B cells followed by
incubation at 37.degree. C. for 72 hours. Infection was quantitated
with Brightglo luciferase assay and read in a Victor3 plate reader
for light output. Human antibody against rabies (17C7) was used as
a negative control.
[0042] FIGS. 3A and 3B are Western blots showing antibody binding
to purified soluble mammalian expressed E2 protein (E2-661) from
HCV genotype 1a and 1b subjected to reducing (FIG. 3A) or
non-reducing (FIG. 3B) SDS-PAGE followed by transfer to PVDF
membrane. Westerns blots were performed with anti-his tag
monoclonal (his), 83-128 and 95-2 using anti-mouse IgG (his) or
anti-human IgG (95-2 and 83-128) conjugated to HRP with enhanced
chemiluminescent detection reagent. The genotype of the soluble E2
is indicated above the blots. The primary antibody used for
detection is listed below the blots. The molecular weight markers
in kilodaltons is listed to the left of each set of blots.
[0043] FIG. 4 is a table showing the affinity of human antibodies
95-2 and 83-128 to HCV E2 412-423 epitope expressed as a bacterial
fusion protein. Goat anti-human IgG Fc was amide coupled to the
Biacore chip. Human antibodies 95-2 and 83-128 were separately
captured on the chip and E2-G bacterially expressed protein
containing E2 412-423 amino acids was flowed over at varying
concentrations. The BIAevaluation.TM. software was used to fit the
curves and calculate the affinity constants.
[0044] FIG. 5 is a map of the HCV polyprotein showing the location
of E1 and E2 mammalian expressed proteins. The E1 and E2
polyprotein is indicated at top by a long white box with amino acid
numbers noted above. The transmembrane domains (TM) are indicated
by light grey boxes and the hypervariable domain 1 (HVR1) of E2 is
indicated by a dark grey box. The mammalian proteins expressed from
engineered constructs are listed below as dark grey bars. Sites of
signal peptidase cleavage are indicated by arrows. The name of each
protein is followed by the encoded amino acids in parenthesis and
total amino acids in the expressed protein.
[0045] FIGS. 6A and 6B are graphs showing the results of lectin
capture ELISA of mammalian expressed C-terminal truncations of E2.
Cell culture supernatants containing mammalian expressed C-terminal
truncations of E2 fusion proteins containing E2 amino acids
(indicated in FIG. 7) were coated on an ELISA plate and probed with
two-fold dilutions of human antibodies 95-2 (FIG. 6A) and 83-128
(FIG. 6B) and mouse six histidine tag antibody (his tag). Bound
antibodies were detected with goat-anti-human (95-2 and 83-128) or
goat-anti-mouse (his tag) conjugated to alkaline phosphatase and
PNPP substrate.
[0046] FIG. 7 is a map showing bacterially expressed proteins
spanning the amino-terminal 80 amino acids of E2. The first 80
amino acids of the E2 protein are represented at top by a white box
with the amino acids numbers above. The 27 amino acid hypervariable
region 1 (HVR1) at the amino-terminus is indicated by a dark grey
box. The proteins expressed from engineered constructs are
indicated by light grey bars with names to the left of the bar and
amino acid numbers for the start and end of the protein listed
below the bar.
[0047] FIGS. 8A, 8B, and 8C are graphs showing binding of human
antibodies 95-2 (FIG. 8A) and 83-128 (FIG. 8B) and mouse six
histidine tag antibody (his tag) (FIG. 8C) to bacterially expressed
pieces of E2 first 80 amino acids. Bacterially expressed fusion
proteins containing E2 amino acids (indicated in FIG. 7) were
coated on an ELISA plate and probed with two-fold dilutions of
anitbody. Bound antibodies were detected with goat-anti-human (95-2
and 83-128) or goat-anti-mouse (his tag) conjugated to alkaline
phosphatase and PNPP substrate.
[0048] FIGS. 9A, 9B, and 9C are graphs showing binding of human
antibodies 95-2 (FIG. 9A) and 83-128 (FIG. 9B) and mouse six
histidine tag antibody (his tag) (FIG. 9C) to bacterially expressed
fusion proteins containing E2 amino acids 412-423 with mutations.
Bacterially expressed fusion proteins containing E2 amino acids
412-423 with the indicated amino acids mutated to an alanine or
containing no mutations (WT) were coated on an ELISA plate and
probed with two-fold dilutions of antibody. Bound antibodies were
detected with goat-anti-human (95-2 and 83-128) or goat-anti-mouse
(his tag) conjugated to alkaline phosphatase and PNPP
substrate.
[0049] FIG. 10 is a table showing E2 amino acids 412-423 alignments
from the Los Alamos HCV Sequence Database supported by NIH. The one
letter amino acid code for the prototype genotype 1a sequence for
amino acids 412-423 is listed at the top left of the chart (1a).
Any amino acids that are identical to the 1a sequence are indicated
with a dash and the amino acid listed for any position that is
different from the prototype genotype 1a sequence. The number of
times that this twelve amino acid sequence is found in the
condensed database is listed under count and the percent of the
total is listed under percent. The sequences of genotypes 1a and 1b
soluble E2 proteins of the present invention are listed in the top
two lines.
[0050] FIG. 11 is a table showing E2 amino acids 412-423 from other
genotypes. The one-letter amino acid code for amino acids 412-423
is listed for isolates that contained changes (underlined) from the
prototype genotype 1a sequence. The genotype name is listed to the
right followed by the Genbank accession number in parenthesis.
[0051] FIGS. 12A, 12B, and 12C are graphs showing binding of human
antibodies 95-2 (FIG. 12A) and 83-128 (FIG. 12B) and mouse six
histidine tag antibody (his tag) (FIG. 12C) to E2 amino acids
412-423 from other genotypes. Bacterially expressed fusion proteins
containing E2 amino acids 412-423 with the sequence from the
indicated genotype were coated on an ELISA plate and probed with
two-fold dilutions of antibody. Bound antibodies were detected with
goat-anti-human (95-2 and 83-128) or goat-anti-mouse (his tag)
conjugated to alkaline phosphatase and PNPP substrate.
[0052] FIG. 13 depicts the E1/E2 codon optimized 1a H77 sequence
(SEQ ID NO:27) including added ends containing restrictions
(represented as bold lowercase), additional overhangs to optimize
restriction enzyme cutting (represented as underlined lowerase),
consensus Kozak sequence (represented as bold underlined lowercase)
and ATG initiation codon (represented as bold underlined
uppercase).
[0053] FIG. 14 depicts the alignment of the heavy chain variable
region of antibodies derived from clones 83-128 (SEQ ID NO:1), 95-2
(SEQ ID NO:3), 95-14 (SEQ ID NO:32), 95-38 (SEQ ID NO:33), 95-25
(SEQ ID NO:34), 95-42 (SEQ ID NO:35), 95-43 (SEQ ID NO:36), 95-49
(SEQ ID NO:37), 95-54 (SEQ ID NO:38), 95-58 (SEQ ID NO:39) and
95-62 (SEQ ID NO:40) and which are reactive against epitope 412-423
of E2-660 HCV glycoprotein.
[0054] FIG. 15 depicts the alignment of light chain variable region
of antibodies derived from clones 83-128 (SEQ ID NO:2), 073-1 (SEQ
ID NO:6), 95-2 (SEQ ID NO:4), 95-14 (SEQ ID NO:44) and 95-38 (SEQ
ID NO:53).
DETAILED DESCRIPTION OF THE INVENTION
[0055] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
DEFINITIONS
[0056] The terms "hepatitis C virus," "HCV," "non-A non-B
hepatitis," or "NANBH" are used interchangeably herein, and include
any "genotype" or "subgenotype" (also termed "subtype") of the
virion, or portion thereof (e.g., a portion of the E2 protein of
genotype 1a of HCV), that is encoded by the RNA of hepatitis C
virus or that occurs by natural allelic variation.
[0057] HCV is organized by a 5'-untranslated region that is
followed by an open reading frame (ORF) that codes for about 3,010
amino acids. The ORF runs from nucleotide base pair 342 to 8,955
followed by another untranslated region at the 3' end. The amino
acids are subdivided into ten proteins in the order from 5' to 3'
as follows: C; E1; E2; NS1; NS2; NS3; NS4 (a and b); and NS5 (a and
b). These proteins are formed from the cleavage of the larger
polyprotein by both host and viral proteases. The C, E1, and E2
proteins are structural and the NS1-NS5 proteins are nonstructural
proteins. The C region codes for the core nucleocapsid protein. E1
and E2 are glycosylated envelope proteins that coat the virus. NS2
may be a zinc metalloproteinase. NS3 is a helicase. NS4a functions
as a serine protease cofactor involved in cleavage between NS4b and
NS5a. NS5a is a serine phosphoprotein whose function is unknown.
The NS5b region has both RNA-dependent RNA polymerase and terminal
transferase activity.
[0058] There are about six (6) distinct genotypes (e.g., genotypes
1, 2, 3, 4, 5, and 6) that are categorized by variations in the
core protein and over 80 subgenotypes which exhibit further
variation within each genotype, some of which include: 1a; 1b; 1c;
2a; 2b; 2c; 3a; 3b; 4a; 4b; 4c; 4d; 4e; 5a; and 6a.
[0059] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chain thereof. An "antibody" refers to a
glycoprotein comprising at least two heavy (H) chains and two light
(L) chains inter-connected by disulfide bonds, or an antigen
binding portion thereof. Each heavy chain is comprised of a heavy
chain variable region (abbreviated herein as V.sub.H) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as V.sub.L) and a light chain constant region. The light chain
constant region is comprised of one domain, C.sub.L. The V.sub.H
and V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies may mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system.
[0060] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., HCV). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a Fd fragment consisting of the V.sub.H and C.sub.H1 domains;
(iv) a Fv fragment consisting of the V.sub.L and V.sub.H domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989)
Nature 341:544-546), which consists of a V.sub.H domain; and (vi)
an isolated complementarity determining region (CDR) or (vii) a
combination of two or more isolated CDRs which may optionally be
joined by a synthetic linker. Furthermore, although the two domains
of the Fv fragment, V.sub.L and V.sub.H, are coded for by separate
genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g.,
Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies. Antigen-binding portions can
be produced by recombinant DNA techniques, or by enzymatic or
chemical cleavage of intact immunoglobulins.
[0061] A "bispecific" or "bifunctional antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0062] The term "monoclonal antibody," as used herein, refers to an
antibody which displays a single binding specificity and affinity
for a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to an antibody which displays a single binding
specificity and which has variable and constant regions derived
from human germline immunoglobulin sequences. In one embodiment,
human monoclonal antibodies are produced by a hybridoma which
includes a B cell obtained from a transgenic non-human animal,
e.g., a transgenic mouse, having a genome comprising a human heavy
chain transgene and a light chain transgene fused to an
immortalized cell.
[0063] The term "recombinant human antibody," as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as (a) antibodies isolated
from an animal (e.g., a mouse) that is transgenic for human
immunoglobulin genes or a hybridoma prepared therefrom, (b)
antibodies isolated from a host cell transformed to express the
antibody, e.g., from a transfectoma, (c) antibodies isolated from a
recombinant, combinatorial human antibody library, and (d)
antibodies prepared, expressed, created or isolated by any other
means that involve splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies can be subjected to in vitro
mutagenesis (or, when an animal transgenic for human Ig sequences
is used, in vivo somatic mutagenesis) and thus the amino acid
sequences of the V.sub.H and V.sub.L, regions of the recombinant
antibodies are sequences that, while derived from and related to
human germline V.sub.H and V.sub.L, sequences, may not naturally
exist within the human antibody germline repertoire in vivo.
[0064] The term "human antibody" includes antibodies having
variable and constant regions (if present) of human germline
immunoglobulin sequences. Human antibodies of the invention can
include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo)
(see, Lonberg, N. et al. (1994) Nature 368(6474): 856-859);
Lonberg, N. (1994) Handbook of Experimental Pharmacology
113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad. Sci 764:536-546). However, the term "human antibody" does not
include antibodies in which CDR sequences derived from the germline
of another mammalian species, such as a mouse, have been grafted
onto human framework sequences (i.e., humanized antibodies).
[0065] As used herein, a "heterologous antibody" is defined in
relation to the transgenic non-human organism producing such an
antibody. This term refers to an antibody having an amino acid
sequence or an encoding nucleic acid sequence corresponding to that
found in an organism not consisting of the transgenic non-human
animal, and generally from a species other than that of the
transgenic non-human animal.
[0066] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds to HCV is substantially free of
antibodies that specifically bind antigens other than HCV). In
addition, an isolated antibody is typically substantially free of
other cellular material and/or chemicals. In one embodiment of the
invention, a combination of "isolated" monoclonal antibodies having
different HCV specificities are combined in a well defined
composition.
[0067] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds (e.g., E1 or E2 of HCV, for example, amino acids
412-464 of E2). Epitopes can be formed both from contiguous amino
acids or noncontiguous amino acids juxtaposed by tertiary folding
of a protein. Epitopes formed from contiguous amino acids are
typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include techniques in the art and those described
herein, for example, x-ray crystallography and 2-dimensional
nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in
Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In
a preferred embodiment, the epitope (or antigenic determinant)
performs as a linear epitope in that, even when in the context of a
larger amino acid segment, e.g., protein having 3-dimensional
structure, no additional increase in affinity for the
epitope/antigenic determinant is observed.
[0068] As used herein, the terms "specific binding," "selective
binding," "selectively binds," and "specifically binds," refer to
antibody binding to an epitope on a predetermined antigen.
Typically, the antibody binds with an affinity (K.sub.D) of
approximately less than 10.sup.-7 M, such as approximately less
than 10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or even lower when
determined by surface plasmon resonance (SPR) technology in a
BIACORE 3000 instrument using recombinant HCV as the analyte and
the antibody as the ligand, and binds to the predetermined antigen
with an affinity that is at least two-fold greater than its
affinity for binding to a non-specific antigen (e.g., BSA, casein)
other than the predetermined antigen or a closely-related antigen.
The phrases "an antibody recognizing an antigen" and "an antibody
specific for an antigen" are used interchangeably herein with the
term "an antibody which binds specifically to an antigen."
[0069] Also encompassed by the present invention are antibodies
that bind the same epitopes as the human antibodies described
herein, i.e., antibodies that compete for binding to HCV.
Antibodies that recognize the same epitope can be identified using
routine techniques such as an immunoassay, for example, by showing
the ability of one antibody to block the binding of another
antibody to a target antigen, i.e., a competitive binding assay.
Competitive binding is determined in an assay in which the
immunoglobulin under test inhibits specific binding of a reference
antibody to a common antigen, such as HCV. Numerous types of
competitive binding assays are known, for example: solid phase
direct or indirect radioimmunoassay (RIA), solid phase direct or
indirect enzyme immunoassay (EIA), sandwich competition assay (see
Stahli et al., Methods in Enzymology 9:242 (1983)); solid phase
direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614
(1986)); solid phase direct labeled assay, solid phase direct
labeled sandwich assay (see Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase
direct label MA using I-125 label (see Morel et al., Mol. Immunol.
25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et
al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer
et al., Scand J. Immunol. 32:77 (1990)). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test immunoglobulin and
a labeled reference immunoglobulin. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test immunoglobulin.
Usually the test immunoglobulin is present in excess. Usually, when
a competing antibody is present in excess, it will inhibit specific
binding of a reference antibody to a common antigen by at least
50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.
[0070] The term "K.sub.D," as used herein, is intended to refer to
the dissociation equilibrium constant of a particular
antibody-antigen interaction. Typically, the human antibodies of
the invention bind to HCV with a dissociation equilibrium constant
(K.sub.D) of less than approximately 10.sup.-7 M, such as less than
approximately 10.sup.-8M, 10.sup.-9 M or 10.sup.-10 M or even lower
when determined by surface plasmon resonance (SPR) technology in a
BIACORE 3000 instrument using recombinant human HCV as the analyte
and the antibody as the ligand.
[0071] The term "K.sub.off," as used herein, is intended to refer
to the off rate constant for the dissociation of an antibody from
the antibody/antigen complex.
[0072] The term "EC50," as used herein, refers to the concentration
of an antibody or an antigen-binding portion thereof, which induces
a response, either in an in vitro or an in vivo assay, which is 50%
of the maximal response, i.e., halfway between the maximal response
and the baseline.
[0073] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes. In one embodiment, a human monoclonal antibody of the
invention is of the IgG1 isotype. In another embodiment, a human
monoclonal antibody of the invention is of the IgG2 isotype.
[0074] As used herein, the terms "neutralizes HCV," "inhibits HCV,"
and "blocks HCV" are used interchangeably to refer to the ability
of an antibody of the invention to prevent HCV from infecting a
given cell.
[0075] As used herein, the term "non-conformational epitope" refers
to a linear epitope which is typically comprised of a continuous
amino acid sequence which is sufficient for binding with an
antibody capable of binding to such an epitope. This is in contrast
to a conformational epitope, which requires discontinuous amino
acid sequences to form a 3-dimensional structure in order for
binding between the epitope and the antibody to occur. A linear
epitope can also be distinguished from a conformational epitope in
that under denaturing conditions, (e.g., in an immunoblot assay as
described herein), the epitope can still be bound by an antibody
that recognizes such an epitope. In one embodiment, the invention
provides an HCV E2 epitope that is linear, for example, non
conformational, for example, comprising residues 412 to 423, or,
consisting of residues 412-423. In a related embodiment, the
antibodies of the invention specifically bind to such a linear
epitope and not a conformational epitope. Still further, the
invention provides such a linear epitope as suitable for the use in
vaccine development, the raising of antibodies thereto, and/or for
the use in active immunotherapy alone or in combination with
passive immunotherapy.
[0076] As used herein, "isotype switching" refers to the phenomenon
by which the class, or isotype, of an antibody changes from one Ig
class to one of the other Ig classes.
[0077] As used herein, "nonswitched isotype" refers to the isotypic
class of heavy chain that is produced when no isotype switching has
taken place; the C.sub.H gene encoding the nonswitched isotype is
typically the first C.sub.H gene immediately downstream from the
functionally rearranged VDJ gene. Isotype switching has been
classified as classical or non-classical isotype switching.
Classical isotype switching occurs by recombination events which
involve at least one switch sequence region in the transgene.
Non-classical isotype switching may occur by, for example,
homologous recombination between human .sigma..sub..mu., and human
.SIGMA..sub..mu. (.delta.-associated deletion). Alternative
non-classical switching mechanisms, such as intertransgene and/or
interchromosomal recombination, among others, may occur and
effectuate isotype switching.
[0078] As used herein, the term "switch sequence" refers to those
DNA sequences responsible for switch recombination. A "switch
donor" sequence, typically a .mu. switch region, will be 5' (i.e.,
upstream) of the construct region to be deleted during the switch
recombination. The "switch acceptor" region will be between the
construct region to be deleted and the replacement constant region
(e.g., .gamma., .epsilon., etc.). As there is no specific site
where recombination always occurs, the final gene sequence will
typically not be predictable from the construct.
[0079] As used herein, "glycosylation pattern" is defined as the
pattern of carbohydrate units that are covalently attached to a
protein, more specifically to an immunoglobulin protein. A
glycosylation pattern of a heterologous antibody can be
characterized as being substantially similar to glycosylation
patterns which occur naturally on antibodies produced by the
species of the nonhuman transgenic animal, when one of ordinary
skill in the art would recognize the glycosylation pattern of the
heterologous antibody as being more similar to said pattern of
glycosylation in the species of the nonhuman transgenic animal than
to the species from which the C.sub.H genes of the transgene were
derived.
[0080] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0081] The term "rearranged" as used herein refers to a
configuration of a heavy chain or light chain immunoglobulin locus
wherein a V segment is positioned immediately adjacent to a D-J or
J segment in a conformation encoding essentially a complete V.sub.H
or V.sub.L domain, respectively. A rearranged immunoglobulin gene
locus can be identified by comparison to germline DNA; a rearranged
locus will have at least one recombined heptamer/nonamer homology
element.
[0082] The term "unrearranged" or "germline configuration" as used
herein in reference to a V segment refers to the configuration
wherein the V segment is not recombined so as to be immediately
adjacent to a D or J segment.
[0083] The term "nucleic acid molecule," as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0084] The term "isolated nucleic acid molecule," as used herein in
reference to nucleic acids encoding antibodies or antibody portions
(e.g., V.sub.H, V.sub.L, CDR3) that bind to HCV, is intended to
refer to a nucleic acid molecule in which the nucleotide sequences
encoding the antibody or antibody portion are free of other
nucleotide sequences encoding antibodies that bind antigens other
than HCV, which other sequences may naturally flank the nucleic
acid in human genomic DNA.
[0085] The present invention also encompasses "conservative
sequence modifications" of the sequences set forth in the SEQ ID
NOs of the present invention, i.e., nucleotide and amino acid
sequence modifications which do not abrogate the binding of the
antibody encoded by the nucleotide sequence or containing the amino
acid sequence, to the antigen. Such conservative sequence
modifications include nucleotide and amino acid substitutions,
additions and deletions. For example, modifications can be
introduced by standard techniques known in the art, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
Conservative amino acid substitutions include ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a human anti-HCV
antibody is preferably replaced with another amino acid residue
from the same side chain family. Methods of identifying nucleotide
and amino acid conservative substitutions which do not eliminate
antigen binding are well-known in the art (see, e.g., Brummell et
al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng.
12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA
94:412-417 (1997))
[0086] Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an anti-HCV antibody
coding sequence, such as by saturation mutagenesis, and the
resulting modified anti-HCV antibodies can be screened for binding
activity.
[0087] A "consensus sequence" is a sequence formed from the most
frequently occurring amino acids (or nucleotides) in a family of
related sequences (See e.g., Winnaker, From Genes to Clones
(Verlagsgesellschaft, Weinheim, Germany 1987). In a family of
proteins, each position in the consensus sequence is occupied by
the amino acid occurring most frequently at that position in the
family. If two amino acids occur equally frequently, either can be
included in the consensus sequence. A "consensus framework" of an
immunoglobulin refers to a framework region in the consensus
immunoglobulin sequence.
[0088] As depicted in FIG. 14, in one embodiment, a consensus
sequence for the heavy chain variable region CDRs is derived by
optimal alignment of the amino acid sequences of the heavy chain
variable region of the antibodies which are reactive against
epitope 412-423 of the E2-660 HCV glycoprotein. For example, the
antibodies derived from clones 83-128 (SEQ ID NO:1), 95-2 (SEQ ID
NO:3), 95-14 (SEQ ID NO:32), 95-38 (SEQ ID NO:33), 95-25 (SEQ ID
NO:34), 95-42 (SEQ ID NO:35), 95-43 (SEQ ID NO:36), 95-49 (SEQ ID
NO:37), 95-54 (SEQ ID NO:38), 95-58 (SEQ ID NO:39) and 95-62 (SEQ
ID NO:40) are optimally aligned to obtain the consensus sequences
for CDR1, CDR2 and CDR3. Accordingly, the consensus sequence for
CDR1 of the heavy chain variable region is found to be "1YGMH," set
forth in SEQ ID NO:87, where "1" represents a small and polar amino
acid residue. The consensus sequence for CDR2 of the heavy chain
variable region is found to be "VIWXDX7NXYYADS1516G," shown in SEQ
ID NO:88, where "X" can be any amino acid residue, "7" represents a
small and polar amino acid residue, "15" represents a hydrophobic
amino acid residue and "16" represents a polar and positively
charged amino acid residue. The consensus sequence for CDR3 of the
heavy chain variable region is found to be "ARDI567XR10X12IYFD17",
shown in SEQ ID NO:89, where "X" represents any amino acid residue,
"5" represents phenylalanine or no amino acid, "6" represents
Serine or Threonine, "7" and "12" represent a hydrophobic amino
acid residue, "10" represents a small amino acid residue and "17"
represents an aromatic amino acid residue.
[0089] Similarly, the consensus sequence for the CDRs of light
chain variable region can be derived by optimal alignment of the
amino acid sequences of antibodies reactive against epitope 412-423
of E2-660 HCV glycoprotein, as shown in FIG. 15. For example, amino
acid sequences of the light chain variable regions of antibodies
derived from clones 83-128 (SEQ ID NO:2), 073-1 (SEQ ID NO:6), 95-2
(SEQ ID NO:4), 95-14 (SEQ ID NO:44) and 95-38 (SEQ ID NO:53) are
optimally aligned to obtain the consensus sequences for CDR1, CDR2
and CDR3 of the light chain variable region. The consensus sequence
for CDR1 of the light chain variable region is found to be
"RASQSVXSYLA," set forth in SEQ ID NO:90, where "X" can be any
amino acid residue. The consensus sequence for CDR2 of the light
chain variable region is found to be "DASNRAT," set forth in SEQ ID
NO:91. The consensus sequence for CDR3 of the light chain variable
region is found to be "QQRSNW7T," shown in SEQ ID NO:92, where "7"
represents a small and hydrophobic amino acid residue.
[0090] For nucleic acids, the term "substantial homology" indicates
that two nucleic acids, or designated sequences thereof, when
optimally aligned and compared, are identical, with appropriate
nucleotide insertions or deletions, in at least about 80% of the
nucleotides, usually at least about 90% to 95%, and more preferably
at least about 98% to 99.5% of the nucleotides. Alternatively,
substantial homology exists when the segments will hybridize under
selective hybridization conditions, to the complement of the
strand.
[0091] The percent identity between two sequences is a function of
the number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of positions.times.100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, as described in the non-limiting examples
below.
[0092] The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package, using
a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80
and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity
between two nucleotide or amino acid sequences can also be
determined using the algorithm of E. Meyers and W. Miller (CABIOS,
4:11-17 (1989)) which has been incorporated into the ALIGN program
(version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4. In addition, the percent
identity between two amino acid sequences can be determined using
the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package, using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6.
[0093] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against public databases to, for example, identify related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., (1997) Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used.
[0094] The nucleic acids may be present in whole cells, in a cell
lysate, or in a partially purified or substantially pure form. A
nucleic acid is "isolated" or "rendered substantially pure" when
purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York (1987).
[0095] The nucleic acid compositions of the present invention,
while often in a native sequence (except for modified restriction
sites and the like), from either cDNA, genomic or mixtures thereof
may be mutated, in accordance with standard techniques to provide
gene sequences. For coding sequences, these mutations, may affect
amino acid sequence as desired. In particular, DNA sequences
substantially homologous to or derived from native V, D, J,
constant, switches and other such sequences described herein are
contemplated (where "derived" indicates that a sequence is
identical or modified from another sequence).
[0096] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence. With
respect to transcription regulatory sequences, operably linked
means that the DNA sequences being linked are contiguous and, where
necessary to join two protein coding regions, contiguous and in
reading frame. For switch sequences, operably linked indicates that
the sequences are capable of effecting switch recombination.
[0097] The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid,"
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors") In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0098] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
[0099] The terms "treat," "treating," and "treatment," as used
herein, refer to therapeutic or preventative measures described
herein. The methods of "treatment" employ administration to a
subject, in need of such treatment, a human antibody of the present
invention, for example, a subject having an HCV-mediated disorder
or a subject who ultimately may acquire such a disorder, in order
to prevent, cure, delay, reduce the severity of, or ameliorate one
or more symptoms of the disorder or recurring disorder, or in order
to prolong the survival of a subject beyond that expected in the
absence of such treatment.
[0100] The term "HCV-mediated disorder," as used herein, includes
disease states and/or symptoms associated with HCV infection. In
general, the term "HCV-mediated disorder" refers to any disorder,
the onset, progression or the persistence of the symptoms of which
requires the participation of HCV. Exemplary HCV-mediated disorders
include, but are not limited to, for example, cirrhosis and liver
cancer.
[0101] The term "effective dose" or "effective dosage" is defined
as an amount sufficient to achieve or at least partially achieve
the desired effect. The term "therapeutically effective dose" is
defined as an amount sufficient to cure or at least partially
arrest the disease and its complications in a patient already
suffering from the disease. Amounts effective for this use will
depend upon the severity of the disorder being treated and the
general state of the patient's own immune system.
[0102] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0103] As used herein, the term "subject" includes any human or
non-human animal. For example, the methods and compositions of the
present invention can be used to treat a subject with an
inflammatory disease, such as arthritis, e.g., rheumatoid
arthritis. The term "non-human animal" includes all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc.
[0104] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the present specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0105] Various aspects of the invention are described in further
detail in the following subsections.
Overview
[0106] HCV causes fatal hepatocyte pathologies in humans. Provided
herein are methods and compositions for treatment and prevention of
HCV infected animals, in particular, human subjects, more
particularly. The compositions include antibodies that recognize
the HCV E2 protein, or portion thereof. In particular, recombinant
fully human monoclonal antibodies are provided. In certain
embodiments, these human monoclonal antibodies are produced in mice
expressing human immunoglobulin gene segments (described below).
Combinations of anti-HCV antibodies are also provided.
[0107] The new methods include administering antibodies (and
antigen-binding portions thereof) that bind to HCV in a subject to
inhibit HCV-mediated disease in the subject. For example, human
monoclonal anti-HCV antibodies described herein can neutralize HCV
and inhibit HCV infection and sequelae thereof, for example,
cirrhosis of the liver and/or liver cancer. In other examples,
combinations of anti-HCV antibodies (e.g., anti-HCV E2 protein
monoclonal antibodies) can be administered to inhibit HCV-mediated
disease. The human monoclonal antibodies can be administered alone
or in combination with other therapies.
I. Production of Human Antibodies to HCV
[0108] The present invention encompasses fully human antibodies
that bind HCV, e.g., human HCV. Exemplary human monoclonal
antibodies that bind HCV include 83-128, 95-2, 95-14, 95-38, and
073-1.
[0109] Human monoclonal antibodies of the invention can be produced
using a variety of known techniques, such as the standard somatic
cell hybridization technique described by Kohler and Milstein,
Nature 256: 495 (1975). Although somatic cell hybridization
procedures are preferred, in principle, other techniques for
producing monoclonal antibodies also can be employed, e.g., viral
or oncogenic transformation of B lymphocytes, phage display
technique using libraries of human antibody genes.
[0110] The preferred animal system for generating hybridomas which
produce human monoclonal antibodies of the invention is the murine
system. Hybridoma production in the mouse is well known in the art,
including immunization protocols and techniques for isolating and
fusing immunized splenocytes.
[0111] In one embodiment, human monoclonal antibodies directed
against HCV are generated using transgenic mice carrying parts of
the human immune system rather than the mouse system. In one
embodiment, the invention employs transgenic mice, referred to
herein as "HuMAb mice" which contain a human immunoglobulin gene
miniloci that encodes unrearranged human heavy (.mu. and .gamma.)
and .kappa. light chain immunoglobulin sequences, together with
targeted mutations that inactivate the endogenous .mu. and .kappa.
chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856-859).
Accordingly, the mice exhibit reduced expression of mouse IgM or
.kappa., and in response to immunization, the introduced human
heavy and light chain transgenes undergo class switching and
somatic mutation to generate high affinity human IgG.kappa.
monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed
in Lonberg, N. (1994) Handbook of Experimental Pharmacology
113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol.
Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y.
Acad. Sci 764:536-546). The preparation of HuMAb mice is described
in detail in Section II below and in Taylor, L. et al. (1992)
Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)
International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.
Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature
Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830;
Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al.,
(1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of
Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994)
International Immunology 6: 579-591; Lonberg, N. and Huszar, D.
(1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and
Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild, D. et
al. (1996) Nature Biotechnology 14: 845-851. See further, U.S. Pat.
Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;
5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to
Lonberg and Kay, and GenPharm International; U.S. Pat. No.
5,545,807 to Surani et al.; International Publication Nos. WO
98/24884, published on Jun. 11, 1998; WO 94/25585, published Nov.
10, 1994; WO 93/1227, published Jun. 24, 1993; WO 92/22645,
published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992.
Immunizations
[0112] To generate fully human monoclonal antibodies to HCV,
transgenic mice containing human immunoglobulin genes (e.g., HCo12,
HCo7) can be immunized with a purified or enriched preparation of
the HCV antigen and/or cells expressing HCV, as described, for
example, by Lonberg et al. (1994) Nature 368(6474): 856-859;
Fishwild et al. (1996) Nature Biotechnology 14: 845-851 and WO
98/24884. As described herein, HuMAb mice are immunized either with
recombinant HCV proteins or cell lines expressing HCV as
immunogens. Alternatively, mice can be immunized with DNA encoding
HCV. Preferably, the mice will be 6-16 weeks of age upon the first
infusion. For example, a purified or enriched preparation (10-100
.mu.g) of the recombinant HCV antigen can be used to immunize the
HuMAb mice intraperitoneally. In the event that immunizations using
a purified or enriched preparation of the HCV antigen do not result
in antibodies, mice can also be immunized with cells expressing HCV
proteins, e.g., a cell line, to promote immune responses. Exemplary
cell lines include HCV-overexpressing stable CHO and Raji cell
lines.
[0113] Cumulative experience with various antigens has shown that
the HuMAb transgenic mice respond best when initially immunized
intraperitoneally (IP) or subcutaneously (SC) with antigen in
complete Freund's adjuvant, followed by every other week IP/SC
immunizations (up to a total of 10) with antigen in incomplete
Freund's adjuvant. The immune response can be monitored over the
course of the immunization protocol with plasma samples being
obtained by retroorbital bleeds. The plasma can be screened by
ELISA (as described below), and mice with sufficient titers of
anti-HCV human immunoglobulin can be used for fusions. Mice can be
boosted intravenously with antigen 3 days before sacrifice and
removal of the spleen.
Antigen Based Vaccines and Conjugates Thereof
[0114] The present invention also provides a conserved
non-conformational epitope of the HCV E2 envelope glycoprotein, for
example, amino acids 412-464, 412-423, or 413-420 of the HCV E2
protein, for use in active immunization against HCV infection. This
epitope can be used alone or be modified, e.g., to enhance an
immune response to the epitope.
[0115] Chemically constructed antigen conjugates can be made using
a variety of well known and readily available cross-linking
reagents. These cross-linking reagents can be homofunctional or
heterofunctional compounds, such as, e.g., SPDP, SATA, SMCC, DTNB,
that form covalent linkages with different reactive amino acid or
carbohydrate side chains on the selected antigen.
[0116] The carrier can be selected from the group consisting of
OMPC (Outer Membrane Protein Complex of Neisseria nieningitidis),
BSA (bovine serum albumin), OVA (ovalbumin), THY (bovine
thyroglobulin), KLH (keyhole limpet hemocyanin), and TT (tetanus
toxoid protein). Other carriers, that have the capability to self
assemble into virus-like particles (VLP's), include HbSAg (surface
antigen protein) and HBcAg (core antigen protein) of Hepatitis B
virus, rotavirus capsid proteins, the L1 protein of the human
papilloma virus, hepatitis E virus particles, polyoma virus protein
and bovine papilloma virus structural proteins. (see J. of Phar.
Sciences 95:70-79 (2005)).
[0117] The conjugating can be achieved genetically or chemically,
for example, using one or more of maleimide/thiol coupling,
bromoacetamide/thiol coupling, and histidine-selective
cross-linking. The conjugating can be achieved using a cross-linker
selected from the group consisting of one of more of
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(sSMCC), N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI), Bis-diazobenzidine (BDB), N-acetyl homocysteine thiolactone
(NAHT), and N-[.epsilon.-maleimidocaproyloxy]sulfosuccinimde ester
(sEMCS). In one embodiment, the cross-linker is a
N-[.epsilon.-maleimidocaproyloxy]sulfosuccinimde ester (sEMCS).
Generation of Hybridomas Producing Human Monoclonal Antibodies to
HCV
[0118] To generate hybridomas producing human monoclonal antibodies
to HCV, splenocytes and lymph node cells from immunized mice can be
isolated and fused to an appropriate immortalized cell line, such
as a mouse myeloma cell line. The resulting hybridomas can then be
screened for the production of antigen-specific antibodies. For
example, single cell suspensions of splenic lymphocytes from
immunized mice can be fused to SP2/0-Ag8.653 nonsecreting mouse
myeloma cells (ATCC, CRL 1580) with 50% PEG (w/v). Cells can be
plated at approximately 1.times.10.sup.5 in flat bottom microtiter
plate, followed by a two week incubation in selective medium
containing besides usual reagents 10% fetal Clone Serum, and
1.times.HAT (Sigma). After approximately two weeks, cells can be
cultured in medium in which the HAT is replaced with HT. Individual
wells can then be screened by ELISA for human anti-HCV monoclonal
IgM and IgG antibodies, or for binding to the surface of cells
expressing HCV proteins, e.g., a CHO cell line expressing HCV, by
FLISA (fluorescence-linked immunosorbent assay). Once extensive
hybridoma growth occurs, medium can be observed usually after 10-14
days. The antibody secreting hybridomas can be replated, screened
again, and if still positive for human IgG, anti-HCV monoclonal
antibodies can be subcloned at least twice by limiting dilution.
The stable subclones can then be cultured in vitro to generate
antibody in tissue culture medium for characterization.
Generation of Transfectomas Producing Human Monoclonal Antibodies
to HCV
[0119] Human antibodies of the invention also can be produced in a
host cell transfectoma using, for example, a combination of
recombinant DNA techniques and gene transfection methods as is well
known in the art (Morrison, S. (1985) Science 229:1202).
[0120] For example, in one embodiment, the gene(s) of interest,
e.g., human antibody genes, can be ligated into an expression
vector such as a eukaryotic expression plasmid such as used by GS
gene expression system disclosed in WO 87/04462, WO 89/01036 and EP
338 841 or other expression systems well known in the art. The
purified plasmid with the cloned antibody genes can be introduced
in eukaryotic host cells such as CHO-cells or NSO-cells or
alternatively other eukaryotic cells like a plant derived cells,
fungi or yeast cells. The method used to introduce these genes can
be methods described in the art such as electroporation,
lipofectine, lipofectamine or other. After introducing these
antibody genes in the host cells, cells expressing the antibody can
be identified and selected. These cells represent the transfectomas
which can then be amplified for their expression level and upscaled
to produce antibodies. Recombinant antibodies can be isolated and
purified from these culture supernatants and/or cells.
[0121] Alternatively these cloned antibody genes can be expressed
in other expression systems such as E. coli or in complete
organisms or can be synthetically expressed.
Use of Partial Antibody Sequences to Express Intact Antibodies
[0122] Antibodies interact with target antigens predominantly
through amino acid residues that are located in the six heavy and
light chain complementarity determining regions (CDRs). For this
reason, the amino acid sequences within CDRs are more diverse
between individual antibodies than sequences outside of CDRs.
Because CDR sequences are responsible for most antibody-antigen
interactions, it is possible to express recombinant antibodies that
mimic the properties of specific naturally occurring antibodies by
constructing expression vectors that include CDR sequences from the
specific naturally occurring antibody grafted onto framework
sequences from a different antibody with different properties (see,
e.g., Riechmann, L. et al, 1998, Nature 332:323-327; Jones, P. et
al, 1986, Nature 321:522-525; and Queen, C. et al, 1989, Proc.
Natl. Acad. See. U.S.A. 86:10029-10033). Such framework sequences
can be obtained from public DNA databases that include germline
antibody gene sequences. These germline sequences will differ from
mature antibody gene sequences because they will not include
completely assembled variable genes, which are formed by V(D)J
joining during B cell maturation. Germline gene sequences will also
differ from the sequences of a high affinity secondary repertoire
antibody at individual evenly across the variable region. For
example, somatic mutations are relatively infrequent in the amino
terminal portion of framework region 1 and in the carboxy-terminal
portion of framework region 4. Furthermore, many somatic mutations
do not significantly alter the binding properties of the antibody.
For this reason, it is not necessary to obtain the entire DNA
sequence of a particular antibody in order to recreate an intact
recombinant antibody having binding properties similar to those of
the original antibody (see PCT/US99/05535 filed on Mar. 12, 1999).
Partial heavy and light chain sequence spanning the CDR regions is
typically sufficient for this purpose. The partial sequence is used
to determine which germline variable and joining gene segments
contributed to the recombined antibody variable genes. The germline
sequence is then used to fill in missing portions of the variable
regions. Heavy and light chain leader sequences are cleaved during
protein maturation and do not contribute to the properties of the
final antibody. To add missing sequences, cloned cDNA sequences can
be combined with synthetic oligonucleotides by ligation or PCR
amplification. Alternatively, the entire variable region can be
synthesized as a set of short, overlapping, oligonucleotides and
combined by PCR amplification to create an entirely synthetic
variable region clone. This process has certain advantages such as
elimination or inclusion or particular restriction sites, or
optimization of particular codons.
[0123] The nucleotide sequences of heavy and light chain
transcripts from a hybridoma are used to design an overlapping set
of synthetic oligonucleotides to create synthetic V sequences with
identical amino acid coding capacities as the natural sequences.
The synthetic heavy and kappa chain sequences can differ from the
natural sequences in three ways: strings of repeated nucleotide
bases are interrupted to facilitate oligonucleotide synthesis and
PCR amplification; optimal translation initiation sites are
incorporated according to Kozak's rules (Kozak, 1991, J. Biol.
Chem. 266:19867-19870); and, HindIII sites are engineered upstream
of the translation initiation sites.
[0124] For both the heavy and light chain variable regions, the
optimized coding, and corresponding non-coding, strand sequences
are broken down into 30-50 nucleotide oligonucleotides starting at
approximately the midpoint of the corresponding non-coding
oligonucleotide. Thus, for each chain, the oligonucleotides can be
assembled into overlapping double stranded sets that span segments
of 150-400 nucleotides. The pools are then used as templates to
produce PCR amplification products of 150-400 nucleotides.
Typically, a single variable region oligonucleotide set will be
broken down into two pools which are separately amplified to
generate two overlapping PCR products. These overlapping products
are then combined by PCR amplification to form the complete
variable region. It may also be desirable to include an overlapping
fragment of the heavy or light chain constant region (including the
BbsI site of the kappa light chain, or the AgeI site if the gamma
heavy chain) in the PCR amplification to generate fragments that
can easily be cloned into the expression vector constructs.
[0125] The reconstructed heavy and light chain variable regions are
then combined with cloned promoter, leader sequence, translation
initiation, leader sequence, constant region, 3' untranslated,
polyadenylation, and transcription termination, sequences to form
expression vector constructs. The heavy and light chain expression
constructs can be combined into a single vector, co-transfected,
serially transfected, or separately transfected into host cells
which are then fused to form a host cell expressing both
chains.
[0126] Plasmids for use in construction of expression vectors can
be constructed so that PCR amplified V heavy and V kappa light
chain cDNA sequences can be used to reconstruct complete heavy and
light chain minigenes. These plasmids can be used to express
completely human IgG.sub.1.kappa. or IgG.sub.4.kappa. antibodies.
Fully human and chimeric antibodies of the present invention also
include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar
plasmids can be constructed for expression of other heavy chain
isotypes, or for expression of antibodies comprising lambda light
chains.
[0127] Thus, in another aspect of the invention, one or more
structural features of a human anti-HCV antibody of the invention
are used to create structurally related human anti-HCV antibodies
that retain at least one functional property of the antibodies of
the invention, such as, for example, binding to HCV or neutralizing
HCV. In one embodiment, one or more CDR regions of antibodies of
the invention can be combined recombinantly with known human
framework regions and CDRs to create additional,
recombinantly-engineered, human anti-HCV antibodies of the
invention. The heavy and light chain variable framework regions can
be derived from the same or different human antibody sequences. The
human antibody sequences can be the sequences of naturally
occurring human antibodies or can be consensus sequences of several
human antibodies. See Kettleborough et al., Protein Engineering
4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993)
and Carter et al., WO 92/22653.
[0128] Accordingly, in another embodiment, the invention provides a
method for preparing a human anti-HCV antibody including: preparing
an antibody including (1) human heavy chain framework regions and
human heavy chain CDRs, where at least one of the human heavy chain
CDRs includes an amino acid sequence selected from the human heavy
chain CDR amino acid sequences described herein; and (2) human
light chain framework regions and human light chain CDRs, where at
least one of the human heavy chain CDRs includes an amino acid
sequence selected from the human light chain CDR amino acid
sequences described herein, where the antibody retains the ability
to bind to HCV. The ability of the antibody to bind HCV can be
determined using standard binding assays, such as those set forth
in the Examples (e.g., an ELISA or a FLISA).
[0129] It is well known in the art that antibody heavy and light
chain CDR3 domains play a particularly important role in the
binding specificity/affinity of an antibody for an antigen (see,
Hall et al., J. Imunol., 149:1605-1612 (1992); Polymenis et al., J.
Immunol., 152:5318-5329 (1994); Jahn et al., Immunobiol.,
193:400-419 (1995); Klimka et al., Brit. J. Cancer, 83:252-260
(2000); Beiboer et al., J. Mol. Biol, 296:833-849 (2000); Rader et
al., Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et
al., J. Am. Chem. Soc., 116:2161-2162 (1994); Ditzel et al., J.
Immunol., 157:739-749 (1996)). Accordingly, the recombinant
antibodies of the invention prepared as set forth above preferably
comprise the heavy and/or light chain CDR3s of antibodies 83-128,
95-2, 95-14, 95-38, and 073-1. The antibodies further can comprise
the CDR2s of antibodies 83-128, 95-2, 95-14, 95-38, and 073-1. The
antibodies further can comprise the CDR1s of antibodies 83-128,
95-2, 95-14, 95-38, and 073-1. The antibodies can further comprise
any combinations of the CDRs.
[0130] Accordingly, in another embodiment, the invention further
provides anti-HCV antibodies comprising: (1) human heavy chain
framework regions, a human heavy chain CDR1 region, a human heavy
chain CDR2 region, and a human heavy chain CDR3 region, wherein the
human heavy chain CDR3 region is selected from the CDR3s of 83-128,
95-2, 95-14, 95-38, and 073-1, for example, a human heavy chain
CDR3 region of 95-2 as shown in the sequence listing described
herein; and (2) human light chain framework regions, a human light
chain CDR1 region, a human light chain CDR2 region, and a human
light chain CDR3 region, wherein the human light chain CDR3 region
is selected from the CDR3s of 83-128, 95-2, 95-14, 95-38, and 073-1
for example, a human light chain CDR3 region of 95-2 as shown in
the sequence listing described herein wherein the antibody binds
HCV. The antibody may further include the heavy chain CDR2 and/or
the light chain CDR2 of antibodies 83-128, 95-2, 95-14, 95-38, and
073-1The antibody may further comprise the heavy chain CDR1 and/or
the light chain CDR1 of 83-128, 95-2, 95-14, 95-38, and 073-1.
[0131] The CDR1, 2, and/or 3 regions of the engineered antibodies
described above can comprise the exact amino acid sequence(s) as
those of antibodies 83-128, 95-2, 95-14, 95-38, and 073-1 disclosed
herein. However, the ordinarily skilled artisan will appreciate
that some deviation from the exact CDR sequences of 83-128, 95-2,
95-14, 95-38, and 073-1 may be possible while still retaining the
ability of the antibody to bind HCV effectively (e.g., conservative
sequence modifications). Accordingly, in another embodiment, the
engineered antibody may be composed of one or more CDRs that are,
for example, 90%, 95%, 98% or 99.5% identical to one or more CDRs
of antibodies 83-128, 95-2, 95-14, 95-38, and 073-1.
[0132] In another embodiment, one or more residues of a CDR may be
altered to modify binding to achieve a more favored on-rate of
binding, a more favored off-rate of binding, or both, such that an
idealized binding constant is achieved. Using this strategy, an
antibody having ultra high binding affinity of, for example, a
K.sub.D10.sup.-10 M or lower, can be achieved. Affinity maturation
techniques, well known in the art and those described herein, can
be used to alter the CDR region(s) followed by screening of the
resultant binding molecules for the desired change in binding.
Accordingly, as CDR(s) are altered, changes in binding affinity as
well as immunogenicity can be monitored and scored such that an
antibody optimized for the best combined binding and low
immunogenicity are achieved.
[0133] In addition to, or instead of, modifications within the
CDRs, modifications can also be made within one or more of the
framework regions, FR1, FR2, FR3 and FR4, of the heavy and/or the
light chain variable regions of a human antibody, so long as these
modifications do not eliminate the binding affinity of the human
antibody. The amino acids at several positions in the framework are
known to be important for determining CDR confirmation (e.g.,
capable of interacting with the CDRs) in many antibodies (Chothia
and Lesk, supra, Chothia et al., supra and Tramontano et al., J.
Mol. Biol. 215:175 (1990), all of which are incorporated herein by
reference). These authors identified conserved framework residues
important for CDR conformation by analysis of the structures of
several known antibodies. The antibodies analyzed fell into a
limited number of structural or "canonical" classes based on the
conformation of the CDRs. Conserved framework residues within
members of a canonical class are referred to as "canonical"
residues. Canonical residues include residues 2, 25, 29, 30, 33,
48, 64, 71, 90, 94 and 95 of the light chain and residues 24, 26,
29, 34, 54, 55, 71 and 94 of the heavy chain. Additional residues
(e.g., CDR structure-determining residues) can be identified
according to the methodology of Martin and Thorton (1996) J. Mol.
Biol. 263:800. Notably, the amino acids at positions 2, 48, 64 and
71 of the light chain and 26-30, 71 and 94 of the heavy chain
(numbering according to Kabat) are known to be capable of
interacting with the CDRs in many antibodies. The amino acids at
positions 35 in the light chain and 93 and 103 in the heavy chain
are also likely to interact with the CDRs. Additional residues
which may effect conformation of the CDRs can be identified
according to the methodology of Foote and Winter (1992) J. Mol.
Biol. 224:487. Such residues are termed "vernier" residues and are
those residues in the framework region closely underlying (i.e.,
forming a "platform" under) the CDRs.
[0134] Residues which "participate in the V.sub.L-V.sub.H
interface" or "packing residues" include those residues at the
interface between V.sub.L and V.sub.H as defined, for example, by
Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985) or
Chothia et al, supra.
[0135] Occasionally, there is some ambiguity about whether a
particular amino acid falls within one or more of the
above-mentioned categories. In such instances, alternative variant
antibodies are produced, one of which has that particular
substitution, the other of which does not. Alternative variant
antibodies so produced can be tested in any of the assays described
herein for the desired activity, and the preferred antibody
selected.
[0136] Additional candidates for substitution within the framework
region are amino acids that are unusual or "rare" for a human
antibody at that position. These amino acids can be substituted
with amino acids from the equivalent position of the human germline
sequence or from the equivalent positions of more typical human
antibodies. For example, substitution may be desirable when the
amino acid in a human framework region of the human antibody is
rare for that position and the corresponding amino acid in the
germline sequence is common for that position in human
immunoglobulin sequences; or when the amino acid in the human
antibody is rare for that position and the corresponding amino acid
in the germline sequence is also rare, relative to other human
sequences. It is contemplated that by replacing an unusual amino
acid with an amino acid from the germline sequence that happens to
be typical for human antibodies, the human antibody may be made
less immunogenic.
[0137] The term "rare", as used herein, indicates an amino acid
occurring at that position in less than about 20%, preferably less
than about 10%, more preferably less than about 5%, even more
preferably less than about 3%, even more preferably less than about
2% and even more preferably less than about 1% of sequences in a
representative sample of sequences, and the term "common", as used
herein, indicates an amino acid occurring in more than about 25%
but usually more than about 50% of sequences in a representative
sample. For example, all human light and heavy chain variable
region sequences are respectively grouped into "subgroups" of
sequences that are especially homologous to each other and have the
same amino acids at certain critical positions (Kabat et al.,
supra). When deciding whether an amino acid in a human antibody
sequence is "rare" or "common" among human sequences, it will often
be preferable to consider only those human sequences in the same
subgroup as the human antibody sequence.
[0138] In general, the framework regions of human antibodies are
usually substantially identical, and more usually, identical to the
framework regions of the human germline sequences from which they
were derived. Of course, many of the amino acids in the framework
region make little or no direct contribution to the specificity or
affinity of an antibody. Thus, many individual conservative
substitutions of framework residues can be tolerated without
appreciable change of the specificity or affinity of the resulting
human immunoglobulin. Thus, in one embodiment the variable
framework region of the human antibody shares at least 85% sequence
identity to a human germline variable framework region sequence or
consensus of such sequences. In another embodiment, the variable
framework region of the human antibody shares at least 90%, 95%,
96%, 97%, 98% or 99% sequence identity to a human germline variable
framework region sequence or consensus of such sequences.
[0139] In addition to simply binding a linear epitope of an HCV E2
protein, a monoclonal antibody may be selected for its retention of
other functional properties of antibodies of the invention, such as
binding to multiple genotypes of HVC E2 and/or binding with an
ultra high affinity such as, for example, a K.sub.D of 10.sup.-9M
or lower.
Characterization of Human Monoclonal Antibodies to HCV
[0140] Human monoclonal antibodies of the invention can be
characterized for binding to HCV using a variety of known
techniques. Generally, the antibodies are initially characterized
by ELISA. Briefly, microtiter plates can be coated with purified
HCV in PBS, and then blocked with irrelevant proteins such as
bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from
HCV-immunized mice are added to each well and incubated for 1-2
hours at 37.degree. C. The plates are washed with PBS/Tween 20 and
then incubated with a goat-anti-human IgG Fc-specific polyclonal
reagent conjugated to alkaline phosphatase for 1 hour at 37.degree.
C. After washing, the plates are developed with ABTS substrate, and
analyzed at OD of 405. Preferably, mice which develop the highest
titers will be used for fusions.
[0141] An ELISA assay as described above can be used to screen for
antibodies and, thus, hybridomas that produce antibodies that show
positive reactivity with the HCV immunogen. Hybridomas that bind,
preferably with high affinity, to HCV can then be subcloned and
further characterized. One clone from each hybridoma, which retains
the reactivity of the parent cell (by ELISA), can then be chosen
for making a cell bank, and for antibody purification.
[0142] To purify human anti-HCV antibodies, selected hybridomas can
be grown in roller bottles, two-liter spinner-flasks or other
culture systems. Supernatants can be filtered and concentrated
before affinity chromatography with protein A-sepharose (Pharmacia,
Piscataway, N.J.) to purify the protein. After buffer exchange to
PBS, the concentration can be determined by OD.sub.280 using 1.43
extinction coefficient or preferably by nephelometric analysis. IgG
can be checked by gel electrophoresis and by antigen specific
method.
[0143] To determine if the selected human anti-HCV monoclonal
antibodies bind to unique epitopes, each antibody can be
biotinylated using commercially available reagents (Pierce,
Rockford, Ill.). Biotinylated MAb binding can be detected with a
streptavidin labeled probe. To determine the isotype of purified
antibodies, isotype ELISAs can be performed using art recognized
techniques. For example, wells of microtiter plates can be coated
with 10 .mu.g/ml of anti-human Ig overnight at 4.degree. C. After
blocking with 5% BSA, the plates are reacted with 10 .mu.g/ml of
monoclonal antibodies or purified isotype controls, at ambient
temperature for two hours. The wells can then be reacted with
either human IgG1 or other human isotype specific conjugated
probes. Plates are developed and analyzed as described above.
[0144] Anti-HCV human IgGs can be further tested for reactivity
with the HCV antigen by Western blotting. Briefly, cell extracts
from cells expressing HCV can be prepared and subjected to sodium
dodecyl sulfate polyacrylamide gel electrophoresis. After
electrophoresis, the separated antigens will be transferred to
nitrocellulose membranes, blocked with 20% mouse serum, and probed
with the monoclonal antibodies to be tested. Human IgG binding can
be detected using anti-human IgG alkaline phosphatase and developed
with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,
Mo.).
II. Production of Transgenic Nonhuman Animals which Generate Human
Monoclonal Anti-HCV Antibodies
[0145] In yet another aspect, the invention provides transgenic
non-human animals, such as transgenic mice, which are capable of
expressing human monoclonal antibodies that specifically bind to
HCV. In a particular embodiment, the invention provides a
transgenic mouse having a genome comprising a human heavy chain
transgene, such that the mouse produces human anti-HCV antibodies
when immunized with HCV antigen and/or cells expressing HCV. The
human heavy chain transgene can be integrated into the chromosomal
DNA of the mouse, as is the case for transgenic, e.g., HuMAb mice,
as described in detail herein and exemplified. Such transgenic mice
are capable of producing multiple isotypes of human monoclonal
antibodies to HCV (e.g., IgG) by undergoing V-D-J recombination and
isotype switching. Isotype switching may occur by, e.g., classical
or non-classical isotype switching.
[0146] The design of a transgenic non-human animal that responds to
foreign antigen stimulation with a heterologous antibody repertoire
requires that the heterologous immunoglobulin transgenes contained
within the transgenic animal function correctly throughout the
pathway of B-cell development. This includes, for example, isotype
switching of the heterologous heavy chain transgene. Accordingly,
transgenes are constructed so as to produce isotype switching and
one or more of the following of antibodies: (1) high level and
cell-type specific expression, (2) functional gene rearrangement,
(3) activation of and response to allelic exclusion, (4) expression
of a sufficient primary repertoire, (5) signal transduction, (6)
somatic hypermutation, and (7) domination of the transgene antibody
locus during the immune response.
[0147] Not all of the foregoing criteria need be met. For example,
in those embodiments wherein the endogenous immunoglobulin loci of
the transgenic animal are functionally disrupted, the transgene
need not activate allelic exclusion. Further, in those embodiments
wherein the transgene comprises a functionally rearranged heavy
and/or light chain immunoglobulin gene, the second criteria of
functional gene rearrangement is unnecessary, at least for that
transgene which is already rearranged. For background on molecular
immunology, see, Fundamental Immunology, 2nd edition (1989), Paul
William E., ed. Raven Press, N.Y.
[0148] In certain embodiments, the transgenic non-human animals
used to generate the human monoclonal antibodies of the invention
contain rearranged, unrearranged or a combination of rearranged and
unrearranged heterologous immunoglobulin heavy and light chain
transgenes in the germline of the transgenic animal. Each of the
heavy chain transgenes comprises at least one C.sub.H gene. In
addition, the heavy chain transgene may contain functional isotype
switch sequences, which are capable of supporting isotype switching
of a heterologous transgene encoding multiple C.sub.H genes in the
B-cells of the transgenic animal. Such switch sequences may be
those which occur naturally in the germline immunoglobulin locus
from the species that serves as the source of the transgene C.sub.H
genes, or such switch sequences may be derived from those which
occur in the species that is to receive the transgene construct
(the transgenic animal). For example, a human transgene construct
that is used to produce a transgenic mouse may produce a higher
frequency of isotype switching events if it incorporates switch
sequences similar to those that occur naturally in the mouse heavy
chain locus, as presumably the mouse switch sequences are optimized
to function with the mouse switch recombinase enzyme system,
whereas the human switch sequences are not. Switch sequences may be
isolated and cloned by conventional cloning methods, or may be
synthesized de novo from overlapping synthetic oligonucleotides
designed on the basis of published sequence information relating to
immunoglobulin switch region sequences (Mills et al., Nucl Acids
Res. 15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642
(1989)). For each of the foregoing transgenic animals, functionally
rearranged heterologous heavy and light chain immunoglobulin
transgenes are found in a significant fraction of the B-cells of
the transgenic animal (at least 10 percent).
[0149] The transgenes used to generate the transgenic animals of
the invention include a heavy chain transgene comprising DNA
encoding at least one variable gene segment, one diversity gene
segment, one joining gene segment and at least one constant region
gene segment. The immunoglobulin light chain transgene comprises
DNA encoding at least one variable gene segment, one joining gene
segment and at least one constant region gene segment. The gene
segments encoding the light and heavy chain gene segments are
heterologous to the transgenic non-human animal in that they are
derived from, or correspond to, DNA encoding immunoglobulin heavy
and light chain gene segments from a species not consisting of the
transgenic non-human animal. In one aspect of the invention, the
transgene is constructed such that the individual gene segments are
unrearranged, i.e., not rearranged so as to encode a functional
immunoglobulin light or heavy chain. Such unrearranged transgenes
support recombination of the V, D, and J gene segments (functional
rearrangement) and preferably support incorporation of all or a
portion of a D region gene segment in the resultant rearranged
immunoglobulin heavy chain within the transgenic non-human animal
when exposed to the HCV antigen.
[0150] In an alternate embodiment, the transgenes comprise an
unrearranged "mini-locus". Such transgenes typically comprise a
substantial portion of the C, D, and J segments as well as a subset
of the V gene segments. In such transgene constructs, the various
regulatory sequences, e.g. promoters, enhancers, class switch
regions, splice-donor and splice-acceptor sequences for RNA
processing, recombination signals and the like, comprise
corresponding sequences derived from the heterologous DNA. Such
regulatory sequences may be incorporated into the transgene from
the same or a related species of the non-human animal used in the
invention. For example, human immunoglobulin gene segments may be
combined in a transgene with a rodent immunoglobulin enhancer
sequence for use in a transgenic mouse. Alternatively, synthetic
regulatory sequences may be incorporated into the transgene,
wherein such synthetic regulatory sequences are not homologous to a
functional DNA sequence that is known to occur naturally in the
genomes of mammals. Synthetic regulatory sequences are designed
according to consensus rules, such as, for example, those
specifying the permissible sequences of a splice-acceptor site or a
promoter/enhancer motif. For example, a minilocus comprises a
portion of the genomic immunoglobulin locus having at least one
internal (i.e., not at a terminus of the portion) deletion of a
non-essential DNA portion (e.g., intervening sequence; intron or
portion thereof) as compared to the naturally-occurring germline Ig
locus.
[0151] In a preferred embodiment of the invention, the transgenic
animal used to generate human antibodies to HCV contains at least
one, typically 2-10, and sometimes 25-50 or more copies of the
transgene described in Example 12 of WO 98/24884 (e.g., pHC1 or
pHC2) bred with an animal containing a single copy of a light chain
transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and
the offspring bred with the J.sub.H deleted animal described in
Example 10 of WO 98/24884. Animals are bred to homozygosity for
each of these three traits. Such animals have the following
genotype: a single copy (per haploid set of chromosomes) of a human
heavy chain unrearranged mini-locus (described in Example 12 of WO
98/24884), a single copy (per haploid set of chromosomes) of a
rearranged human K light chain construct (described in Example 14
of WO 98/24884), and a deletion at each endogenous mouse heavy
chain locus that removes all of the functional J.sub.H segments
(described in Example 10 of WO 98/24884). Such animals are bred
with mice that are homozygous for the deletion of the J.sub.H
segments (Examples 10 of WO 98/24884) to produce offspring that are
homozygous for the J.sub.H deletion and hemizygous for the human
heavy and light chain constructs. The resultant animals are
injected with antigens and used for production of human monoclonal
antibodies against these antigens.
[0152] B cells isolated from such an animal are monospecific with
regard to the human heavy and light chains because they contain
only a single copy of each gene. Furthermore, they will be
monospecific with regards to human or mouse heavy chains because
both endogenous mouse heavy chain gene copies are nonfunctional by
virtue of the deletion spanning the J.sub.H region introduced as
described in Example 9 and 12 of WO 98/24884. Furthermore, a
substantial fraction of the B cells will be monospecific with
regards to the human or mouse light chains because expression of
the single copy of the rearranged human .kappa. light chain gene
will allelically and isotypically exclude the rearrangement of the
endogenous mouse .kappa. and lambda chain genes in a significant
fraction of B-cells.
[0153] Transgenic mice employed in the present invention exhibit
immunoglobulin production with a significant repertoire, ideally
substantially similar to that of a native mouse. Thus, for example,
in embodiments where the endogenous Ig genes have been inactivated,
the total immunoglobulin levels will range from about 0.1 to 10
mg/ml of serum, preferably 0.5 to 5 mg/ml, ideally at least about
1.0 mg/ml. When a transgene capable of effecting a switch to IgG
from IgM has been introduced into the transgenic mouse, the adult
mouse ratio of serum IgG to IgM is preferably about 10:1. The IgG
to IgM ratio will be much lower in the immature mouse. In general,
greater than about 10%, preferably 40 to 80% of the spleen and
lymph node B cells express exclusively human IgG protein.
[0154] The repertoire will ideally approximate that shown in a
native mouse, usually at least about 10% as high, preferably 25 to
50% or more. Generally, at least about a thousand different
immunoglobulins (ideally IgG), preferably 10.sup.4 to 10.sup.6 or
more, will be produced, depending primarily on the number of
different V, J and D regions introduced into the mouse genome.
These immunoglobulins will typically recognize about one-half or
more of highly antigenic proteins, e.g., staphylococcus protein A.
Typically, the immunoglobulins will exhibit an affinity (K.sub.D)
for preselected antigens of below 10.sup.-7 M, such as of below
10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or even lower.
[0155] It may be preferable to generate mice with predetermined
repertoires to limit the selection of V genes represented in the
antibody response to a predetermined antigen type. A heavy chain
transgene having a predetermined repertoire may comprise, for
example, human V.sub.H genes which are preferentially used in
antibody responses to the predetermined antigen type in humans.
Alternatively, some V.sub.H genes may be excluded from a defined
repertoire for various reasons (e.g., have a low likelihood of
encoding high affinity V regions for the predetermined antigen;
have a low propensity to undergo somatic mutation and affinity
sharpening; or are immunogenic to certain humans). Thus, prior to
rearrangement of a transgene containing various heavy or light
chain gene segments, such gene segments may be readily identified,
e.g. by hybridization or DNA sequencing, as being from a species of
organism other than the transgenic animal.
[0156] Transgenic mice as described above can be immunized with,
for example, a purified or enriched preparation of HCV antigen
and/or cells expressing HCV proteins. Alternatively, the transgenic
mice can be immunized with DNA encoding HCV proteins. The mice will
then produce B cells which undergo class-switching via
intratransgene switch recombination (cis-switching) and express
immunoglobulins reactive with HCV. The immunoglobulins can be human
antibodies (also referred to as "human sequence antibodies"),
wherein the heavy and light chain polypeptides are encoded by human
transgene sequences, which may include sequences derived by somatic
mutation and V region recombinatorial joints, as well as
germline-encoded sequences; these human antibodies can be referred
to as being substantially identical to a polypeptide sequence
encoded by a human V.sub.L or V.sub.H gene segment and a human
J.sub.L or D.sub.H and J.sub.H segment, even though other
non-germline sequences may be present as a result of somatic
mutation and differential V-J and V-D-J recombination joints. The
variable regions of each antibody chain are typically at least 80
percent encoded by human germline V, J, and, in the case of heavy
chains, D, gene segments; frequently at least 85 percent of the
variable regions are encoded by human germline sequences present on
the transgene; often 90 or 95 percent or more of the variable
region sequences are encoded by human germline sequences present on
the transgene. However, since non-germline sequences are introduced
by somatic mutation and VJ and VDJ joining, the human sequence
antibodies will frequently have some variable region sequences (and
less frequently constant region sequences) which are not encoded by
human V, D, or J gene segments as found in the human transgene(s)
in the germline of the mice. Typically, such non-germline sequences
(or individual nucleotide positions) will cluster in or near CDRs,
or in regions where somatic mutations are known to cluster.
[0157] Human antibodies which bind to the predetermined antigen can
result from isotype switching, such that human antibodies
comprising a human sequence .gamma. chain (such as .gamma.1,
.gamma.2a, .gamma.2B, or .gamma.3) and a human sequence light chain
(such as kappa) are produced. Such isotype-switched human
antibodies often contain one or more somatic mutation(s), typically
in the variable region and often in or within about 10 residues of
a CDR) as a result of affinity maturation and selection of B cells
by antigen, particularly subsequent to secondary (or subsequent)
antigen challenge. These high affinity human antibodies may have
binding affinities (K.sub.D) of below 10.sup.-7 M, such as of below
10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M or even lower.
[0158] Another aspect of the invention includes B cells derived
from transgenic mice as described herein. The B cells can be used
to generate hybridomas expressing human monoclonal antibodies which
bind with high affinity (e.g., K.sub.D lower than 10.sup.-7 M) to
human HCV. Thus, in another embodiment, the invention provides a
hybridoma which produces a human antibody having an affinity
(K.sub.D) of below 10.sup.-7 M, such as of below 10.sup.-8 M,
10.sup.-9 M or 10.sup.-10 M or even lower when determined by
surface plasmon resonance (SPR) technology in a BIACORE 3000
instrument using recombinant human HCV as the analyte and the
antibody as the ligand for binding human HCV, wherein the antibody
comprises:
[0159] a human sequence light chain composed of (1) a light chain
variable region having a polypeptide sequence which is
substantially identical to a polypeptide sequence encoded by a
human V.sub.L gene segment and a human J.sub.L segment, and (2) a
light chain constant region having a polypeptide sequence which is
substantially identical to a polypeptide sequence encoded by a
human C.sub.L gene segment; and
[0160] a human sequence heavy chain composed of a (1) a heavy chain
variable region having a polypeptide sequence which is
substantially identical to a polypeptide sequence encoded by a
human V.sub.H gene segment, optionally a D region, and a human
J.sub.H segment, and (2) a constant region having a polypeptide
sequence which is substantially identical to a polypeptide sequence
encoded by a human C.sub.H gene segment.
[0161] The development of high affinity human monoclonal antibodies
against HCV can be facilitated by a method for expanding the
repertoire of human variable region gene segments in a transgenic
mouse having a genome comprising an integrated human immunoglobulin
transgene, said method comprising introducing into the genome a V
gene transgene comprising V region gene segments which are not
present in said integrated human immunoglobulin transgene. Often,
the V region transgene is a yeast artificial chromosome comprising
a portion of a human V.sub.H or V.sub.L (V.sub.K) gene segment
array, as may naturally occur in a human genome or as may be
spliced together separately by recombinant methods, which may
include out-of-order or omitted V gene segments. Often at least
five or more functional V gene segments are contained on the YAC.
In this variation, it is possible to make a transgenic mouse
produced by the V repertoire expansion method, wherein the mouse
expresses an immunoglobulin chain comprising a variable region
sequence encoded by a V region gene segment present on the V region
transgene and a C region encoded on the human Ig transgene. By
means of the V repertoire expansion method, transgenic mice having
at least 5 distinct V genes can be generated; as can mice
containing at least about 24 V genes or more. Some V gene segments
may be non-functional (e.g., pseudogenes and the like); these
segments may be retained or may be selectively deleted by
recombinant methods available to the skilled artisan, if
desired.
[0162] Once the mouse germline has been engineered to contain a
functional YAC having an expanded V segment repertoire,
substantially not present in the human Ig transgene containing the
J and C gene segments, the trait can be propagated and bred into
other genetic backgrounds, including backgrounds where the
functional YAC having an expanded V segment repertoire is bred into
a mouse germline having a different human Ig transgene. Multiple
functional YACs having an expanded V segment repertoire may be bred
into a germline to work with a human Ig transgene (or multiple
human Ig transgenes). Although referred to herein as YAC
transgenes, such transgenes when integrated into the genome may
substantially lack yeast sequences, such as sequences required for
autonomous replication in yeast; such sequences may optionally be
removed by genetic engineering (e.g., restriction digestion and
pulsed-field gel electrophoresis or other suitable method) after
replication in yeast is no longer necessary (i.e., prior to
introduction into a mouse ES cell or mouse prozygote). Methods of
propagating the trait of human sequence immunoglobulin expression,
include breeding a transgenic mouse having the human Ig
transgene(s), and optionally also having a functional YAC having an
expanded V segment repertoire. Both V.sub.H and V.sub.L gene
segments may be present on the YAC. The transgenic mouse may be
bred into any background desired by the practitioner, including
backgrounds harboring other human transgenes, including human Ig
transgenes and/or transgenes encoding other human lymphocyte
proteins. The invention also provides a high affinity human
sequence immunoglobulin produced by a transgenic mouse having an
expanded V region repertoire YAC transgene. Although the foregoing
describes a preferred embodiment of the transgenic animal of the
invention, other embodiments are contemplated which have been
classified in four categories:
[0163] I. Transgenic animals containing an unrearranged heavy and
rearranged light immunoglobulin transgene;
[0164] II. Transgenic animals containing an unrearranged heavy and
unrearranged light immunoglobulin transgene;
[0165] III. Transgenic animal containing rearranged heavy and an
unrearranged light immunoglobulin transgene; and
[0166] IV. Transgenic animals containing rearranged heavy and
rearranged light immunoglobulin transgenes.
[0167] Of these categories of transgenic animal, the preferred
order of preference is as follows II>I>III>IV where the
endogenous light chain genes (or at least the K gene) have been
knocked out by homologous recombination (or other method) and
I>II>III>IV where the endogenous light chain genes have
not been knocked out and must be dominated by allelic
exclusion.
III. Antibody Conjugates/Immunotoxins
[0168] In another aspect, the present invention features a human
anti-HCV monoclonal antibody conjugated to a therapeutic moiety,
such as a cytotoxin, a drug (e.g., an immunosuppressant) or a
radioisotope. When conjugated to a cytotoxin, these antibody
conjugates are referred to as "immunotoxins." A cytotoxin or
cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine). An antibody of the present invention
can be conjugated to a radioisotope, e.g., radioactive iodine, to
generate cytotoxic radiopharmaceuticals for treating a HCV-related
disorder, such as a cancer.
[0169] The antibody conjugates of the invention can be used to
modify a given biological response. The therapeutic moiety is not
to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other cytokines or growth factors.
[0170] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
IV. Pharmaceutical Compositions
[0171] In another aspect, the present invention provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of human monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present invention, formulated together
with a pharmaceutically acceptable carrier. In a preferred
embodiment, the compositions include a combination of multiple
(e.g., two or more) isolated human antibodies of the invention.
Preferably, each of the antibodies of the composition binds to a
distinct, pre-selected epitope of HCV.
[0172] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include a
composition of the present invention with at least one or more
additional therapeutic agents, such as anti-inflammatory agents,
DMARDs (disease-modifying anti-rheumatic drugs), immunosuppressive
agents, chemotherapeutics, and psoriasis agents. The pharmaceutical
compositions of the invention can also be administered in
conjunction with radiation therapy. Co-administration with other
antibodies, such as CD4 specific antibodies and IL-2 specific
antibodies, are also encompassed by the invention. Such
combinations with CD4 specific antibodies or IL-2 specific
antibodies are considered particularly useful for treating
autoimmune diseases and transplant rejections.
[0173] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
Preferably, the carrier is suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody, bispecific and
multispecific molecule, may be coated in a material to protect the
compound from the action of acids and other natural conditions that
may inactivate the compound.
[0174] A "pharmaceutically acceptable salt" refers to a salt that
retains the desired biological activity of the parent compound and
does not impart any undesired toxicological effects (see e.g.,
Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of
such salts include acid addition salts and base addition salts.
Acid addition salts include those derived from nontoxic inorganic
acids, such as hydrochloric, nitric, phosphoric, sulfuric,
hydrobromic, hydroiodic, phosphorous and the like, as well as from
nontoxic organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
aromatic acids, aliphatic and aromatic sulfonic acids and the like.
Base addition salts include those derived from alkaline earth
metals, such as sodium, potassium, magnesium, calcium and the like,
as well as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0175] A composition of the present invention can be administered
by a variety of methods known in the art. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. The active compounds can
be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0176] To administer a compound of the invention by certain routes
of administration, it may be necessary to coat the compound with,
or co-administer the compound with, a material to prevent its
inactivation. For example, the compound may be administered to a
subject in an appropriate carrier, for example, liposomes, or a
diluent. Pharmaceutically acceptable diluents include saline and
aqueous buffer solutions. Liposomes include water-in-oil-in-water
CGF emulsions as well as conventional liposomes (Strejan et al.
(1984) J. Neuroimmunol. 7:27).
[0177] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0178] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0179] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0180] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. For example, the human antibodies of the invention may
be administered once or twice weekly by subcutaneous injection or
once or twice monthly by subcutaneous injection.
[0181] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0182] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0183] For the therapeutic compositions, formulations of the
present invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal and/or
parenteral administration. The formulations may conveniently be
presented in unit dosage form and may be prepared by any methods
known in the art of pharmacy. The amount of active ingredient which
can be combined with a carrier material to produce a single dosage
form will vary depending upon the subject being treated, and the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the composition which
produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 0.001 percent to about
ninety percent of active ingredient, preferably from about 0.005
percent to about 70 percent, most preferably from about 0.01
percent to about 30 percent.
[0184] Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate. Dosage forms for the
topical or transdermal administration of compositions of this
invention include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0185] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and
infusion.
[0186] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0187] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0188] When the compounds of the present invention are administered
as pharmaceuticals, to humans and animals, they can be given alone
or as a pharmaceutical composition containing, for example, 0.001
to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of
active ingredient in combination with a pharmaceutically acceptable
carrier.
[0189] Regardless of the route of administration selected, the
compounds of the present invention, which may be used in a suitable
hydrated form, and/or the pharmaceutical compositions of the
present invention, are formulated into pharmaceutically acceptable
dosage forms by conventional methods known to those of skill in the
art.
[0190] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts. A physician or veterinarian having ordinary skill in
the art can readily determine and prescribe the effective amount of
the pharmaceutical composition required. For example, the physician
or veterinarian can start doses of the compounds of the invention
employed in the pharmaceutical composition at levels lower than
that required in order to achieve the desired therapeutic effect
and gradually increase the dosage until the desired effect is
achieved. In general, a suitable daily dose of a compositions of
the invention will be that amount of the compound which is the
lowest dose effective to produce a therapeutic effect. Such an
effective dose will generally depend upon the factors described
above. It is preferred that administration be intravenous,
intramuscular, intraperitoneal, or subcutaneous, preferably
administered proximal to the site of the target. If desired, the
effective daily dose of a therapeutic compositions may be
administered as two, three, four, five, six or more sub-doses
administered separately at appropriate intervals throughout the
day, optionally, in unit dosage forms. While it is possible for a
compound of the present invention to be administered alone, it is
preferable to administer the compound as a pharmaceutical
formulation (composition).
[0191] Therapeutic compositions can be administered with medical
devices known in the art. For example, in a preferred embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices
disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,
5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of
well-known implants and modules useful in the present invention
include: U.S. Pat. No. 4,487,603, which discloses an implantable
micro-infusion pump for dispensing medication at a controlled rate;
U.S. Pat. No. 4,486,194, which discloses a therapeutic device for
administering medicants through the skin; U.S. Pat. No. 4,447,233,
which discloses a medication infusion pump for delivering
medication at a precise infusion rate; U.S. Pat. No. 4,447,224,
which discloses a variable flow implantable infusion apparatus for
continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses
an osmotic drug delivery system having multi-chamber compartments;
and U.S. Pat. No. 4,475,196, which discloses an osmotic drug
delivery system. Many other such implants, delivery systems, and
modules are known to those skilled in the art.
[0192] In certain embodiments, the human monoclonal antibodies of
the invention can be formulated to ensure proper distribution in
vivo. For example, the blood-brain barrier (BBB) excludes many
highly hydrophilic compounds. To ensure that the therapeutic
compounds of the invention cross the BBB (if desired), they can be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties which
are selectively transported into specific cells or organs, thus
enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J.
Clin. Pharmacol. 29:685). Exemplary targeting moieties include
folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et
al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res.
Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS
Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.
39:180); surfactant protein A receptor (Briscoe et al. (1995) Am.
J. Physiol. 1233:134), different species of which may comprise the
formulations of the inventions, as well as components of the
invented molecules; p 120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In
one embodiment of the invention, the therapeutic compounds of the
invention are formulated in liposomes; in a more preferred
embodiment, the liposomes include a targeting moiety. In a most
preferred embodiment, the therapeutic compounds in the liposomes
are delivered by bolus injection to a site proximal to the tumor or
infection. The composition must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and
fungi.
[0193] A "therapeutically effective dosage" for rheumatoid
arthritis preferably will result in an ACR20 Preliminary Definition
of Improvement in the patients, more preferred in an ACR50
Preliminary Definition of Improvement and even more preferred in an
ARCD70 Preliminary Definition of Improvement.
[0194] ACR20 Preliminary Definition of Improvement is defined
as:
.gtoreq.20% improvement in: Tender Joint Count (TCJ) and Swollen
Joint Count (SWJ)
[0195] and .gtoreq.20% improvement in 3 of following 5 assessments:
Patient Pain Assessment (VAS), Patient Global assessment (VAS),
Physician Global Assessment (VAS), Patent Self-Assessed Disability
(HAQ), Acute Phase Reactant (CRP or ESR).
[0196] ACR50 and ACR70 are defined in the same way with .gtoreq.50%
and .gtoreq.70% improvements, respectively. For further details see
Felson et al. in American College of Rheumatology Preliminary
Definition of Improvement in Rheumatoid Arthritis; Arthritis
Rheumatism (1995) 38: 727-735.
[0197] The ability of a compound to inhibit cancer can be evaluated
in an animal model system predictive of efficacy in human tumors.
Alternatively, this property of a composition can be evaluated by
examining the ability of the compound to inhibit, such inhibition
in vitro by assays known to the skilled practitioner. A
therapeutically effective amount of a therapeutic compound can
decrease tumor size, or otherwise ameliorate symptoms in a subject.
One of ordinary skill in the art would be able to determine such
amounts based on such factors as the subject's size, the severity
of the subject's symptoms, and the particular composition or route
of administration selected.
[0198] The ability of the antibodies to treat or prevent cirhossis
can also be evaluated according to methods well known in the
art.
[0199] The composition must be sterile and fluid to the extent that
the composition is deliverable by syringe. In addition to water,
the carrier can be an isotonic buffered saline solution, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyetheylene glycol, and the like), and suitable mixtures thereof.
Proper fluidity can be maintained, for example, by use of coating
such as lecithin, by maintenance of required particle size in the
case of dispersion and by use of surfactants. In many cases, it is
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in
the composition. Long-term absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate or gelatin.
[0200] When the active compound is suitably protected, as described
above, the compound may be orally administered, for example, with
an inert diluent or an assimilable edible carrier.
V. Uses and Methods of the Invention
[0201] Human anti-HCV antibodies to HCV of the present invention
(including derivatives and conjugates of the antibodies) and
compositions containing the antibodies can be used in a variety of
in vitro and in vivo diagnostic and therapeutic applications. For
example, the human monoclonal antibodies of the invention can be
used for treating HCV-mediated disorders, including but not limited
to, liver disorders, such as cirrhosis, hepatocellular carcinoma,
and hepatocarcinogenesis.
[0202] Accordingly, in yet another embodiment, the present
invention provides a method for treating or preventing a disorder
mediated by HCV (e.g., a liver disorder), by administering to a
subject a human antibody of the invention in an amount effective to
treat or prevent the disorder. The antibody can be administered
alone or along with another therapeutic agent, such as alpha-IFN or
a cytotoxin which acts in conjunction with or synergistically with
the antibody to treat or prevent the HCV mediated disease. Other
disorders suitable for treatment using the antibodies of the
invention include HCV-mediated hepatocyte pathologies, HCV-mediated
cirrhosis of the liver, and for HCV-mediated liver cancer.
[0203] Additionally, the antibodies of the invention can be used in
vitro or in vivo to diagnose a variety of diseases mediated by HCV.
Specifically, the antibodies can be used to detect levels of HCV,
or levels of cells which contain HCV. Alternatively, the antibodies
can be used to inhibit or neutralize HCV function which, in turn,
can prevent or ameliorate disease symptoms caused by HCV
function.
[0204] Also within the scope of the present invention are kits
comprising human anti-HCV antibodies of the invention and,
optionally, instructions for use. The kit can further contain one
ore more additional reagents, such as alpha-IFN or one or more
additional human antibodies of the invention (e.g., a human
antibody having a complementary activity which binds to an epitope
of an HCV antigen distinct from the first human antibody).
[0205] Accordingly, patients treated with antibodies of the
invention can be additionally administered (prior to,
simultaneously with, or following administration of a human
antibody of the invention) with another therapeutic agent which
enhances or augments the therapeutic effect of the human
antibodies.
[0206] Other embodiments of the present invention are described in
the following Examples.
[0207] The present invention is further illustrated by the
following examples which should not be construed as further
limiting. The contents of Sequence Listing, figures and all
references, patents and published patent applications cited
throughout this application are expressly incorporated herein by
reference.
EXAMPLES
[0208] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
[0209] Materials and Methods
[0210] Throughout the examples, the following materials and methods
were used unless otherwise stated.
[0211] In general, the practice of the present invention employs,
unless otherwise indicated, conventional techniques of chemistry,
molecular biology, recombinant DNA technology, immunology
(especially, e.g., antibody technology), and standard techniques in
polypeptide preparation. See, e.g., Sambrook, Fritsch and Maniatis,
Molecular Cloning: Cold Spring Harbor Laboratory Press (1989);
Antibody Engineering Protocols (Methods in Molecular Biology), 510,
Paul, S., Humana Pr (1996); Antibody Engineering: A Practical
Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr
(1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L.
Press, Pub. (1999); and Current Protocols in Molecular Biology,
eds. Ausubel et al., John Wiley & Sons (1992). In vitro and in
vivo model systems for assaying HCV biology are described, for
example, in Cell culture models and animal models of viral
hepatitis. Part II: hepatitis C, Lab. Anim. (NY).; 34(2):39-47
(2005) and in The chimpanzee model of hepatitis C virus infections,
ILAR J.; 42(2):117-26 (2001).
[0212] Assembly of Codon Optimized HCV Genotype 1a E1/E2 Expression
Construct
[0213] Briefly, a nucleic acid sequence encoding an E2 genotype 1a,
strain H77 (prototype 1a sequence) protein sequence (Genbank
accession number NC004102) was constructed and engineered into
pCDNA vector. The nucleotide sequence was altered for optimized
codon usage for improved mammalian expression. The sequence was
assembled using overlapping PCR with oligonucleotides purchased
from IDT and cloned into the pCDNA3.1-myc, his expression vector
with HindIII and XbaI in frame with the C-terminal myc and 6
histidine tags. The vector was then sequenced to confirm the
construct was correct.
[0214] The E1/E2 codon optimized 1a H77 sequence with added ends
containing restrictions sites (bold lowercase), additional
overhangs to insure restriction enzyme cutting (underlined
lowercase), and consensus Kozak sequence (bold underlined
lowercase) with ATG initiation codon (bold underlined uppercase)
(see SEQ ID NO: 27 and FIG. 13).
[0215] Assembly of Codon-Optimized HCV Genotype 1a E2-661
Expression Construct to Express Soluble E2 Protein
(pCDNA-1a-CO-E2-661)
[0216] The portion of the E2 protein encoding amino acids 364 to
661 was PCR amplified using pCDNA-1a-CO-E1/E2 as template and
cloned into pCDNA3.1-his expression vector with HindIII and XbaI in
frame with the C-terminal 6 histidine tag. The vector was sequenced
to confirm the construct was correct.
[0217] Expression and Purification of Codon Optimized HCV Genotype
1a E2-661
[0218] 293T human tissue culture cells were transiently transfected
using Lipofectamine 2000 (Invitrogen). Supernatant from the cells
containing secreted proteins was collected and 1a E2-661 was
purified from the supernatant using nickel affinity chromatography.
The protein concentration was determined based on OD 280 nm and
further evaluated by Coomassie stained SDS-PAGE and Western blot
with commercially available E2 mouse antibody from Biodesign
International
[0219] Assembly of Wildtype HCV Genotype 1a E1/E2 Expression Vector
(pCDNA-1a-E1/E2)
[0220] The E1/E2 of HCV1a H77 encoding sequence (amino acids
165-746) was PCR amplified with primers to introduce HindIII and
XbaI cloning sites, a consensus Kozak sequence and an initiation
codon, and was then cloned into the pCDNA3.1-his expression vector
with HindIII and XbaI in frame with the C-terminal 6 histidine tag.
The vector was sequenced to confirm the construct was correct.
[0221] Assembly of Wildtype HCV Genotype 1a E2-660 Expression
Vector (pCDNA-1a-E2-660)
[0222] The portion of E2 protein encoding amino acids 364 to 660
was PCR amplified using pCDNA-1a-E1/E2 as template and cloned into
pCDNA3.1-his expression vector with HindIII and XbaI in frame with
the C-terminal 6 histidine tag. The vector was sequenced to confirm
the construct was correct.
[0223] Assembly of HCV Genotype 1b, 2b, 3a and 4a E1/E2 Expression
Vectors (pCDNA-E1/E2)
[0224] High titer genotype 1b, 2b, 3a and 4a HCV positive patient
serum was obtained and RNA was isolated from the serum. RT-PCR was
performed on the isolated RNA with genotype specific primers
complimentary to the end of the core gene and for the p7 gene that
flanks the E1/E2 genes. PCR was used to amplify the E1/E2 encoding
sequence (amino acids 165-746) using primers to introduce HindIII
and XbaI cloning sites, a consensus Kozak sequence and an
initiation codon. The sequence was cloned into pCDNA3.1-his
expression vector with HindIII and XbaI in frame with the
C-terminal 6 histidine tag and then sequenced. A blast search
confirmed that the sequences were the expected genotype, but the
sequences had no exact match in the database.
[0225] Assembly of HCV Genotype 1b E2-661 Expression Vector
(pCDNA-1b-E2-661)
[0226] PCR was used to amplify the portion of E2 protein encoding
amino acids 364 to 661 using pCDNA-1b-E1/E2 as template. The
sequence was then cloned into pCDNA3.1-his expression vector with
HindIII and XbaI in frame with the C-terminal 6 histidine tag. The
vector was then sequenced to confirm the construct was correct.
Expression and purification was carried out essentially as
described above for 1a E2-661.
[0227] Assembly of HCV Genotype 1a E2 HVR1 Deletion Expression
Vector
[0228] In order to generate an E1/E2 encoding construct with a
deletion of the nucleotides encoding the hypervariable region 1
(HVR1) of E2, E1 was PCR amplified using a 3' primer containing an
overhang for a region of E2 beginning with amino acids 411 (skipped
over amino acids 384 to 410--HVR1) using pCDNA-1a-E1/E2 as
template. In a separate reaction, the E2 region encoding amino
acids 411-746 was PCR amplified using a 5' primer containing an
overhang for the 3' end of E1 using pCDNA-1a-E1/E2 as template. The
E1 and E2 PCR products that contained complementary overhangs at
the E1-E2 junction without the region encoding the HVR1 for E2 were
mixed, and then PCR amplified using a primer specific for the 5'
end of E1 with a HindIII site and 3' end of E2 with an XbaI site.
The sequence was cloned into pCDNA3.1-his expression vector with
HindIII and XbaI in frame with the C-terminal 6 histidine tag. The
vector was then sequenced to confirm that the construct was
correct.
[0229] Assembly of Codon Optimized HCV Genotype 1a E2 Trunction
Expression Vectors
[0230] Truncations for mammalian expression were engineered in
which amino acids from the C-terminus of E2 were removed. The
portion of E2 protein encoding desired amino acids was PCR
amplified using pCDNA-1a-CO-E1/E2 as template and cloned into
pCDNA3.1-his expression vector with HindIII and XbaI in frame with
the C-terminal 6 histidine tag. The vector was then sequenced to
confirm that the construct was correct. The following constructs
were made: [0231] pCDNA-1a-CO-E2-624 (E2 amino acids 384 to 624)
[0232] pCDNA-1a-CO-E2-584 (E2 amino acids 384 to 584) [0233]
pCDNA-1a-CO-E2-544 (E2 amino acids 384 to 544) [0234]
pCDNA-1a-CO-E2-504 (E2 amino acids 384 to 504) [0235]
pCDNA-1a-CO-E2-464 (E2 amino acids 384 to 464) Expression and
purification from each construct was carried out essentially as
described above for 1a E2-661.
[0236] Lectin ELISA
[0237] Lectin capture ELISA was carried out with the E2 truncations
to determine antibody reactivity. Briefly, 96-well plates were
coated with Galanthus nivalis (GNA) lectin. E2 truncations from the
supernatants of transiently transfected 293 cells were added to the
96-well plates coated with GNA lectin in order to capture the
protein. Hybridoma supernatant was then added to the 96 well plates
to determine protein reactivity. Bound antibody was detected with
anti-human alkaline phosphatase secondary antibody and PNPP
substrate.
[0238] Assembly of Bacterial Expression Constructs Spanning Amino
Acids 384 to 464 of HCV Genotype 1a E2 Glycoprotein
[0239] Next, fusion proteins were engineered with N-terminal
thioredoxin fusion for bacterial expression in order to allow
production of small portions of E2.
[0240] For the first set of proteins, the portion of E2 protein
encoding the desired amino acids was PCR amplified using
pCDNA-1a-E1/E2 as template and cloned into pET32 expression vector
with EcoRI and HindIII in frame with the C-terminal myc and 6
histidine tags. The vector was then sequenced to confirm that the
construct was correct. The following constructs were made: [0241]
pET32-E2-A (amino acids 384 to 463) [0242] pET32-E2-B (amino acids
411 to 463) [0243] pET32-E2-C (amino acids 432 to 463) [0244]
pET32-E2-D (amino acids 436 to 463) [0245] pET32-E2-E (amino acids
384 to 431) The vectors were transformed into BL21-DE3 E. coli
bacteria (Invitrogen) and expression was induced with IPTG.
Bacteria were lysed and the proteins of interest were purified with
nickel affinity chromatography. Protein concentration was
determined based on OD 280 nm and further evaluated by Coomassie
stained SDS-PAGE and Western blot using mouse antibody specific for
myc and histidine tags.
[0246] For the second set of proteins, complementary phosphorylated
oligonucleotides were prepared that, when annealed, would have
EcoRI and HindIII overhangs and encode the desired amino acids. The
DNA was cloned into pET32 expression vector with EcoRI and HindIII
in frame with the C-terminal myc and 6 histidine tags. The vector
was then sequenced to confirm that the construct was correct. The
following constructs were made: [0247] pET32-E2-G (amino acids 412
to 423) [0248] pET32-E2-H (amino acids 432 to 443) [0249]
pET32-E2-I (amino acids 436 to 447) Expression and purification was
carried out as described above for the first set of proteins.
[0250] Generation of Alanine Scanning Mutants of HCV E2-412-423
Epitope
[0251] Each amino acid in the 412-423 epitope was separately
mutated to an alanine in the bacterial expression construct to
determine the important amino acid contacts for the antibodies.
Complementary phosphorylated oligonucleotides were prepared that,
when annealed, would have EcoRI and HindIII overhangs and encode
the E2 412-423 region with the desired changes. The DNA was cloned
into the pET32 expression vector with EcoRI and HindIII in frame
with the C-terminal myc and 6 histidine tags. The vector was then
sequenced to confirm that the construct was correct.
The following constructs were made: [0252] pET32-E2-G-Q412A (amino
acid 412 from a Q to an A) [0253] pET32-E2-G-L413A [0254]
pET32-E2-G-I414A [0255] pET32-E2-G-N415A [0256] pET32-E2-G-T416A
[0257] pET32-E2-G-N417A [0258] pET32-E2-G-G418A [0259]
pET32-E2-G-S419A [0260] pET32-E2-G-W420A [0261] pET32-E2-G-H421A
[0262] pET32-E2-G-I422A [0263] pET32-E2-G-N423A Expression and
purification was carried out as described above for the other
bacterial proteins.
[0264] Pseudovirus Generation and Neutralization Assay
[0265] A pseudovirus was generated having an HIV backbone that
contained a mutation to prevent HIV envelope glycoprotein
expression and a luciferase gene to direct luciferase expression in
target cells (pNL4-3.Luc.R-E-). HCV E1/E2 glycoproteins were
provided in trans by cotransfection of 293 cells with pcDNA E1/E2
derived from the various genotypes (1a, 1b, 2b, 3a and 4a) with
pNL4-3.Luc.R-E-. Supernatant containing secreted virus particles
was harvested from cells from 48 to 72 hrs post-transfection and
concentrated using Centricon 70 concentrators and/or
ultracentrifugation. Pseudovirus was then used to infect Hep3B
cells (ATCC) in the presence or absence of antibody. Pseudovirus
was preincubated with antibody for 1 hr at room temperature before
adding to Hep3B cells. After incubation for 72 hrs, infection was
quantitated by luciferase detection with BrightGlo luciferase assay
(Promega) and read in a Victor3 plate reader (Perkin Elmer)
[0266] Biacore Affinity Determination
[0267] Goat-anti-human IgG (Fc) was amide coupled to a CM5 sensor
chip from Biacore in flow cell 1 and 2. Human antibody (95-2 or
83-128) was captured in flow cell 2 only using HBS-P buffer
(Biacore). E2-G was flowed over both flow cell 1 (for background)
and flow cell 2 (for specific interaction with human antibody) at
125, 62.5, 31.3, and 15.6 nM in HBS-P buffer at 50 .mu.l/min for
200 sec. After injection was stopped, buffer was flowed over both
flow cells to collect dissociation data. Data was evaluated and
curves fitted to determine the affinity constant using the
BIAevaluation software
[0268] Mouse Immunizations
[0269] Mice transgenic for human IgG genes (HuMab mice) from
Medarex were immunized with soluble E2 protein to generate an
immune response. Mouse 85083 was immunized with 1a E2-661 with
Freunds' adjuvant for 1 injection and MPL+TDM adjuvant (Sigma) for
14 injections. Mouse 97895 was immunized with 1a E2-661 with
MPL+TDM+CWS adjuvant (Sigma) for 1 injection and MPL+TDM adjuvant
for 5 injections
[0270] Splenic Fusions and Hybridoma Selection
[0271] Mouse spleens were removed and spleen cells were isolated.
Spleen cells were fused to mouse myeloma cells following a standard
PEG fusion protocol to generate hybridomas. Hybridoma supernatants
were then screened for production of antibody reactive to 1a E2-661
by ELISA and positive cells were moved forward for further
characterization.
[0272] Isolation and Sequencing of Hybridoma Antibody Genes
[0273] RNA was isolated from hybridoma cells using a Qiagen RNeasy
kit. RT-PCR was performed for the heavy chain variable region with
gene-specific primers containing restrictions sites, the resulting
sequence was cloned and the construct was sequenced. RACE was
performed for the light chain variable region with gene-specific
primers. The sequence was cloned into the TOPO vector and the
vector was sequenced. Gene specific primers were designed and used
to PCR amplify and add restriction sites.
Example 1
Generation of Anti-HCV Monoclonal Antibodies
[0274] Mouse Immunizations, Hybridoma Production and Selection
[0275] Transgenic mice comprising human immunoglobulin genes
generated as described above in the section entitled "Generation of
Human Monoclonal Antibodies in HuMab Mice" and supplied by Medarex,
Milpitas, Calif., were immunized with a soluble version of the HCV
genotype 1a E2 envelope glycoprotein. The antigen was administered
in combination with Freund's complete or RMI adjuvant. Mouse sera
responses were monitored by enzyme linked immunosorbent assay
(ELISA) to soluble E2 protein (1a E2-661). Splenic B cells were
isolated from the immunized animal and fused to mouse myeloma
(P3X-AG8.653) cells using standard spleen cells fusion methods.
Clonal hybridomas were generated and screened using ELISA for
production of antibody reactive to E2-661. Positive hybridomas were
tested for equivalent reactivity to HCV genotype 1a and genotype 1b
E2-661 by ELISA with decreasing amounts of hybridoma supernatant.
Hybridomas which exhibited equivalent reactivity to genotypes 1a
E2-661 and 1b E2-661 were tested for capacity to neutralize HCV
pseudovirus infectivity of Hep3B cells. This selection process
yielded 20 hybridomas that neutralized HCV pseudovirus and had
equivalent reactivity to 1a and 1b E2 soluble protein by ELISA.
Specifically, nineteen (19) hybridomas were from mouse #97895 and
produced IgG1 antibody as follows: 95-2; 95-14; 95-15; 95-18;
95-20; 95-21; 95-25; 95-26; 95-30; 95-38; 95-39; 95-42; 95-43;
95-48; 95-49; 95-52; 95-54; 95-58; and 95-62. One (1) hybridoma,
83-128, that neutralized HCV pseudovirus and had equivalent
reactivity to 1a and 1b E2 soluble protein by ELISA was also
identified from mouse #85083 and produced IgG3 antibody. One (1)
hybridoma, 073-1, that neutralized HCV pseudovirus and had
equivalent reactivity to 1a and 1b E2 soluble protein by ELISA was
also identified from mouse #113073 and produced IgG3 antibody.
These twentyone (21) hybridomas were further characterized.
[0276] Antibody Sequencing and Cloning
[0277] The heavy and light chain variable sequences of the human
antibodies 83-128 (from the hybridoma from mouse #85083) and 073-1
(from the hybridoma from mouse #113073) were sequenced and cloned
into vectors containing the heavy and light chain constant regions
for human IgG1/kappa antibody. This changed human antibodies 83-128
and 073-1 from an IgG3 antibody to an IgG1 antibody.
[0278] The heavy chain variable sequences of the human antibodies
from the nineteen (19) hybridomas from mouse #97895 were sequenced
and compared. All nineteen (19) antibodies were found to share
substantial sequence identity (i.e., >96% identical). Three (3)
hybridomas, 95-2, 95-14, and 95-38, were chosen based on
neutralization assay data and their light chain variable region
sequences were determined. The heavy and light chain variable
region sequences of human antibodies 95-2, 95-14, and 95-38 were
cloned into vectors containing the heavy and light chain constant
regions for human IgG1/kappa antibody. Antibody was expressed by
transient transfection, purified, and directly compared in a HCV
pseudovirus neutralization assay. Human antibody 95-2 from mouse
#97895 was selected to be further characterized.
[0279] The amino acid sequences of the heavy chain variable region
of antibodies produced by clones 95-14, 95-49, 95-62, 95-42, 95-58,
95-25, 95-43, 95-54, 95-2, 95-38, 83-128, 073-1 is set forth in SEQ
ID NOs:32, 37, 40, 35, 39, 34, 36, 38, 3, 33, 1 and 5 respectively.
The amino acid sequences of the light chain variable region of
antibodies produced by clones 95-2, 95-14, 95-38, 073-1 and 83-128
is set forth in SEQ ID NOs:4, 44, 53, 6 and 2, respectively.
[0280] The nucleotide sequences encoding the heavy chain variable
region of antibodies produced by clones 95-2, 95-14, 95-38, 073-1
and 83-128 is set forth in SEQ ID NOs:27, 48, 57, 29 and 25,
respectively; and the nucleotide sequences encoding the light chain
variable region of antibodies produced by clones 95-2, 95-14,
95-38, 073-1 and 83-128 is set forth in SEQ ID NOs:28, 49, 58, 30
and 26, respectively.
Example 2
Antibody Characterization
[0281] HCV Genotype 1a and 1b--Soluble E2 ELISA
[0282] The reactivity of human antibody 95-2 (FIG. 1A) and human
antibody 83-128 (FIG. 1B) to soluble HCV genotype 1a E2-660
(triangles) and soluble HCV genotype 1b E2-661 (squares) was
compared. ELISA plates were coated with 2 .mu.g/ml of antigen and
probed with antibody in two-fold dilutions. Bound antibody was
detected with goat-anti-human secondary antibody conjugated to
alkaline phosphatase and PNPP substrate. Both human antibodies,
95-2 and 83-128 were found to react equivalently to both genotypes
(see FIG. 1). These experiments were also performed with the 95-14,
95-38 and 073-1 antibodies and all three recognized genotype 1a and
1b equivalently (data not shown).
[0283] Neutralization of HCV Pseudovirus from Multiple
Genotypes
[0284] The capacity of human antibodies 95-2 and 83-128 to
neutralize both multiple genotypes of HCV pseudovirus was
determined using Hep3B cells. Five-fold dilutions of antibody were
incubated with HCV pseudovirus for one hour at room temperature.
The virus-antibody mixture was added to Hep3B cells followed by
incubation at 37.degree. C. for 72 hours. Infection was quantitated
with Brightglo luciferase assay and read in a Victor3 plate reader
for light output. An isotype-matched irrelevant human antibody was
used as a negative control. Both human antibodies, 95-2 and 83-128,
were found to neutralize HCV genotypes 1a, 1b, 2b, 3a and 4a
pseudovirus (see FIGS. 2A, 2B, 2C, 2D and 2E, respectively).
Antibody 073-1 was also shown to neutralize all of the above listed
pseudovirus (data not shown).
[0285] Reducing and Non-Reducing Western Blots of Genotype 1a and
1b E2 Soluble Protein
[0286] The reactivity of human antibodies 95-2 and 83-128 to
soluble E2 protein (E2-661) from HCV genotype 1a and 1b subjected
to reducing or non-reducing SDS-PAGE was analyzed. Westerns blots
were performed with anti-his tag monoclonal (his), 83-128 and 95-2
using anti-mouse IgG (his) or anti-human IgG (95-2 and 83-128)
conjugated to HRP with enhanced chemiluminescent detection reagent.
Both human antibodies 95-2 and 83-128 were found to recognize
E2-661 subjected to denaturing, reducing gel followed by transfer
to PVDF membrane (see FIG. 3).
[0287] Binding Affinity of Human Antibodies 95-2 and 83-128 for E2
412-423 Epitope Expressed as a Bacterial Fusion Protein
[0288] Binding affinity of human antibodies 95-2 and 83-128 for E2
412-423 epitope expressed as a bacterial fusion protein was
determined and summarized in FIG. 4. Goat anti-human IgG Fc was
amide coupled to the Biacore chip. Human antibodies 95-2 and 83-128
were separately captured on the chip and E2-G bacterially expressed
protein containing E2 412-423 amino acids was flowed over at
varying concentrations. The BIAevaluation.TM. software was used to
fit the curves and calculate the affinity constants.
Example 3
Epitope Determination
[0289] Determination of which Region of the E2 Protein Human
Antibodies 83-128 and 95-2 Recognize
[0290] To determine which region of the E2 protein human antibodies
83-128 and 95-2 recognize, carboxy-terminal truncations of
mammalian expressed E2 protein were captured by lectin ELISA and
probed with 83-128 and 95-2 (see FIG. 5 for map of constructs and
FIG. 6 for ELISA data). Based on the data, the epitope for these
antibodies is within amino acids 412-464 of E2. As expected, all
antibodies (95-14, 95-49, 95-62, 95-42, 95-58, 95-25, 95-43, 95-54,
95-2, 95-38, and 073-1) also mapped to this region of the E2
protein.
[0291] To further define the epitope, bacterially expressed fusion
protein was used because the smaller pieces of E2 did not express
well in the mammalian system. The purified proteins were coated on
an ELISA plate and probed with human antibodies 83-128 and 95-2
(see FIG. 7 for map of constructs and FIG. 8 for ELISA data). Both
human antibodies 83-128 and 95-2 recognized all constructs
containing E2 amino acids 412-423, thus, identifying amino acids
412-423 as the epitope. This experiment was also performed using
the 073-1 antibody and it also recognized the 412-423 epitope (data
not shown).
[0292] To determine the important contacts within the 412-423
epitope, alanine scanning mutagenesis was used to individually
change each amino acid in the 412-423 epitope to an alanine in the
E2-G bacterial fusion protein. The purified proteins were coated on
an ELISA plate and probed with both human antibodies (83-128 and
95-2). An anti-his monoclonal antibody specific for the (His).sub.6
epitope tag on the fusion proteins was used to control for coating
of the plate (see FIG. 9). Based on the data, residues 413 and 420
were found to be critical for binding for human antibody 95-2;
residues 413, 418 and 420 were found to be critical for binding for
human antibody 83-128.
[0293] To determine the frequency of naturally occurring changes
within the 412-423 epitope, this region of E2 was analyzed for
sequences reported in the Los Alamos HCV sequence database that
were condensed by manual optimization with only complete sequences
and similar sequences reduced to one sequence. Based on the
alignment (FIG. 10), 77.7% of the sequences in the condensed
database are identical to the sequences in this region of the 1a
and 1b expressed E2 proteins used in the present examples.
Accordingly, human antibodies 95-2 and 83-128 should recognize
these proteins.
[0294] To determine if some of the reported sequence differences in
this region will affect human antibody 95-2 and 83-128 recognition,
sequence changes were engineered into the E2-G bacterially
expressed fusion protein containing the 412-423 epitope (FIG. 11).
A sequence from a genotype 5 strain reported in Tan et al. (2006)
Hepatology (genbank accession number AY785283) was included that
was not in the database. The purified proteins were coated on an
ELISA plate that was probed with both antibodies (83-128 and 95-2).
An anti-his monoclonal antibody specific for the his tag on the
fusion proteins was used to control for coating of the plate (FIG.
12). Human antibody 95-2 recognized all constructs, with the
exception of the genotype 6a, with a 10-fold reduction of this
specific genotype 5 sequence. Human antibody 83-128 recognized all
constructs, except the those derived from specific genotype 5 and
6a sequence, and had weak recognition of 5a.
Example 4
In Vitro Model
[0295] Human antibodies of the present invention are tested in in
vitro models for antiviral activity and for the ability to inhibit
HCV infection. Numerous in vitro models for HCV infection are known
in the art and are used to test the human antibodies of the present
invention, including, e.g., (i) infection of primary hepatocytes
with HCV; (ii) stable transfection of hepatocyte cell lines; and
(iii) transfection with full-length or subgenomic replicons, as
described, e.g., in C. Guha et al. ("Cell culture models and animal
models of viral hepatitis. Part II: hepatitis C" Lab Animal (NY)
2005, 34(2):39-47). Cultured cells in these in vitro models are
exposed to human antibodies of the present invention, or to
appropriate control antibodies, and the effect of the antibodies on
HCV infection and viral activity are monitored by using standard
techniques known in the art, e.g., by detecting HCV viral RNA,
e.g., by using quantitative, real-time reverse
transcription/polymerase chain reaction (RT/PCR).
Example 5
In Vivo Animal Model
[0296] Human antibodies of the present invention are tested in an
in vivo animal model for the ability to inhibit hepatitis C viral
infection. In particular, the human antibodies of the present
invention are tested in a chimpanzee animal model as described in
R. E. Lanford et al. (ILAR J. 2001; 42(2):117-26). As described in
Lanford et al., the chimpanzee (Pan troglodytes) is the only
experimental animal susceptible to infection with hepatitis C virus
(HCV) and was instrumental in the initial studies on non-A, non-B
hepatitis, including observations on the clinical course of
infection, determination of the physical properties of the virus,
and eventual cloning of the HCV nucleic acid. The chimpanzee model
has been critical for the analysis of early events in HCV infection
because it represents a population for which samples are available
from the time of exposure and all exposed animals are examined. For
this reason, the chimpanzee represents a truly nonselected
population. In contrast, human cohorts are often selected for
disease status or antibody reactivity and typically include
individuals that have been infected for decades. The chimpanzee
model is essential to an improved understanding of the factors
involved in viral clearance, analysis of the immune response to
infection, and the development of vaccines. The development of
infectious cDNA clones of HCV was dependent on the use of
chimpanzees, and they will continue to be needed in the use of
reverse genetics to evaluate critical sequences for viral
replication. In addition, chimpanzees have been used in conjunction
with DNA microarray technology to probe the entire spectrum of
changes in liver gene expression during the course of HCV
infection. The chimpanzee will continue to provide a critical
aspect to the understanding of HCV disease and the development of
therapeutic modalities.
[0297] Accordingly, chimpanzees are inoculated with HCV and are
additionally administered a human antibody of the present
invention. Control animals are inoculated with HCV and are either
not administered a human antibody of the present invention or are
administered an appropriate control antibody. Human antibodies of
the invention are administered to the chimpanzees either prior to
HCV inoculation, at the time of HCV inoculation or subsequent to
HCV inoculation. The effects of the human antibody on HCV
infection, progression and clearance are then monitored by using
assays standard in the art. For example, HCV infection and/or
clearance can be monitored by: (i) measuring increases in serum
alanine amino transferase (ALT) levels, indicating liver damage;
(ii) detecting HCV viral RNA in serum by, e.g., quantitative,
real-time reverse transcription/polymerase chain reaction (RT/PCR);
and (iii) evaluation of anti-HCV antibody response, e.g., by
enzyme-linked immunosorbent and recombinant immunoblot assays.
These assays are known to those or ordinary skill in the art and
are carried out, for example, as described in Lanford et al.
Additionally, in experiments similar to those described above, the
effects of the antibodies of the invention on the response to the
HCV E1 and/or E2 proteins are evaluated, e.g., as described in
Lanford et al. Downmodulation of HCV infection, a decrease in HCV
progression or an increase in HCV clearance in the animals exposed
to a human antibody of the invention, as compared to control
animals, is indicative of the effectiveness of the antibody to
prevent or treat HCV infection.
[0298] Additionally, DNA microarrays are used to monitor the
effects of the human antibodies of the invention on the progressive
changes in HCV infection in the chimpanzee by monitoring changes in
gene expression in the liver of infected animals at multiple time
points during the acute phase of infection and through viral
clearance. DNA microarray analysis is conducted on liver tissue
obtained before infection and at multiple time points throughout
infection and clearance, e.g., up to week 16 after infection, in
animals exposed to the antibody and in control animals not exposed
to antibody. These studies may be conducted, for example, with the
Affymetrix Human FL DNA microarray, which contains oligonucleotides
representative of approximately 6800 genes, essentially as
described in Lanford et al.
Example 6
Production of Anti-HCV Antibodies for Administration in Humans
[0299] Human antibodies of the present invention can be cloned and
recombinantly expressed to facilitate or increase their production
using known techniques.
[0300] Nucleic acid sequences encoding the variable heavy chain and
light chains of an antibody clone of the invention can be cloned
into a pIE-Ugamma1F vector using standard recombinant DNA
methodology. The vector is amplified in E. coli, purified, and
transfected into CHO cells. Transfected cells are plated at
4.times.10.sup.5 cells per well in a 96-well dish and selected for
vector transfection with G418. Resistant clones selected by G418
resistance, are then assayed along with other transfectomas for
production of IgG. The expression of an antibody can be amplified
by growth in the presence of increasing concentrations of
methotrexate. A culture capable of growth in 175 nM methotrexate is
chosen for cloning single cells for further development. Plating
the culture in 96 well plates at low density allowed generation of
cultures arising from a single cell or clones. The cultures are
screened for production of human IgG, and the cell that produces
the highest level of IgG is typically selected for further use. The
methotrexate-amplified clone is expanded to produce a cell bank
including multiple frozen vials of cells. Alternatively, glutamine
synthetase (GS) vectors can be used with cell selection achieved
using, e.g., methionine sulphoximine (see, e.g., U.S. Pat. Nos.
5,827,739; 5,122,464; 5,879,936; and 5,891,693).
[0301] To prepare antibodies from transfected cells, cells from a
clone isolated in the previous steps are cultured and expanded as
inoculum for a bioreactor. The bioreactor typically holds a 500
liter volume of culture medium. The cells are cultured in the
bioreactor until cell viability drops, which indicates a maximal
antibody concentration has been produced in the culture. The cells
are removed by filtration. The filtrate is applied to a protein A
column. Antibodies bind to the column, and are eluted with a low pH
wash. Next, the antibodies are applied to a Q-Sepharose column to
remove residual contaminants, such as CHO cell proteins, DNA, and
other contaminants (e.g., viral contaminants, if present).
Antibodies are eluted from the Q-Sepharose column, nano-filtered,
concentrated, and washed in a buffer such as PBS. The preparation
is then aseptically aliquoted into vials for administration.
EQUIVALENTS
[0302] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. Any combination of the embodiments disclosed in
the dependent claims are contemplated to be within the scope of the
invention.
INCORPORATION BY REFERENCE
[0303] All publications, patents, and pending patent applications
referred to herein are hereby incorporated by reference in their
entirety.
TABLE-US-00001 SUMMARY OF SEQUENCE LISTING SEQ ID NO Type
Description of Sequence 1 AA VH of clone 83-128 2 AA VL of clone
83-128 3 AA VH of clone 95-2 4 AA VL of clone 95-2 5 AA VH of clone
073-1 6 AA VL of clone 073-1 7 AA CDR1 of VH of clone 83-128 8 AA
CDR2 of VH of clone 83-128 9 AA CDR3 of VH of clone 83-128 10 AA
CDR1 of VH of clone 95-2 11 AA CDR2 of VH of clone 95-2 12 AA CDR3
of VH of clone 95-2 13 AA CDR1 of VH of clone 073-1 14 AA CDR2 of
VH of clone 073-1 15 AA CDR3 of VH of clone 073-1 16 AA CDR1 of VL
of clone 83-128 17 AA CDR2 of VL of clone 83-128 18 AA CDR3 of VL
of clone 83-128 19 AA CDR1 of VL of clone 95-2 20 AA CDR2 of VL of
clone 95-2 21 AA CDR3 of VL of clone 95-2 22 AA CDR1 of VL of clone
073-1 23 AA CDR2 of VL of clone 073-1 24 AA CDR3 of VL of clone
073-1 25 NA VH of clone 83-128 26 NA VL of clone 83-128 27 NA VH of
clone 95-2 28 NA VL of clone 95-2 29 NA VH of clone 073-1 30 NA VL
of clone 073-1 31 NA E1/E2 1a H77 (FIG. 13) 32 AA VH of clone 95-14
(FIG. 14) 33 AA VH of clone 95-38 (FIG. 14) 34 AA VH of clone 95-25
(FIG. 14) 35 AA VH of clone 95-42 (FIG. 14) 36 AA VH of clone 95-43
(FIG. 14) 37 AA VH of clone 95-49 (FIG. 14) 38 AA VH of clone 95-54
(FIG. 14) 39 AA VH of clone 95-58 (FIG. 14) 40 AA VH of clone 95-62
(FIG. 14) 41 AA CDR1 of VH of clone 95-14 42 AA CDR2 of VH of clone
95-14 43 AA CDR3 of VH of clone 95-14 44 AA VL of clone 95-14 45 AA
CDR1 of VL of clone 95-14 46 AA CDR2 of VL of clone 95-14 47 AA
CDR3 of VL of clone 95-14 48 NA VH of clone 95-14 49 NA VL of clone
95-14 50 AA CDR1 of VH of clone 95-38 51 AA CDR2 of VH of clone
95-38 52 AA CDR3 of VH of clone 95-38 53 AA VL of clone 95-38 54 AA
CDR1 of VL of clone 95-38 55 AA CDR2 of VL of clone 95-38 56 AA
CDR3 of VL of clone 95-38 57 NA VH of clone 95-38 58 NA VL of clone
95-38 59 AA E2 412-423 (1a) QLINTNGSWHIN (FIG. 10) 60 AA (1b)
QLVNTNGSWHIN (FIG. 10) 61 AA QLVNSNGSWHVN (FIG. 10) 62 AA
QLINSNGSWHIN (FIG. 10) 63 AA HLINTNGSWHIN (FIG. 10) 64 AA
QLIKTNGSWHIN (FIG. 10) 65 AA QLVNTNGSWHVN (FIG. 10) 66 AA
QFVNTNGSWHIN (FIG. 10) 67 AA QLIKNGSSWHIN (FIG. 10) 68 AA
QLVKTNGSWHIN (FIG. 10) 69 AA HLVNTNGSWHIN (FIG. 10) 70 AA
HLVNSNGSWHIN (FIG. 10) 71 AA QLIHTNGSWHIN (FIG. 10) 72 AA
QLVKTEGNWHIN (FIG. 10) 73 AA NLIKTNGSWHIN (FIG. 10) 74 AA
QLIYTNGSWHIN (FIG. 10) 75 AA QLINTNGSWHLN (FIG. 10) 76 AA
YLINTNGSWHIN (FIG. 10) 77 AA SLINTNGSWHIN (FIG. 10) 78 AA
NLINTNGSWHIN (FIG. 10) 79 AA HLVNSNGSWHIN genotype 2b (FIG. 11) 80
AA QLVNSSGSWHIN genotype 3a (FIG. 11) 81 AA QLINSNGSWHIN genotype
4a (FIG. 11) 82 AA QLIQNGSSWHIN genotype 5 (FIG. 11) 83 AA
QFVNTNGSWHIN genotype 5a (FIG. 11) 84 AA QLIKNGSSWHIN genotype 6a
(FIG. 11) 85 AA QLIKTNGSWHIN genotype 6g (FIG. 11) 86 AA
QLINSNGSWHVN genotype 6k (FIG. 11) 87 AA VH CDR1 consensus sequence
88 AA VH CDR2 consensus sequence 89 AA VH CDR3 consensus sequence
90 AA VL CDR1 consensus sequence 91 AA VL CDR2 consensus sequence
92 AA VL CDR3 consensus sequence
Sequence CWU 1
1
931123PRTArtificial SequenceSynthetic Construct 1Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30
Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Leu 35
40 45 Ala Val Ile Trp Phe Asp Glu Asn Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Ser Leu Val Arg Asp
Ala Phe Ile Tyr Phe Asp Phe 100 105 110 Trp Gly Leu Gly Thr Leu Val
Thr Val Ser Ser 115 120 2106PRTArtificial SequenceSynthetic
Construct 2Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Ile Thr 85 90 95 Phe Gly
Gln Gly Thr Arg Leu Glu Ile Lys 100 105 3124PRTArtificial
SequenceSynthetic Construct 3Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Trp Phe Asp Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Ile Phe Thr Val Val Arg Gly Phe Phe Ile
Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 4106PRTArtificial SequenceSynthetic Construct 4Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Tyr 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Arg Ser Asn Trp Val Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys 100 105 5126PRTArtificial SequenceSynthetic Construct
5Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45 Ala Val Ile Trp Tyr Asp Glu Asn Asn Arg Tyr
Ser Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Thr
Asn Tyr His Gly Ser Gly Ser Tyr Tyr Ile Tyr 100 105 110 Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125
6106PRTArtificial SequenceSynthetic Construct 6Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Val Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 75PRTArtificial SequenceSynthetic peptide 7Asn Tyr Gly Met
His 1 5 817PRTArtificial SequenceSynthetic peptide 8Val Ile Trp Phe
Asp Glu Asn Asn Lys Tyr Tyr Ala Asp Ser Val Arg 1 5 10 15 Gly
916PRTArtificial SequenceSynthetic peptide 9Ala Arg Asp Ile Ser Leu
Val Arg Asp Ala Phe Ile Tyr Phe Asp Phe 1 5 10 15 105PRTArtificial
SequenceSynthetic peptide 10Ser Tyr Gly Met His 1 5
1117PRTArtificial SequenceSynthetic peptide 11Val Ile Trp Phe Asp
Gly Asn Asn Gln Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
1217PRTArtificial SequenceSynthetic peptide 12Ala Arg Asp Ile Phe
Thr Val Val Arg Gly Phe Phe Ile Tyr Phe Asp 1 5 10 15 Tyr
135PRTArtificial SequenceSynthetic peptide 13Asn Tyr Gly Met His 1
5 1417PRTArtificial SequenceSynthetic peptide 14Val Ile Trp Tyr Asp
Glu Asn Asn Arg Tyr Ser Ala Asp Ser Val Lys 1 5 10 15 Gly
1519PRTArtificial SequenceSynthetic peptide 15Ala Arg Asp Pro Thr
Asn Tyr His Gly Ser Gly Ser Tyr Tyr Ile Tyr 1 5 10 15 Phe Asp Tyr
1611PRTArtificial SequenceSynthetic peptide 16Arg Ala Ser Gln Ser
Val Ser Ser Tyr Leu Ala 1 5 10 177PRTArtificial SequenceSynthetic
peptide 17Asp Ala Ser Asn Arg Ala Thr 1 5 188PRTArtificial
SequenceSynthetic peptide 18Gln Gln Arg Ser Asn Trp Ile Thr 1 5
1911PRTArtificial SequenceSynthetic peptide 19Arg Ala Ser Gln Ser
Val Gly Ser Tyr Leu Ala 1 5 10 207PRTArtificial SequenceSynthetic
peptide 20Asp Ala Ser Asn Arg Ala Thr 1 5 218PRTArtificial
SequenceSynthetic peptide 21Gln Gln Arg Ser Asn Trp Val Thr 1 5
2211PRTArtificial SequenceSynthetic peptide 22Arg Ala Ser Gln Ser
Val Ser Ser Tyr Leu Ala 1 5 10 237PRTArtificial SequenceSynthetic
peptide 23Asp Ala Ser Asn Arg Ala Thr 1 5 248PRTArtificial
SequenceSynthetic peptide 24Gln Gln Arg Ser Asn Trp Val Thr 1 5
25369DNAArtificial SequenceSynthetic Construct 25caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggagatc cctgagactc 60tcctgtacag
cgtctggatt caccttcaat aactatggca tgcactgggt ccgccagact
120ccaggcaagg ggctggagtg gctggcagtt atatggtttg atgaaaataa
taagtactat 180gcagactccg tgaggggccg attcaccatc tccagagaca
attccaagaa cacgctgttt 240ctgcaaatga acagcctgaa aaccgaagac
acggctatgt attattgtgc gagagatatt 300tctctggttc gggatgcttt
tatctacttt gacttctggg gcctgggaac cctggtcacc 360gtctcctca
36926318DNAArtificial SequenceSynthetic Construct 26gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggatcacctt cggccaaggg 300acacgactgg agatcaaa
31827372DNAArtificial SequenceSynthetic Construct 27caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggaatg ggtggcagtt atatggtttg atggaaataa
tcaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagacatc 300tttactgtgg ttcggggatt
ttttatctac tttgactact ggggccaggg aaccctggtc 360accgtctcct ca
37228318DNAArtificial SequenceSynthetic Construct 28gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttggc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact gggtcacctt cggccaaggg 300acacgactgg agatcaaa
31829378DNAArtificial SequenceSynthetic Construct 29caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag
cgtctggatt caccttcagt aactatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg gatggcagtt atctggtatg atgaaaataa
tagatactct 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagatcct 300acgaattacc atggttcggg
gagttattat atctactttg actactgggg ccagggaacc 360ctggtcaccg tctcctca
37830318DNAArtificial SequenceSynthetic Construct 30gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcac cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact gggtcacctt cggccaaggg 300acacgactgg agatcaaa
318311785DNAArtificial SequenceSynthetic Construct 31cgctatagaa
gcttgccgcc accatggcca ccggcaacct gcccggctgc tccttctcca 60tcttcctgct
ggccctgctg tcctgcctga ccgtgcccgc ctccgcctac caggtgcgca
120actcctccgg cctgtaccac gtgaccaacg actgccctaa ctcctccatc
gtgtacgagg 180ccgccgacgc catcctgcac actcctggct gcgtgccctg
cgtgcgcgag ggcaacgcct 240cccgctgctg ggtggccgtg actcctaccg
tggccacccg cgacggcaag ctgcccacca 300cccagctgcg ccgccacatc
gacctgctgg tgggctccgc caccctgtgc tccgccctgt 360acgtgggcga
cctgtgcggc tccgtgttcc tggtgggcca gctgttcacc ttctctcctc
420gccgccactg gaccacccag gactgcaact gctccatcta ccctggccac
atcaccggcc 480accgcatggc ctgggacatg atgatgaact ggtctcccac
cgccgccctg gtggtggccc 540agctgctgcg catccctcag gccatcatgg
acatgatcgc cggcgcccac tggggcgtcc 600tggccggcat cgcctacttc
tccatggtgg gcaactgggc caaggtgctg gtggtgctgc 660tgctgttcgc
cggcgtggac gccgagaccc acgtgacggg cggctccgcc ggccgcacca
720ccgccggcct ggtgggcctg ctgacacccg gcgccaagca gaacatccag
ctgatcaaca 780ccaacggctc ctggcacatc aactccaccg ccctgaactg
caacgagtcc ctgaacaccg 840gctggctggc cggcctgttc taccagcaca
agttcaactc ctccggctgt cccgagcgcc 900tggcctcctg ccgccgcctg
accgacttcg cccagggctg gggccctatc tcctacgcca 960acggctccgg
cctggacgag cggccctact gctggcacta ccctcctcgc ccttgcggca
1020tcgtgcccgc caagtccgtg tgcggccctg tgtactgctt cactccctct
cccgtggtgg 1080tgggcaccac cgaccgctcc ggcgctccca cctactcctg
gggcgccaac gacaccgacg 1140tgttcgtgct gaacaacacc cgccctcctc
tgggcaactg gttcggctgc acctggatga 1200actccaccgg cttcaccaag
gtgtgcggcg cgcctccctg cgtgatcggc ggcgtgggca 1260acaacaccct
gctgtgccct accgactgct tccgcaagca tcccgaggcc acctactccc
1320gctgcggctc cgggccctgg atcactcctc gctgcatggt ggactatccc
taccgcctgt 1380ggcactatcc ctgcaccatc aactacacca tcttcaaggt
gcgcatgtac gtgggcggcg 1440tggagcaccg cctggaggcg gcgtgcaact
ggacccgcgg cgagcgctgc gacctggagg 1500accgcgaccg ctccgagctg
tctcctctgc tgctgtccac cacccagtgg caggtgctgc 1560cctgctcctt
caccaccctg cccgcgctgt ccaccggcct gatccacctg caccagaaca
1620tcgtggacgt gcagtacctg tacggcgtgg gctcctccat cgcgtcctgg
gcgatcaagt 1680gggagtacgt ggtgctgctg ttcctgctgc tggcggacgc
gcgcgtgtgc tcctgcctgt 1740ggatgatgct gctgatctcc caggcggagg
cgtctagagg agtca 178532124PRTArtificial SequenceSynthetic Construct
32Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Asn Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Phe
Thr Met Val Arg Gly Val Phe Ile Tyr Phe Asp 100 105 110 Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 33124PRTArtificial
SequenceSynthetic Construct 33Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Trp Leu Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Ile Phe Thr Val Ala Arg Gly Val Ile Ile
Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 34124PRTArtificial SequenceSynthetic Construct 34Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Phe Asp Gly Asn Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Phe Thr
Met Asp Arg Gly Val Phe Ile Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 35124PRTArtificial
SequenceSynthetic Construct 35Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Trp Tyr Asp Glu Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Ile Phe Thr Met Val Arg Gly Val Phe Ile
Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 36124PRTArtificial SequenceSynthetic Construct 36Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala
Val Ile Trp Phe Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Met 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Ile Phe Thr Met Val Arg Gly Val Phe
Ile Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 37124PRTArtificial SequenceSynthetic Construct
37Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Asn Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Phe
Thr Met Val Arg Gly Val Phe Ile Tyr Phe Asp 100 105 110 Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 38124PRTArtificial
SequenceSynthetic Construct 38Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Trp Phe Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Ile Phe Thr Met Val Arg Gly Val Phe Ile
Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 39124PRTArtificial SequenceSynthetic Construct 39Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Glu Asn Asn Lys Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Asn Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Phe Thr
Met Val Arg Gly Val Phe Ile Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 40124PRTArtificial
SequenceSynthetic Construct 40Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Leu Ser Ser Tyr 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala Val Ile
Trp Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Ile Phe Thr Met Val Arg Gly Val Phe Ile
Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 415PRTArtificial SequenceSynthetic peptide 41Ser Tyr
Gly Met His 1 5 4217PRTArtificial SequenceSynthetic peptide 42Val
Ile Trp Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 4317PRTArtificial SequenceSynthetic peptide 43Ala Arg Asp
Ile Phe Thr Met Val Arg Gly Val Phe Ile Tyr Phe Asp 1 5 10 15 Tyr
44106PRTArtificial SequenceSynthetic Construct 44Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Val Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105 4511PRTArtificial SequenceSynthetic peptide 45Arg Ala
Ser Gln Ser Val Gly Ser Tyr Leu Ala 1 5 10 467PRTArtificial
SequenceSynthetic peptide 46Asp Ala Ser Asn Arg Ala Thr 1 5
478PRTArtificial SequenceSynthetic peptide 47Gln Gln Arg Ser Asn
Trp Val Thr 1 5 48372DNAArtificial SequenceSynthetic Construct
48caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt catcctcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaaataa
taaatactat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagacatc 300tttactatgg ttcggggagt
ttttatctac tttgactact ggggccaggg aaccctggtc 360accgtctcct ca
37249318DNAArtificial SequenceSynthetic Construct 49gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttggc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact gggtcacctt cggccaaggg 300acacgactgg agatcaaa
318505PRTArtificial SequenceSynthetic peptide 50Ser Tyr Gly Met His
1 5 5117PRTArtificial SequenceSynthetic peptide 51Val Ile Trp Leu
Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
5217PRTArtificial SequenceSynthetic peptide 52Ala Arg Asp Ile Phe
Thr Val Ala Arg Gly Val Ile Ile Tyr Phe Asp 1 5 10 15 Tyr
53106PRTArtificial SequenceSynthetic Construct 53Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35
40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser Asn Trp Val Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105 5411PRTArtificial SequenceSynthetic peptide 54Arg Ala
Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 557PRTArtificial
SequenceSynthetic peptide 55Asp Ala Ser Asn Arg Ala Thr 1 5
568PRTArtificial SequenceSynthetic peptide 56Gln Gln Arg Ser Asn
Trp Val Thr 1 5 57372DNAArtificial SequenceSynthetic Construct
57caggtgcaac tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct
120ccaggcaagg ggctggagtg ggtggcagtt atatggcttg atggaagtaa
cacatactat 180gcagactccg tgaagggccg attcaccatt tctagagaca
attccaagaa cacgctgttt 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgc gagagacatc 300tttactgtgg ctcggggagt
tattatctac tttgactact ggggccaggg aaccctggtc 360accgtctcct ca
37258318DNAArtificial SequenceSynthetic Construct 58gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact gggtcacctt cggccaaggg 300acacgactgg agatcaaa
3185912PRTHepatitis C virus 59Gln Leu Ile Asn Thr Asn Gly Ser Trp
His Ile Asn 1 5 10 6012PRTHepatitis C virus 60Gln Leu Val Asn Thr
Asn Gly Ser Trp His Ile Asn 1 5 10 6112PRTHepatitis C virus 61Gln
Leu Val Asn Ser Asn Gly Ser Trp His Ile Asn 1 5 10 6212PRTHepatitis
C virus 62Gln Leu Ile Asn Ser Asn Gly Ser Trp His Ile Asn 1 5 10
6312PRTHepatitis C virus 63His Leu Ile Asn Thr Asn Gly Ser Trp His
Ile Asn 1 5 10 6412PRTHepatitis C virus 64Gln Leu Ile Lys Thr Asn
Gly Ser Trp His Ile Asn 1 5 10 6512PRTHepatitis C virus 65Gln Leu
Val Asn Thr Asn Gly Ser Trp His Val Asn 1 5 10 6612PRTHepatitis C
virus 66Gln Phe Val Asn Thr Asn Gly Ser Trp His Ile Asn 1 5 10
6712PRTHepatitis C virus 67Gln Leu Ile Lys Asn Gly Ser Ser Trp His
Ile Asn 1 5 10 6812PRTHepatitis C virus 68Gln Leu Val Lys Thr Asn
Gly Ser Trp His Ile Asn 1 5 10 6912PRTHepatitis C virus 69His Leu
Val Asn Thr Asn Gly Ser Trp His Ile Asn 1 5 10 7012PRTHepatitis C
virus 70His Leu Val Asn Ser Asn Gly Ser Trp His Ile Asn 1 5 10
7112PRTHepatitis C virus 71Gln Leu Ile His Thr Asn Gly Ser Trp His
Ile Asn 1 5 10 7212PRTHepatitis C virus 72Gln Leu Val Lys Thr Glu
Gly Asn Trp His Ile Asn 1 5 10 7312PRTHepatitis C virus 73Asn Leu
Ile Lys Thr Asn Gly Ser Trp His Ile Asn 1 5 10 7412PRTHepatitis C
virus 74Gln Leu Ile Tyr Thr Asn Gly Ser Trp His Leu Asn 1 5 10
7512PRTHepatitis C virus 75Gln Leu Ile Asn Thr Asn Gly Ser Trp His
Leu Asn 1 5 10 7612PRTHepatitis C virus 76Tyr Leu Ile Asn Thr Asn
Gly Ser Trp His Ile Asn 1 5 10 7712PRTHepatitis C virus 77Ser Leu
Ile Asn Thr Asn Gly Ser Trp His Ile Asn 1 5 10 7812PRTHepatitis C
virus 78Asn Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn 1 5 10
7912PRTHepatitis C virus 79His Leu Val Asn Ser Asn Gly Ser Trp His
Ile Asn 1 5 10 8012PRTHepatitis C virus 80Gln Leu Val Asn Ser Ser
Gly Ser Trp His Ile Asn 1 5 10 8112PRTHepatitis C virus 81Gln Leu
Ile Asn Ser Asn Gly Ser Trp His Ile Asn 1 5 10 8212PRTHepatitis C
virus 82Gln Leu Ile Gln Asn Gly Ser Ser Trp His Ile Asn 1 5 10
8312PRTHepatitis C virus 83Gln Phe Val Asn Thr Asn Gly Ser Trp His
Ile Asn 1 5 10 8412PRTHepatitis C virus 84Gln Leu Ile Lys Asn Gly
Ser Ser Trp His Ile Asn 1 5 10 8512PRTHepatitis C virus 85Gln Leu
Ile Lys Thr Asn Gly Ser Trp His Ile Asn 1 5 10 8612PRTHepatitis C
virus 86Gln Leu Ile Asn Ser Asn Gly Ser Trp His Val Asn 1 5 10
875PRTArtificial SequenceSynthetic peptide 87Xaa Tyr Gly Met His 1
5 8817PRTArtificial SequenceSynthetic peptide 88Val Ile Trp Xaa Asp
Xaa Xaa Asn Xaa Tyr Tyr Ala Asp Ser Xaa Xaa 1 5 10 15 Gly
8917PRTArtificial SequenceSynthetic peptide 89Ala Arg Asp Ile Xaa
Xaa Xaa Xaa Arg Xaa Xaa Xaa Ile Tyr Phe Asp 1 5 10 15 Xaa
9011PRTArtificial SequenceSynthetic peptide 90Arg Ala Ser Gln Ser
Val Xaa Ser Tyr Leu Ala 1 5 10 917PRTArtificial SequenceSynthetic
peptide 91Asp Ala Ser Asn Arg Ala Thr 1 5 928PRTArtificial
SequenceSynthetic peptide 92Gln Gln Arg Ser Asn Trp Xaa Thr 1 5
93383PRTHepatitis C virusMISC_FEATURE(1)..(383)Amino Acid 1 is
amino acid 364 of the HCV E2 protein 93Met Val Gly Asn Trp Ala Lys
Val Leu Val Val Leu Leu Leu Phe Ala 1 5 10 15 Gly Val Asp Ala Glu
Thr His Val Thr Gly Gly Ser Ala Gly Arg Thr 20 25 30 Thr Ala Gly
Leu Val Gly Leu Leu Thr Pro Gly Ala Lys Gln Asn Ile 35 40 45 Gln
Leu Ile Asn Thr Asn Gly Ser Trp His Ile Asn Ser Thr Ala Leu 50 55
60 Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Leu Ala Gly Leu Phe Tyr
65 70 75 80 Gln His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala
Ser Cys 85 90 95 Arg Arg Leu Thr Asp Phe Ala Gln Gly Trp Gly Pro
Ile Ser Tyr Ala 100 105 110 Asn Gly Ser Gly Leu Asp Glu Arg Pro Tyr
Cys Trp His Tyr Pro Pro 115 120 125 Arg Pro Cys Gly Ile Val Pro Ala
Lys Ser Val Cys Gly Pro Val Tyr 130 135 140 Cys Phe Thr Pro Ser Pro
Val Val Val Gly Thr Thr Asp Arg Ser Gly 145 150 155 160 Ala Pro Thr
Tyr Ser Trp Gly Ala Asn Asp Thr Asp Val Phe Val Leu 165 170 175 Asn
Asn Thr Arg Pro Pro Leu Gly Asn Trp Phe Gly Cys Thr Trp Met 180 185
190 Asn Ser Thr Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Cys Val Ile
195 200 205 Gly Gly Val Gly Asn Asn Thr Leu Leu Cys Pro Thr Asp Cys
Phe Arg 210 215 220 Lys His Pro Glu Ala Thr Tyr Ser Arg Cys Gly Ser
Gly Pro Trp Ile 225 230 235 240 Thr Pro Arg Cys Met Val Asp Tyr Pro
Tyr Arg Leu Trp His Tyr Pro 245 250 255 Cys Thr Ile Asn Tyr Thr Ile
Phe Lys Val Arg Met Tyr Val Gly Gly 260 265 270 Val Glu His Arg Leu
Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg 275 280 285 Cys Asp Leu
Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu 290 295 300 Ser
Thr Thr Gln Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro 305 310
315 320 Ala Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp
Val 325 330 335 Gln Tyr Leu Tyr Gly Val Gly Ser Ser Ile Ala Ser Trp
Ala Ile Lys 340 345 350 Trp Glu Tyr Val Val Leu Leu Phe Leu Leu Leu
Ala Asp Ala Arg Val 355 360 365 Cys Ser Cys Leu Trp Met Met Leu Leu
Ile Ser Gln Ala Glu Ala 370 375 380
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