U.S. patent application number 12/457447 was filed with the patent office on 2009-12-31 for humanized antibodies against west nile virus and therapeutic and prophylactic uses thereof.
This patent application is currently assigned to MacroGenics Inc.. Invention is credited to Ling Huang, Leslie S. Johnson.
Application Number | 20090324593 12/457447 |
Document ID | / |
Family ID | 36060647 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090324593 |
Kind Code |
A1 |
Johnson; Leslie S. ; et
al. |
December 31, 2009 |
Humanized antibodies against west nile virus and therapeutic and
prophylactic uses thereof
Abstract
The present invention relates to compositions comprising
humanized antibodies or fragments thereof that immunospecifically
bind to one or more antigens of a flavivirus, particularly of West
Nile Virus (WNV) and methods for preventing, treating or
ameliorating symptoms associated with a flavivirus, particularly of
West Nile Virus (WNV) infection utilizing said compositions. In
particular, the present invention relates to methods for
preventing, treating or ameliorating symptoms associated with WNV
infection, said methods comprising administering to a human subject
an effective amount of one or more humanized antibodies or
fragments thereof that immunospecifically bind to a WNV antigen.
The present invention also relates to detectable or diagnostic
compositions comprising humanized antibodies or fragments thereof
that immunospecifically bind to a WNV. antigen and methods for
detecting or diagnosing WNV infection utilizing said
compositions.
Inventors: |
Johnson; Leslie S.;
(Darnestown, MD) ; Huang; Ling; (Bethesda,
MD) |
Correspondence
Address: |
DRINKER BIDDLE & REATH (DC)
1500 K STREET, N.W., SUITE 1100
WASHINGTON
DC
20005-1209
US
|
Assignee: |
MacroGenics Inc.
Rockville
MD
|
Family ID: |
36060647 |
Appl. No.: |
12/457447 |
Filed: |
June 11, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11226886 |
Sep 13, 2005 |
7572456 |
|
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12457447 |
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60609766 |
Sep 13, 2004 |
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Current U.S.
Class: |
424/133.1 ;
435/5; 530/387.3 |
Current CPC
Class: |
G01N 33/56983 20130101;
C07K 2317/24 20130101; A61P 31/12 20180101; C07K 2317/567 20130101;
Y02A 50/60 20180101; Y02A 50/30 20180101; C07K 16/1081 20130101;
C07K 2317/565 20130101; G01N 2333/18 20130101; Y02A 50/388
20180101; A61K 2039/505 20130101; Y02A 50/396 20180101; Y02A 50/394
20180101; Y02A 50/386 20180101; Y02A 50/53 20180101; G01N 2469/20
20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3; 435/5 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; C12Q 1/70 20060101
C12Q001/70; A61P 31/12 20060101 A61P031/12 |
Claims
1-16. (canceled)
17. A humanized antibody or an epitope binding fragment thereof
comprising three VH complementary determining regions (CDRs) or
three VL CDRs, wherein the antibody or epitope binding fragment
thereof specifically binds a West Nile Virus (WNV) epitope defined
by monoclonal antibody E16, E24, or E34, wherein said three VH or
VL CDRs are from monoclonal antibody E16, E24, or E34 or differ by
one amino acid substitution from a CDR from monoclonal antibody
E16, E24, or E34.
18. The humanized antibody of claim 17 comprising a humanized
variable heavy region comprising the amino acid sequence of SEQ ID
NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 37,
or SEQ ID NO: 46.
19. The humanized antibody of claim 17 comprising a humanized
variable light chain region comprising the amino acid sequence of
SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 38, or SEQ ID NO: 47.
20. The humanized antibody of claim 17 comprising a VH CDR1
comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:
27, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2,
SEQ ID NO: 28, or SEQ ID NO: 39, and a VH CDR3 comprising the amino
acid sequence of SEQ ID NO: 3, SEQ ID NO: 29, or SEQ ID NO: 40.
21. The humanized antibody of claim 17 comprising a VL CDR1 having
the amino acid sequence of SEQ ID NO: 11, a VL CDR2 comprising the
amino acid sequence of SEQ ID NO: 12, and a VL CDR3 comprising the
amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 34.
22. The humanized antibody of claim 17 comprising a framework
region comprising at least one amino acid modification in heavy
chain FR3 or light chain FR2.
23. The humanized antibody of claim 22, wherein the at least one
amino acid modification in heavy chain FR3 comprises a substitution
at position 5, 6, 9, 11, 12, 19, 20, 25, 30, 38, 40, 43, 48, 66,
67, 69, 71, 75, 76, 79, 81, 82A, 83, 85, 87, 105, or 109.
24. The humanized antibody of claim 22, wherein the at least one
amino acid modification in light chain FR2 comprises a substitution
at position 8, 9, 10, 11, 12, 13, 15, 17, 19, 20, 22, 43, 49, 63,
71, 78, 83, 85, or 100.
25. The humanized antibody of claim 17, wherein the humanized
antibody is an epitope-binding antibody fragment.
26. The humanized antibody of claim 25, wherein the epitope-binding
antibody fragment is a Fab, F(ab').sub.2, or scFv fragment.
27. A pharmaceutical composition comprising (i) a therapeutically
effective amount of the humanized antibody of claim 17; and (ii) a
pharmaceutically acceptable carrier.
28. A method of treating a WNV infection in a patient, said method
comprising administering to said patient a therapeutically
effective amount of the humanized antibody of claim 17.
29. The method of claim 28 further comprising administering an
anti-viral agent.
30. The method of claim 29, wherein the anti-viral agent is
selected from the group consisting of protease inhibitors,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, nucleoside analogs, and
alpha-interferons.
31. The method of claim 29, wherein the anti-viral agent is
selected from the group consisting of zidovudine, acyclovir,
gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin,
foscarnet, amantadine, rimantadine, saquinavir, indinavir,
amprenavir, lopinavir, ritonavir, adefovir, clevadine, entecavir,
and pleconaril.
32. The method of claim 28, wherein said patient is human.
33. A method of diagnosis of WNV infection in a subject comprising:
(a) contacting a biological sample from said subject with an
effective amount of the humanized antibody of claim 17; and (b)
detecting binding of said antibody, wherein detection of said
detectable marker above a background or standard level indicates
that said subject has a WNV infection.
34. The method of claim 33, wherein said detectable marker is a
chemiluminescent, enzymatic, fluorescent, or radioactive label.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/609,766, filed on Sep. 13, 2004, which is
incorporated herein by reference in its entirety.
1. FIELD OF THE INVENTION
[0002] The present invention relates to humanized antibodies,
fragments, and variants thereof, that immunospecifically bind to
one or more antigens of a flavivirus, particularly of West Nile
Virus (WNV). The invention also relates to pharmaceutical
compositions comprising the humanized antibodies of the invention
and methods of use for preventing, treating or ameliorating
symptoms associated with a flaviviral, particularly a WNV,
infection. The invention also encompasses diagnostic compositions
comprising humanized WNV antibodies and methods for diagnosing a
WNV infection using the humanized antibodies of the invention.
2. BACKGROUND OF THE INVENTION
[0003] WNV cycles between mosquitoes and birds but also infects
humans, horses, and other vertebrate species. It is endemic in
parts of Africa, Europe, the Middle East, and Asia, and outbreaks
throughout the United States during the past four years indicate
that it has established its presence in the Western Hemisphere.
Humans develop a febrile illness that can progress rapidly to a
meningitis or encephalitis syndrome (Hubalek et al., 1999, Emerg
Inf Dis 5:643-650), and no specific therapy or vaccine has been
approved for use in humans.
[0004] 2.1 Virology
[0005] A member of the Flavivirus genus of the Flaviviridae family,
WNV is a neurotropic enveloped virus with a single-stranded,
positive-polarity 11-kilobase RNA genome. It is translated in the
cytoplasm as a polyprotein, and cleaved into structural (C, M, and
E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5)
proteins by virus- and host-encoded proteases. The structural
proteins include a capsid protein (C), a transmembrane protein (M)
that regulates fusion of the virus with the host membrane, and an
envelope protein (E) that functions in receptor binding, membrane
fusion, and viral assembly. The role of nonstructural proteins is
not fully delineated but these proteins form the viral protease
(NS2B, NS3), NTPase (NS3), RNA helicase (NS3), and RNA-dependent
RNA polymerase (NS5) (Chambers et al. 1990, Annu. Rev. Microbiol.
44: 649-88). After the E protein of WNV binds to an uncharacterized
cell surface receptor, viral uptake is believed to occur through
receptor-mediated endocytosis (Chambers et al., 1990, Annu Rev
Microbiol 44:649-88). In the endosome, an acid-catalyzed
conformational change in E (Gollins et al., 1986, J. Gen. Virol.
67:1941-1950; Kimura et al., 1986, J. Gen. Virol. 67:2423-33)
releases the nucleocapsid into the cytoplasm. At the endoplasmic
reticulum (ER) membrane, the structural proteins and NS1 undergo
co-translational translocation, glycosylation, and
membrane-associated cleavage, while the other nonstructural
proteins are translated in the cytoplasm (Falgout et al., 1995, J
Virol 69:7232-43; Markoff et al., 1994, Virology 204:526-40).
Assembly occurs at the ER, and viral particles are exocytosed.
[0006] 2.2 WNV Immunology
[0007] Host factors including immune status influence the
expression of WNV disease in humans (Camenga et al., 1974, J Infect
Dis 130:634-41). Infants, the elderly, and patients with impaired
immune systems are at greatest risk for severe neurological disease
(Asnis et al., 2000, Clin Infect Dis 30:413-8; Hubalek et al.,
1999, Emerg Inf Dis 5:643-650; Tsai et al., 1998, Lancet
352:767-71). Investigations are beginning to elucidate the
molecular basis of WNV infection and the protective immune system
response. Maturation of the immune system correlates with
resistance to WNV infection (Eldadah et al., 1967, Am J Epidemiol
86:776-90; Eldadah et al., 1967, Am J Epidemiol 86:765-75; Weiner
et al., 1970, J Hyg (Lond) 68:435-46). Depletion of macrophages
increases the neuro-invasiveness and virulence of an attenuated
strain (Ben-Nathan et al., 1996, Arch Virol 141:459-69).
Lymphocytes are critical for protection against WNV infection as
SCID and RAG1 mice uniformly succumb to infection with WNV (Diamond
et al., 2003, J Virol 77:2578-2586; Halevy et al., 1994, Arch Virol
137:355-70). Recent studies demonstrate that components of humoral
immunity (IgM, IgG, and complement) have essential functions early
in the course of infection and prevent dissemination to the central
nervous system (CNS) (Diamond et al., 2003, J Virol 77:2578-2586;
Diamond et al., 2003, Viral Immunology 16:259-278). The cellular
basis of immunity against WNV is beginning to be delineated.
Several studies suggest a protective role for cytotoxic and helper
T cells. In vitro, T cells kill targets, proliferate, and release
inflammatory cytokines after exposure to WNV-infected cells
(Douglas et al., 1994, Immunology 82:561-70; Kesson et al., 1987, J
Gen Virol 68:2001-6; Kulkarni et al., 1991, Viral Immunol 4:73-82;
Liu et al., 1989, J Gen Virol 70:565-73). In vivo, antigen-specific
helper and cytotoxic T cell responses are generated in mice after
administration of a candidate vaccine strain of WNV (Yang et al.,
2001, J Infect Dis 184:809-16). Although the precise contribution
of T cell-mediated immunity in vivo to viral clearance and
long-term immunity has yet to be established, recent studies
demonstrate an essential role for T cells in the control of WNV
infection. Mice that lack CD8.sup.+ T cells or classical class I
MHC molecules show increased mortality and viral loads, and
long-term viral persistence in the CNS after WNV infection
(Shrestha et al., 2004, J Virol. 78:8312-21), and an absence of
.gamma..delta.T cells results in increased mortality after WNV
infection (Wang et al., 2003, J Immunol 171:2524-2531).
[0008] 2.3 Antivirals
[0009] At present, treatment for all flavivirus infections,
including WNV, is supportive. Ribavirin has been suggested as a
candidate agent because it inhibits WNV infection in cells (Jordan
et al., 2000, J Infect Dis 182:1214-7); however, its activity was
modest at concentrations that are achievable in the CNS (Anderson
et al., 2002, Emerg Infect Dis 8:107-8; Jordan et al., 2000, J
Infect Dis 182:1214-7). The limited in vivo experience with
ribavirin against flaviviruses has not been promising, as it failed
to attenuate infection of the closely related Dengue (DEN) virus in
mice (Koff et al., 1983, Antimicrob Agents Chemother 24:134-6) and
monkeys (Malinoski et al., 1990, Antiviral Res 13:139-49). Based on
preliminary cell culture studies (Anderson et al., 2002, Emerg
Infect Dis 8:107-8), interferon (IFN) .alpha..sub.2b was recently
proposed as a possible therapy for WNV. Although in vivo studies
have not been performed with WNV, based on experiments with related
flaviviruses, IFNs may inhibit WNV dissemination (Harinasuta et
al., 1985, Southeast Asian J Trop Med Public Health 16:332-6). Mice
that are deficient in IFN .alpha., .beta., and .gamma. receptors
succumb to dengue (DEN) virus infection (Johnson et al., 1999, J
Virol 73:783-6) or Murray Valley encephalitis (Lobigs et al., 2003,
J Gen Virol 84:567-72) virus infection and mice deficient in IFN
.gamma. produced higher viral loads after yellow fever virus
infection (Liu et al., 2001, J Virol 75:2107-18). IFN a was
effective as prophylaxis and therapy against Saint Louis
encephalitis virus in mice (Brooks et al., 1999, Antiviral Res
41:57-64) although clinical benefit was achieved only when therapy
was initiated within 24 hours of infection. Indeed, clinical trials
on patients with serologically confirmed Japanese encephalitis
virus demonstrated no benefit of IFN therapy (Solomon et al., 2003,
Lancet 361:821-6). Thus, the window of opportunity for IFN a
therapy against WNV infection may be too narrow to be clinically
relevant.
[0010] The present invention is aimed at addressing the concerns
and shortcomings of currents prophylactic and therapeutic methods
against flaviviral, particularly WNV, infections.
3. SUMMARY OF THE INVENTION
[0011] The instant invention provides humanized antibodies, or
fragments thereof, that immunospecifically bind a WNV antigen,
e.g., the E protein. In a specific embodiment, the humanized
antibodies of the invention bind to the ectodomain of the WNV E
protein, e.g., domain III of the WNV E protein comprising amino
acids 290 to 415. In most preferred embodiments, the present
invention relates to humanized versions of E16, E24, or E34 mouse
monoclonal antibodies or fragments thereof, preferably antigen
binding fragments thereof. Hybridomas producing antibodies E16,
E24, or E34 have been deposited with the American Type Culture
Collection (10801 University Blvd., Manassas, Va. 20110-2209) on
Jun. 4, 2004 under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of patent Procedures, and assigned accession numbers
PTA-6050, PTA-6051, and PTA-6052, respectively, and are
incorporated herein by reference. Representative plasmids encoding
humanized antibodies of the invention, e.g., pMGX623-humanized E16
light chain version 1, the vector is pCINeo (Invitrogen), the
insert consists of human germline sequence VKB2 and JK2 as
framework, human kappa as constant region and mouse E16 CDRs;
pMGX624--humanized E16 light chain version 2, same description as
pMGX623 except a Y49S mutation in the variable region;
pMGX625--humanized E16 heavy chain version 1, the vector is pCINeo
(Invitrogen), the insert consists of human germline sequence VH1-18
and JH6 as framework, human IgG1 as constant region, and mouse E16
CDRs; pMGX626--humanized E16 heavy chain version 2, same
description as pMGX625 except V67A, M69F, and T71A mutations in the
variable region; and pMGX627--humanized E16 heavy chain version 3,
same description as pMGX625 except a T71A mutation in the variable
region; having ATCC Accession numbers PTA-6199, PTA-6200, PTA-6201,
PTA-6202, and PTA-6203, respectively, were deposited under the
provisions of the Budapest Treaty with the American Type Culture
Collection (10801 University Blvd., Manassas, Va. 20110-2209) on
Sep. 10, 2004, and are incorporated herein by reference.
[0012] The humanized antibodies of the invention may comprise one
or more CDRs of E16, E24, or E34, i.e., have a heavy chain variable
region (VH) comprising the amino acid sequence of CDR1 (SEQ ID NO:
1 or SEQ ID NO: 27) and/or CDR2 (SEQ ID NO: 2, SEQ ID NO: 28 or SEQ
ID NO: 39) and/or CDR3 (SEQ ID NO: 3, SEQ ID NO: 29 or SEQ ID NO:
40) of E16, E24 or E34 and/or a light chain variable region (VL)
comprising the amino acid sequence of CDR1 (SEQ ID NO: 11) and/or a
CDR2 (SEQ ID NO: 12) and/or CDR3 (SEQ ID NO: 13 or SEQ ID NO: 34)
of E16, E24 or E34. The sequences of the CDRs for E16, E24, and E34
heavy and light chain variable regions are provided in Table 1.
[0013] In yet other preferred embodiments, the humanized antibodies
of the invention comprise a heavy chain variable region comprising
an amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID
NO: 23, and/or a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26, and/or amino acid
sequence variants thereof.
[0014] In particular, the invention provides a humanized antibody
that immunospecifically binds to a WNV antigen, preferably a WNV E
antigen, said humanized antibody comprising (or alternatively,
consisting of) a VH CDR1 and a VL CDR1; a VH CDR1 and a VL CDR2; a
VH CDR1 and a VL CDR3; a VH CDR2 and a VL CDR1; VH CDR2 and VL
CDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VH CDR1; a VH CDR3
and a VL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, a VH CDR2 and a
VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, a VH CDR2
and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, a VH
CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; a VH CDR1,
a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; a VH
CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3;
a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL
CDR3; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a
VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3
and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1 and a VL CDR2; a VH
CDR1, a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR1, a VH CDR3, a
VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR3, a VL CDR1 and a VL
CDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR2, a
VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR2
and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL
CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a
VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR1,
a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VH
CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combination
thereof of the VH CDRs and VL CDRs disclosed herein.
[0015] In one specific embodiment, the invention provides a
humanized E16 antibody,
[0016] wherein the VH region consists of the framework region (FR)
segments from the human germline VH segment VH1-18 and JH6, as
depicted in FIG. 1a, and the CDR regions of the E16 VH. In another
specific embodiment, the humanized E16 antibody further comprises a
VL region, which consists of the FR segments of the human germline
VL segment VKB-3, as depicted in FIG. 1b, and the CDR regions of
E16 VL.
[0017] In certain embodiments, the heavy chain comprises one or
more substitutions at Kabat numbers 5, 6, 9, 11, 12, 19, 20, 25,
30, 38, 40, 43, 48, 66, 67, 69, 71, 75, 76, 79, 81, 82A, 83, 85,
87, 105, 109. In certain embodiments, the light chain comprises one
or more substitutions at Kabat numbers 8, 9, 10, 11, 12, 13, 15,
17, 19, 20, 22, 43, 49, 63, 71, 78, 83, 85, or 100. In another
embodiment, the heavy chain FR3 may consist of the amino acid
sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In another
embodiment, the light chain FR2 may consist of the amino acid
sequence of SEQ ID NO: 16 or SEQ ID NO: 17. Humanized E16
antibodies comprising a VH FR3 sequence of SEQ ID NO: 7, SEQ ID NO:
8, or SEQ ID NO: 9 and a VH FR2 sequence of SEQ ID NO: 16 or SEQ ID
NO: 17 are provided in Table 2 as HuE16-1.1, HuE16-1.2, HuE16-2.1,
HuE16-2.2, HuE16-3.1, and HuE16-3.2.
[0018] The present invention provides humanized antibody molecules
specific for WNV in which one or more regions of one or more CDRs
of the heavy and/or light chain variable regions of a human
antibody (the recipient antibody) have been substituted by
analogous parts of one or more CDRs of a donor monoclonal antibody
which specifically binds a WNV antigen, e.g., a monoclonal antibody
produced by clones E16, E24, or E34. In a most preferred
embodiment, the humanized antibody can specifically bind to the
same epitope as the donor murine antibody. It will be appreciated
by one skilled in the art that the invention encompasses CDR
grafting of antibodies in general. Thus, the donor and acceptor
antibodies may be derived from animals of the same species and even
the same antibody class or sub-class. More usually, however, the
donor and acceptor antibodies are derived from animals of different
species. Typically the donor antibody is a non-human antibody, such
as a rodent MAb, and the acceptor antibody is a human antibody.
[0019] In some embodiments, at least one CDR from the donor
antibody is grafted onto the human antibody. In other embodiments,
at least two and preferably all three CDRs of each of the heavy
and/or light chain variable regions are grafted onto the human
antibody. The CDRs may comprise the Kabat CDRs, the structural loop
CDRs or a combination thereof. In some embodiments, the invention
encompasses a humanized WNV antibody comprising at least one CDR
grafted heavy chain and at least one CDR-grafted light chain.
[0020] In a preferred embodiment, the CDR regions of the humanized
WNV specific antibody are derived from a murine antibody against
WNV. In some embodiments, the humanized antibodies described herein
comprise alterations, including, but not limited to, amino acid
deletions, insertions, and modifications, of the acceptor antibody,
i.e., human, heavy and/or light chain variable domain framework
regions that are necessary for retaining binding specificity of the
donor monoclonal antibody. In some embodiments, the framework
regions of the humanized antibodies described herein do not
necessarily consist of the precise amino acid sequence of the
framework region of a natural occurring human antibody variable
region, but contain various alterations, including, but not limited
to, amino acid deletions, insertions, modifications that alter the
property of the humanized antibody, for example, improve the
binding properties of a humanized antibody region that is specific
for the same target as the murine WNV specific antibody. In most
preferred embodiments, a minimal number of alterations are made to
the framework region in order to avoid large-scale introductions of
non-human framework residues and to ensure minimal immunogenicity
of the humanized antibody in humans. In some embodiments, the
framework residues are derived from the human germline VH segment
VH1-18 and JH6 and/or the human germline VL segment VK-B3. In
another embodiment, the heavy chain FR3 may consist of the amino
acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In
another embodiment, the light chain FR2 may consist of the amino
acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17. In some
embodiments of the invention, there are no alterations made to the
framework regions. The donor monoclonal antibody is preferably a
monoclonal antibody produced by clones E16, E24, or E34, which bind
WNV E antigen.
[0021] The humanized antibodies of the present invention include
complete antibody molecules having full length heavy and light
chains, or any fragment thereof, such as the Fab or (Fab').sub.2
fragments, a heavy chain and light chain dimer, or any minimal
fragment thereof such as an Fv, an SCA (single chain antibody), and
the like, specific for a WNV antigen.
[0022] The invention encompasses the production of humanized
anti-WNV specific antibodies. The invention encompasses any method
known in the art useful for the production of polypeptides, e.g.,
in vitro synthesis, recombinant DNA production, and the like.
Preferably, the humanized antibodies are produced by recombinant
DNA technology. The humanized WNV specific antibodies of the
invention may be produced using recombinant immunoglobulin
expression technology. Exemplary methods for the production of
recombinant humanized antibodies of the invention may comprise the
following: a) constructing, by conventional molecular biology
methods, an expression vector comprising an operon that encodes an
antibody heavy chain in which the CDRs and a minimal portion of the
variable region framework that are required to retain donor
antibody binding specificity are derived from a non-human
immunoglobulin, such as the murine WNV E antigen specific
monoclonal antibody, e.g., monoclonal antibody produced by clones
E16, E24, or E34, which bind WNV E antigen, and the remainder of
the antibody is derived from a human immunoglobulin, thereby
producing a vector for the expression of a humanized antibody heavy
chain; b) constructing, by conventional molecular biology methods,
an expression vector comprising an operon that encodes an antibody
light chain in which the CDRs and a minimal portion of the variable
region framework that are required to retain donor antibody binding
specificity are derived from a non-human immunoglobulin, such as
the murine WNV E antigen specific monoclonal antibody, e.g.,
monoclonal antibody produced by clones E16, E24, or E34, which
binds WNV E antigen, and the remainder of the antibody is derived
from a human immunoglobulin, thereby producing a vector for the
expression of humanized antibody light chain; c) transferring the
expression vectors to a host cell by conventional molecular biology
methods to produce a transfected host cell for the expression of
humanized anti-WNV antibodies; and d) culturing the transfected
cell by conventional cell culture techniques so as to produce
humanized anti-WNV antibodies. Host cells may be cotransfected with
two expression vectors of the invention, the first vector
containing an operon encoding a heavy chain derived polypeptide and
the second containing an operon encoding a light chain derived
polypeptide. The two vectors may contain different selectable
markers but, with the exception of the heavy and light chain coding
sequences, are preferably identical. This procedure provides for
equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes both heavy
and light chain polypeptides. The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA or both. The host
cell used to express the recombinant humanized antibodies of the
invention may be either a bacterial cell such as Escherichia coli,
or, preferably, a eukaryotic cell. Preferably, a mammalian cell
such as a chinese hamster ovary cell or HEK-293 may be used. The
choice of expression vector is dependent upon the choice of host
cell, and may be selected so as to have the desired expression and
regulatory characteristics in the selected host cell. The general
methods for construction of the vector of the invention,
transfection of cells to produce the host cell of the invention,
culture of cells to produce the humanized antibodies of the
invention are all conventional molecular biology methods. Likewise,
once produced, the recombinant humanized antibodies of the
invention may be purified by standard procedures of the art,
including cross-flow filtration, ammonium sulphate precipitation,
affinity column chromatography, gel electrophoresis and the
like.
[0023] In some embodiments, cell fusion methods for making
monoclonal antibodies may be used in the methods of the invention
such as those disclosed in U.S. Pat. No. 5,916,771, incorporated
herein by reference in its entirety. Briefly, according to this
method, DNA encoding the desired heavy chain (or a fragment of the
heavy chain) is introduced into a first mammalian host cell, while
DNA encoding the desired light chain (or a fragment of the light
chain) is introduced into a second mammalian host cell. The first
transformed host cell and the second transformed host cell are then
combined by cell fusion to form a third cell. Prior to fusion of
the first and second cells, the transformed cells may be selected
for specifically desired characteristics, e.g., high levels of
expression. After fusion, the resulting hybrid cell contains and
expresses both the DNA encoding the desired heavy chain and the DNA
encoding the desired light chain, resulting in production of the
multimeric antibody.
[0024] The invention encompasses using the humanized antibodies of
the present invention in conjunction with, or attached to, other
antibodies or fragments thereof such as human or humanized
antibodies. These other antibodies may be reactive with other
markers (epitopes) characteristic for the disease against which the
humanized antibodies of the invention are directed or may have
different specificities chosen, for example, to recruit molecules
or cells of the human immune system to the diseased cells. The
humanized antibodies of the invention (or parts thereof) may be
administered with such antibodies (or parts thereof) as separately
administered compositions or as a single composition with the two
agents linked by conventional chemical or by molecular biological
methods. Additionally, the diagnostic and therapeutic value of the
humanized antibodies of the invention may be augmented by labelling
the humanized antibodies with labels that produce a detectable
signal (either in vitro or in vivo) or with a label having a
therapeutic property. Some labels, e.g., radionucleotides, may
produce a detectable signal and have a therapeutic property.
Examples of radionuclide labels include .sup.125I, .sup.131I, and
.sup.14C. Examples of other detectable labels include a fluorescent
chromophore such as fluorescein, phycobiliprotein or tetraethyl
rhodamine for fluorescence microscopy; an enzyme which produces a
fluorescent or colored product for detection by fluorescence,
absorbance, visible color or agglutination, or which produces an
electron dense product for demonstration by electron microscopy; or
an electron dense molecule such as ferritin, peroxidase or gold
beads for direct or indirect electron microscopic visualization.
Labels having therapeutic properties include drugs for the
treatment of cancer, such as methotrexate and the like.
[0025] The methods of the invention also encompass polynucleotides
that encode the humanized antibodies of the invention. In one
embodiment, the invention provides an isolated nucleic acid
sequence encoding a heavy chain or a light chain of a humanized
antibody or a fragment thereof that specifically binds a WNV virus
antigen, preferably a WNV E antigen. The invention also relates to
a vector comprising said nucleic acid. The invention further
provides a vector comprising a first nucleic acid molecule encoding
a heavy chain and a second nucleic acid molecule encoding a light
chain, said heavy chain and light chain being of a humanized
antibody or a fragment thereof that specifically binds a WNV virus
antigen. In one specific embodiment, said vector is an expression
vector. The invention further provides host cells containing the
vectors or polynucleotides encoding the humanized antibodies of the
invention. Preferably, the invention encompasses polynucleotides
encoding heavy and light chains of the humanized antibodies of the
invention.
[0026] The present invention provides methods of preventing,
treating and ameliorating one or more symptoms associated with
flaviviral infection, particularly WNV infection, in a subject
comprising administering to said subject one or more humanized
antibodies or fragments thereof which immunospecifically bind to
one or more flaviviral antigens, particularly WNV antigens, with
high affinity and/or high avidity. The humanized antibodies of the
invention are useful for prevention or treatment of a flaviviral
infection, for example, as a single agent therapy. Alternatively,
the humanized antibodies of the invention may be used in a
combination therapy for the treatment or prevention of a flaviviral
infection with new drugs as they become available. The invention
also provides a method of treating a WNV infection in a patient in
need thereof, said method further comprising administering to said
patient a therapeutically effective amount of one or more
anti-viral agents.
[0027] In most preferred embodiments, the invention encompasses
humanized antibodies (e.g., anti-E antibodies) or fragments thereof
that have potent neutralizing activity as measured for example
using standard methods known in the art, e.g., in vitro plaque
reduction neutralization titer (PRNT) assay. Although not intending
to be bound by a particular mechanism of action, the humanized
antibodies of the invention may directly neutralize virus, block
entry of the virus into the cell, or block fusion and uncoating of
the virus inside the cell, thus treating or preventing viral
infections. In some embodiments, the invention encompasses
humanized antibodies which immunospecifically bind WNV-E protein
such that the PRNT.sub.50 values are at least 1/500, preferably at
least 1/10,000 at a concentration of 1 mg/mL.
[0028] In yet other preferred embodiments, humanized antibodies of
the invention have enhanced antibody-dependent complement mediated
neutralization of WNV infected virions and trigger lysis of
WNV-infected cells more effectively, as determined using standard
methods known in the art and exemplified herein. Humanized
antibodies are added to virus particles in the presence of
complement. Subsequently, inhibition of virus activity is
determined by plaque reduction assay. For complement-dependent cell
lysis, humanized antibodies are added to infected cells in the
presence of complement. Subsequently, cell lysis is evaluated by
standard methods (e.g., propidium iodide staining and flow
cytometry). Although not intending to be bound by a particular
mechanism of action the humanized antibodies of the invention have
enhanced clinical efficacy, therapeutically and prophylactically,
as they have enhanced effector functions, neutralize virus
attachment, trigger complement mediated lysis, promote clearance
from the circulatory systems and prevent emergence of viral
resistance. The humanized antibodies of the invention preferably
have a potent in vivo inhibitory activity, i.e., protect against
WNV infection by at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 99%. In vivo inhibitory activity as
used herein refers to the activity of the humanized antibodies of
the invention to neutralize virus activity, for example, by
inhibiting a step in the viral life cycle, e.g., virus attachment.
In vivo inhibitory activity may also refer to the ability of the
antibody to reduce morbidity and mortality in an animal model of
infection.
[0029] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens, particularly WNV antigens, and have an
apparent dissociation constant of less than 100 ng/mL as determined
by a sandwich ELISA. The present invention provides humanized
antibodies or fragments thereof which immunospecifically bind to
one or more flaviviral antigens, particularly WNV antigens, and
have an apparent dissociation constant of about 1-10 nM as measured
by surface plasmon resonance (SPR) using a BIAcore sensor. The
present invention provides humanized antibodies or fragments
thereof which immunospecifically bind to one or more flaviviral
antigens, particularly WNV antigens, and have an on rate of about
1.times.10.sup.4, about 5.times.10.sup.4, about 1.times.10.sup.5,
about 5.times.10.sup.5, about 1.times.10.sup.6, or about
5.times.10.sup.6 and an off rate of about 1.times.10.sup.-3, about
5.times.10.sup.-4, about 1.times.10.sup.-4, about
5.times.10.sup.-5, about 1.times.10.sup.-5, about
5.times.10.sup.-6, about 1.times.10.sup.-6, as measured by surface
plasmon resonance (SPR) using a BIAcore sensor.
[0030] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens and have a median
effective concentration (EC.sub.50) of less than 100 ng/mL, in an
in vitro microneutralization assay. In particular, the present
invention provides compositions for use in the prevention,
treatment, or amelioration of one or more symptoms associated with
a flaviviral infection, said compositions comprising one or more
humanized antibodies or fragments thereof which immunospecifically
bind to one or more one or more flaviviral antigens, particularly
WNV antigens, and have an EC.sub.50 of less than 0.01 nM, less than
0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM,
less than 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25
nM, less than 1.5 nM, less than 1.75 nM, or less than 2 nM, in an
in vitro microneutralization assay.
[0031] In some embodiments, the invention encompasses humanized
antibodies comprising variant Fc regions that bind FcRn with an
enhanced affinity, resulting in an increased antibody half life,
e.g., a half-life of greater than 15 days, preferably greater than
20 days, greater than 25 days, greater than 30 days, greater than
35 days, greater than 40 days, greater than 45 days, greater than 2
months, greater than 3 months, greater than 4 months, or greater
than 5 months. Although not intending to be bound by a particular
mechanism of action the neonatal Fc receptor (FcRn) plays an
important role in regulating the serum half-lives of IgG
antibodies. A correlation has been established between the
pH-dependent binding affinity of IgG antibodies to FcRn and their
serum half-lives in mice. The increased half-lives of the humanized
antibodies of the present invention or fragments thereof in a
mammal, preferably a human, results in a higher serum titer of said
humanized antibodies or antibody fragments in the mammal, and thus,
reduces the frequency of the administration of said humanized
antibodies or antibody fragments and/or reduces the concentration
of said humanized antibodies or antibody fragments to be
administered. For example, humanized antibodies or fragments
thereof with increased in vivo half-lives can be generated by
modifying (e.g., substituting, deleting or adding) amino acid
residues identified as involved in the interaction between the Fc
domain and the FcRn receptor. For example, the invention
encompasses humanized antibodies comprising variant Fc regions
which have at least one or more modification that enhances the
affinity to FcRn, e.g., a modification of one or more amino acid
residues 251-256, 285-290, 308-314, 385-389, and 428-436, or a
modification at positions 250 and 428, see, e.g., Hinton et al.,
2004, J. Biol. Chem. 279(8): 6213-6; PCT Publication No. WO
97/34631; and WO 02/060919, all of which are incorporated herein by
reference in its entirety.
[0032] The invention encompasses the use of the humanized
antibodies of the invention to detect the presence of one or more
flaviviral antigens specifically in a biological sample. In one
embodiment, the invention provides a method of diagnosis of a WNV
infection in a subject comprising: (i) contacting a biological
sample from said subject with an effective amount of a humanized
antibody of the invention; and (ii) detecting binding of said
humanized antibody or a fragment thereof, wherein detection of said
detectable marker above a background or standard level indicates
that said subject has a WNV infection.
[0033] The invention further provides a pharmaceutical composition
comprising (i) a therapeutically or prophylactically effective
amount of the humanized antibody or a fragment thereof that
specifically binds one or more flaviviral antigens, e.g., WNV
antigen; and (ii) a pharmaceutically acceptable carrier.
3.1 DEFINITIONS
[0034] As used herein, the term "analog" refers to a polypeptide
that possesses a similar or identical function as a flaviviral,
including WNV, polypeptide, a fragment of a flaviviral, including
WNV polypeptide, an antibody, or antibody fragment but does not
necessarily comprise a similar or identical amino acid sequence of
a flaviviral, including WNV polypeptide, a fragment of a
flaviviral, including WNV polypeptide, an antibody, or antibody
fragment, or possess a similar or identical structure of a
flaviviral, including WNV polypeptide, a fragment of a flaviviral,
including WNV polypeptide, an antibody, or antibody fragment. A
polypeptide that has a similar amino acid sequence refers to a
polypeptide that satisfies at least one of the following: (a) a
polypeptide having an amino acid sequence that is at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95% or at least 99%
identical to the amino acid sequence of a flaviviral, including WNV
polypeptide, a fragment of a flaviviral, including WNV polypeptide,
an antibody, or antibody fragment described herein; (b) a
polypeptide encoded by a nucleotide sequence that hybridizes under
stringent conditions to a nucleotide sequence encoding a
flaviviral, including WNV, polypeptide, a fragment of a flaviviral,
including WNV, polypeptide, an antibody, or antibody fragment
described herein of at least 5 amino acid residues, at least 10
amino acid residues, at least 15 amino acid residues, at least 20
amino acid residues, at least 25 amino acid residues, at least 30
amino acid residues at least 40 amino acid residues, at least 50
amino acid residues, at least 60 amino residues, at least 70 amino
acid residues, at least 80 amino acid residues, at least 90 amino
acid residues, at least 100 amino acid residues, at least 125 amino
acid residues, or at least 150 amino acid residues; and (c) a
polypeptide encoded by a nucleotide sequence that is at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95% or at least 99%
identical to the nucleotide sequence encoding a flaviviral,
including WNV, polypeptide, a fragment of a flaviviral, including
WNV, polypeptide, an antibody, or antibody fragment described
herein. A polypeptide with similar structure to a flaviviral,
including WNV, polypeptide, a fragment of a flaviviral, including
WNV, polypeptide, an antibody, or antibody fragment described
herein refers to a polypeptide that has a similar secondary,
tertiary or quaternary structure of a WNV polypeptide, a fragment
of a flaviviral, including WNV, an antibody, or antibody fragment
described herein. The structure of a polypeptide can determined by
methods known to those skilled in the art, including but not
limited to, X-ray crystallography, nuclear magnetic resonance, and
crystallographic electron microscopy.
[0035] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino acid or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=number of identical overlapping
positions/total number of positions.times.100%). In one embodiment,
the two sequences are the same length. The determination of percent
identity between two sequences can also be accomplished using a
mathematical algorithm. A preferred, non-limiting example of a
mathematical algorithm utilized for the comparison of two sequences
is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad.
Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993,
Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et
al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be
performed with the NBLAST nucleotide program parameters set, e.g.,
for score=100, wordlength=12 to obtain nucleotide sequences
homologous to a nucleic acid molecules of the present invention.
BLAST protein searches can be performed with the XBLAST program
parameters set, e.g., to score-50, wordlength=3 to obtain amino
acid sequences homologous to a protein molecule of the present
invention. To obtain gapped alignments for comparison purposes,
Gapped BLAST can be utilized as described in Altschul et al., 1997,
Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be
used to perform an iterated search which detects distant
relationships between molecules (Id.). When utilizing BLAST, Gapped
BLAST, and PSI-Blast programs, the default parameters of the
respective programs (e.g., of XBLAST and NBLAST) can be used.
Another preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated
in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be
used. The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0036] As used herein, the terms "antibody" and "antibodies" refer
to monoclonal antibodies, multispecific antibodies, human
antibodies, humanized antibodies, synthetic antibodies, chimeric
antibodies, camelized antibodies, single-chain Fvs (scFv), single
chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked
Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies
(including, e.g., anti-Id and anti-anti-Id antibodies to antibodies
of the invention), bispecific, and epitope-binding fragments of any
of the above. In particular, antibodies include immunoglobulin
molecules and immunologically active fragments of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site.
Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA, and IgA.sub.2) or subclass.
[0037] As used herein, the term "antibodies or fragments that
immunospecifically bind to a flaviviral antigen" refers to
antibodies or fragments thereof that specifically bind to a
flaviviral polypeptide or a fragment of a flaviviral polypeptide
and do not non-specifically bind to other polypeptides. Antibodies
or fragments that immunospecifically bind to a flaviviral
polypeptide or fragment thereof may have cross-reactivity with
other antigens. Preferably, antibodies or fragments that
immunospecifically bind to a flaviviral polypeptide or fragment
thereof do not cross-react with other antigens. Antibodies or
fragments that immunospecifically bind to a flaviviral polypeptide
can be identified, for example, by immunoassays or other techniques
known to those of skill in the art.
[0038] As used herein, the term "derivative" as used herein refers
to a polypeptide that comprises an amino acid sequence of a
flaviviral polypeptide, including WNV polypeptide, a fragment of a
flaviviral polypeptide, including WNV polypeptide, an antibody that
immunospecifically binds to a flaviviral polypeptide, including WNV
polypeptide, or an antibody fragment that immunospecifically binds
to a flaviviral polypeptide, including WNV polypeptide, which has
been altered by the introduction of amino acid residue
substitutions, deletions or additions. The term "derivative" as
used herein also refers to a flaviviral polypeptide, including WNV
polypeptide, a fragment of a flaviviral polypeptide, including WNV
polypeptide, an antibody that immunospecifically binds to a
flaviviral polypeptide, including WNV polypeptide, or an antibody
fragment that immunospecifically binds to a flaviviral polypeptide,
including WNV polypeptide, which has been modified, i.e, by the
covalent attachment of any type of molecule to the polypeptide. For
example, but not by way of limitation, a flaviviral polypeptide,
including WNV polypeptide, a fragment of a flaviviral polypeptide,
including WNV polypeptide, an antibody, or antibody fragment may be
modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. A derivative of a flaviviral
polypeptide, including WNV polypeptide, a fragment of a flaviviral
polypeptide, including WNV polypeptide, an antibody, or antibody
fragment may be modified by chemical modifications using techniques
known to those of skill in the art, including, but not limited to,
specific chemical cleavage, acetylation, formylation, metabolic
synthesis of tunicamycin, etc. Further, a derivative of a
flaviviral polypeptide, including WNV polypeptide, a fragment of a
flaviviral polypeptide, including WNV polypeptide, an antibody, or
antibody fragment may contain one or more non-classical amino
acids. A polypeptide derivative possesses a similar or identical
function as a flaviviral polypeptide, including WNV polypeptide, a
fragment of a flaviviral polypeptide, including WNV polypeptide, an
antibody, or antibody fragment described herein.
[0039] As used herein, the terms "disorder" and "disease" are used
interchangeably to refer to a condition in a subject.
[0040] As used herein, the term "effective neutralizing titer" as
used herein refers to the amount of antibody which corresponds to
the amount present in the serum of animals that has been shown to
be either clinically efficacious (in humans) or to reduce virus by
50%, 80%, 90% or 99% in, for example, mice. The 99% reduction is
defined by a specific challenge of, e.g., 10.sup.3 pfu, 10.sup.4
pfu, 10.sup.5 pfu, 10.sup.6 pfu, 10.sup.7 pfu, 10.sup.8 pfu, or
10.sup.9 pfu of a flavivirus, e.g., a WNV, or by the relative
amount of virus present in the blood of an animal before and after
therapeutic intervention. The terms "effective neutralizing titer"
or "neutralizing titer" also refers to the titer of antibody that
results in a given (e.g., 90%) reduction in the number of cells
producing infectious virus using the plaque reduction assay, which
is an in vitro assay and evaluates the ability of a given
concentration of antibody to inhibit 50 (PRNT50) or 90 (PRNT90) %
of infection in BHK21 or Vero cells.
[0041] As used herein, the term "epitopes" refers to portions of a
flavivirus, including WNV polypeptide having antigenic or
immunogenic activity in an animal, preferably a mammal, and most
preferably in a human. An epitope having immunogenic activity is a
portion of a flavivirus, including WNV, polypeptide that elicits an
antibody response in an animal. An epitope having antigenic
activity is a portion of a flavivirus, including WNV, polypeptide
to which an antibody immunospecifically binds as determined by any
method well known in the art, for example, by the immunoassays
described herein. Antigenic epitopes need not necessarily be
immunogenic.
[0042] As used herein, the term "flaviviral antigen" refers to a
flaviviral polypeptide or fragment thereof to which an antibody or
antibody fragment immunospecifically binds. A flaviviral antigen
also refers to an analog or derivative of a flaviviral polypeptide
or fragment thereof to which an antibody or antibody fragment
immunospecifically binds. In a preferred embodiment, a flaviviral
antigen is a WNV E, a fragment, an analog or a derivative thereof
to which an antibody or antibody fragment immunospecifically
binds.
[0043] As used herein, the term "fragment" refers to a peptide or
polypeptide comprising an amino acid sequence of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 30 contiguous amino acid residues, at least 35
contiguous amino acid residues, at least 40 contiguous amino acid
residues, at least 50 contiguous amino acid residues, at least 60
contiguous amino acid residues, at least 70 contiguous amino acid
residues, at least contiguous 80 amino acid residues, at least
contiguous 90 amino acid residues, at least contiguous 100 amino
acid residues, at least contiguous 125 amino acid residues, at
least 150 contiguous amino acid residues, at least contiguous 175
amino acid residues, at least contiguous 200 amino acid residues,
or at least contiguous 250 amino acid residues of the amino acid
sequence of a flavivirus, including WNV, polypeptide or an antibody
that immunospecifically binds to a flavivirus, including WNV,
polypeptide. In certain embodiments, a fragment refers to a peptide
or polypeptide comprising an amino acid sequence of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 40 contiguous amino acid residues, or at least
50 contiguous amino acid residues of a WNV structural or
non-structural protein. In other embodiments, a fragment refers to
a peptide or polypeptide comprising an amino acid of at least 5
contiguous amino acid residues, at least 10 contiguous amino acid
residues, at least 15 contiguous amino acid residues, at least 20
contiguous amino acid residues, at least 25 contiguous amino acid
residues, at least 30 contiguous amino acid residues, at least 35
contiguous amino acid residues, at least 40 contiguous amino acid
residues, or at least 50 contiguous amino acid residues of a VH
and/or VL domain of an antibody that immunospecifically binds to a
flavivirus, including WNV, polypeptide. Preferably, a fragment of a
flavivirus, including WNV, polypeptide or a fragment of an antibody
that immunospecifically binds to a flavivirus, including WNV,
polypeptide retains at least one function of said flavivirus,
including WNV, polypeptide or antibody.
[0044] As used herein, the term "fusion protein" refers to a
peptide, polypeptide or protein that comprises an amino acid
sequence of an antibody or fragment thereof that immunospecifically
binds to a flavivirus, including WNV, antigen and an amino acid
sequence of a heterologous peptide, polypeptide or protein. In
certain embodiments, a fusion protein retains the ability to
immunospecifically bind to a flavivirus, including WNV, antigen. In
other embodiments, a fusion protein does not retain the ability to
immunospecifically bind to a flavivirus, including WNV,
antigen.
[0045] As used herein, the term "host" as used herein refers to a
mammal, preferably a human.
[0046] As used herein, the term "host cell" refers to the
particular subject cell transfected with a nucleic acid molecule
and the progeny or potential progeny of such a cell. Progeny of
such a cell may not be identical to the parent cell transfected
with the nucleic acid molecule due to mutations or environmental
influences that may occur in succeeding generations or integration
of the nucleic acid molecule into the host cell genome.
[0047] As used herein, the term "humanized antibody" refers to an
immunoglobulin comprising a human framework region and one or more
CDR's from a non-human (usually a mouse or rat) immunoglobulin. The
non-human immunoglobulin providing the CDR's is called the "donor"
and the human immunoglobulin providing the framework is called the
"acceptor". Constant regions need not be present, but if they are,
they must be substantially identical to human immunoglobulin
constant regions, i.e., at least about 85-90%, preferably about 95%
or more identical. Hence, all parts of a humanized immunoglobulin,
except possibly the CDR's, are substantially identical to
corresponding parts of natural human immunoglobulin sequences. A
"humanized antibody" is an antibody comprising a humanized light
chain and a humanized heavy chain immunoglobulin. For example, a
humanized antibody would not encompass a typical chimeric antibody,
because, e.g., the entire variable region of a chimeric antibody is
non-human. The purpose of humanization is to construct an antibody
that has the binding characteristics of a previously generated
antibody that binds to a desired target but is immunologically
recognized as a self antigen by the immune system of the human
patient to whom it is administered. For the most part, humanized
antibodies are human immunoglobulins (recipient or acceptor
antibody) in which hypervariable region residues of the recipient
are replaced by hypervariable region residues from a non-human
species (donor antibody) such as mouse, rat, rabbit or a non-human
primate having the desired specificity, affinity, and capacity. In
some instances, Framework Region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are
not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FRs are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin, that immunospecifically binds to one or more
flaviviral antigens, that has been altered by the introduction of
amino acid residue substitutions, deletions or additions (i.e.,
mutations). In some embodiments, a humanized antibody is a
derivative. Such a humanized antibody comprises amino acid residue
substitutions, deletions or additions in one or more non-human
CDRs. The humanized antibody derivative may have substantially the
same binding, better binding, or worse binding when compared to a
non-derivative humanized antibody. In specific embodiments, one,
two, three, four, or five amino acid residues of the CDR have been
substituted, deleted or added (i.e., mutated). For further details
in humanizing antibodies, see European Patent Nos. EP 239,400, EP
592,106, and EP 519,596; International Publication Nos. WO 91/09967
and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101, 5,565,332,
5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular
Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein
Engineering 7(6):805-814; Roguska et al., 1994, Proc Natl Acad Sci
USA 91:969-973; Tan et al., 2002, J. Immunol. 169:1119-25; Caldas
et al., 2000, Protein Eng. 13:353-60; Morea et al., 2000, Methods
20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-84; Roguska
et al., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer
Res. 55 (23 Supp):5973s-5977s; Couto et al., 1995, Cancer Res.
55:1717-22; Sandhu, 1994, Gene 150:409-10; Pedersen et al., 1994,
J. Mol. Biol. 235:959-73; Jones et al., 1986, Nature 321:522-525;
Reichmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr.
Op. Struct. Biol. 2:593-596.
[0048] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody which are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "Complementarity Determining Region" or "CDR"
(i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in
the heavy chain variable domain (Kabat et al., Sequences of
proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e., residues 26-32 (L1),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and
26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable
domain (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).
"Framework Region" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0049] As used herein, the term "in combination" refers to the use
of more than one prophylactic and/or therapeutic agents. The use of
the term "in combination" does not restrict the order in which
prophylactic and/or therapeutic agents are administered to a
subject with a disorder. A first prophylactic or therapeutic agent
can be administered prior to (e.g., 1 minute, 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second prophylactic or therapeutic agent to a
subject which had, has, or is susceptible to a disorder. The
prophylactic or therapeutic agents are administered to a subject in
a sequence and within a time interval such that the agent of the
invention can act together with the other agent to provide an
increased benefit than if they were administered otherwise. Any
additional prophylactic or therapeutic agent can be administered in
any order with the other additional prophylactic or therapeutic
agents.
[0050] An "isolated" or "purified" antibody or fragment thereof is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free of chemical precursors or other
chemicals when chemically synthesized. The language "substantially
free of cellular material" includes preparations of an antibody or
antibody fragment in which the antibody or antibody fragment is
separated from cellular components of the cells from which it is
isolated or recombinantly produced. Thus, an antibody or antibody
fragment that is substantially free of cellular material includes
preparations of antibody or antibody fragment having less than
about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein
(also referred to herein as a "contaminating protein"). When the
antibody or antibody fragment is recombinantly produced, it is also
preferably substantially free of culture medium, i.e., culture
medium represents less than about 20%, 10%, or 5% of the volume of
the protein preparation. When the antibody or antibody fragment is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
antibody or antibody fragment have less than about 30%, 20%, 10%,
5% (by dry weight) of chemical precursors or compounds other than
the antibody or antibody fragment of interest. In a preferred
embodiment, humanized antibodies of the invention or fragments
thereof are isolated or purified.
[0051] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
In a preferred embodiment, nucleic acid molecules encoding
humanized antibodies of the invention or fragments thereof are
isolated or purified.
[0052] As used herein, the terms "manage," "managing" and
"management" refer to the beneficial effects that a subject derives
from administration of a prophylactic or therapeutic agent, which
does not result in a cure of the disease. In certain embodiments, a
subject is administered one or more prophylactic or therapeutic
agents to "manage" a disease so as to prevent the progression or
worsening of the disease.
[0053] As used herein, the terms "nucleic acids" and "nucleotide
sequences" include DNA molecules (e.g., cDNA or genomic DNA), RNA
molecules (e.g., mRNA), combinations of DNA and RNA molecules or
hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such
analogs can be generated using, for example, nucleotide analogs,
which include, but are not limited to, inosine or tritylated bases.
Such analogs can also comprise DNA or RNA molecules comprising
modified backbones that lend beneficial attributes to the molecules
such as, for example, nuclease resistance or an increased ability
to cross cellular membranes. The nucleic acids or nucleotide
sequences can be single-stranded, double-stranded, may contain both
single-stranded and double-stranded portions, and may contain
triple-stranded portions, but preferably is double-stranded
DNA.
[0054] As used herein, the phrases "a peptide, polypeptide or
protein comprising a variable or hypervariable region of an
antibody of the invention", "a peptide, polypeptide or protein
comprising a VH or VL domain of an antibody of the invention", "a
peptide, polypeptide or protein comprising one or more CDRs having
an amino acid sequence of one or more of the CDRs listed in SEQ ID
NOS: 1, 2, 3, 11, 12, 13, 27, 28, 29, 34, 39, and 40, and analogous
phrases, refer to fusion proteins.
[0055] As used herein, the terms "prevent", "preventing" and
"prevention" refer to the prevention of the occurrence and/or
recurrence or onset of one or more symptoms of a disorder in a
subject resulting from the administration of a prophylactic or
therapeutic agent.
[0056] As used herein, the terms "prophylactic agent" and
"prophylactic agents" refer to any agent(s) which can be used in
the prevention of a disorder, or prevention of recurrence or spread
of a disorder. A prophylactically effective amount may also refer
to the amount of the prophylactic agent that provides a
prophylactic benefit in the prevention of disease. Further, a
prophylactically effective amount with respect to a prophylactic
agent of the invention means that amount of prophylactic agent
alone, or in combination with other agents, that provides a
prophylactic benefit in the prevention of disease. Used in
connection with an amount of an antibody of the invention, the term
can encompass an amount that improves overall prophylaxis or
enhances the prophylactic efficacy of or synergizes with another
prophylactic agent, such as, but not limited to, a therapeutic
antibody.
[0057] In certain embodiments of the invention, a "prophylactically
effective serum titer" is the serum titer in a mammal, preferably a
human, that reduces the incidence of a flaviviral infection in said
mammal. Preferably, the prophylactically effective serum titer
reduces the incidence of flaviviral infections in humans with the
greatest probability of complications resulting from flaviviral
infection (e.g., a human infant, or an elderly human or a human
with an impaired immune system). In certain other embodiments of
the invention, a "prophylactically effective serum titer" is the
serum titer in a mouse model that results in a flaviviral titer 3
days after challenge with 10.sup.3 pfu that is 99% lower than the
flaviviral titer 3 days after challenge with 10.sup.3 pfu of
flaviviral in the same strain of mouse not administered an antibody
or antibody fragment that immunospecifically binds to a flaviviral
antigen.
[0058] As used herein, the phrase "side effects" encompasses
unwanted and adverse effects of a prophylactic or therapeutic
agent. Adverse effects are always unwanted, but unwanted effects
are not necessarily adverse. An adverse effect from a prophylactic
or therapeutic agent might be harmful or uncomfortable or risky.
Side effects from chemotherapy include, but are not limited to,
gastrointestinal toxicity such as, but not limited to, early and
late-forming diarrhea and flatulence, nausea, vomiting, anorexia,
leukopenia, anemia, neutropenia, asthenia, abdominal cramping,
fever, pain, loss of body weight, dehydration, alopecia, dyspnea,
insomnia, dizziness, mucositis, xerostomia, and kidney failure, as
well as constipation, nerve and muscle effects, temporary or
permanent damage to kidneys and bladder, flu-like symptoms, fluid
retention, and temporary or permanent infertility. Side effects
from radiation therapy include, but are not limited to, fatigue,
dry mouth, and loss of appetite. Side effects from biological
therapies/immunotherapies include, but are not limited, to rashes
or swellings at the site of administration, flu-like symptoms such
as fever, chills and fatigue, digestive tract problems and allergic
reactions. Side effects from hormonal therapies include, but are
not limited to, nausea, fertility problems, depression, loss of
appetite, eye problems, headache, and weight fluctuation.
Additional undesired effects typically experienced by patients are
numerous and known in the art, see, e.g., the Physicians' Desk
Reference (56.sup.th ed., 2002), which is incorporated herein by
reference in its entirety.
[0059] As used herein, the terms "single-chain Fv" or "scFv" refer
to antibody fragments that comprise the VH and VL domains of the
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the
scFv to form the desired structure for antigen binding. For a
review of scFv, see Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New
York, pp. 269-315 (1994). In specific embodiments, scFvs include
bi-specific scFvs and humanized scFvs.
[0060] As used herein, the term "specifically binds to a flaviviral
antigen" and analogous terms refer to antibodies or fragments
thereof that specifically bind to a flaviviral antigen or fragment
thereof and do not specifically bind to other viral antigens.
Examples of flaviviral antigens include, but are not limited to,
structural proteins, e.g., C, M, and E, and non-structural
proteins, e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. An
antibody that specifically binds to a flaviviral antigen or
fragment thereof may bind to other peptides or polypeptides with
lower affinity as determined by, e.g., immunoassays, BIAcore, or
other assays known in the art. Preferably, antibodies or fragments
that specifically bind to to a flaviviral antigen or fragment
thereof do not cross-react with other antigens. Antibodies or
fragments that specifically bind to a flaviviral antigen or
fragment thereof can be identified, for example, by immunoassays,
BIAcore, or other techniques known to those of skill in the art. An
antibody or a fragment thereof binds specifically to a flaviviral
antigen or fragment thereof with higher affinity than to any
cross-reactive antigen as determined using experimental techniques,
such as western blots, radioimmunoassays (RIA) and enzyme-linked
immunosorbent assays (ELISAs). See, e.g., Paul, ed., 1989,
Fundamental Immunology Second Edition, Raven Press, New York at
pages 332-336 for a discussion regarding antibody specificity.
[0061] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, a subject is preferably a mammal
such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats
etc.) and a primate (e.g., monkey and human), most preferably a
human.
[0062] As used herein, a "therapeutically effective amount" refers
to that amount of the therapeutic agent sufficient to treat or
manage flaviviral infection or to enhance the therapeutic efficacy
of another therapy, e.g., therapeutic antibody, vaccine therapy,
etc. A therapeutically effective amount may refer to the amount of
therapeutic agent sufficient to delay or minimize the onset of
disease. A therapeutically effective amount may also refer to the
amount of the therapeutic agent that provides a therapeutic benefit
in the treatment or management of a disease. Further, a
therapeutically effective amount with respect to a therapeutic
agent of the invention means that amount of therapeutic agent
alone, or in combination with other therapies, that provides a
therapeutic benefit in the treatment or management of a disease,
e.g., sufficient to enhance the therapeutic efficacy of a
therapeutic antibody sufficient to treat or manage a disease. Used
in connection with an amount of an antibody of the invention, the
term can encompass an amount that improves overall therapy, reduces
or avoids unwanted side effects, or enhances the therapeutic
efficacy of or synergizes with another therapeutic agent.
[0063] In certain embodiments of the invention, a "therapeutically
effective serum titer" is the serum titer in a mammal, preferably a
human, that reduces the severity, the duration and/or the symptoms
associated with a flaviviral infection in said mammal. Preferably,
the therapeutically effective serum titer reduces the severity, the
duration and/or the number of symptoms associated with flaviviral
infections in humans with the greatest probability of complications
resulting from a flaviviral infection (e.g., a human infant, an
elderly human or a human with an impaired immune system). In
certain other embodiments of the invention, a "therapeutically
effective serum titer" is the serum titer in a mouse model that
results in a flaviviral titer 3 days after challenge with 10.sup.2,
10.sup.3 or 10.sup.4 pfu that is 99% lower than the flaviviral
titer 3 days after challenge with 10.sup.2, 10.sup.3 or 10.sup.4
pfu of flaviviral in the same strain of mouse not administered an
antibody or antibody fragment that immunospecifically binds to a
flaviviral antigen.
[0064] As used herein, the terms "treat," "treating" and
"treatment" refer to the eradication, reduction or amelioration of
symptoms of a disease or disorder related to a flaviviral
infection, e.g., a WNV infection.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIGS. 1A and B AMINO ACID ALIGNMENTS OF E16 HEAVY AND LIGHT
CHAINS
[0066] A. The comparison of the murine WNV E16 VH (muE16VH) and the
humanized WNV E16 VH (huE16VH-1). Framework regions from the human
VH1-18 segment used in the humanization scheme are indicated.
[0067] B. The comparison of the murine WNV E16 VL (muE16VL) and the
humanized WNV E16 VL (huE16VL-1). Framework regions from the human
VK-B3 segment used in the humanization scheme are indicated.
[0068] FIGS. 2A AND B BINDING OF CHIMERIC, HUMANIZED OR HYBRID
ANTI-WNV MAB E16 TO ANTIGEN
[0069] A. Antibody binding in direct antigen ELISA. Chimeric,
humanized, chE16LC/huE16HC or huE16LC/chE16HC E16 antibody was
obtained from conditioned media of transfected HEK-293 cultures.
Conditioned media was serially diluted into wells of a 96 well
plate previously coated with 100 ng/well of WNV E-protein Domain
III. Binding was detected by HRP conjugated F(ab').sub.2 goat anti
human IgG F(ab').sub.2 specific secondary antibody, and the
OD.sub.450 nm was read by SOFTmax program.
[0070] B. Antibody binding in antigen capture ELISA. Chimeric,
humanized, chE16LC/huE16HC or huE16LC/chE16HC E16 antibody was
obtained from conditioned media of transfected HEK-293 cultures. A
96 well plate was prepared by coating each well with murine
anti-WNV E protein antibody E9 followed by incubation with 2.5
ng/well of WNV E-protein Domain III. Conditioned media was serially
diluted into the prepared wells and binding detected by HRP
conjugated F(ab').sub.2 goat anti human IgG F(ab').sub.2 specific
secondary antibody. The OD.sub.450 nm was read by SOFTmax
program.
[0071] FIGS. 3A AND B BINDING OF HUMANIZED ANTI-WNV MAB E16 LIGHT
CHAIN VARIANTS TO ANTIGEN
[0072] A. Antibody binding in direct antigen ELISA. Chimeric and
light-chain variants of humanized E16 antibody, huE16HC/huE16LC
(huE16-1.1) or huE16HC/huE16LC-2 Y49S (huE16-1.2), were obtained
from conditioned media of transfected HEK-293 cultures. Conditioned
media was serially diluted into wells of a 96 well plate previously
coated with 100 ng/well of WNV E-protein Domain III. Binding was
detected by HRP conjugated F(ab').sub.2 goat anti human IgG
F(ab').sub.2 specific secondary antibody, and the OD.sub.450 nm was
read by SOFTmax program.
[0073] B. Antibody binding in antigen capture ELISA. Chimeric and
light-chain variants of humanized E16 antibody, huE16HC/huE16LC
(huE16-1.1) or huE16HC/huE16LC-2 Y49S (huE16-1.2), were obtained
from conditioned media of transfected HEK-293 cultures. A 96 well
plate was prepared by coating each well with murine anti-WNV E
protein antibody E9 followed by incubation with 2.5 ng/well of WNV
E-protein Domain III. Conditioned media was serially diluted into
the prepared wells and binding detected by HRP conjugated
F(ab').sub.2 goat anti human IgG F(ab').sub.2 specific secondary
antibody. The OD.sub.450 nm was read by SOFTmax program.
[0074] FIGS. 4 AND 5 BINDING OF HUMANIZED ANTI-WNV MAB E16 HEAVY
CHAIN VARIANTS TO ANTIGEN
[0075] Antibody binding in antigen capture ELISA. A 96 well plate
was prepared by coating each well with murine anti-WNV E protein
antibody E9 followed by incubation with 2.5 ng/well of WNV
E-protein Domain III. Conditioned media was serially diluted into
the prepared wells and binding detected by HRP conjugated
F(ab').sub.2 goat anti human IgG F(ab').sub.2 specific secondary
antibody. The OD.sub.450 nm was read by SOFTmax program. Results
from FIG. 3B are included for purposes of comparison.
[0076] 4. Chimeric and heavy-chain variants of humanized E16
antibody; huE16HC-2 V67A, M69F, T71A/huE16LC (huE16-2.1) or
huE16HC-2 V67A, M69F, T71A/huE16LC2 Y49S (huE16-2.2); obtained from
conditioned media of transfected HEK-293 cultures. Results from
FIG. 3B are included for purposes of comparison.
[0077] 5. Chimeric and heavy-chain variants of humanized E16
antibody, huE16HC-2 T71A/huE16LC (huE16-3.1) or huE16HC-2
T71A/huE16LC2 Y49S (huE16-3.2), obtained from conditioned media of
transfected HEK-293 cultures. Results from FIG. 3B are included for
purposes of comparison.
[0078] FIGS. 6, 7, 8, 9, AND 10 PROPHYLAXIS AND THERAPEUTIC STUDIES
OF ANTI-WNV ANTIBODIES IN A MURINE MODEL OF WNV
[0079] To establish the WNV disease model, 5-week old mice were
inoculated with 10.sup.2 PFU WNV via footpad injection.
[0080] 6. Prophylaxis of immune human .gamma.-globulin. Treatment
with a single IP 15 mg dose of purified immune human
.gamma.-globulin against WNV immediately prior to inoculation (day
0) or at days 1, 2, 3, 4, 5 (DO, D1, D2, D3, D4, D5, respectively)
post-infection.
[0081] 7A. Prophylaxis of murine E16, E24 and E34. Treatment at day
2 post-infection with a single IP 0.5 mg dose of murine anti-WNV
mAb E16, E24 or E34. A single IP 0.5 mg dose of irrelevant
humanized IgG, anti-SARSORF7a, served as control.
[0082] 7B. Dose response of murine anti-WNV mAb E16 prophylaxis.
Treatment at day 4 post-infection with a single 0.8, 4, 20, 100 or
500 .mu.g IP dose of murine anti-WNV mAb E16.
[0083] 8. Prophylaxis of murine E16, E24 and E34. Treatment at day
5 post-infection with a single IP 5 mg dose of murine anti-WNV mAb
E16 or E24.
[0084] 9A. Dose response of humanized anti-WNV mAb E16H-173
(huE16-1.2) therapy. Treatment at day 2 post-infection with a
single 4, 20 or 100 .mu.g IP dose of humanized anti-WNV mAb
E16H-173.
[0085] 9B. Dose response of humanized anti-WNV mAb E16H-167
(huE16-1.1) therapy. Treatment at day 2 post-infection with a
single 4, 20 or 100 .mu.g IP dose of humanized anti-WNV mAb
E16H-167.
[0086] 10. Dose response of humanized anti-WNV mAB hE16-3.2
prophylaxis. Prophylaxis at one day pre-infection with a 0.03, 0.1,
0.3, 1.0 or 3.0 mg/kg IP dose of humanized anti-WNV mAB
hE16-3.2.
5. DETAILED DESCRIPTION OF THE INVENTION
[0087] The present invention provides humanized antibodies, or
antigen binding fragments thereof that immunospecifically bind to
one or more flaviviral antigens, preferably WNV antigens.
Preferably, the humanized antibodies of the invention or fragments
thereof immunospecifically bind to one or more flaviviral antigens,
preferably WNV antigens, regardless of the strain of the virus. In
some embodiments, the humanized antibodies of the invention bind
with similar affinities and/or avidities to all WNV strains
including lineage I and II strains such as North American West Nile
strains including those related to the New York 1999 strain.
[0088] The present invention provides numerous humanized antibodies
specific for WNV based on the discovery that the CDR regions of the
murine monoclonal antibody could be spliced into a human acceptor
framework so as to produce a humanized recombinant antibody
specific for the WNV. Preferred humanized WNV specific antibodies
contain one or more additional changes in the framework region (or
in other regions) to increasing binding for WNV. Particularly
preferred embodiments of the invention are the exemplified
humanized antibody molecules that have superior binding properties
for WNV.
[0089] In most preferred embodiments, the present invention
provides humanized antibodies that immunospecifically bind a
structural protein of WNV, e.g., E protein, for prevention and/or
treatment of WNV infections in mammals. In a specific embodiment,
the humanized antibodies of the invention bind to the ectodomain of
WNV E protein, as determined by standard methods known to one
skilled in the art and exemplified herein, e.g., yeast two hybrid
system, ELISA, immunoprecipitation, immunoblotting. In another
specific embodiment, the humanized antibodies of the invention bind
to domain III of the WNV E protein, comprising amino acids 290 to
415. In other specific embodiments, the humanized antibodies of the
invention bind to the viral fusion peptide in domain II, comprising
amino acids 98-109, or to other regions in domain I (e.g., amino
acids 1-52, 132-193, and 280-290), or domain II (e.g., amino acids
52-132 and 193-280).
[0090] In some embodiments, the humanized antibodies of the
invention bind to one or more epitopes of a structural protein
and/or one or more epitopes of a non-structural protein of a WNV.
In other embodiments, the present invention also provides humanized
antibodies or fragments thereof that differentially or
preferentially bind to flaviviral antigens from one strain of the
flavivirus versus another strain.
[0091] In most preferred embodiments, the invention encompasses
humanized antibodies or fragments thereof that have potent
neutralizing activity as measured for example using standard
methods known in the art, e.g., in vitro plaque reduction
neutralization titer (PRNT) assay. Although not intending to be
bound by a particular mechanism of action the humanized antibodies
of the invention may directly neutralize virus or block entry of
the virus into the cell, thus treating or preventing viral
infections. In some embodiments, the invention encompasses
humanized antibodies which immunospecifically bind WNV-E protein
such that the PRNT.sub.50 values are at least 1/500, preferably at
least 1/10,000 at a concentration of 1 mg/mL. PRNT assays may be
done using any method known to one skilled in the art, such as
those described in Diamond et al., 2003, J. Virol. 77: 2578-2586,
which is incorporated herein by reference in its entirety.
[0092] In yet other preferred embodiments, humanized antibodies of
the invention have enhanced antibody-dependent complement mediated
neutralization of WNV virions and trigger lysis of WNV-infected
cells more effectively, as determined using standard methods known
in the art and exemplified herein, such as complement fixation and
cell viability assays. Although not intending to be bound by a
particular mechanism of action the humanized antibodies of the
invention have enhanced clinical efficacy, therapeutically and
prophylactically as they have enhanced effector functions,
neutralize virus attachment, trigger complement mediated lysis,
promote clearance from the circulatory systems and prevent
emergence of viral resistance. The humanized antibodies of the
invention preferably have a potent in vivo inhibitory activity,
i.e., protect against WNv infection by at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 99%.
[0093] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens and have an apparent
dissociation constant of about 1-10 nM, as determined by a sandwich
ELISA. The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens and have an K.sub.on
rate of about 1.times.10.sup.4, about 5.times.10.sup.4, about
1.times.10.sup.5, about 5.times.10.sup.5, about 1.times.10.sup.6,
or about 5.times.10.sup.6 and a K.sub.off rate of about
1.times.10.sup.-3, about 5.times.10.sup.-4, about
1.times.10.sup.-4, about 5.times.10.sup.-5 about 1.times.10.sup.-5,
about 5.times.10.sup.-6, or about 1.times.10.sup.-6 as measured by
surface plasmon resonance (SPR) using a BIAcore sensor.
[0094] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens and have a median
effective concentration (EC.sub.50) of less than 1 .mu.g/ml, in an
in vitro microneutralization assay. In particular, the present
invention provides compositions for use in the prevention,
treatment or amelioration of one or more symptoms associated with a
flaviviral infection, said compositions comprising one or more
humanized antibodies or fragments thereof which immunospecifically
bind to one or more one or more flaviviral antigens particularly
WNV antigens and have an EC.sub.50 of less than 0.01 nM, less than
0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM,
less than 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25
nM, less than 1.5 nM, less than 1.75 nM, or less than 2 nM, in an
in vitro microneutralization assay.
[0095] The present invention also provides humanized antibodies
which immunospecifically bind to one or more flaviviral antigens,
particularly WNV antigens, and have increased in vivo half-lives
(by for example 30 days) relative to known antibodies. In
particular, the present invention encompasses humanized antibodies
which immunospecifically bind to one or more flaviviral antigens,
particularly WNV antigens, and have increased in vivo half-lives
relative to known antibodies, said increased half-lives resulting
from one or more modifications (e.g., substitutions, deletions, or
insertions) in amino acid residues identified to be involved in the
interaction of the Fc domain of said antibodies and the FcRn
receptor. The present invention also encompasses pegylated
humanized antibodies and fragments thereof which immunospecifically
bind to one or more flaviviral antigens, particularly WNV antigens,
and have increased in vivo half-lives relative to known antibodies.
The increased in vivo half-lives of humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens reduce the dosage
and/or frequency of administration of said humanized antibodies or
fragments thereof to a subject.
[0096] In one specific preferred embodiment, the humanized
antibodies of the invention bind to the WNV E protein. In another
specific embodiment, the humanized antibodies of the invention
specifically or selectively recognize one or more epitopes of WNV E
protein. Another embodiment of the invention encompasses the use of
phage display technology, DNA shuffling, or any other similar
method known to one skilled in the art, to increase the affinity of
the humanized antibodies of the invention for WNV E protein. Any
screening method known in the art can be used to identify mutant
antibodies with increased avidity for WNV E protein (e.g., ELISA).
In another specific embodiment, humanized antibodies of the
invention are screened using antibody screening assays well known
in the art (e.g., BIACORE assays) to identify antibodies with
K.sub.off rate of about 1.times.10.sup.-3, about 5.times.10.sup.-4,
about 1.times.10.sup.-4, about 5.times.10.sup.-5, about
1.times.10.sup.-5, about 5.times.10.sup.-6, or about
1.times.10.sup.-6.
[0097] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens and have an
association rate constant or k, rate (antibody (Ab)+antigen
(Ag).sup.5.sup.on .fwdarw.Ab-Ag) of at least 1.times.10.sup.4,
about 5.times.10.sup.4, about 1.times.10.sup.5, about
5.times.10.sup.5, about 1.times.10.sup.6, or about
5.times.10.sup.6. In particular, the present invention provides
compositions for use in the prevention, treatment or amelioration
of one or more symptoms associated with a flaviviral infection,
said compositions comprising one or more humanized antibodies or
fragments thereof which immunospecifically bind to one or more one
or more flaviviral antigens particularly WNV antigens and have an a
k.sub.on rate of at least 1.times.10.sup.4, about 5.times.10.sup.4,
about 1.times.10.sup.5, about 5.times.10.sup.5, about
1.times.10.sup.6, or about 5.times.10.sup.6.
[0098] The present invention provides methods for treating,
preventing, or ameliorating a flaviviral infection by
administration of one or more humanized antibodies of the
invention. The present invention also provides methods of
preventing, treating and ameliorating one or more symptoms
associated with flaviviral infection, particularly WNV infection,
in a subject comprising administering to said subject one or more
humanized antibodies or fragments thereof which immunospecifically
bind to one or more flaviviral antigens particularly WNV antigens
with high affinity and/or high avidity. The humanized antibodies of
the invention are useful for prevention or treatment of a
flaviviral infection for example, in one embodiment, as a single
agent therapy. These methods can be used for achieving or inducing
a prophylactically and/or therapeutically effective response
against flaviviral infections including, but not limited to,
Japanese Encephalitis (JE, e.g., JE SA14-14-2), Dengue (DEN, e.g.,
any of the Dengue serotypes 1-4); Murray Valley encephalitis, St
Louis Encephalitis, West Nile, Tick borne encephalitis, Hepatitis C
viruses, Kunjin virus, Powassan virus, Kyasanur Forest Disease
virus, yellow fever virus, and Omsk Hemorrhagic Fever Virus. The
methods of the instant invention are more effective
prophylactically and therapeutically compared to conventional modes
of treatment or prophylaxis of flaviviral infections, particularly
WNV infections, including, but not limited to, passive
administration of immune serum or purified polyclonal antibody,
administration of .gamma.-globulin, interferon alpha therapy and
IVIG therapies. The methods and compositions of the present
invention are particularly effective for prophylaxis against
flaviviral infections in a human population which is at an
increased risk of flaviviral infections. In specific preferred
embodiments, the methods and compositions of the present invention
are particularly useful to a human population which is at an
increased risk for WNV infection including, but not limited to,
human infants, elderly humans, and human patients with an impaired
immune system.
[0099] Although not intending to be bound by a particular mechanism
of action, humanized antibodies of the invention are more effective
than current treatments against flaviviral infections such as, for
example, treatment using IVIG for WNV infections from donors with
high neutralizing titres. Because IVIG is made from human blood
plasma, it has an inherent risk of transmitting an infectious
agent. Although the source plasma donors are screened and the
plasma is solvent/detergent treated to inactivate viruses such as
HIV, virus removal and inactivation must be validated to remove a
wide variety of agents as a precaution; and the list of agents that
can be transmitted by blood grows with every emerging infection.
Even with all these precautions, there is never 100% assurance of
elimination of infectious agents. Finally, most preparations have
excipients such as human albumin, another blood product, and
sucrose, which can increase the risk of adverse events. Another
limitation of IVIG can be the large volumes needed, especially in
patients with cardiac or renal co-morbidities. In using a specific
immune globulin from vaccinated donors, while enriched for
antibodies to the target agent, most of the preparation contains
unrelated antibodies. The present invention cures the deficiency of
current IVIG regimens. Humanized antibodies of the instant
invention offer an inherently safer and potentially more
efficacious alternative to IVIG for the prevention and treatment of
flaviviral infections such as those caused by WNV. Additional
benefits of the humanized antibodies of the invention include, but
are not limited to, their ability to be grown in tissue culture
under defined conditions with chemically defined medium without the
addition of animal or human-derived proteins; unlike polyclonal
serum, they can be selected for desired properties including
epitope specificity, affinity and neutralizing capacity, allowing
lower doses; and they can be formulated at high concentration to
reduce the volume of administration.
[0100] In a specific embodiment, the invention encompasses methods
for treating, preventing, or ameliorating a WNV infection
comprising administering a first antibody that immunospecifically
binds a structural protein of WNV, e.g., E protein, and a second
antibody that binds a non-structural protein of WNV, e.g., NS1
protein. In other specific embodiments, the invention encompasses
methods for treating, preventing, or ameliorating a WNV infection
comprising administering a first antibody that immunospecifically
binds an epitope of a structural protein of WNV, e.g., E protein,
and a second antibody that binds the same structural protein of WNV
but binds at a distinct site.
[0101] The invention also encompasses polynucleotides that encode
the humanized antibodies of the invention. In one embodiment, the
invention provides an isolated nucleic acid sequence encoding a
heavy chain or a light chain of a humanized antibody or a fragment
thereof that specifically binds one or more flaviviral antigens,
particularly WNV antigens. The invention also relates to a vector
comprising said nucleic acid. The invention further provides a
vector comprising a first nucleic acid molecule encoding a heavy
chain and a second nucleic acid molecule encoding a light chain,
said heavy chain and light chain being of a humanized antibody or a
fragment thereof that specifically binds one or more flaviviral
antigens, particularly WNV antigens. In one specific embodiment,
said vector is an expression vector. The invention further provides
host cells containing vectors containing polynucleotides encoding
the humanized antibodies of the invention. Preferably, the
invention encompasses polynucleotides encoding heavy and light
chains of the humanized antibodies of the invention.
[0102] The invention further provides methods for the production of
humanized antibodies of the invention or fragments thereof. The
humanized antibodies of the invention or fragments thereof can be
produced by any method known in the art for the production of
humanized antibodies, in particular, by secretion from cultured
hybridoma cells, chemical synthesis or by recombinant expression
techniques known in the art. In one specific embodiment, the
invention relates to a method for recombinantly producing the
humanized antibodies of the invention, said method comprising: (i)
culturing under conditions suitable for the expression of said
antibody in a medium, a host cell containing a first nucleic acid
molecule, operably linked to a heterologous promoter and a second
nucleic acid operably linked to the same or a different
heterologous promoter, said first nucleic acid and second nucleic
acid encoding a heavy chain and a light chain, respectively, of an
antibody or a fragment thereof that specifically binds one or more
flaviviral antigens; and (ii) recovery of said antibody from said
medium.
[0103] The invention further provides a pharmaceutical composition
comprising (i) a therapeutically or prophylactically effective
amount of a humanized antibody of the invention; and (ii) a
pharmaceutically acceptable carrier.
[0104] In another embodiment, the invention provides a method of
diagnosis of a flaviviral infection in a subject comprising: (i)
contacting a biological sample from said subject with an effective
amount of a humanized antibody of the invention; and (ii) detecting
binding of said humanized antibody or a fragment thereof, wherein
detection of said detectable marker above a background or standard
level indicates that said subject has a flaviviral infection.
[0105] 5.1 Antibodies
[0106] The present invention encompasses humanized antibodies, or
antigen binding fragments thereof, that immunospecifically bind to
one or more flaviviral antigens, preferably WNV antigens.
Preferably, the humanized antibodies of the invention or fragments
thereof immunospecifically bind to one or more flaviviral antigens,
preferably WNV antigens regardless of the strain of the virus. In
some embodiments, the humanized antibodies of the invention bind
with similar affinities and/or avidities to all WNV strains
including lineage I and II strains such as North American strains
(e.g., the New York 1999 and related strains).
[0107] In most preferred embodiments, the present invention
provides humanized antibodies that immunospecifically bind a
structural protein of WNV, e.g., E protein, for prevention and/or
treatment of WNV infections in avians or mammals, particularly
humans. In a specific embodiment, the isolated humanized antibodies
of the invention bind to the ectodomain of WNV E protein, as
determined by standard methods known to one skilled in the art and
exemplified herein, e.g., ELISA, flow cytometry,
immunoprecipitation, immunoblot. In another specific embodiment,
the isolated humanized antibodies of the invention bind to domain
III of the WNV E protein, comprising amino acids 290 to 415, as
determined by standard methods known to one skilled in the art and
exemplified herein, e.g., ELISA, immunoprecipitation,
immunoblotting.
[0108] In some embodiments, the humanized antibodies of the
invention bind to one or more epitopes of a structural protein
and/or one or more epitopes of a non-structural protein of an WNV.
In other embodiments, the present invention also provides humanized
antibodies or fragments thereof that differentially or
preferentially bind to flaviviral antigens from one strain of the
flavivirus versus another strain.
[0109] In one particular embodiment, the humanized antibodies of
the invention are derived from a mouse monoclonal antibody produced
by clones E16, E24, or E34. Hybridomas producing antibodies E16,
E24, or E34 have been deposited with the American Type Culture
Collection (10801 University Blvd., Manassas, Va. 20110-2209) on
Jun. 4, 2004 under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of patent Procedures, and assigned accession numbers
PTA-6050, PTA-6051, and PTA-6052, respectively, and are
incorporated herein by reference. Representative plasmids encoding
humanized antibodies of the invention, e.g., pMGX623--humanized E16
light chain version 1, the vector is pCINeo (Invitrogen), the
insert consists of human germline sequence VKB2 and JK2 as
framework, human kappa as constant region and mouse E16 CDRs;
pMGX624--humanized E16 light chain version 2, same description as
pMGX623 except a Y49S mutation in the variable region;
pMGX625--humanized E16 heavy chain version 1, the vector is pCINeo
(Invitrogen), the insert consists of human germline sequence VH1-18
and JH6 as framework, human IgG1 as constant region, and mouse E16
CDRs; pMGX626--humanized E16 heavy chain version 2, same
description as pMGX625 except V67A, M69F, and T71A mutations in the
variable region; and pMGX627--humanized E16 heavy chain version 3,
same description as pMGX625 except a T71A mutation in the variable
region; having ATCC Accession numbers PTA-6199, PTA-6200, PTA-6201,
PTA-6202, and PTA-6203, respectively, were deposited under the
provisions of the Budapest Treaty with the American Type Culture
Collection (10801 University Blvd., Manassas, Va. 20110-2209) on
Sep. 10, 2004, and are incorporated herein by reference.
[0110] In preferred embodiments, the invention encompasses
humanized antibodies comprising the CDRs of E16, E24, or E34. In
some embodiments, the present invention provides humanized
antibodies or fragments thereof that immunospecifically bind to one
or more WNV antigens, said antibodies or antibody fragments
comprising a variable heavy ("VH") chain having an amino acid
sequence of any one of the VH domains listed in SEQ ID NOS: 21, 22,
or 23. The present invention also provides isolated humanized
antibodies or fragments thereof that immunospecifically bind to one
or more WNV antigens, said humanized antibodies or antibody
fragments comprising a VL domain having an amino acid sequence of
any one of the VL domains listed in SEQ ID NOS: 25 or 26.
[0111] In a specific embodiment, the invention encompasses a
humanized antibody comprising the CDRs of E16, E24, or E34. The
humanized WNV antibodies of the invention may have a heavy chain
variable region comprising the amino acid sequence of CDR1 (SEQ ID
NO: 1 or SEQ ID NO: 27) and/or CDR2 (SEQ ID NO: 2, SEQ ID NO: 28 or
SEQ ID NO: 39) and/or CDR3 (SEQ ID NO: 3, SEQ ID NO: 29 or SEQ ID
NO: 40) and/or a light chain variable region comprising the amino
acid sequence of CDR1 (SEQ ID NO: 11) and/or a CDR2 (SEQ ID NO: 12)
and/or CDR3 (SEQ ID NO: 13 or SEQ ID NO: 34). The sequences of the
CDRs for E16, E24, and E34 heavy and light chain variable regions
are provided in Table 1.
TABLE-US-00001 TABLE 1 SEQ SEQ Kabat ID ID Segment Number E16 NO.
E24 NO. VH FR1 1-30 QVQLQQSGSELMKPGASVQISCKATGYTFS 4
QVQLQQSGPELVKPGALVKISCKASGHTFT 30 CDR H1 31-35 DYWIE 1 SYDIN 27 VH
FR2 36-49 WVKQRPGHGLEWIG 5 WVKQRPGQGLEWIG 31 CDR H2 50-65
DILCGTGRTRYNEKLKA 2 WIYPGDGRIKYNEKFKG 28 VH FR3 66-94
MATFTADTSSNTAFMQLSSLTSEDSAVYYCAR 6 KAILTADKSSSTAYMQLSSLTSENSAVYFCAR
32 CDR H3 95-102 SASYGDYADY 3 GGSSGTYFDY 29 VH FR4 103-113
WGHGTTLTVSS 10 WGQGTTLTVSS 33 VL FR1 1-23 DIVMTQSHKFMSTSVGDRVSITC
14 DIVMTQSHKFMSTSVGDRVSITC 14 CDR L1 24-34 KASQDVSTAVA 11
KASQDVSTAVA 11 VL FR2 35-49 WYQQKPGQSPKLLIS 15 WYQQKPGQSPKVLIY 35
CDR L2 50-56 WASTRHT 12 WASTRHT 12 VL FR3 57-88
GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC 18
GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC 18 CDR L3 89-97 QQHYTTPLT 13
QQHYSNPPT 34 VL FR4 98-107 FGAGTKLELK 19 FGGGTKLEIK 36 SEQ Kabat ID
Segment Number E34 NO. VH FR1 1-30 QVQLQQSGPELVKPGTLVKISCKTSGYTFT
41 CDR H1 31-35 SYDIN 27 VH FR2 36-49 WVKQRPGQGLEWIG 31 CDR H2
50-65 WIFPGDGRIKYNEQIKD 39 VH FR3 66-94
KATLTADKSSSTAYMELSSLTSENSAVYFCAR 42 CDR H3 95-102 ASYYGSIFDY 40 VH
FR4 103-113 WGQGTTLTVSS 33 VL FR1 1-23 DIVMTQSHKFMSTSVGDRVNITC 43
CDR L1 24-34 KASQDVSTAVA 11 VL FR2 35-49 WYQQKPGQSPKLLIY 44 CDR L2
50-56 WASTRHT 12 VL FR3 57-88 GVPDRFTGSGSGTHYTLTISSVQAEDLALYYC 45
CDR L3 89-97 QQHYTTPLT 13 VL FR4 98-107 FGAGTKLELK 19
TABLE-US-00002 TABLE 2 SEQ SEQ Kabat ID ID Segment Number HuE16-1.1
NO. HuE16-1.2 NO. VH FR1 1-30 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 48
QVQLVQSGAEVKKPGASVKVSCKASGYTFT 48 CDR H1 31-35 DYWIE 1 DYWIE 1 VH
FR2 36-49 WVRQAPGQGLEWMG 49 WVRQAPGQGLEWMG 49 CDR H2 50-65
DILCGTGRTRYNEKLKA 2 DILCGTGRTRYNEKLKA 2 VH FR3 66-94
RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR 7 RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR
7 CDR H3 95-102 SASYGDYADY 3 SASYGDYADY 3 VH FR4 103-113
WGQGTTVTVSS 50 WGQGTTVTVSS 50 VL FR1 1-23 DIVMTQSPDSLAVSLGERATINC
51 DIVMTQSPDSLAVSLGERATINC 51 CDR L1 24-34 KASQDVSTAVA 11
KASQDVSTAVA 11 VL FR2 35-49 WYQQKPGQPPKLLIY 16 WYQQKPGQPPKLLIS 17
CDR L2 50-56 WASTRHT 12 WASTRHT 12 VL FR3 57-88
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 52
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 52 CDR L3 89-97 QQHYTTPLT 13
QQHYTTPLT 13 VL FR4 98-107 FGQGTKLEIK 53 FGQGTKLEIK 53 SEQ SEQ
Kabat ID ID Segment Number HuE16-2.1 NO. HuE16-2.2 NO. VH FR1 1-30
QVQLVQSGAEVKKPGASVKVSCKASGYTFT 48 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 48
CDR H1 31-35 DYWIE 1 DYWIE 1 VH FR2 36-49 WVRQAPGQGLEWMG 49
WVRQAPGQGLEWMG 49 CDR H2 50-65 DILCGTGRTRYNEKLKA 2
DILCGTGRTRYNEKLKA 2 VH FR3 66-94 RATFTADTSTSTAYMELRSLRSDDTAVYYCAR 8
RATFTADTSTSTAYMELRSLRSDDTAVYYCAR 8 CDR H3 95-102 SASYGDYADY 3
SASYGDYADY 3 VH FR4 103-113 WGQGTTVTVSS 50 WGQGTTVTVSS 50 VL FR1
1-23 DIVMTQSPDSLAVSLGERATINC 51 DIVMTQSPDSLAVSLGERATINC 51 CDR L1
24-34 KASQDVSTAVA 11 KASQDVSTAVA 11 VL FR2 35-49 WYQQKPGQPPKLLIY 16
WYQQKPGQPPKLLIS 17 CDR L2 50-56 WASTRHT 12 WASTRHT 12 VL FR3 57-88
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 52
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 52 CDR L3 89-97 QQHYTTPLT 13
QQHYTTPLT 13 VL FR4 98-107 FGQGTKLEIK 53 FGQGTKLEIK 53 SEQ SEQ
Kabat ID ID Segment Number HuE16-3.1 NO. HuE16-3.2 NO. VH FR1 1-30
QVQLVQSGAEVKKPGASVKVSCKASGYTFT 48 QVQLVQSGAEVKKPGASVKVSCKASGYTFT 48
CDR H1 31-35 DYWIE 1 DYWIE 1 VH FR2 36-49 WVRQAPGQGLEWMG 49
WVRQAPGQGLEWMG 49 CDR H2 50-65 DILCGTGRTRYNEKLKA 2
DILCGTGRTRYNEKLKA 2 VH FR3 66-94 RVTMTADTSTSTAYMELRSLRSDDTAVYYCAR 9
RVTMTADTSTSTAYMELRSLRSDDTAVYYCAR 9 CDR H3 95-102 SASYGDYADY 3
SASYGDYADY 3 VH FR4 103-113 WGQGTTVTVSS 50 WGQGTITVTVSS 50 VL FR1
1-23 DIVMTQSPDSLAVSLGERATINC 51 DIVMTQSPDSLAVSLGERATINC 51 CDR L1
24-34 KASQDVSTAVA 11 KASQDVSTAVA 11 VL FR2 35-49 WYQQKPGQPPKLLIY 16
WYQQKPGQPPKLLIS 17 CDR L2 50-56 WASTRHT 12 WASTRHT 12 VL FR3 57-88
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 52
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 52 CDR L3 89-97 QQHYTTPLT 13
QQHYTTPLT 13 VL FR4 98-107 FGQGTKLEIK 53 FGQGTKLEIK 53
[0112] In one specific embodiment, the invention provides a
humanized E16, E24, or E34 antibody, wherein the VH region consists
of the FR segments from the human germline VH segment VH1-18
(Matsuda et al., 1998, J. Exp. Med. 188:2151062) and JH6 (Ravetch
et al., 1981, Cell 27(3 Pt. 2): 583-91), and one or more CDR
regions of a E16, E24, or E34 VH, having the amino acid sequence of
SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO:
28, SEQ ID NO: 29, SEQ ID NO: 39, or SEQ ID NO: 40. In one
embodiment, the E16 VH has the amino acid sequence of SEQ ID NO:
21, SEQ ID NO: 22, or SEQ ID NO: 23. In another specific
embodiment, the humanized E16 antibody further comprises a VL
region, which consists of the FR segments of the human germline VL
segment VK-B3, and one or more CDR regions of E16, E24, or E34 VL,
having the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ
ID NO: 13, or SEQ ID NO: 34. In one embodiment, the E16 VL has the
amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26. In another
embodiment, the heavy chain FR3 may consist of the amino acid
sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In another
embodiment, the light chain FR2 may consist of the amino acid
sequence of SEQ ID NO: 16 or SEQ ID NO: 17. Humanized E16
antibodies comprising a VH FR3 sequence of SEQ ID NO: 7, SEQ ID NO:
8, or SEQ ID NO: 9 and a VH FR2 sequence of SEQ ID NO: 16 or SEQ ID
NO: 17 are provided in Table 2 as HuE16-1.1, HuE16-1.2, HuE16-2.1,
HuE16-2.2, HuE16-3.1, and HuE16-3.2.
[0113] In preferred embodiments, the humanized antibodies of the
invention comprise the FR regions of the human germline VH segment
VH1-18 and JH6 and the FR regions of the human germline VL segment
VK-B3, but have one or more of the following back mutations: V67A,
M69F, T71A in the heavy chain and Y49S in the light chain.
[0114] In particular, the invention provides a humanized antibody a
WNV virus antigen, said antibody comprising (or alternatively,
consisting of) CDR sequences of E16, E24, or E34, in any of the
following combinations: a VH CDR1 and a VL CDR1; a VH CDR1 and a VL
CDR2; a VH CDR1 and a VL CDR3; a VH CDR2 and a VL CDR1; VH CDR2 and
VL CDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VH CDR1; a VH
CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, a VH CDR2
and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, a VH
CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2,
a VH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; a VH
CDR1, a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3;
a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL
CDR3; a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and
a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH
CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a
VH CDR3 and a VL CDR3; a VH CDR1, a VH CDR1, a VL CDR1 and a VL
CDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR1, a
VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR3, a VL CDR1
and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VH
CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a
VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1
and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL
CDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a
VH CDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR2,
a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combination
thereof of the VH CDRs and VL CDRs disclosed herein.
[0115] The present invention provides humanized antibody molecules
specific for WNV in which one or more regions of one or more CDRs
of the heavy and/or light chain variable regions of a human
antibody (the recipient antibody) have been substituted by
analogous parts of one or more CDRs of a donor monoclonal antibody
which specifically binds a WNV antigen, e.g., a monoclonal antibody
produced by clone E16, E24, or E34. In other embodiments, the
humanized antibodies of the invention bind to the same epitope as
E16, E24, or E34. In a most preferred embodiment, the humanized
antibody specifically binds to the same epitope as the donor murine
antibody. It will be appreciated by one skilled in the art that the
invention encompasses CDR grafting of antibodies in general. Thus,
the donor and acceptor antibodies may be derived from animals of
the same species and even same antibody class or sub-class. More
usually, however, the donor and acceptor antibodies are derived
from animals of different species. Typically the donor antibody is
a non-human antibody, such as a rodent MAb, and the acceptor
antibody is a human antibody.
[0116] In some embodiments, at least one CDR from the donor
antibody is grafted onto the human antibody. In other embodiments,
at least two and preferably all three CDRs of each of the heavy
and/or light chain variable regions are grafted onto the human
antibody. The CDRs may comprise the Kabat CDRs, the structural loop
CDRs or a combination thereof. In some embodiments, the invention
encompasses a humanized WNV antibody comprising at least one CDR
grafted heavy chain and at least one CDR-grafted light chain.
[0117] In a preferred embodiment, the CDR regions of the humanized
WNV specific antibody are derived from a murine antibody specific
for WNV. In some embodiments, the humanized antibodies described
herein comprise alterations, including, but not limited to, amino
acid deletions, insertions, modifications, of the acceptor
antibody, i.e., human, heavy and/or light chain variable domain
framework regions that are necessary for retaining binding
specificity of the donor monoclonal antibody. In some embodiments,
the framework regions of the humanized antibodies described herein
does not necessarily consist of the precise amino acid sequence of
the framework region of a natural occurring human antibody variable
region, but contains various alterations, including, but not
limited to, amino acid deletions, insertions, modifications that
alter the property of the humanized antibody, for example, improve
the binding properties of a humanized antibody region that is
specific for the same target as the murine WNV specific antibody.
In most preferred embodiments, a minimal number of alterations are
made to the framework region in order to avoid large-scale
introductions of non-human framework residues and to ensure minimal
immunogenicity of the humanized antibody in humans. The donor
monoclonal antibody is preferably a monoclonal antibody produced by
clones E16, E24, or E34 which bind the WNV E antigen.
[0118] In specific embodiments of the invention, the humanized
antibodies of the invention comprise one or more of the
substitutions in the VH region as depicted in Table 3 and/or one or
more of the substitutions in the VL region as depicted in Table 4
(substitutions are from mouse to human).
TABLE-US-00003 TABLE 3 Framework Mouse Human Position region Amino
Acid Amino Acid 5 FR1 Q V 6 FR1 Q E 9 FR1 S A 11 FR1 L V 12 FR1 M K
19 FR1 Q K 20 FR1 I V 25 FR1 T S 30 FR1 S T 38 FR2 K R 40 FR2 R A
43 FR2 H Q 48 FR2 I M 66 FR3 M R 67 FR3 A V 69 FR3 F M 71 FR3 A T
75 FR3 S T 76 FR3 N S 79 FR3 F Y 81 FR3 Q E .sup. 82A FR3 S R 83
FR3 T R 85 FR3 E D 87 FR3 S T 105 FR4 H Q 109 FR4 L V
TABLE-US-00004 TABLE 4 Framework Mouse Human Position region Amino
Acid Amino Acid 8 FR1 H P 9 FR1 K D 10 FR1 F S 11 FR1 M L 12 FR1 S
A 13 FR1 T V 15 FR1 V L 17 FR1 D E 19 FR1 V A 20 FR1 S T 22 FR1 T N
43 FR2 S P 49 FR2 S Y 63 FR3 T S 71 FR3 Y F 78 FR3 V L 83 FR3 L V
85 FR3 L V 100 FR4 A Q
[0119] In a specific embodiment, the invention encompasses a
CDR-grafted antibody which specifically binds a WNV antigen,
wherein the CDR-grafted antibody comprises a heavy chain variable
region domain comprising framework residues of the recipient
antibody and residues from the donor monoclonal antibody, which
specifically binds WNV, e.g., monoclonal antibody produced from
clones E16, E24, or E34. In another specific embodiment, the
invention encompasses a CDR-grafted antibody which specifically
binds a WNV antigen, wherein the CDR-grafted antibody comprises a
light chain variable region domain comprising framework residues of
the recipient antibody and residues from the donor monoclonal
antibody, which specifically binds a WNV antigen, e.g., a
monoclonal antibody produced from one of clones E16, E24, or
E34.
[0120] Humanized WNV specific antibodies of the invention may
comprise substantially all of at least one, and typically two,
variable domains in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin (i.e.,
donor antibody) and all or substantially all of the framework
regions are those of a human immunoglobulin consensus sequence.
Preferably, a humanized antibody of the invention also comprises at
least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. The constant domains of
the humanized antibodies of the invention may be selected with
respect to the proposed function of the antibody, in particular the
effector function which may be required. In some embodiments, the
constant domains of the humanized antibodies of the invention are
human IgA, IgE, IgG or IgM domains. In a specific embodiment, human
IgG constant domains, especially of the IgG1 and IgG3 isotypes, are
used when the humanized antibodies of the invention are intended
for therapeutic uses and antibody effector functions are needed. In
alternative embodiments, IgG2 and IgG4 isotypes are used when the
humanized antibodies of the invention are intended for therapeutic
purposes and antibody effector function is not required. The
invention encompasses Fc constant domains comprising one or more
amino acid modifications which alter antibody effector functions
such as those disclosed in U.S. application Ser. No. 10/754,922,
filed Jan. 9, 2004; U.S. Provisional Application Nos. 60/439,498;
60/456,041; 60/514,549; 60/569,882, 60/582,045; and 60/582,043
filed on Jan. 9, 2003; Mar. 19, 2003; Oct. 23, 2003; May 10, 2004;
Jun. 21, 2004; and Jun. 21, 2004, respectively; all of which are
incorporated herein by reference in their entireties.
[0121] In some embodiments, humanized antibodies of the invention
contain both the light chain as well as at least the variable
domain of a heavy chain. In other embodiments, humanized antibodies
of the invention may further comprise one or more of the CHI,
hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized
antibody can be selected from any class of immunoglobulins,
including IgM, IgG, IgD, IgA and IgE, and any isotype, including
IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4. In some embodiments,
the constant domain is a complement fixing constant domain where it
is desired that the humanized antibody exhibit cytotoxic activity,
and the class is typically IgG.sub.1. In other embodiments, where
such cytotoxic activity is not desirable, the constant domain may
be of the IgG.sub.2 class. Humanized antibodies of the invention
may comprise sequences from more than one class or isotype, and
selecting particular constant domains to optimize desired effector
functions is within the ordinary skill in the art.
[0122] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
CDR or the consensus framework may be mutagenized by substitution,
insertion or deletion of at least one residue so that the CDR or
framework residue at that site does not correspond to either the
consensus or the donor antibody. Such mutations, however, are
preferably not extensive. Usually, at least 75% of the humanized
antibody residues will correspond to those of the parental
framework region (FR) and CDR sequences, more often 90%, and most
preferably greater than 95%.
[0123] The humanized antibodies used in the methods of the
invention include derivatives that are modified, i.e, by the
covalent attachment of any type of molecule to the antibody such
that covalent attachment. For example, but not by way of
limitation, the antibody derivatives include antibodies that have
been modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Antibody derivatives may
also include aglycosylated forms. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis of tunicamycin, etc. Additionally,
the derivative may contain one or more non-classical amino
acids.
[0124] Further, the humanized antibodies of the invention can, in
turn, be utilized to generate anti-idiotype antibodies using
techniques well known to those skilled in the art. (See, e.g.,
Greenspan & Bona, 1989, FASEB J. 7:437-444; and Nissinoff,
1991, J. Immunol. 147:2429-2438).
[0125] The present invention encompasses single domain antibodies,
including camelized single domain antibodies (See e.g., Muyldermans
et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000,
Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J.
Immunol. Meth. 231:25; International Publication Nos. WO 94/04678
and WO 94/25591; U.S. Pat. No. 6,005,079; which are incorporated
herein by reference in their entireties). In one embodiment, the
present invention provides single domain antibodies comprising two
VH domains with modifications such that single domain antibodies
are formed.
[0126] The methods of the present invention also encompass the use
of humanized antibodies or fragments thereof that have half-lives
(e.g., serum half-lives) in a mammal, preferably a human, of
greater than 15 days, preferably greater than 20 days, greater than
25 days, greater than 30 days, greater than 35 days, greater than
40 days, greater than 45 days, greater than 2 months, greater than
3 months, greater than 4 months, or greater than 5 months. The
increased half-lives of the humanized antibodies of the present
invention or fragments thereof in a mammal, preferably a human,
results in a higher serum titer of said humanized antibodies or
antibody fragments in the mammal, and thus, reduces the frequency
of the administration of said humanized antibodies or antibody
fragments and/or reduces the concentration of said humanized
antibodies or antibody fragments to be administered. Humanized
antibodies or fragments thereof having increased in vivo half-lives
can be generated by techniques known to those of skill in the art.
For example, humanized antibodies or fragments thereof with
increased in vivo half-lives can be generated by modifying (e.g.,
substituting, deleting or adding) amino acid residues identified as
involved in the interaction between the Fc domain and the FcRn
receptor. The humanized antibodies of the invention may be
engineered by methods described in Ward et al. to increase
biological half-lives (See U.S. Pat. No. 6,277,375 B1). For
example, humanized antibodies of the invention may be engineered in
the Fc-hinge domain to have increased in vivo or serum
half-lives.
[0127] Humanized antibodies or fragments thereof with increased in
vivo half-lives can be generated by attaching to said humanized
antibodies or antibody fragments polymer molecules such as high
molecular weight polyethyleneglycol (PEG). PEG can be attached to
said humanized antibodies or antibody fragments with or without a
multifunctional linker either through site-specific conjugation of
the PEG to the N- or C-terminus of said humanized antibodies or
antibody fragments or via epsilon-amino groups present on lysine
residues. Linear or branched polymer derivatization that results in
minimal loss of biological activity will be used. The degree of
conjugation will be closely monitored by SDS-PAGE and mass
spectrometry to ensure proper conjugation of PEG molecules to the
humanized antibodies. Unreacted PEG can be separated from
antibody-PEG conjugates by, e.g., size exclusion or ion-exchange
chromatography.
[0128] The humanized antibodies of the invention may also be
modified by the methods and coupling agents described by Davis et
al. (See U.S. Pat. No. 4,179,337) in order to provide compositions
that can be injected into the mammalian circulatory system with
substantially no immunogenic response.
[0129] The invention also provides humanized antibodies with
altered oligosaccharide content. Oligosaccharides, as used herein,
refer to carbohydrates containing two or more simple sugars and the
two terms may be used interchangeably herein. Carbohydrate moieties
of the instant invention will be described with reference to
commonly used nomenclature in the art. For a review of carbohydrate
chemistry, see, e.g., Hubbard et al., 1981 Ann. Rev. Biochem., 50:
555-583, which is incorporated herein by reference in its entirety.
This nomenclature includes, for example, Man which represents
mannose; GlcNAc which represents 2-N-acetylglucosamine; Gal which
represents galactose; Fuc for fucose and Glc for glucose. Sialic
acids are described by the shorthand notation NeuNAc for
5-N-acetylneuraminic acid, and NeuNGc for 5-glycolneuraminic.
[0130] In general, antibodies contain carbohydrate moeities at
conserved positions in the constant region of the heavy chain, and
up to 30% of human IgGs have a glycosylated Fab region. IgG has a
single N-linked biantennary carbohydrate structure at Asn 297 which
resides in the CH2 domain (Jefferis et al., 1998, Immunol. Rev.
163: 59-76; Wright et al., 1997, Trends Biotech 15: 26-32). Human
IgG typically has a carbohydrate of the following structure;
GlcNAc(Fucose)-GlcNAc-Man-(ManGlcNAc).sub.2. However variations
among IgGs in carbohydrate content does occur which leads to
altered function, see, e.g., Jassal et al., 2001, Biochem. Biophys.
Res. Commun. 288: 243-9; Groenink et al., 1996, J. Immunol. 26:
1404-7; Boyd et al., 1995, Mol. Immunol. 32: 1311-8; Kumpel et al.,
1994, Human Antibody Hybridomas, 5:143-51. In one embodiment, the
carbohydrate moiety has a galactose and/or galactose-sialic acid at
one or both of the terminal GlcNAc and/or a third GlcNac arm
(bisecting GlcNAc).
[0131] In some embodiments, the humanized antibodies of the
invention are substantially free of one or more selected sugar
groups, e.g., one or more sialic acid residues, one or more
galactose residues, one or more fucose residues. A humanized
antibody that is substantially free of one or more selected sugar
groups may be prepared using common methods known to one skilled in
the art, including, for example, recombinantly producing an
humanized antibody of the invention in a host cell that is
defective in the addition of the selected sugar groups(s) to the
carbohydrate moiety of the antibody, such that about 90-100% of the
humanized antibody in the composition lacks the selected sugar
group(s) attached to the carbohydrate moiety. Alternative methods
for preparing such antibodies include, for example, culturing cells
under conditions which prevent or reduce the addition of one or
more selected sugar groups, or post-translational removal of one or
more selected sugar groups.
[0132] In a specific embodiment, the invention encompasses a method
of producing a substantially homogenous humanized antibody
preparation, wherein about 80-100% of the humanized antibody in the
composition lacks a fucose on its carbohydrate moiety. The antibody
may be prepared, for example, by (a) use of an engineered host cell
that is deficient in fucose metabolism such that it has a reduced
ability to fucosylate proteins expressed therein; (b) culturing
cells under conditions which prevent or reduce fusocylation; (c)
post-translational removal of fucose, e.g., with a fucosidase
enzyme; or (d) purification of the antibody so as to select for the
product which is not fucosylated. Most preferably, a nucleic acid
encoding the desired antibody is expressed in a host cell that has
a reduced ability to fucosylate the antibody expressed therein.
Preferably, the host cell is a Lec 13 CHO cell (lectin resistant
CHO mutant cell line; U.S. Patent Application Publication No.
2003/0115614; PCT Publication No. WO 00/61739; European Patent
Application EP 1 229 125; Ribka & Stanley, 1986, Somatic Cell
& Molec. Gen. 12(1): 51-62; Ripka et al., 1986 Arch. Biochem.
Biophys. 249(2): 533-45), CHO-K1 cell, DUX-B11 cell, CHO-DP12 cell
or CHO-DG44 cell, which has been modified so that the antibody is
not substantially fucosylated. Thus, the cell may display altered
expression and/or activity for the fucoysltransferase enzyme, or
another enzyme or substrate involved in adding fucose to the
N-linked oligosaccharide so that the enzyme has a diminished
activity and/or reduced expression level in the cell. For methods
to produce antibodies with altered fucose content, see, e.g., WO
03/035835 and Shields et al., 2002, J. Biol. Chem. 277(30):
26733-40; both of which are incorporated herein by reference in
their entirety.
[0133] In some embodiments, the altered carbohydrate modifications
modulate one or more of the following: solubilization of the
antibody, facilitation of subcellular transport and secretion of
the antibody, promotion of antibody assembly, conformational
integrity, and antibody-mediated effector function. In a specific
embodiment the altered carbohydrate modifications enhance antibody
mediated effector function relative to the antibody lacking the
carbohydrate modification. Carbohydrate modifications that lead to
altered antibody mediated effector function are well known in the
art (for example, see Shields R. L. et al., 2001, J. Biol. Chem.
277(30): 26733-40; Davies J. et al., 2001, Biotechnology &
Bioengineering, 74: 288-294). In another specific embodiment, the
altered carbohydrate modifications enhance the binding of humanized
antibodies of the invention to a flaviviral antigen. Altering
carbohydrate modifications in accordance with the methods of the
invention includes, for example, increasing the carbohydrate
content of the antibody or decreasing the carbohydrate content of
the antibody. Methods of altering carbohydrate contents are known
to those skilled in the art, see, e.g., Wallick et al., 1988, J
Exp. Med. 168(3): 1099-1109; Tao et al., 1989, Journal of
Immunology, 143(8): 2595-2601; Routledge et al., 1995,
Transplantation, 60(8): 847-53; Elliott et al., 2003, Nature
Biotechnology 21:414-21; Shields et al., 2002, J Biol Chem,
277(30): 26733-40; all of which are incorporated herein by
reference in their entirety.
[0134] In some embodiments, the invention encompasses humanized
antibodies comprising one or more glycosylation sites, so that one
or more carbohydrate moieties are covalently attached to the
antibody. In other embodiments, the invention encompasses humanized
antibodies comprising one or more glycosylation sites. In some
embodiments, the invention further comprises humanized antibodies
comprising one or more modifications of amino acids that are
directly or indirectly known to interact with a carbohydrate moiety
of the antibody. Amino acids that directly or indirectly interact
with a carbohydrate moiety of an antibody are known in the art,
see, e.g., Jefferis et al., 1995, Immunology Letters 44: 111-7,
which is incorporated herein by reference in its entirety.
[0135] The invention encompasses humanized antibodies that have
been modified by introducing one or more glycosylation sites into
one or more sites of the antibodies, preferably without altering
the functionality of the antibody. Glycosylation sites may be
introduced into the variable and/or constant region of the
humanized antibodies of the invention. As used herein,
"glycosylation sites" include any specific amino acid sequence in
an antibody to which an oligosaccharide (i.e., carbohydrates
containing two or more simple sugars linked together) will
specifically and covalently attach. Oligosaccharide side chains are
typically linked to the backbone of an antibody via either N- or
O-linkages. N-linked glycosylation refers to the attachment of an
oligosaccharide moiety to the side chain of an asparagine residue.
O-linked glycosylation refers to the attachment of an
oligosaccharide moiety to a hydroxyamino acid, e.g., serine,
threonine. The humanized antibodies of the invention may comprise
one or more glycosylation sites, including N-linked and O-linked
glycosylation sites. Any glycosylation site for N-linked or
O-linked glycosylation known in the art may be used in accordance
with the instant invention. An exemplary N-linked glycosylation
site that is useful in accordance with the methods of the present
invention, is the amino acid sequence: Asn-X-Thr/Ser, wherein X may
be any amino acid and Thr/Ser indicates a threonine or a serine.
Such a site or sites may be introduced into humanized antibodies of
the invention using methods well known in the art to which this
invention pertains. See, for example, "In vitro Mutagenesis,"
Recombinant DNA: A Short Course, J. D. Watson, et al. W.H. Freeman
and Company, New York, 1983, chapter 8, pp. 106-116, which is
incorporated herein by reference in its entirety. An exemplary
method for introducing a glycosylation site into humanized
antibodies of the invention may comprise: modifying or mutating an
amino acid sequence of the antibody so that the desired
Asn-X-Thr/Ser sequence is obtained.
[0136] In some embodiments, the invention encompasses methods of
modifying the carbohydrate content of humanized antibodies of the
invention by adding or deleting a glycosylation site. Methods for
modifying the carbohydrate content of antibodies are well known in
the art and encompassed within the invention, see, e.g., U.S. Pat.
No. 6,218,149; EP 0 359 096 B1; U.S. Patent Application Publication
No. US 2002/0028486; WO 03/035835; U.S. Publication No.
2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all
of which are incorporated herein by reference in their entirety. In
other embodiments, the invention encompasses methods of modifying
the carbohydrate content of humanized antibodies of the invention
by deleting one or more endogenous carbohydrate moieties of the
antibody.
[0137] The invention further encompasses methods of modifying an
effector function of humanized antibodies of the invention, wherein
the method comprises modifying the carbohydrate content of the
antibody using the methods disclosed herein or known in the
art.
[0138] Standard techniques known to those skilled in the art can be
used to introduce mutations in the nucleotide sequence encoding an
antibody, or fragment thereof, including, e.g., site-directed
mutagenesis and PCR-mediated mutagenesis, which results in amino
acid substitutions. Preferably, the derivatives include less than
15 amino acid substitutions, less than 10 amino acid substitutions,
less than 5 amino acid substitutions, less than 4 amino acid
substitutions, less than 3 amino acid substitutions, or less than 2
amino acid substitutions relative to the original antibody or
fragment thereof. In a preferred embodiment, the derivatives have
conservative amino acid substitutions made at one or more predicted
non-essential amino acid residues.
[0139] The present invention also encompasses humanized antibodies
or fragments thereof comprising an amino acid sequence of a
variable heavy chain and/or variable light chain that is at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, or at least 99% identical to the amino acid sequence
of the variable heavy chain and/or light chain of the mouse
monoclonal antibody produced by clone E16, E24, or E34. The present
invention further encompasses humanized antibodies or fragments
thereof that specifically bind WNV, said humanized antibodies or
antibody fragments comprising an amino acid sequence of one or more
CDRs that is at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 99% identical to
the amino acid sequence of one or more CDRs of the mouse monoclonal
antibody produced by clone E16, E24, or E34. The determination of
percent identity of two amino acid sequences can be determined by
any method known to one skilled in the art, including BLAST protein
searches.
[0140] The present invention also encompasses the use of humanized
antibodies or antibody fragments that specifically bind WNV,
wherein said humanized antibodies or antibody fragments are encoded
by a nucleotide sequence that hybridizes to the nucleotide sequence
of the mouse monoclonal antibody produced by clone E16, E24, or
E34, under stringent conditions. In a preferred embodiment, the
invention provides humanized antibodies or fragments thereof that
specifically bind WNV, said humanized antibodies or antibody
fragments comprising a variable light chain and/or variable heavy
chain encoded by a nucleotide sequence that hybridizes under
stringent conditions to the nucleotide sequence of the variable
light chain and/or variable heavy chain of the mouse monoclonal
antibody produced by clone E16, E24, or E34, under stringent
conditions. In another preferred embodiment, the invention provides
humanized antibodies or fragments thereof that specifically bind
WNV, said humanized antibodies or antibody fragments comprising one
or more CDRs encoded by a nucleotide sequence that hybridizes under
stringent conditions to the nucleotide sequence of one or more CDRs
of the mouse monoclonal antibody produced by clone E16, E24, or
E34. Stringent hybridization conditions include, but are not
limited to, hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., highly stringent conditions such as hybridization to
filter-bound DNA in 6.times.SSC at about 45.degree. C. followed by
one or more washes in 0.1.times.SSC/0.2% SDS at about 60.degree.
C., or any other stringent hybridization conditions known to those
skilled in the art (see, for example, Ausubel, F. M. et al., eds.
1989 Current Protocols in Molecular Biology, vol. 1, Green
Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at
pages 6.3.1 to 6.3.6 and 2.10.3, incorporated herein by
reference).
[0141] The antibody or antibody fragment generated by introducing
substitutions in the VH domain, VH CDRs, VL domain and/or VL CDRs
of the humanized antibodies of the invention can be tested in vitro
and in vivo, for example, for its ability to bind to flaviviral
antigens, particularly WNV antigens, for its ability to neutralize
a flavivirus, particularly WNV, or for its ability to prevent,
treat or ameliorate one or more symptoms associated with a
flavivirus, particularly WNV infection.
[0142] In most preferred embodiments, the invention encompasses
humanized antibodies or fragments thereof that have potent
neutralizing activity as measured for example using standard
methods known in the art and exemplified herein, e.g., in vivo
plaque reduction neutralization titer (PRNT) assay. Although not
intending to be bound by a particular mechanims of action the
humanized antibodies of the invention may directly neutralize virus
or block entry of the virus into the cell, thus preventing viral
infections. In some embodiments, the invention encompasses
humanized antibodies which immunospecifically bind WNV-E protein
such that the PRNT.sub.50 values are at least 1/500, preferably at
least 1/10,000 at a concentration of 1 mg/mL.
[0143] In yet other preferred embodiments, humanized antibodies of
the invention have enhanced antibody-dependent complement mediated
neutralization of WNV infected virions and trigger lysis of
WNV-infected cells more effectively, as determined using standard
methods known in the art and exemplified herein such as complement
fixation and viability assays Although not intending to be bound by
a particular mechanism of action, the humanized antibodies of the
invention have enhanced clinical efficacy, therapeutically and
prophylactically as they have enhanced effector functions,
neutralize virus attachment, trigger complement mediated lysis,
promote clearance from the circulatory systems and prevent
emergence of viral resistance. The humanized antibodies of the
invention preferably have a potent in vivo inhibitory activity,
i.e., protect against WNV infection by at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 99%.
[0144] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens particularly WNV antigens and have an apparent
dissociation constant of about 1-10 nM, as determined by a sandwich
ELISA. The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens, particularly WNV antigens, and have an
apparent dissociation constant of about 1-10 nM as measured by
surface plasmon resonance (SPR) using a BIAcore sensor.
[0145] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens, particularly WNV antigens, and have a
k.sub.off rate (antibody (Ab)+antigen
(Ag).sup.K.sup.off.fwdarw.Ab-Ag of less than 10.sup.-1 s.sup.-1,
less than 5.times.10.sup.-1s.sup.-1, less than 10.sup.-2 s.sup.-1,
less than 5.times.10.sup.-2 s.sup.-1, less than 10.sup.-3 s.sup.-1,
less than 5.times.10.sup.-3 s.sup.-1, less than 10.sup.-4 s.sup.-1,
less than 5.times.10.sup.-4 s.sup.-1, less than 10.sup.--5
s.sup.-1, less than 5.times.10.sup.-5 s.sup.-1, less than 10.sup.-6
s.sup.-1, less than 5.times.10.sup.-6 s.sup.-1, less than 10.sup.-7
s.sup.-1, less than 5.times.10.sup.-7 s.sup.-1, less than 10.sup.-8
s.sup.-1, less than 5.times.10.sup.-8 s.sup.-1, less than 10.sup.-9
s.sup.-1, less than 5.times.10.sup.-9 s.sup.-1, or less than
10.sup.-1 s.sup.-1. The present invention provides humanized
antibodies or fragments thereof which immunospecifically bind to
one or more flaviviral antigens, particularly WNV antigens, and
have a k.sub.off rate (antibody (Ab)+antigen
(Ag).sup.Koff.fwdarw.Ab-Ag of about 1.times.10.sup.-3, about
5.times.10.sup.-4, about 1.times.10.sup.-4, about
5.times.10.sup.-5, about 1.times.10.sup.-5, about
5.times.10.sup.-6, or about 1.times.10.sup.-6. The present
invention provides humanized antibodies or fragments thereof which
immunospecifically bind to one or more flaviviral antigens,
particularly WNV antigens, and have a k.sub.on rate of about
1.times.10.sup.4, about 5.times.10.sup.4, about 1.times.10.sup.5,
about 5.times.10.sup.5, about 1.times.10.sup.6, or about
5.times.10.sup.6.
[0146] The present invention provides humanized antibodies or
fragments thereof which immunospecifically bind to one or more
flaviviral antigens, particularly WNV antigens, and have a median
effective concentration (EC.sub.50) of less than 1 .mu.g/ml, in an
in vitro microneutralization assay. In particular, the present
invention provides compositions for use in the prevention,
treatment, or amelioration of one or more symptoms associated with
a flaviviral infection, said compositions comprising one or more
humanized antibodies or fragments thereof which immunospecifically
bind to one or more one or more flaviviral antigens particularly
WNV antigens and have an EC.sub.50 of less than 0.01 nM, less than
0.025 nM, less than 0.05 nM, less than 0.1 nM, less than 0.25 nM,
less than 0.5 nM, less than 0.75 nM, less than 1 nM, less than 1.25
nM, less than 1.5 nM, less than 1.75 nM, or less than 2 nM, in an
in vitro microneutralization assay.
[0147] 5.1.1 ANTIBODY CONJUGATES
[0148] The present invention encompasses humanized antibodies, or
fragments thereof, recombinantly fused or chemically conjugated
(including both covalently and non-covalently conjugations) to
heterologous polypeptides (i.e., an unrelated polypeptide; or
portion thereof, preferably at least 10, at least 20, at least 30,
at least 40, at least 50, at least 60, at least 70, at least 80, at
least 90 or at least 100 amino acids of the polypeptide) to
generate fusion proteins. The fusion does not necessarily need to
be direct, but may occur through linker sequences. Humanized
antibodies may be used, for example, to target heterologous
polypeptides to particular cell types (e.g., respiratory epithelial
cells), either in vitro or in vivo, by fusing or conjugating the
humanized antibodies to antibodies specific for particular cell
surface receptors. Antibodies fused or conjugated to heterologous
polypeptides may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g., PCT
Publication No. WO 93/21232; EP 439,095; Naramura et al., 1994
Immunol. Lett., 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al.,
1992, Proc. Natl. Acad. Sci. USA, 89:1428-1432; and Fell et al.,
1991, J. Immunol., 146:2446-2452, all of which are incorporated
herein by reference in their entireties.
[0149] The present invention further includes compositions
comprising heterologous polypeptides fused or conjugated to
antibody fragments. For example, the heterologous polypeptides may
be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment,
F(ab).sub.2 fragment, or portion thereof. Methods for fusing or
conjugating polypeptides to antibody portions are known in the art.
See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, and 5,112,946; EP 307,434; EP 367,166;
International Publication Nos. WO 96/04388 and WO 91/06570;
Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539;
Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al.,
1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (said references
incorporated by reference in their entireties).
[0150] Additional fusion proteins may be generated through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA
shuffling"). DNA shuffling may be employed to alter the activities
of humanized antibodies of the invention or fragments thereof
(e.g., antibodies or fragments thereof with higher affinities and
lower dissociation rates). See, generally, U.S. Pat. Nos.
5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and
Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-1-33;
Harayama, 1998, Trends Biotechnol. 16:76; Hansson, et al., 1999, J.
Mol. Biol. 287:265; and Lorenzo and Blasco, 1998, BioTechniques
24:308 (each of these patents and publications are hereby
incorporated by reference in its entirety). Antibodies or fragments
thereof, or the encoded antibodies or fragments thereof, may be
altered by being subjected to random mutagenesis by error-prone
PCR, random nucleotide insertion or other methods prior to
recombination. One or more portions of a polynucleotide encoding an
antibody or antibody fragment, which portions immunospecifically
bind to a flaviviral antigen may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of
one or more heterologous molecules.
[0151] Moreover, the humanized antibodies of the present invention
or fragments thereof can be fused to marker sequences, such as a
peptide to facilitate purification. In preferred embodiments, the
marker amino acid sequence is a hexa-histidine peptide, such as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al., 1989, Proc.
Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine
provides for convenient purification of the fusion protein. Other
peptide tags useful for purification include, but are not limited
to, the hemagglutinin "HA" tag, which corresponds to an epitope
derived from the influenza hemagglutinin protein (Wilson et al.,
1984, Cell 37:767) and the "flag" tag (Knappik et al., 1994,
Biotechniques, 17:754-761).
[0152] The present invention also encompasses humanized antibodies,
or fragments thereof, conjugated to a diagnostic or therapeutic
agent. The humanized antibodies can be used diagnostically to, for
example, monitor the development or progression of a flaviviral
infection as part of a clinical testing procedure to, e.g.,
determine the efficacy of a given treatment regimen. Detection can
be facilitated by coupling the antibody, or a fragment thereof, to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
materials, positron emitting metals, and nonradioactive
paramagnetic metal ions. The detectable substance may be coupled or
conjugated either directly to the antibody or indirectly, through
an intermediate (such as, for example, a linker known in the art)
using techniques known in the art. See, for example, U.S. Pat. No.
4,741,900 for metal ions which can be conjugated to antibodies for
use as diagnostics according to the present invention. Such
diagnosis and detection can be accomplished by coupling the
antibody to detectable substances including, but not limited to,
various enzymes, enzymes including, but not limited to, horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; prosthetic group complexes such as, but not
limited to, streptavidin/biotin and avidin/biotin; fluorescent
materials such as, but not limited to, umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; luminescent material
such as, but not limited to, luminol; bioluminescent materials such
as, but not limited to, luciferase, luciferin, and aequorin;
radioactive material such as, but not limited to, bismuth
(.sup.213Bi), carbon (.sup.14C), chromium (.sup.51Cr), cobalt
(.sup.57Co), fluorine (.sup.18F), gadolinium (.sup.153Gd,
.sup.159Gd), gallium (.sup.68Ga, .sup.67Ga), germanium (.sup.68Ge),
holmium (.sup.166Ho), indium (.sup.115In, .sup.113In, .sup.112In,
.sup.111In), iodine (.sup.131I, .sup.125I, .sup.123I, .sup.121I),
lanthanium (.sup.140La), lutetium (.sup.177Lu), manganese
(.sup.54Mn), molybdenum (.sup.99Mo), palladium (.sup.103Pd),
phosphorous (.sup.32P), praseodymium (.sup.142Pr), promethium
(.sup.149Pm), rhenium (.sup.186Re, .sup.188Re), rhodium
(.sup.105Rh), ruthemium (.sup.97Ru), samarium (.sup.153Sm),
scandium (.sup.47Sc), selenium (.sup.75Se), strontium (.sup.85Sr),
sulfur (.sup.35S), technetium (.sup.99Tc), thallium (.sup.201Ti),
tin (.sup.113Sn, .sup.117Sn), tritium (.sup.3H), xenon
(.sup.133Xe), ytterbium (.sup.169Yb, .sup.175Yb), yttrium
(.sup.90Y), zinc (.sup.65Zn); positron emitting metals using
various positron emission tomographies, and nonradioactive
paramagnetic metal ions.
[0153] An antibody, or fragment thereof, may be conjugated to a
therapeutic moiety such as a cytotoxin (e.g., a cytostatic or
cytocidal agent), a therapeutic agent or a radioactive element
(e.g., alpha-emitters, gamma-emitters, etc.). Cytotoxins or
cytotoxic agents include any agent that is detrimental to cells.
Examples include paclitaxol, 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), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cisdichlorodiamine 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).
[0154] Further, a humanized antibody, or fragment thereof, may be
conjugated to a therapeutic agent or drug moiety that modifies a
given biological response. Therapeutic agents or drug moieties are
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, a toxin such as abrin, ricin A,
pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin,
gelonin, and pokeweed antiviral protein, a protein such as tumor
necrosis factor, interferons including, but not limited to,
.alpha.-interferon (IFN-.alpha.), .beta.-interferon (IFN-.beta.),
nerve growth factor (NGF), platelet derived growth factor (PDGF),
tissue plasminogen activator (TPA), an apoptotic agent (e.g.,
TNF-.alpha., TNF-.beta., AIM I as disclosed in PCT Publication No.
WO 97/33899), AIM II (see, e.g., PCT Publication No. WO 97/34911),
Fas Ligand (Takahashi et al., 1994, J. Immunol. 6:1567-1574), and
VEGI (PCT Publication No. WO 99/23105), a thrombotic agent or an
anti-angiogenic agent (e.g., angiostatin or endostatin), or a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), and granulocyte colony stimulating factor
("G-CSF")), macrophage colony stimulating factor, ("M-CSF"), or a
growth factor (e.g., growth hormone ("GH")); a protease, or a
ribonuclease.
[0155] Moreover, a humanized antibody can be conjugated to
therapeutic moieties such as radioactive materials or macrocyclic
chelators useful for conjugating radiometal ions (see above for
examples of radioactive materials). In certain embodiments, the
macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA)
which can be attached to the antibody via a linker molecule. Such
linker molecules are commonly known in the art and described in
Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al.,
1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl.
Med. Biol. 26:943-50 each of which is herein incorporated by
reference in their entirety.
[0156] Techniques for conjugating such therapeutic moieties to
antibodies are well known; see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
1985, pp. 243-56, Alan R. Liss, Inc.; Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc.; Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), 1985, pp. 475-506; "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.), 1985, pp.
303-16, Academic Press; and Thorpe et al, 1982, Immunol. Rev.,
62:119-58.
[0157] An antibody or fragment thereof, with or without a
therapeutic moiety conjugated to it, administered alone or in
combination with cytotoxic factor(s) and/or cytokine(s) can be used
as a therapeutic. Alternatively, an antibody can be conjugated to a
second antibody to form an antibody heteroconjugate as described by
Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0158] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0159] 5.2 Preparation of Humanized Antibodies
[0160] The invention encompasses nucleotide sequences that encode
the CDR-grafted heavy and light chains, cloning and expression
vectors containing the nucleotide sequences, host cells transformed
with the nucleotide sequences, and methods for the production of
the CDR-grafted chains and antibody molecules comprising the
nucleotide sequences in the transformed host cells.
[0161] The invention encompasses donor amino acid sequences, which
encode antibodies that immunospecifically bind a West Nile Virus
antigen, such as those disclosed in U.S. Provisional Application
No. 60/581,819, filed on Jun. 21, 2004, incorporated herein by
reference in its entirety. In a specific embodiment, the donor
amino acid sequence encodes for the monoclonal antibody produced
from clone E16, E24 and E34, or other monoclonal antibodies
produced by immunization methods of the invention as disclosed in
U.S. Provisional Application No. 60/581,819 filed on Jun. 21, 2004,
incorporated herein by reference in its entirety. In another
specific embodiment, the donor amino acid sequence encodes for the
antibody produced from clone E16-1.1, E16-1.2, E16-2.1, E16-2.2,
E16-3.1, or E16-3.2. Donor murine antibodies may be produced using
any method known in the art, including those disclosed in U.S.
Provisional Application No. 60/581,819, filed on Jun. 21, 2004,
incorporated herein by reference in its entirety.
[0162] The invention also encompass polynucleotides that encode for
donor amino acid sequences that hybridize under various stringency,
e.g., high stringency, intermediate or low stringency conditions,
to polynucleotides that encode for the monoclonal antibody produced
from clone E16, E24 and E34, or other monoclonal antibodies
produced by immunization methods of the invention as disclosed in
U.S. Provisional Application No. 60/581,819 filed on Jun. 21, 2004.
The hybridization can be performed under various conditions of
stringency. By way of example and not limitation, procedures using
conditions of low stringency are as follows (see also Shilo and
Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792).
Filters containing DNA are pretreated for 6 h at 40.degree. C. in a
solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH
7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml
denatured salmon sperm DNA. Hybridizations are carried out in the
same solution with the following modifications: 0.02% PVP, 0.02%
Ficoll, 0.2% BSA, 100 .mu.g/ml salmon sperm DNA, 10% (wt/vol)
dextran sulfate, and 5-20.times.10.sup.6 cpm .sup.32P-labeled probe
is used. Filters are incubated in hybridization mixture for 18-20 h
at 40.degree. C., and then washed for 1.5 h at 55.degree. C. in a
solution containing 2.times.SSC, 25 mM Tris-HCl (pH 7.4), 5 mM
EDTA, and 0.1% SDS. The wash solution is replaced with fresh
solution and incubated an additional 1.5 h at 60.degree. C. Filters
are blotted dry and exposed for autoradiography. If necessary,
filters are washed for a third time at 65-68.degree. C. and
re-exposed to film. Other conditions of low stringency which may be
used are well known in the art (e.g., as employed for cross-species
hybridizations). By way of example and not limitation, procedures
using conditions of high stringency are as follows.
Prehybridization of filters containing DNA is carried out for 8 h
to overnight at 65.degree. C. in buffer composed of 6.times.SSC, 50
mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02%
BSA, and 500 .mu.g/ml denatured salmon sperm DNA. Filters are
hybridized for 48 h at 65.degree. C. in prehybridization mixture
containing 100 .mu.g/ml denatured salmon sperm DNA and
5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Washing of
filters is done at 37.degree. C. for 1 h in a solution containing
2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is
followed by a wash in 0.1.times.SSC at 50.degree. C. for 45 min
before autoradiography. Other conditions of high stringency which
may be used are well known in the art. Selection of appropriate
conditions for such stringencies is well known in the art (see
e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory
Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; see also, Ausubel et al., eds., in the Current
Protocols in Molecular Biology series of laboratory technique
manuals, .COPYRGT. 1987-1997, Current Protocols, .COPYRGT.
1994-1997 John Wiley and Sons, Inc.; see especially, Dyson, 1991,
"Immobilization of nucleic acids and hybridization analysis," In:
Essential Molecular Biology: A Practical Approach, Vol. 2, T. A.
Brown, ed., pp. 11'-156, IRL Press at Oxford University Press,
Oxford, UK).
[0163] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art.
[0164] DNA sequences which encode the acceptor amino acid sequences
may be obtained by any method known to one skilled in the art. For
example, DNA sequences coding for preferred human acceptor
framework sequences include, but are not limited to, FR segments
from the human germline VH segment VH1-8 and JH6 and the human
germline VL segment VK-B3, as depicted in Table 5.
TABLE-US-00005 TABLE 5 VH1-18 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGA
SEQ ID AGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTC NO: 54
TGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTG
CGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAT
GGATCAGCGCTTACAATGGTAACACAAACTATGCACA
GAAGCTCCAGGGCAGAGTCACCATGACCACAGACACA
TCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGA
GATCTGACGACACGGCCGTGTATTACTGTGCGAGAGA JH6
ATTACTACTACTACTACGGTATGGACGTCTGGGGGCA SEQ ID
AGGGACCACGGTCACCGTCTCCTCAG NO: 55 VKB-3
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTG SEQ ID
TGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTC NO: 56
CAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAAC
TACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTC
CTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATC
CGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGG
ACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTG
AAGATGTGGCAGTTTATTACTGTCAGCAATATTATAG TACTGCTCC
[0165] A polynucleotide encoding an antibody may be generated from
nucleic acid from a suitable source (e.g., a cDNA library generated
from, or nucleic acid, preferably poly A+ RNA, isolated from, any
tissue or cells expressing the antibody, such as hybridoma cells
selected to express a humanized antibody of the invention) by
hybridization with Ig specific probes and/or PCR amplification
using synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0166] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., 1990, Molecular Cloning A Laboratory Manuals 2d
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and
Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY, which are both incorporated by reference
herein in their entireties), to generate antibodies having a
different amino acid sequence, for example to create amino acid
substitutions, deletions, and/or insertions.
[0167] In a specific embodiment, one or more of the CDRs are
inserted within framework regions using routine recombinant DNA
techniques. The framework regions may be naturally occurring or
consensus framework regions, and preferably human framework regions
(see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a
listing of human framework regions). Preferably, the polynucleotide
generated by the combination of the framework regions and CDRs
encodes an antibody that specifically binds to a flaviviral
antigen. Preferably, as discussed supra, one or more amino acid
substitutions may be made within the framework regions, and,
preferably, the amino acid substitutions improve binding of the
humanized antibodies of the invention to a flaviviral antigen.
[0168] The humanized antibodies of the present invention may be
produced by any method known in the art useful for the production
of polypeptides, e.g., in vitro synthesis, recombinant DNA
production, and the like. Preferably, the humanized antibodies are
produced by recombinant DNA technology. The humanized WNV specific
antibodies of the invention may be produced using recombinant
immunoglobulin expression technology. The recombinant production of
immunoglobulin molecules, including humanized antibodies are
described in U.S. Pat. No. 4,816,397 (Boss et al.), U.S. Pat. Nos.
6,331,415 and 4,816,567 (both to Cabilly et al.), U.K. patent GB
2,188,638 (Winter et al.), and U.K. patent GB 2,209,757; all of
which are incorporated herein by reference in their entireties.
Techniques for the recombinant expression of immunoglobulins,
including humanized immunoglobulins, can also be found, in Goeddel
et al., Gene Expression Technology Methods in Enzymology Vol. 185
Academic Press (1991), and Borreback, Antibody Engineering, W. H.
Freeman (1992). Additional information concerning the generation,
design and expression of recombinant antibodies can be found in
Mayforth, Designing Antibodies, Academic Press, San Diego
(1993).
[0169] An exemplary process for the production of the recombinant
humanized antibodies of the invention may comprise the following:
a) constructing, by conventional molecular biology methods, an
expression vector comprising an operon that encodes an antibody
heavy chain in which the CDRs and a minimal portion of the variable
region framework that are required to retain donor antibody binding
specificity are derived from a non-human immunoglobulin, such as
the murine WNV monoclonal antibody, and the remainder of the
antibody is derived from a human immunoglobulin, thereby producing
a vector for the expression of a humanized antibody heavy chain; b)
constructing, by conventional molecular biology methods, an
expression vector comprising an operon that encodes an antibody
light chain in which the CDRs and a minimal portion of the variable
region framework that are required to retain donor antibody binding
specificity are derived from a non-human immunoglobulin, such as
the murine WNV monoclonal antibody, and the remainder of the
antibody is derived from a human immunoglobulin, thereby producing
a vector for the expression of humanized antibody light chain; c)
transferring the expression vectors to a host cell by conventional
molecular biology methods to produce a transfected host cell for
the expression of humanized anti-WNV antibodies; and d) culturing
the transfected cell by conventional cell culture techniques so as
to produce humanized anti-WNV antibodies. Host cells may be
cotransfected with two expression vectors of the invention, the
first vector containing an operon encoding a heavy chain derived
polypeptide and the second containing an operon encoding a light
chain derived polypeptide. The two vectors may contain different
selectable markers but, with the exception of the heavy and light
chain coding sequences, are preferably identical. This procedure
provides for equal expression of heavy and light chain
polypeptides. Alternatively, a single vector may be used which
encodes both heavy and light chain polypeptides. The coding
sequences for the heavy and light chains may comprise cDNA or
genomic DNA or both. The host cell used to express the recombinant
humanized antibodies of the invention may be either a bacterial
cell such as Escherichia coli, or preferably a eukaryotic cell.
Preferably, a mammalian cell such as a chinese hamster ovary cell
or HEK-293 cells, may be used. The choice of expression vector is
dependent upon the choice of host cell, and may be selected so as
to have the desired expression and regulatory characteristics in
the selected host cell. Other cell lines that may be used include,
but are not limited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden,
Netherlands).
[0170] In a specific embodiment the method for producing a
humanized WNV antibody comprises the following: RNA from hybridoma
cells of E16 is converted to cDNA and the VH and VL segrnents are
PCR amplified using, for example, the RLM-RACE kit (Ambion, Inc.).
Gene specific primers for the VH are used. Examples of such primers
for VH include: SJ15R, SEQ ID NO: 57 (5' GGT CAC TGT CAC TGG CTC
AGG G 3') and SJ16R, SEQ ID NO: 58 (5' AGG CGG ATC CAG GGG CCA GTG
GAT AGA C 3'), and for VL include SJ17R, SEQ ID NO: 59 (5' GCA CAC
GAC TGA GGC ACC TCC AGA TG 3') and SJ18R, SEQ ID NO: 60 (5'CGG CGG
ATC CGA TGG ATA CAG TTG GTG CAG CAT C3'). The RACE product is
inserted into a plasmid, e.g., pCR2.1--TOPO using a TOPO TA Cloning
kit (Invitrogen, Inc.). The resulting plasmids are then subjected
to DNA sequencing to determine the VH and VL sequences for E16. The
resulting sequences are translated and the predicted amino acid
sequence determined for each. From these sequences the framework
(FR) and complementarity determining (CDR) regions are identified
as defined by Kabat. The mouse VH is then joined to a human
C-Gamma1 constant region and an Ig leader sequence and inserted
into pCI-neo for mammalian expression. The mouse VL is joined to a
human C-kappa segment and an Ig leader sequence and also cloned
into pCI-neo for mammalian expression. The humanized E16 VH
consists of the FR segments from the human germline VH segment
VH1-18 and JH6, and the CDR regions of the E16 VH. The humanized
E16 VL consists of the FR segments of the human germline VL segment
VK-B3, and the CDR regions of E16 VL. The humanized VH and VL
segments are assembled de novo from oligonucleotides combined and
amplified by PCR. The resulting fragment is then combined by PCR
with a leader sequence and the appropriate constant region segment
cloned into the expression vector pCI-neo. The DNA sequence of the
resulting plasmids is confirmed by sequence analysis. After this
procedure light chain segments having predicted humanized E16 VL
sequence are identified. Humanized E24 and humanized E34 antibodies
are made in a similar manner.
[0171] The general methods for construction of the vectors of the
invention, transfection of cells to produce the host cell of the
invention, culture of cells to produce the humanized antibodies of
the invention are all conventional molecular biology methods.
Likewise, once produced, the recombinant humanized antibodies of
the invention may be purified by standard procedures of the art,
including cross-flow filtration, ammonium sulphate precipitation,
affinity column chromatography, gel electrophoresis and the
like.
[0172] The humanized WNV specific antibodies of the present
invention may be used in conjunction with, or attached to, other
antibodies (or parts thereof) such as human or humanized monoclonal
antibodies. These other antibodies may be reactive with other
markers (epitopes) characteristic for the disease against which the
humanized antibodies of the invention are directed or may have
different specificities chosen, for example, to recruit molecules
or cells of the human immune system to the infected cells. The
humanized antibodies of the invention (or parts thereof) may be
administered with such antibodies (or parts thereof) as separately
administered compositions or as a single composition with the two
agents linked by conventional chemical or by molecular biological
methods. Additionally the diagnostic and therapeutic value of the
humanized antibodies of the invention may be augmented by labelling
the humanized antibodies with labels that produce a detectable
signal (either in vitro or in vivo) or with a label having a
therapeutic property. Some labels, e.g., radionucleotides, may
produce a detectable signal and have a therapeutic property.
Examples of radionuclide labels include, but are not limited to,
.sup.125I, .sup.131I, and .sup.14C. Examples of other detectable
labels include a fluorescent chromophore such as fluorescein,
phycobiliprotein or tetraethyl rhodamine for fluorescence
microscopy, an enzyme which produces a fluorescent or colored
product for detection by fluorescence, absorbance, visible color or
agglutination, which produces an electron dense product for
demonstration by electron microscopy; or an electron dense molecule
such as ferritin, peroxidase or gold beads for direct or indirect
electron microscopic visualization.
[0173] The invention encompasses standard recombinant DNA methods
for preparing DNA sequences which code for the CDR-grafted
humanized antibodies of the invention. DNA sequences may be
synthesized completely or in part using oligonucleotide synthesis
techniques. Methods for oligonucleotide directed synthesis are well
known in the art. The invention further encompasses site-directed
mutagenesis methods such as those known in the art.
[0174] Any suitable host cell/vector system may be used for
expression of the DNA sequences coding for the CDR-grafted heavy
and light chains. Bacterial, e.g., E. coli, and other microbial
systems may be used, in particular for expression of antibody
fragments such as Fab and (Fab').sub.2 fragments, and especially FV
fragments and single chain antibody fragments, e.g., single chain
FVs. Eucaryotic systems, e.g., mammalian host cell expression
systems, may be used for production of larger CDR-grafted antibody
products, including complete antibody molecules. Suitable mammalian
host cells include CHO cells and myeloma or hybridoma cell lines.
Other cell lines that may be used include, but are not limited to,
CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).
[0175] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the complete light chain, and the variable
region, the CH1 region and at least a portion of the hinge region
of the heavy chain.
[0176] Humanized antibodies of the invention can also be generated
using various phage display methods known in the art. In phage
display methods, functional antibody domains are displayed on the
surface of phage particles which carry the polynucleotide sequences
encoding them. In a particular embodiment, such phage can be
utilized to display antigen binding domains, such as Fab and Fv or
disulfide-bond stabilized Fv, expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage,
including fd and M13. The antigen binding domains are expressed as
a recombinantly fused protein to either the phage gene III or gene
VIII protein. Examples of phage display methods that can be used to
make the immunoglobulins, or fragments thereof, of the present
invention include those disclosed in Brinkman et al., 1995, J.
Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods
184:177-186; Kettleborough et al., 1994, Eur. J. Immunol.,
24:952-958; Persic et al., 1997., Gene 187:9-18; Burton et al.,
1994, Advances in Immunology 57:191-280; PCT Application No.
PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0177] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired fragments, and expressed in any desired host,
including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as described in detail below. For example,
techniques to recombinantly produce Fab, Fab' and F(ab').sub.2
fragments can also be employed using methods known in the art such
as those disclosed in PCT Publication WO 92/22324; Mullinax et al.,
BioTechniques, 12(6):864-869, 1992; and Sawai et al., AJRI,
34:26-34, 1995; and Better et al., Science, 240:1041-1043, 1988
(each of which is incorporated by reference in its entirety).
Examples of techniques which can be used to produce single-chain
Fvs and antibodies include those described in U.S. Pat. Nos.
4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology
203:46-88; Shu et al., 1993, Proc. Natl. Acad. Sci. USA
90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.
[0178] Phage display technology can be used to increase the
affinity of humanized antibodies of the invention for its cognate
antigen, e.g., flaviviral antigen. This technique would be useful
in obtaining high affinity antibodies that could be used in the
combinatorial methods of the invention. This technology, referred
to as affinity maturation, employs mutagenesis or CDR walking and
re-selection using a flaviviral antigen or an antigenic fragment
thereof to identify antibodies that bind with higher affinity to
the antigen when compared with the initial or parental antibody
(See, e.g., Glaser et al., 1992, J Immunology 149:3903).
Mutagenizing entire codons rather than single nucleotides results
in a semi-randomized repertoire of amino acid mutations. Libraries
can be constructed consisting of a pool of variant clones each of
which differs by a single amino acid alteration in a single CDR and
which contain variants representing each possible amino acid
substitution for each CDR residue. Mutants with increased binding
affinity for the antigen can be screened by contacting the
immobilized mutants with labeled antigen. Any screening method
known in the art can be used to identify mutant antibodies with
increased avidity to the antigen (e.g., ELISA) (See Wu et al.,
1998, Proc Natl. Acad. Sci. USA 95:6037; Yelton et al., 1995, J
Immunology 155:1994). CDR walking which randomizes the light chain
is also possible (See Schier et al., 1996, J. Mol. Bio.
263:551).
[0179] 5.3 Screening for Biological Properties
[0180] The humanized antibodies of the invention may be
characterized for specific binding to a WNV antigen using any
immunological or biochemical based method known in the art for
characterizing, including quantitating the interaction of the
antibody to a WNV antigen. Specific binding of the humanized
antibodies of the invention to a WNV antigen may be determined, for
example, using immunological or biochemical based methods
including, but not limited to, an ELISA assay, surface plasmon
resonance assays, immunoprecipitation assay, affinity
chromatography, fluorescence activated cell sorting (FACS), and
equilibrium dialysis. Immunoassays which can be used to analyze
immunospecific binding and cross-reactivity of the humanized
antibodies of the invention include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety).
[0181] Humanized antibodies of the invention may be characterized
by epitope mapping, so that humanized antibodies may be selected
that have the greatest specificity for a WNV antigen, e.g., E
protein. Epitope mapping methods of antibodies are well known in
the art and encompassed within the methods of the invention. In
certain embodiments fusion proteins comprising one or more regions
of an WNV antigen may be used in mapping epitopes of the humanized
antibodies of the invention.
[0182] To define distinct structural epitopes that are present on
WNV protein, e.g., E proteins of WNV, the invention encompasses
competition-binding studies using an ELISA and/or surface plasmon
resonance based assays such as those disclosed in (Lanciotti et
al., 2000, J Clin Microbiol 38:4066-71; Modis et al., 2003, Proc
Natl Acad Sci USA 100:6986-91).
[0183] ELISA based assays are well known in the art and encompassed
within the instant invention. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). In an exemplary assay, in the ELISA format, small
quantities of individual purified monoclonal antibodies will be
labeled with biotin. Competing unlabeled monoclonal antibodies will
be bound to recombinant E proteins in microtiter plates.
Subsequently, biotinylated monoclonal antibodies will be added, and
after washing, detected with peroxidase-conjugated streptavidin.
Competition for an individual structural epitope will be defined as
a >40% decrease in the mean OD.sub.450 across multiple
experiments after comparing binding of biotinylated monoclonal
antibodies plus competing monoclonal antibodies with binding of
biotinylated monoclonal antibodies alone.
[0184] Surface plasmon resonance based assays are known in the art
and encompassed within the instant invention. For a review of
SPR-based technology see Mullet et al., 2000, Methods 22: 77-91;
Dong et al., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et
al., 1998, Current Opinion in Biotechnology 9: 97-101; Rich et al.,
2000, Current Opinion in Biotechnology 11: 54-61; all of which are
incorporated herein by reference in their entirety. Additionally,
any of the SPR instruments and SPR based methods for measuring
protein-protein interactions described in U.S. Pat. Nos. 6,373,577;
6,289,286; 5,322,798; 5,341,215; 6,268,125 are contemplated in the
methods of the invention, all of which are incorporated herein by
reference in their entirety. In an exemplary assay, in the BIAcore
format, monoclonal antibodies are reacted sequentially with a
surface onto which the antigen WNV E protein has been coupled,
leading to an increase in the SPR signal. After saturation of all
of the available sites by a first antibody, the addition of a
competing monoclonal antibody will should not increase the SPR
signal appreciably. A non-competing monoclonal antibody, on the
other hand will increase the overall signal independent of the
first binding level achieved. Since the maximum signal obtained
with different mAbs may vary, each assay will be repeated in the
reverse order of monoclonal antibody addition. Preferably the
invention encompasses characterizing the humanized antibodies of
the invention using both an ELISA and a BIAcore based assay to
define a functional epitope map using the panel of mAbs
obtained.
[0185] The invention encompasses epitope mapping using one or more
of the following three strategies: (1) directed evolution of an WNV
antigen, e.g., E protein on the surface of yeast; (2) synthetic
peptides; (3) WNV protein chimeras. An exemplary yeast display
system for epitope mapping of the humanized WNV specific antibodies
of the invention may comprise the following: expressing the entire
ectodomain of WNV E protein or domain III alone on the surface of
yeast; using the yeast displaying these proteins to identify
humanized antibodies that are domain III-specific; a combinatorial
library of E variants will be generated by error-prone PCR and used
to map antibody epitopes at the amino acid level. The entire
ectodomain or domain III of the WNV E protein will be mutagenized
by error-prone PCR; importantly, an N-terminal Xpress.TM. peptide
tag will be added to track E protein surface expression
independently. Mutagenesis will be achieved by changing the
Mg.sup.2+:Mn.sup.2+ ratio (to .about.6.6:1) in the initial PCR
reaction to obtain a nucleotide error rate of approximately 0.5%
using a method such as that disclosed in Chothia et al., 1989,
Nature 342:877-83, or on average 1 amino acid change per variant.
These variants will be cloned into a yeast expression vector, e.g.,
pYD1, with the goal of generating .about.10.sup.5 independent
transformants. Libraries will be constructed by cloning or
homologous recombination of PCR-mutagenized segments with the
parental vector in yeast cells, a technique that gives rise to
libraries of high diversity (See, Chothia et al., 1989, Nature
342:877-83; Holgate et al., 2001, Curr Med Res Opin 17:233-40). To
isolate variants that have lost a particular mAb epitope, an
initial depletion step will be performed with protein G-coated
magnetic beads using a method disclosed in Pogodina et al., 1983,
Arch Virol 75:71-86. The remaining yeast cells will be sorted by
two-color flow cytometry using a directly conjugated mAb to the
Xpress tag and the individual antibody to the E protein that is
being mapped. Yeast cells that are Xpress.sup.HI and anti-E low or
null will be collected, cultivated and subjected to repeated rounds
of sorting and then immunostained with other anti-E mAbs to confirm
that large-scale structural changes have not occurred. Finally, the
E protein variants from individual clones will be sequenced;
plasmids can be recovered from yeast by E. coli rescue using a
commercially available kit (Zymo Research, Orange, Calif.) and used
to prepare DNA for sequencing. Under optimal screening conditions,
flow cytometry sorting should allow fine discrimination between
mutants with antibody specificity changes. In some instances, a
single amino acid change may not be sufficient to abrogate mAb
recognition. For mAbs that show decreased but detectable expression
after the initial screen, serial mutagenesis will be
undertaken.
[0186] In other embodiments, the invention encompasses methods
whereby mAb binding sites may be mapped by analysis of binding to
synthetic peptides or recombinant E protein fragments. Initially,
about 30 overlapping peptides (e.g., 15-20 amino acids in length)
will be synthesized; these peptides will be designed based on
previous mapping studies with the related DEN (see, e.g., Kulkarni
et al., 1991, Viral Immunol 4:73-82; Kurane et al., 1984, J Virol
52:223-30) and Murray Valley encephalitis viruses (see, e.g.,
Kurane et al., 1992, Semin Immunol 4:121-7) and the
three-dimensional crystal structure of DEN (see, e.g., Kacani et
al., 2001, Mol Immunol 38:241-7), tick-borne encephalitis (see,
e.g., Kramer et al., 2001, Ann N Y Acad Sci 951:84-93), and WNV E
proteins. mAbs will be mapped on the basis of their ability to bind
peptides adsorbed to microtiter plates using a standard ELISA
assay.
[0187] Because some of the mAbs may bind non-linear epitopes or
epitopes not correctly displayed by the yeast cells, the invention
further encompasses an alternate strategy using recombinantly
derived fragments of the E protein. The extracellular domain of DEN
and WNV E protein will each be expressed and secreted in mammalian
cells (HEK-293) using a mammalian expression vector (e.g.,
pcDNA3.1). E protein chimera will be generated such that
sub-domains of the WNV E protein are replaced by the equivalent
regions of DEN (or vice versa). Finally, WNV and DEN E proteins
chimera will be made in which specific segments or amino acid
residues of domain III are substituted. Binding of the antibodies
to this each of these proteins will be determined by ELISA and used
for fine structural mapping.
[0188] The invention encompasses characterization of the humanized
antibodies produced by the methods of the invention using certain
characterization assays for identifying the function of the
humanized antibodies of the invention, particularly the activity to
inhibit a flaviviral infection using in vitro and in vivo based
assays. The characterization assays of the invention can be
cell-based or cell-free assays.
[0189] The invention encompasses characterizing the humanized
antibodies of the invention using qualitative based screens, e.g.,
an ELISA assay, preferably as a primary screen for characterizing
the humanized antibodies of the invention. The invention provides
an ELISA that detects humanized antibodies against adsorbed
purified E protein as the primary screen. An exemplary ELISA based
assay for characterizing the humanized antibodies of the invention
comprises the following: when intact virus is used as an immunogen,
lysates from WNV-infected BHK21 cells will be substituted to insure
that additional E protein epitopes are present during the screen;
Positive clones will be confirmed for immunoreactivity with
WNV-infected cells by flow cytometry. To obtain mnAbs that
recognize conserved WNV epitopes, immunoreactivity with other
(lineage I and II) WNV strains will be confirmed. To avoid possible
complications associated with flavivirus cross-reactive antibodies
(e.g., ADE associated with heterologous flavivirus infection),
candidate mAbs that positively react with WNV proteins will be
tested for binding to Vero cells infected with DEN, yellow fever,
or St. Louis encephalitis viruses; only WNV-specific mAbs will be
used for further studies. Because different mAb isotypes may
display different effector functions in vivo, isotypes will be
determined using a commercially available ELISA kit.
[0190] In other embodiments, the invention encompasses quantitative
functional screens to characterize the potential mechanisms of
mAb-mediated inhibition of WNV infection. A scoring system will be
generated from each assay to identify mAbs with the greatest
inhibitory activity. The invention encompasses characterization of
the anitbodies of the invention using virus neutralization assays
using methods known in the art and encompassed herein. In an
exemplary assay, the ability to neutralize WNV infection in cell
culture will be determined using a plaque reduction neutralization
assay (PRNT) with BHK21 cells. For the anti-E mAbs, a neutralizing
index will be generated. Using a standard concentration (e.g., 100
.mu.g/ml) of purified antibody, a point scale will be assigned from
the PRNT.sub.50 value: <1/10=0 points, 1/10-1/100=1 point,
>1/100=2 points. The invention encompasses characterization of
the antibodies of the invention using complement-mediated cytolysis
assays using methods known in the art and encompassed herein. The
ability of antibodies to trigger complement-mediated lysis of
WNV-infected cells will be assessed by a standard target cell lysis
assay (see, e.g., Stanley et al., 1986, J Virol 58:107-115). BHK21
cells will be infected with WNV for 24 hours and labeled with
.sup.51Cr. Washed cells will be incubated with purified mAbs and
guinea pig complement (1 h at 37.degree. C.). Supernatants will be
harvested and antibody-dependent complement-mediated cell lysis
will be measured by scintillation counting. A point scale will be
assigned based on the percentage of cells that are specifically
lysed by mAb and complement: <10%=0 points, 10-40%=1 point,
>40%=2 points. In yet other embodiments, the invention
encompasses characterization of the antibodies of the invention
using Complement-fixation on virus. The ability of mAbs to bind to
virus and fix complement directly in solution will be evaluated by
detecting cleavage products of C3 that occur after fixation using
methods known in the art such as those disclosed in Manderson et
al., 2001, J Exp Med 194:747-56). WNV or DEN virus (negative
control) will be incubated with anti-WNV mAbs against E in the
presence of serum from wild type mice at 37.degree. C. to enable C3
binding. Samples will be denatured with detergent,
immunoprecipitated with goat anti-mouse C3, and subjected to
Western blot analysis with rabbit polyclonal antibodies against C3.
If complement fixation occurs, the C3a chain (M, of 100) will be
cleaved and increased levels of C3d (M, of 40) will be detected. As
an additional control, mAbs and WNV will also be incubated with
factor B -/- and Clq -/- serum. If complement fixation on virus
requires antibodies (and uses the classical pathway of complement
activation), a deficiency of Clq but not factor B will prevent
conversion of C3 to C3d. The use of these complement-deficient sera
will confirm that antibody binding triggers C3 activation directly
and rule out C3 activation that occurs spontaneously in solution
(Manderson et al., 2001, J Exp Med 194:747-56) or via the
alternative pathway. A point scale will be assigned based on
whether mAbs facilitate direct complement-fixation on WNV: no C3
fixation=0 points, C3 fixation=2 points.
[0191] In yet other embodiments, the invention encompasses
characterization of the humanized antibodies of the invention using
Antibody-dependent cell-mediated cytotoxicity (ADCC) assays known
in the art and encompassed herein. The ability of mAbs to promote
ADCC of WNV-infected cells will be evaluated according to
previously described assays (Kurane et al., 1984, J Virol
52:223-30; Meguro et al., 1979, J Immunol 122:2521-6; Zhang et al.,
1992, Acta Virol 36:533-40). MC57GL mouse fibroblasts will be
infected with WNV for 24 hours, labeled with .sup.51Cr, incubated
with purified anti-WNV or control mAbs, and mixed with different
concentrations of washed syngeneic peripheral blood mononuclear
cells (PBMC) isolated from WNV-naive mice. After incubation (12 to
16 h at 37.degree. C.), supernatants will be harvested and ADCC
activity will be measured by scintillation counting. A point scale
will be assigned based on the percentage of cells that are
specifically lysed in the presence of mAb with an effector:target
ratio of 50:1: <10%=0 points, 10-40%=1 point, >40%=2
points.
[0192] Because passive administration of high-affinity
non-neutralizing mAbs can prevent lethal encephalitis caused by
Sindbis virus (Schmaljohn et al., 1982, Nature 297:70-2), mAbs will
also be evaluated for their relative avidity. Avidity will be
assessed by the constant antigen varying antibody method (Tyler et
al., 1993, J Virol 67:3446-53; Virgin et al., 1991, J Virol
65:6772-81). A fixed quantity of recombinant E protein will be
adsorbed to a microtiter well, incubated with varying
concentrations of I.sup.125-labeled purified mAb, and evaluated for
reactivity by scintillation counting. Competition studies will be
performed with a 100-fold excess of unlabeled antibody so that a
K.sub.D can be determined by Scatchard analysis. A point scale will
be assigned based on the relative avidity of the bivalent mAbs for
purified WNV proteins: >10.sup.-6 M=0 points,
10.sup.-6-10.sup.-8 M=1 point, <10.sup.-8 M=2 points.
[0193] As mentioned, the point system is designed to facilitate
ranking and selection of the mAbs with the greatest potential
inhibitory activity of three categories will be selected for
further competition binding and in vivo studies.
[0194] 5.4 Prophylactic and Therapeutic Methods
[0195] The present invention encompasses antibody-based therapies
which involve administering one or more of the humanized antibodies
of the invention to an animal, preferably a mammal, and most
preferably a human, for preventing, treating, or ameliorating one
or more symptoms associated with a flaviviral infection,
particularly an WNV infection. Prophylactic and therapeutic
compounds of the invention include, but are not limited to, the
humanized antibodies of the invention (including fragments, analogs
and derivatives thereof as described herein) and nucleic acids
encoding the humanized antibodies of the invention (including
fragments, analogs and derivatives thereof) and anti-idiotypic
antibodies as described herein. Humanized antibodies of the
invention or fragments thereof may be provided in pharmaceutically
acceptable compositions as known in the art or as described
herein.
[0196] Humanized antibodies of the present invention or fragments
thereof that function as antagonists of a flaviviral infection can
be administered to a mammal, preferably a human, to treat, prevent
or ameliorate one or more symptoms associated with a flaviviral
infection. For example, humanized antibodies or fragments thereof
which disrupt or prevent the interaction between a flaviviral
antigen and its host cell receptor may be administered to a mammal,
preferably a human, to treat, prevent or ameliorate one or more
symptoms associated with a flaviviral infection.
[0197] The present invention provides methods for treating,
preventing, or ameliorating a flaviviral infection by
administration of one or more humanized antibodies of the
invention. In a specific embodiment, the invention encompasses
methods for treating, preventing, or ameliorating a WNV infection
comprising administering a humanized antibody that
immunospecifically binds a structural protein of WNV, e.g., E
protein. In another embodiment, the invention encompasses methods
for treating, preventing, or ameliorating a WNV infection
comprising administering a first humanized antibody that
immunospecifically binds a structural protein of WNV, e.g., E
protein, and a second antibody that binds a non-structural protein
of WNV, e.g., NS1 protein. Although not intending to be bound by a
particular mechanism of action such combination regimens are more
effective than single antibody treatment regimens because the
RNA-dependent RNA polymerase of WNV has a high error rate and thus
a potential to rapidly alter immunodominant residues. In other
specific embodiments, the invention encompasses methods for
treating, preventing, or ameliorating a WNV infection comprising
administering a first antibody that immunospecifically binds an
epitope of a structural protein of WNV, e.g., E protein, and a
second antibody that binds the same structural protein of WNV but
binds a different epitope.
[0198] In a specific embodiment, a humanized antibody or fragment
thereof prevents flavivirus, e.g. WNV from binding to its host cell
receptor by at least 99%, at least 95%, at least 90%, at least 85%,
at least 80%, at least 75%, at least 70%, at least 60%, at least
50%, at least 45%, at least 40%, at least 45%, at least 35%, at
least 30%, at least 25%, at least 20%, or at least 10% relative to
flaviviral binding to its host cell receptor in the absence of said
humanized antibodies or antibody fragments. In another embodiment,
a combination of humanized antibodies, a combination of antibody
fragments, or a combination of humanized antibodies and antibody
fragments prevent flaviviral from binding to its host cell receptor
by at least 99%, at least 95%, at least 90%, at least 85%, at least
80%, at least 75%, at least 70%, at least 60%, at least 50%, at
least 45%, at least 40%, at least 45%, at least 35%, at least 30%,
at least 25%, at least 20%, or at least 10% relative to WNV binding
to its host cell receptor in the absence of said antibodies and/or
antibody fragments.
[0199] One or more humanized antibodies of the present invention or
fragments thereof that immunospecifically bind to one or more
flaviviral antigens, particularly a WNV antigen, may be used
locally or systemically in the body as a therapeutic. The humanized
antibodies of this invention or fragments thereof may also be
advantageously utilized in combination with other monoclonal or
chimeric antibodies, or with lymphokines or hematopoietic growth
factors (such as, e.g., IL-2, IL-3 and IL-7), which, for example,
serve to increase the number or activity of effector cells which
interact with the antibodies or serve to increase the immune
response. The humanized antibodies of this invention or fragments
thereof may also be advantageously utilized in combination with one
or more drugs used to treat flaviviral infections, particularly WNV
infections, such as, for example anti-viral agents. Examples of
anti-viral agents include, but are not limited to, protease
inhibitors, nucleoside reverse transcriptase inhibitors,
non-nucleoside reverse transcriptase inhibitors and nucleoside
analogs, zidovudine, acyclovir, gangcyclovir, vidarabine,
idoxuridine, trifluridine, and ribavirin, as well as foscarnet,
amantadine, rimantadine, saquinavir, indinavir, amprenavir,
lopinavir, ritonavir, the alpha-interferons; adefovir, clevadine,
entecavir, and pleconaril. The invention encompasses any other
anti-viral agent being developed and known to those skilled in the
art.
[0200] The humanized antibodies of the invention may be
administered alone or in combination with other types of treatments
(e.g., hormonal therapy, immunotherapy, and anti-inflammatory
agents). Generally, administration of products of a species origin
or species reactivity (in the case of antibodies) that is the same
species as that of the patient is preferred. Thus, in a preferred
embodiment, human or humanized antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0201] It is preferred to use high affinity and/or potent in vivo
inhibiting antibodies and/or neutralizing antibodies that
immunospecifically bind to a flaviviral antigen, particularly WNV
antigen, for prevention of flaviviral infection, particularly WNV
infection and therapy for flaviviral infection, particularly WNV
infection. It is also preferred to use polynucleotides encoding
high affinity and/or potent in vivo inhibiting antibodies and/or
neutralizing antibodies that immunospecifically bind to a
flaviviral antigen, particularly WNV antigen, for both immunoassays
directed to WNV and therapy for WNV infection. Such antibodies or
fragments thereof will preferably have an affinity for the WNV E
protein. In a specific embodiment, a mammal, preferably a human, is
administered a therapeutic or pharmaceutical composition comprising
one or more humanized antibodies of the present invention or
fragments thereof for the treatment, prevention or amelioration of
one or more symptoms associated with a flavirial infection,
particularly WNF infection.
[0202] Prophylactic and therapeutic compounds that may be used in
combination with the humanized antibodies of the invention include,
but are not limited to, proteinaceous molecules, including, but not
limited to, peptides, polypeptides, proteins, including
post-translationally modified proteins, antibodies, etc.; small
molecules (less than 1000 daltons), inorganic or organic compounds;
nucleic acid molecules including, but not limited to,
double-stranded or single-stranded DNA, double-stranded or
single-stranded RNA, as well as triple helix nucleic acid
molecules. Prophylactic and therapeutic compounds can be derived
from any known organism (including, but not limited to, animals,
plants, bacteria, fungi, and protista, or viruses) or from a
library of synthetic molecules.
[0203] In certain embodiments, one or more humanized antibodies of
the invention are administered to a mammal, preferably, a human,
concurrently with one or more other therapeutic agents, e.g.,
anti-viral agents, useful for the treatment or prevention of a
flaviviral infection, particularly, a WNV infection. The term
"concurrently" is not limited to the administration of prophylactic
or therapeutic agents at exactly the same time, but rather it is
meant that humanized antibodies of the invention and the other
agent are administered to a subject in a sequence and within a time
interval such that the humanized antibodies of the invention can
act together with the other agent to provide an increased benefit
than if they were administered otherwise. For example, each
prophylactic or therapeutic agent may be administered at the same
time or sequentially in any order at different points in time;
however, if not administered at the same time, they should be
administered sufficiently close in time so as to provide the
desired therapeutic or prophylactic effect. Each therapeutic agent
can be administered separately, in any appropriate form and by any
suitable route.
[0204] In various embodiments, the prophylactic or therapeutic
agents are administered less than 1 hour apart, at about 1 hour
apart, at about 1 hour to about 2 hours apart, at about 2 hours to
about 3 hours apart, at about 3 hours to about 4 hours apart, at
about 4 hours to about 5 hours apart, at about 5 hours to about 6
hours apart, at about 6 hours to about 7 hours apart, at about 7
hours to about 8 hours apart, at about 8 hours to about 9 hours
apart, at about 9 hours to about 10 hours apart, at about 10 hours
to about 11 hours apart, at about 11 hours to about 12 hours apart,
no more than 24 hours apart or no more than 48 hours apart. In
preferred embodiments, two or more components are administered
within the same patient visit.
[0205] The dosage amounts and frequencies of administration
provided herein are encompassed by the terms therapeutically
effective and prophylactically effective. The dosage and frequency
further will typically vary according to factors specific for each
patient depending on the specific therapeutic or prophylactic
agents administered, the severity and type of cancer, the route of
administration, as well as age, body weight, response, and the past
medical history of the patient. Suitable regimens can be selected
by one skilled in the art by considering such factors and by
following, for example, dosages reported in the literature and
recommended in the Physician's Desk Reference (56.sup.th ed.,
2002).
[0206] 5.5 Compositions and Methods of Administering
[0207] The invention provides methods of treatment, prophylaxis,
and amelioration of one or more symptoms associated with flaviviral
infection, particularly WNV infection, by administrating to a
subject of an effective amount of a humanized antibody of the
invention or fragment thereof, or pharmaceutical composition
comprising a humanized antibody of the invention or fragment
thereof. In a preferred aspect, an antibody or fragment thereof is
substantially purified (i.e., substantially free from substances
that limit its effect or produce undesired side-effects). The
subject is preferably a mammal such as non-primate (e.g., cows,
pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey
such as a cynomolgous monkey and a human). In a preferred
embodiment, the subject is a human, particularly a human who is at
an increased risk of flaviviral infection, particularly WNV
infection. In another preferred embodiment, the subject is a human
infant, an elderly human, or a human with an impaired immune
system.
[0208] Various delivery systems are known and can be used to
administer a composition comprising humanized antibodies of the
invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or fusion protein, receptor-mediated endocytosis (see, e.g., Wu and
Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic
acid as part of a retroviral or other vector, etc.
[0209] In some embodiments, the humanized antibodies of the
invention are formulated in liposomes for targeted delivery of the
humanized antibodies of the invention. Liposomes are vesicles
comprised of concentrically ordered phopsholipid bilayers which
encapsulate an aqueous phase. Liposomes typically comprise various
types of lipids, phospholipids, and/or surfactants. The components
of liposomes are arranged in a bilayer configuration, similar to
the lipid arrangement of biological membranes. Liposomes are
particularly preferred delivery vehicles due, in part, to their
biocompatibility, low immunogenicity, and low toxicity. Methods for
preparation of liposomes are known in the art and are encompassed
within the invention, see, e.g., Epstein et al., 1985, Proc. Natl.
Acad. Sci. USA, 82: 3688; Hwang et al., 1980 Proc. Natl. Acad. Sci.
USA, 77: 4030-4; U.S. Pat. Nos. 4,485,045 and 4,544,545; all of
which are incorporated herein by reference in their entirety.
[0210] The invention also encompasses methods of preparing
liposomes with a prolonged serum half-life, i.e., enhanced
circulation time, such as those disclosed in U.S. Pat. No.
5,013,556. Preferred liposomes used in the methods of the invention
are not rapidly cleared from circulation, i.e., are not taken up
into the mononuclear phagocyte system (MPS). The invention
encompasses sterically stabilized liposomes which are prepared
using common methods known to one skilled in the art. Although not
intending to be bound by a particular mechanism of action,
sterically stabilized liposomes contain lipid components with bulky
and highly flexible hydrophilic moieties, which reduces the
unwanted reaction of liposomes with serum proteins, reduces
oposonization with serum components and reduces recognition by MPS.
Sterically stabilized liposomes are preferably prepared using
polyethylene glycol. For preparation of liposomes and sterically
stabilized liposome, see, e.g., Bendas et al., 2001 BioDrugs,
15(4): 215-224; Allen et al., 1987 FEBS Lett. 223: 42-6; Klibanov
et al., 1990 FEBS Lett., 268: 235-7; Blum et al., 1990, Biochim.
Biophys. Acta., 1029: 91-7; Torchilin et al., 1996, J Liposome Res.
6: 99-116; Litzinger et al., 1994, Biochim. Biophys. Acta, 1190:
99-107; Maruyama et al., 1991, Chem. Pharm. Bull., 39: 1620-2;
Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8; Allen et
al., 1994, Adv. Drug Deliv. Rev, 13: 285-309; all of which are
incorporated herein by reference in their entirety. The invention
also encompasses liposomes that are adapted for specific organ
targeting, see, e.g., U.S. Pat. No. 4,544,545. Particularly useful
liposomes for use in the compositions and methods of the invention
can be generated by reverse phase evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG
derivatized phosphatidylethanolamine (PEG-PE). Liposomes are
extruded through filters of defined pore size to yield liposomes
with the desired diameter. In some embodiments, a fragment of a
humanized antibody of the invention, e.g., F(ab'), may be
conjugated to the liposomes using previously described methods,
see, e.g., Martin et al., 1982, J. Biol. Chem. 257: 286-288, which
is incorporated herein by reference in its entirety.
[0211] The humanized antibodies of the invention may also be
formulated as immunoliposomes. Immunoliposomes refer to a liposomal
composition, wherein a humanized antibody of the invention or a
fragment thereof is linked, covalently or non-covalently to the
liposomal surface. The chemistry of linking an antibody to the
liposomal surface is known in the art and encompassed within the
invention, see, e.g., Allen et al., 1995, Stealth Liposomes, Boca
Raton: CRC Press, 233-44; Hansen et al., 1995, Biochim. Biophys.
Acta, 1239: 133-44; which are incorporated herein by reference in
their entirety. In most preferred embodiments, immunoliposomes for
use in the methods and compositions of the invention are further
sterically stabilized. Preferably, the humanized antibodies of the
invention are linked covalently or non-covalently to a hydrophobic
anchor, which is stably rooted in the lipid bilayer of the
liposome. Examples of hydrophobic anchors include, but are not
limited to, phospholipids, e.g., phosoatidylethanolamine (PE),
phospahtidylinositol (PI). To achieve a covalent linkage between an
antibody and a hydrophobic anchor, any of the known biochemical
strategies in the art may be used, see, e.g., J. Thomas August,
ed., 1997, Gene Therapy: Advances in Pharmacology, Volume 40,
Academic Press, San Diego, Calif., p. 399-435, which is
incorporated herein by reference in its entirety. For example, a
functional group on an antibody molecule may react with an active
group on a liposome associated hydrophobic anchor, e.g., an amino
group of a lysine side chain on an antibody may be coupled to
liposome associated N-glutaryl-phosphatidylethanolamine activated
with water-soluble carbodiimide; or a thiol group of a reduced
antibody can be coupled to liposomes via thiol reactive anchors,
such as pyridylthiopropionyl-phosphatidylethanolamine. See, e.g.,
Dietrich et al., 1996, Biochemistry, 35: 1100-1105; Loughrey et
al., 1987, Biochim. Biophys. Acta, 901: 157-160; Martin et al.,
1982, J. Biol. Chem. 257: 286-288; Martin et al., 1981,
Biochemistry, 20: 4429-38; all of which are incorporated herein by
reference in their entirety.
[0212] The invention encompasses immunoliposomes comprising a
humanized antibody of the invention or a fragment thereof. In some
embodiments, the immunoliposomes further comprise one or more
additional therapeutic agents, such as those disclosed herein.
[0213] The immunoliposomal compositions of the invention comprise
one or more vesicle forming lipids, a humanized antibody of the
invention or a fragment or derivative thereof, and, optionally, a
hydrophilic polymer. A vesicle forming lipid is preferably a lipid
with two hydrocarbon chains, such as acyl chains and a polar head
group. Examples of vesicle forming lipids include phospholipids,
e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic
acid, phosphatidylinositol, sphingomyelin, and glycolipids, e.g.,
cerebrosides, gangliosides. Additional lipids useful in the
formulations of the invention are known to one skilled in the art
and encompassed within the invention. In some embodiments, the
immunoliposomal compositions further comprise a hydrophilic
polymer, e.g., polyethylene glycol, and gnaglioside GM1, which
increases the serum half life of the liposome. Methods of
conjugating hydrophilic polymers to liposomes are well known in the
art and encompassed within the invention. For a review of
immunoliposomes and methods of preparing them, see, e.g., PCT
International Publication No. WO 97/38731, Vingerhoeads et al.,
1994, Immunomethods, 4: 259-72; Maruyama, 2000, Biol. Pharm. Bull.
23(7): 791-799; Abra et al., 2002, Journal of Liposome Research,
12(1&2): 1-3; Park, 2002, Bioscience Reports, 22(2): 267-281;
Bendas et al., 2001 BioDrugs, 14(4): 215-224, J. Thomas August,
ed., 1997, Gene Therapy: Advances in Pharmacology, Volume 40,
Academic Press, San Diego, Calif., p. 399-435, all of which are
incorporated herein by reference in their entireties.
[0214] Methods of administering the humanized antibodies of the
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural, and mucosal (e.g.,
intranasal and oral routes). In a specific embodiment, the
humanized antibodies of the invention are administered
intramuscularly, intravenously, or subcutaneously. The compositions
may be administered by any convenient route, for example, by
infusion or bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.) and may be administered together with other
biologically active agents. Administration can be systemic or
local. In addition, pulmonary administration can also be employed,
e.g., by use of an inhaler or nebulizer, and formulation with an
aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,20;
5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and
4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO
97/44013; WO 98/31346; and WO 99/66903, each of which is
incorporated herein by reference in its entirety.
[0215] The invention also provides that the humanized antibodies of
the invention are packaged in a hermetically sealed container, such
as an ampoule or sachette, indicating the quantity of antibody. In
one embodiment, the humanized antibodies of the invention are
supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted, e.g., with water or saline to the appropriate
concentration for administration to a subject. Preferably, the
humanized antibodies of the invention are supplied as a dry sterile
lyophilized powder in a hermetically sealed container at a unit
dosage of at least 5 mg, more preferably at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, or at least 75 mg. The lyophilized humanized antibodies of the
invention should be stored at between 2 and 8.degree. C. in their
original container and the humanized antibodies should be
administered within 12 hours, preferably within 6 hours, within 5
hours, within 3 hours, or within 1 hour after being reconstituted.
In an alternative embodiment, humanized antibodies of the invention
are supplied in liquid form in a hermetically sealed container
indicating the quantity and concentration of the antibody, fusion
protein, or conjugated molecule. Preferably, the liquid form of the
humanized antibodies are supplied in a hermetically sealed
container at least 1 mg/ml, more preferably at least 2.5 mg/ml, at
least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15
mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at
least 150 mg/ml, at least 200 mg/ml of the humanized
antibodies.
[0216] The amount of the composition of the invention which will be
effective in the treatment, prevention or amelioration of one or
more symptoms associated with a flaviviral infection, particularly,
a WNV infection, can be determined by standard clinical techniques.
The precise dose to be employed in the formulation will also depend
on the route of administration, and the seriousness of the
condition, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0217] For humanized antibodies encompassed by the invention, the
dosage administered to a patient is typically 0.0001 mg/kg to 100
mg/kg of the patient's body weight. Preferably, the dosage
administered to a patient is between 0.0001 mg/kg and 20 mg/kg,
0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2
mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001
mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15
mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg
or 0.01 to 0.10 mg/kg of the patient's body weight. In one
embodiment, the dosage of the humanized antibodies of the invention
administered to a patient are 0.01 mg to 1000 mg/day, when used as
single agent therapy. In other embodiments, the therapeutically or
prophylactically effective dosage administered to a subject is
typically 0.1 mg/kg to 200 mg/kg of the subject's body weight.
Preferably, the dosage administered to a subject is between 0.1
mg/kg and 20 mg/kg of the subject's body weight and more preferably
the dosage administered to a subject is between 1 mg/kg to 10 mg/kg
of the subject's body weight. Generally, human antibodies have a
longer half-life within the human body than antibodies from other
species due to the immune response to the foreign polypeptides.
Thus, lower dosages of human antibodies and less frequent
administration is often possible. Further, the dosage and frequency
of administration of humanized antibodies of the invention or
fragments thereof may be reduced by enhancing uptake and tissue
penetration of the antibodies by modifications such as, for
example, lipidation.
[0218] In another embodiment the antibodies of the invention are
used in combination with other therapeutic compositions and the
dosage administered to a patient are lower than when said humanized
antibodies are used as a single agent therapy.
[0219] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion, by injection, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. Preferably, when administering a humanized
antibody of the invention, care must be taken to use materials to
which the antibody or the fusion protein does not absorb.
[0220] In another embodiment, the compositions can be delivered in
a vesicle, in particular a liposome (See Langer, Science
249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.).
[0221] In yet another embodiment, the compositions can be delivered
in a controlled release or sustained release system. Any technique
known to one of skill in the art can be used to produce sustained
release formulations comprising one or more humanized antibodies of
the invention. See, e.g., U.S. Pat. No. 4,526,938; PCT publication
WO 91/05548; PCT publication WO 96/20698; Ning et al., 1996,
"Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft
Using a Sustained-Release Gel," Radiotherapy & Oncology
39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397; Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854; and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in its entirety. In one
embodiment, a pump may be used in a controlled release system (See
Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20;
Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N.
Engl. J. Med. 321:574). In another embodiment, polymeric materials
can be used to achieve controlled release of antibodies (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; See also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 71:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253). Examples of
polymers used in sustained release formulations include, but are
not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl
methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet
another embodiment, a controlled release system can be placed in
proximity of the therapeutic target (e.g., the lungs), thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)). In another embodiment, polymeric compositions
useful as controlled release implants are used according to Dunn et
al. (See U.S. Pat. No. 5,945,155). This particular method is based
upon the therapeutic effect of the in situ controlled release of
the bioactive material from the polymer system. The implantation
can generally occur anywhere within the body of the patient in need
of therapeutic treatment. In another embodiment, a non-polymeric
sustained delivery system is used, whereby a non-polymeric implant
in the body of the subject is used as a drug delivery system. Upon
implantation in the body, the organic solvent of the implant will
dissipate, disperse, or leach from the composition into surrounding
tissue fluid, and the non-polymeric material will gradually
coagulate or precipitate to form a solid, microporous matrix (See
U.S. Pat. No. 5,888,533).
[0222] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938; International
Publication Nos. WO 91/05548 and WO 96/20698; Ning et al., 1996,
Radiotherapy & Oncology 39:179-189; Song et al., 1995, PDA
Journal of Pharmaceutical Science & Technology 50:372-397;
Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.
Bioact. Mater. 24:759-760, each of which is incorporated herein by
reference in its entirety.
[0223] In a specific embodiment where the composition of the
invention is a nucleic acid encoding an antibody, the nucleic acid
can be administered in vivo to promote expression of its encoded
humanized antibody, by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by use of a retroviral vector (See
U.S. Pat. No. 4,980,286), or by direct injection, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, or by administering it in linkage to a homeobox-like
peptide which is known to enter the nucleus (See e.g., Joliot et
al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.
Alternatively, a nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression by homologous
recombination.
[0224] Treatment of a subject with a therapeutically or
prophylactically effective amount of humanized antibodies of the
invention can include a single treatment or, preferably, can
include a series of treatments. In a preferred example, a subject
is treated with humanized antibodies of the invention in the range
of between about 0.1 to 30 mg/kg body weight, one time per week for
between about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. In other embodiments, the pharmaceutical
compositions of the invention are administered once a day, twice a
day, or three times a day. In other embodiments, the pharmaceutical
compositions are administered once a week, twice a week, once every
two weeks, once a month, once every six weeks, once every two
months, twice a year or once per year. It will also be appreciated
that the effective dosage of the humanized antibodies used for
treatment may increase or decrease over the course of a particular
treatment.
[0225] 5.5.1 Pharmaceutical Compositions
[0226] The compositions of the invention include bulk drug
compositions useful in the manufacture of pharmaceutical
compositions (e.g., impure or non-sterile compositions) and
pharmaceutical compositions (i.e., compositions that are suitable
for administration to a subject or patient) which can be used in
the preparation of unit dosage forms. Such compositions comprise a
prophylactically or therapeutically effective amount of a
prophylactic and/or therapeutic agent disclosed herein or a
combination of those agents and a pharmaceutically acceptable
carrier. Preferably, compositions of the invention comprise a
prophylactically or therapeutically effective amount of humanized
antibodies of the invention and a pharmaceutically acceptable
carrier.
[0227] In one particular embodiment, the pharmaceutical composition
comprises of a therapeutically effective amount of a humanized
antibody, or a fragment thereof, that binds one or more flaviviral
antigens, particularly WNV antigens, and a pharmaceutically
acceptable carrier. In another embodiment, said pharmaceutical
composition further comprises one or more additional prophylactic
or therapeutic agents.
[0228] In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant (e.g., Freund's adjuvant (complete and incomplete),
excipient, or vehicle with which the therapeutic is administered.
Such pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. The composition, if desired, can also contain minor
amounts of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions, suspensions,
emulsion, tablets, pills, capsules, powders, sustained-release
formulations and the like.
[0229] Generally, the ingredients of compositions of the invention
are supplied either separately or mixed together in unit dosage
form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0230] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include,
but are not limited to, those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0231] 5.6 Characterization and Demonstration of Therapeutic
Utility
[0232] Humanized antibodies of the present invention or fragments
thereof may be characterized in a variety of ways. In particular,
humanized antibodies of the invention or fragments thereof may be
assayed for the ability to immunospecifically bind to a WNV
antigen. Such an assay may be performed in solution (e.g.,
Houghten, 1992, Bio/Techniques 13:412-421), on beads (Lam, 1991,
Nature 354:82-84), on chips (Fodor, 1993, Nature 364:555-556), on
bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. Nos.
5,571,698; 5,403,484; and 5,223,409), on plasmids (Cull et al.,
1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott
and Smith, 1990, Science 249:386-390; Devlin, 1990, Science
249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310) (each of
these references is incorporated herein in its entirety by
reference). Humanized antibodies or fragments thereof that have
been identified to immunospecifically bind to a flaviviral antigen
or a fragment thereof can then be assayed for their specificity and
affinity for a flaviviral antigen.
[0233] The humanized antibodies of the invention or fragments
thereof may be assayed for immunospecific binding to a flaviviral
antigen, particularly WNV antigen and cross-reactivity with other
antigens by any method known in the art. Immunoassays which can be
used to analyze immunospecific binding and cross-reactivity
include, but are not limited to, competitive and non-competitive
assay systems using techniques such as western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0234] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1 to 4 hours)
at 40.degree. C., adding protein A and/or protein G sepharose beads
to the cell lysate, incubating for about an hour or more at
40.degree. C., washing the beads in lysis buffer and resuspending
the beads in SDS/sample buffer. The ability of the antibody of
interest to immunoprecipitate a particular antigen can be assessed
by, e.g., western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g, pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0235] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sup.32P or .sup.125I diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0236] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g. Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
[0237] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., .sup.3H or .sup.125I) with the antibody of interest
in the presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the humanized antibodies of the present invention or
fragments thereof for a WNV antigen and the binding off-rates can
be determined from the data by scatchard plot analysis. Competition
with a second antibody can also be determined using
radioimmunoassays. In this case, a WNV antigen is incubated with a
humanized antibody of the present invention or a fragment thereof
conjugated to a labeled compound (e.g., .sup.3H or .sup.125I) in
the presence of increasing amounts of an unlabeled second
antibody.
[0238] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of humanized antibodies
or fragments thereof to a WNV antigen. BIAcore kinetic analysis
comprises analyzing the binding and dissociation of a WNV antigen
from chips with immobilized humanized antibodies or fragments
thereof on their surface.
[0239] The humanized antibodies of the invention or fragments
thereof can also be assayed for their ability to inhibit the
binding of a flaviviral antigen to its host cell receptor using
techniques known to those of skill in the art and exemplified
herein. For example, cells expressing the receptor for WNV can be
contacted with WNV in the presence or absence of an antibody or
fragment thereof and the ability of the antibody or fragment
thereof to inhibit WNV's binding can measured by, for example, flow
cytometry or a scintillation assay. WNV (e.g., WNV antigen such as
E protein) or the antibody or antibody fragment can be labeled with
a detectable compound such as a radioactive label (e.g., .sup.32P,
.sup.35S, and .sup.125I) or a fluorescent label (e.g., fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine) to enable
detection of an interaction between WNV and its host cell receptor.
Alternatively, the ability of humanized antibodies or fragments
thereof to inhibit WNV from binding to its receptor can be
determined in cell-free assays. For example, WNV or a WNV antigen
can be contacted with an antibody or fragment thereof and the
ability of the antibody or antibody fragment to inhibit WNV or the
WNV antigen from binding to its host cell receptor can be
determined. Preferably, the antibody or the antibody fragment is
immobilized on a solid support and WNV or a WNV antigen is labeled
with a detectable compound. Alternatively, WNV or a WNV antigen is
immobilized on a solid support and the antibody or fragment thereof
is labeled with a detectable compound. WNV or a WNV antigen may be
partially or completely purified (e.g., partially or completely
free of other polypeptides) or part of a cell lysate. Further, an
WNV antigen may be a fusion protein comprising the WNV antigen and
a domain such as glutathionine-5-transferase. Alternatively, an WNV
antigen can be biotinylated using techniques well known to those of
skill in the art (e.g., biotinylation kit, Pierce Chemicals;
Rockford, Ill.).
[0240] Several aspects of the pharmaceutical compositions or
prophylactic or therapeutic agents of the invention are preferably
tested in vitro, e.g., in a cell culture system, and then in vivo,
e.g., in an animal model organism, such as a rodent animal model
system, for the desired therapeutic or prophylatic activity, prior
to use in humans. For example, in vitro assays which can be used to
determine whether administration of a specific pharmaceutical
composition is indicated, include cell culture assays in which a
patient tissue sample is grown in culture, and exposed to or
otherwise contacted with a pharmaceutical composition, and the
effect of such composition upon the tissue sample is observed. In
various specific embodiments, in vitro assays can be carried out
with representative cells of cell types involved in a WNV infection
to determine if a pharmaceutical composition of the invention has a
desired effect upon such cell types. Preferably, the humanized
antibodies or compositions of the invention are also tested in in
vitro assays and animal model systems prior to administration to
humans. In a specific embodiment, mice are administered a humanized
antibody the invention or fragment thereof, or a composition of the
invention, challenged with 100 to 1000 pfu of WNV, and four or more
days later the mice are sacrificed and WNV titer and anti-WNV
antibody serum titer is determined.
[0241] Efficacy in treating or preventing viral infection may be
demonstrated by detecting the ability of a humanized antibody or
composition of the invention to inhibit the replication of the
virus, to inhibit transmission or prevent the virus from
establishing itself in its host, to reduce the incidence of WNV
nfection, or to prevent, ameliorate or alleviate one or more
symptoms associated with WNV infection. The treatment is considered
therapeutic if there is, for example, a reduction is viral load,
amelioration of one or more symptoms, a reduction in the duration
of a WNV infection, or a decrease in mortality and/or morbidity
following administration of a humanized antibody or composition of
the invention. Further, the treatment is considered therapeutic if
there is an increase in the immune response following the
administration of one or more humanized antibodies or fragments
thereof which immunospecifically bind to one or more WNV
antigens.
[0242] Humanized antibodies or compositions of the invention can be
tested in vitro and in vivo for the ability to induce the
expression of cytokines such as IFN-.alpha., IFN-.beta.,
IFN-.gamma., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-12 and IL-15. Techniques known to those of skill in the art can
be used to measure the level of expression of cytokines. For
example, the level of expression of cytokines can be measured by
analyzing the level of RNA of cytokines by, for example, RT-PCR and
Northern blot analysis, and by analyzing the level of cytokines by,
for example, immunoprecipitation followed by western blot analysis
and ELISA.
[0243] Humanized antibodies or compositions of the invention can be
tested in vitro and in vivo for their ability to modulate the
biological activity of immune cells, preferably human immune cells
(e.g., T-cells, B-cells, and Natural Killer cells). The ability of
a humanized antibody or composition of the invention to modulate
the biological activity of immune cells can be assessed by
detecting the expression of antigens, detecting the proliferation
of immune cells, detecting the activation of signaling molecules,
detecting the effector function of immune cells, or detecting the
differentiation of immune cells. Techniques known to those of skill
in the art can be used for measuring these activities. For example,
cellular proliferation can be assayed by .sup.3H-thymidine
incorporation assays and trypan blue cell counts. Antigen
expression can be assayed, for example, by immunoassays including,
but are not limited to, competitive and non-competitive assay
systems using techniques such as western blots,
immunohistochemistry radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays and FACS analysis. The activation of signaling
molecules can be assayed, for example, by kinase assays and
electrophoretic shift assays (EMSAs).
[0244] Humanized antibodies or compositions of the invention can
also be tested for their ability to inhibit viral replication or
reduce viral load in in vitro, ex vivo and in vivo assays.
Humanized antibodies or compositions of the invention can also be
tested for their ability to decrease the time course of WNV
infection. Humanized antibodies or compositions of the invention
can also be tested for their ability to increase the survival
period of humans suffering from WNV infection by at least 25%,
preferably at least 50%, at least 60%, at least 75%, at least 85%,
at least 95%, or at least 99%. Further, humanized antibodies or
compositions of the invention can be tested for their ability
reduce the hospitalization period of humans suffering from WNV
infection by at least 60%, preferably at least 75%, at least 85%,
at least 95%, or at least 99%. Techniques known to those of skill
in the art can be used to analyze the function of the humanized
antibodies or compositions of the invention in vivo.
[0245] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including, but not
limited to, in rats, mice, chicken, cows, monkeys, pigs, dogs,
rabbits, hamsters, etc., for example, the animal models described
above. Any animal system well-known in the art may be used.
[0246] Combinations of prophylactic and/or therapeutic agents can
be tested in suitable animal model systems prior to use in humans.
In a specific embodiment of the invention, combinations of
prophylactic and/or therapeutic agents are tested in a mouse model
system. Such model systems are widely used and well-known to the
skilled artisan. Prophylactic and/or therapeutic agents can be
administered repeatedly. Several aspects of the procedure may vary
such as the temporal regime of administering the prophylactic
and/or therapeutic agents, and whether such agents are administered
separately or as an admixture.
[0247] Once the prophylactic and/or therapeutic agents of the
invention have been tested in an animal model they can be tested in
clinical trials to establish their efficacy. Establishing clinical
trials will be done in accordance with common methodologies known
to one skilled in the art, and the optimal dosages and routes of
administration as well as toxicity profiles of the compositions of
the invention can be established using routine experimentation.
[0248] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of the
prophylactic and/or therapeutic agents for use in humans. The
dosage of such agents lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
any agent used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0249] Further, any assays known to those skilled in the art can be
used to evaluate the prophylactic and/or therapeutic utility of the
combinatorial therapies disclosed herein for a WNV infection.
[0250] 5.7 Diagnostic Methods
[0251] Labeled antibodies, fragments, derivatives and analogs
thereof, which immunospecifically bind to a WNV antigen can be used
for diagnostic purposes to detect, diagnose, or monitor a WNV
infection. The invention provides for the detection of a WNV
infection, comprising: (a) assaying the expression of a WNV antigen
in cells or a tissue sample of a subject using one or more
humanized antibodies or fragments thereof that immunospecifically
bind to the WNV antigen; and (b) comparing the level of the WNV
antigen with a control level, e.g., levels in normal tissue samples
not infected with WNV, whereby an increase in the assayed level of
WNV antigen compared to the control level of the WNV antigen is
indicative of a WNV infection.
[0252] The invention provides a diagnostic assay for diagnosing a
WNV infection, comprising: (a) assaying for the level of a WNV
antigen in cells or a tissue sample of an individual using one or
more humanized antibodies or fragments thereof that
immunospecifically bind to a WNV antigen; and (b) comparing the
level of the WNV antigen with a control level, e.g., levels in
normal tissue samples not infected with WNV, whereby an increase in
the assayed WNV antigen level compared to the control level of the
WNV antigen is indicative of a WNV infection. A more definitive
diagnosis of WNV infection may allow health professionals to employ
preventative measures or aggressive treatment earlier thereby
preventing the development or further progression of WNV
infection.
[0253] Humanized antibodies of the invention, or fragments thereof,
can be used to assay WNV antigen levels in a biological sample
using classical immunohistological methods as described herein or
as known to those of skill in the art (e.g., see Jalkanen et al.,
1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J.
Cell. Biol. 105:3087-3096). Other antibody-based methods useful for
detecting protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA). Suitable antibody assay labels are known in the art and
include enzyme labels, such as, alkaline phosphatase, glucose
oxidase; radioisotopes, such as iodine (.sup.125I, .sup.131I),
carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.121In), and technetium (.sup.99mTc); luminescent labels, such
as luminol; and fluorescent labels, such as fluorescein and
rhodanine, and biotin.
[0254] One aspect of the invention is the detection and diagnosis
of a WR.sup.V infection in a human. In one embodiment, diagnosis
comprises: a) administering (for example, parenterally,
subcutaneously, or intraperitoneally) to a subject an effective
amount of a labeled antibody or fragment thereof that
immunospecifically binds to a WNV antigen; b) waiting for a time
interval following the administering for permitting the labeled
antibody or fragment thereof to preferentially concentrate at sites
in the subject where the WNV antigen is expressed (and for unbound
labeled molecule to be cleared to background level); c) determining
background level; and d) detecting the labeled antibody or fragment
thereof in the subject, such that detection of labeled antibody or
fragment thereof above the background level indicates that the
subject has a WNV infection. In accordance with this embodiment,
the antibody may be labeled with an imaging moiety which is
detectable using an imaging system known to one of skill in the
art. Background level can be determined by various methods
including, comparing the amount of labeled molecule detected to a
standard value previously determined for a particular system.
[0255] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of .sup.99mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain the specific protein. In vivo tumor imaging is
described in S. W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor
Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982).
[0256] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0257] In one embodiment, monitoring of a WNV infection is carried
out by repeating the method for diagnosing the WNV infection, for
example, one month after initial diagnosis, six months after
initial diagnosis, one year after initial diagnosis, etc.
[0258] Presence of the labeled molecule can be detected in the
subject using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0259] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patient using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
[0260] 5.8 Kits
[0261] The invention provides a pharmaceutical pack or kit
comprising one or more containers filled with humanized antibodies
of the invention. In an alternative embodiment, a kit comprises an
antibody fragment that immunospecifically binds to a WNV antigen.
Additionally, one or more other prophylactic or therapeutic agents
useful for the treatment of a disease can also be included in the
pharmaceutical pack or kit. The invention also provides a
pharmaceutical pack or kit comprising one or more containers filled
with one or more of the ingredients of the pharmaceutical
compositions of the invention. Optionally associated with such
container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0262] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises one or more
humanized antibodies of the invention. In another embodiment, a kit
further comprises one or more other prophylactic or therapeutic
anti-viral agents, in one or more containers. In another
embodiment, a kit further comprises one or more cytotoxic
antibodies.
[0263] In a specific embodiment, the kits of the present invention
contain a substantially isolated WNV antigen as a control.
Preferably, the kits of the present invention further comprise a
control antibody which does not react with the WNV antigen. In
another specific embodiment, the kits of the present invention
contain a means for detecting the binding of an antibody to a WNV
antigen (e.g., the antibody may be conjugated to a detectable
substrate such as a fluorescent compound, an enzymatic substrate, a
radioactive compound or a luminescent compound, or a second
antibody which recognizes the first antibody may be conjugated to a
detectable substrate). In specific embodiments, the kit may include
a recombinantly produced or chemically synthesized WNV antigen. The
WNV antigen provided in the kit may also be attached to a solid
support. In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which WNV antigen
is attached. Such a kit may also include a non-attached
reporter-labeled anti-human antibody. In this embodiment, binding
of the antibody to the WNV antigen can be detected by binding of
the said reporter-labeled antibody.
[0264] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing WNV antigens.
The diagnostic kit includes a substantially isolated humanized
antibody specifically immunoreactive with a WNV antigen, and means
for detecting the binding of the WNV antigen to the antibody. In
one embodiment, the antibody is attached to a solid support. The
detecting means of the kit may include a second, labeled monoclonal
antibody. Alternatively, or in addition, the detecting means may
include a labeled, competing antigen.
[0265] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound WNV antigen obtained
by the methods of the present invention. After the WNV antigen
binds to a specific antibody, the unbound serum components are
removed by washing, reporter-labeled anti-human antibody is added,
unbound anti-human antibody is removed by washing, and a reagent is
reacted with reporter-labeled anti-human antibody to bind reporter
to the reagent in proportion to the amount of bound anti-WNV
antigen antibody on the solid support. Typically, the reporter is
an enzyme which is detected by incubating the solid phase in the
presence of a suitable fluorometric, luminescent or colorimetric
substrate (Sigma, St. Louis, Mo.).
[0266] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0267] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant W antigen, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-WNV antigen antibody.
6. EXAMPLES
6.1 Humanization of Mouse Anti West Nile Virus Mab E16
[0268] RNA was converted to cDNA and the VH and VL segments were
PCR amplified using the 5' RACE kit (Invitrogen, Inc.). Gene
specific primers for the VH were SJ15R, SEQ ID NO. 57 (5' GGT CAC
TGT CAC TGG CTC AGG G 3') and SJ16R, SEQ ID NO. 58 (5' AGG CGG ATC
CAG GGG CCA GTG GAT AGA C 3'). Gene specific primers for the VL
were SJ17R, SEQ ID NO. 59 (5'GCA CAC GAC TGA GGC ACC TCC AGA TG 3')
and SJ18R, SEQ ID NO. 60 (5'CGG CGG ATC CGA TGG ATA CAG TTG GTG CAG
CAT C 3'). The RACE product was inserted into the plasmid
pCR2.1--TOPO using a TOPO TA Cloning kit (Invitrogen, Inc.). The
resulting plasmids were then subjected to DNA sequencing to
determine the VH and VL sequences for E16 and E34. The resulting
sequences were then translated and the predicted amino acid
sequence determined for each. From these sequences the framework
(FR) and complementarity determining (CDR) regions were identified
as defined by Kabat. The mouse VH was then joined to a human
C-Gamma1 constant region and an Ig leader sequence and inserted
into pCI-neo for mammalian expression. The mouse VL was joined to a
human C-kappa segment and an Ig leader sequence and also cloned
into pCI-neo for mammalian expression.
[0269] The humanized E16 VH consists of the CDR regions of E16 VH
and the FR segments from the human germline VH1-18 VH segment and
JH6. The humanized E16 VL consists of the CDR regions of E16 VL and
the FR segments of the human germline VK-B3 VL segment and JK4. The
humanized VH segments were assembled de novo from oligonucleotides
combined and amplified by PCR. The humanized VL segments were
assembled by PCR and overlapping PCR. The resulting fragment was
then combined by PCR with a leader sequence and the appropriate
constant region segment cloned into the expression vector pCI-neo
as a Nhe I-EcoRI fragment. The DNA sequence of the resulting
plasmids was confirmed by sequence analysis.
[0270] The alignment of the amino acid sequences of mouse E16 VH
and humanized E16 VH is shown in FIG. 1A. The alignment of the
amino acid sequences of mouse E16 VL and humanized E16 VL is shown
in FIG. 1B.
6.2 Expression and Characterization of the Humanized E16 Heavy and
Light Chains
[0271] Chimeric E16 (chE16), humanized E16 (huE16) and hybrid E16
antibodies were expressed in HEK-293 cells by co-transfecting the
following combinations of E16 heavy chain (HC) and E16 light chain
expression plasmids: huE161HC/huE16LC, huE116HC/chE16LC,
chE116HC/huE16LC and chE116HC/chE16LC. After three days in culture
the amount of antibody expressed having a human IgG constant domain
was quantitated by ELISA. Binding to WNV E-protein domain III
(dIII) was determined by ELISA and antibody-capture ELISA as
described below.
[0272] Protocol for ELISA assay: 100 ng/well of dIII was coated
directly on 96-well Maxisorp plates at 4.degree. C. overnight. A
series of two-fold dilutions of conditioned medium of chE16, huE16,
huE16HC/chE16LC or chE16HC/huE16LC starting from 5 ng/well was
added to each well. The plate was incubated at room temperature for
1 hour, then binding was detected by HRP conjugated F(ab').sub.2
goat anti human IgG F(ab)'.sub.2 specific secondary antibody
(Jackson ImmunoResearch, Inc.) at 1:10,000 dilution. After
incubation with the secondary antibody for approximately 45
minutes, the plate was developed using a TMB substrate. After 5
minutes incubation, the reaction was stopped by adding 1%
H.sub.2SO.sub.4. The OD.sub.450 nm was read by SOFTmax program.
Between each step, the plates were washed 3 times with PBS/0.1%
Tween 20. Plates were blocked by 0.5% BSA in PBS/0.1% Tween 20 for
30 mins at room temperature before adding conditioned medium.
[0273] Protocol for antibody-capture ELISA assay: 2.5 ng/well of
dIII was captured on 96-well Maxisorp plates by mouse anti-WNV
E-protein antibody E9 at room temperature for 2 hours. A serial of
two-fold dilution of conditioned medium of chE16, huE16,
huE16HC/ch2B6LC or chE16HC/huE16LC starting from 5 ng/well was
added to each well. The plate was incubated at room temperature for
1 hour, then binding was detected by HRP conjugated F(ab').sub.2
goat anti human IgG F(ab)'.sub.2 specific secondary antibody
(Jackson ImmunoResearch, Inc.) at 1:10,000 dilution. After
incubation with the secondary antibody for approximately 45
minutes, the plate was developed using a TMB substrate. After 5
minutes incubation, the reaction was stopped by 1% H.sub.2SO.sub.4.
The OD.sub.450 nm was read by SOFTmax program. Between each step,
the plates were washed 3 times with PBS/0.1% Tween20. Plates were
blocked by 0.5% BSA in PBS/0.1% Tween 20 for 30 mins at room
temperature before adding conditioned medium.
[0274] Results: The results of the ELISA assay depicted in FIG. 2A
indicated that all mAbs bound to the receptor with similar
affinity/avidity. In the dIII capture ELISA, depicted in FIG. 2B,
huE16 exhibited lower binding levels than chE16 or either hybrid
E16. One interpretation of this result is that the humanized E16
may shift the binding epitope slightly so that it now competes with
the E9 antibody used to originally capture dIII.
6.3 Generation, Expression and Characterization of Humanized E16.
Heavy and Light Chain Variants.
[0275] To further improve the binding affinity/avidity of humanized
E16 antibody, variants of huE16LC and huE16HC were created using
site directed mutagenesis (Stratagene kit). For example, HuE16LC-2
(Y49S) was formed by mutating the tyrosine (Y) at huE16LC position
49 to serine (S).
[0276] Mutated huE16 antibody was expressed by co-transfection of
HEK-293 cells with the variant huE16HC and huE16LC according to the
following combinations:
TABLE-US-00006 Expressed Antibody Heavy Chain Light Chain huE16-1.1
huE16HC huE16LC huE16-1.2 huE16HC huE16LC-2 (Y49S) huE16-2.1
huE16HC-2 (V67A, M69F, T71A) huE16LC huE16-2.2 huE16HC-2 (V67A,
M69F, T71A) huE16LC-2 (Y49S) huE16-3.1 huE16-3 (T71A) huE16LC
huE16-3.2 huE16-2 (T71A) huE16LC-2 (Y49S)
After three days in culture the amount of human IgG expressed was
quantitated by ELISA. Binding to WNV E-protein domain III (dIII)
was determined by ELISA and antibody-capture ELISA assays as
described above.
[0277] Results: The effects of mutating huE16LC on binding affinity
are depicted in FIGS. 3A and 3B. The results indicate that the Y49S
change in the light chain improved the binding of E16 to the
antigen. This improvement was further enhanced when combined with
the described mutations in huE16HC as shown in FIGS. 4 and 5. A
comparison of the last two figures also shows that the single
(T71A) mutation in huE16HC is functionally equivalent to the triple
mutation (V67A, M69F, T71A) in huE16HC in terms of mAb binding to
the receptor.
6.4 Model of WNV Encephalitis in Mice and Therapeutic and
Prophylaxis Studies of Humanized Anti West Nile Virus Mab E16
[0278] Murine model: A WNV infection model was established in
C57BL/6 (wild type) mice that closely paralleled the human disease.
One week after subcutaneous inoculation, wild-type mice developed
systemic and central nervous system (CNS) infection with a subset
progressing to paralysis and death. Younger mice were found to have
consistently higher mortality rates and thus offered the
possibility for greater mortality benefit in response to treatment.
Five week-old mice were selected for all therapeutic studies; for
this group, footpad inoculation with 10.sup.2 plaque-forming units
(PFU) of WNV resulted in 87% mortality in the absence of
therapy.
[0279] Prophylaxis study of human .gamma.-globulin: To confirm that
antibodies mediated protection against WNV, the efficacy of
purified immune human .gamma.-globulin against WNV infection was
evaluated in the mouse model described above. Purified immune human
.gamma.-globulin, specifically human .gamma.-globulin with
immunoreactivity against WNV, was obtained from pooled donors in
Israel: an area of sporadic outbreaks of WNV such that 10-20% of
the population carries antibodies against WNV. A single 15 mg dose
of purified immune human .gamma.-globulin against WNV was
administered via intraperitoneal (IP) injection immediately prior
(day (0) to or at the indicated days after footpad inoculation with
10.sup.2 PFU of WNV.
[0280] The results depicted in FIG. 6 indicate that treatment with
immune .gamma.-globulin at day 1, 2, 3, 4 or 5 (D1, D2, D3, D4 and
D5, respectively) post infection increased the average survival
time and decreased mortality rates. The beneficial effect of
therapy at day 5 is interesting because it suggests that antibody
may be able to limit the disease even after it has spread to the
CNS.
[0281] Post exposure therapeutic studies with murine anti-WNV mAb:
Several murine anti-WNV protein E mAbs and a control mAb against
SARS ORF7a were evaluated for therapeutic effect in the described
mouse model, FIG. 7A. At 4 days post infection, intra-peritoneal
administration of 0.5 mg of mAb E16 produced the greatest increase
in both mean survival time and mortality benefit.
[0282] The dose response to this antibody was further examined as
depicted in FIG. 7B. At 2 days post infection, intra-peritoneal
administration of 0.8 .mu.g improved survival from 5% to 30% at day
30. More dramatic improvements were seen at doses of at least 4
.mu.g mAb E16; at this dose, day 30 survival improved by at least
75% as compared to control. Although higher doses did correlate
with improved survival, the highly comparable survival curves for
doses ranging from 4 to 500 .mu.g indicate only minimal gains from
a substantially increased concentration of circulating antibody.
This may suggest a threshold of antigen saturation beyond which the
administered antibody has no available target.
[0283] The more effective of the murine anti-WNV protein E
antibodies, mAb E16 and mAb E24, were also tested at the higher
viral load at 5 days post infection and at an increased dose of 2
mg, FIG. 8. Administration of either mAb E16 and mAb E24 increased
the average survival over the control with mAb16 the seemingly more
effective of the two in the short term.
[0284] Post exposure therapeutic studies with humanized anti-WNVmAb
E16: Two humanized versions of murine mAb E16 have been tested in
the mouse model, mAb E16H-173 (huE16-1.2) and mAb E16H-167
(huE16-1.1). As shown in FIGS. 9A and 9B, respectively, doses of at
least 4 .mu.g at 2 days post-infection substantially increased 30
day survival. For either version of the humanized mAb E16, higher
doses improved survival; however, clear differences in their
functioning are apparent.
[0285] Prophylaxis studies with humanized anti-WNV mAB E16: A
humanized version of murine mAB E16, hE16-3.2, was tested in the
above mouse model in a prophylaxis study.
[0286] Groups of 10 mice each were administered PBS or hE16-3.2 at
doses of 0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg via an intraperitoneal
route. Approximately 24 hr later animals were bled and administered
10.sup.2 PFU of WNV via footpad inoculation. Survival was monitored
over a 24 day period. Antibody levels were determined by ELISA.
Data reflects 10 mice per condition. FIG. 10 and Table 6 depict the
survival of mice administered varying doses of hE16-3.2.
Significant protection was seen at all doses.
TABLE-US-00007 TABLE 6 Dose (mg/kg) Survival (day 24) Percent
Survival 0 1/10 10% 0.03 9/10 90% 0.1 9/10 90% 0.3 6/10 60% 1.0
10/10 100% 3.0 10/10 100%
6.5 Clinical Trial of Humanized Anti West Nile Virus Mab E16
[0287] This study is a phase I trial of humanized mAb E16
administration to patients with active West Nile virus infection,
and is designed to evaluate both its effect on WNV infection and
its possible toxicity. Patients with suspected WNV infection may be
enrolled after positive identification of viral DNA/RNA or
infectious virus in serum or cerebrospinal fluid. Accepted
diagnostic tests include immunohistochemistry with anti-WNV
antibodies and detection of the WNV genome through polymerase chain
reaction (PCR), Southern blot or in situ hybridization
analysis.
[0288] No specific therapy currently exists for West Nile viral
illness, limiting treatment to supportive care. Thus no alternative
WNV treatments are given in association with the anti-WNV antibody.
However, because sub-neutralizing concentrations of antibody
enhance flatavirus replication in myeloid cells in vitro, acute or
unusual progression of the disease will halt administration of
anti-WNV antibody.
[0289] Anti-WNV dose and administration: Initial patients receive
0.5 mg/kg humanized mAb E16 administered as a 1 hour intravenous
infusion. Given adequate tolerance, the dose will be increased
stepwise in subsequent patients to 5 mg/kg. Additionally, the
method of administration may be changed to bolus injection.
[0290] Study Protocol and Criteria: Toxicity of humanized mAb E16
is evaluated in patients according to the World Health Organization
Toxicity Criteria: blood pressure, temperature and heart rate are
monitored every 10 minutes during mAb E16 infusion, then every hour
for 3 hours and finally every 3 hours for 24 hours. Hematologic,
renal and liver function tests are conducted every other day for
one week and on day 15, 30, 60 and 120 post injection.
[0291] Serum and/or tissue samples are obtained once a day for two
weeks so that the effects of mAb E16 on viremia and viral load may
be determined by plaque and fluorogenic RT-PCR assays. Virologic
analysis will quantitatively define the effect of mAb E16 on the
progression of WNV infection, and pathologic studies will assess
their effect on related tissue damage and leukocyte
infiltration.
[0292] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0293] Various references are cited herein, the disclosure of which
are incorporated by reference in their entirety.
Sequence CWU 1
1
6015PRTmus.Heavy chain variable CDR1 from clone E16 1Asp Tyr Trp
Ile Glu1 5217PRTmus.Heavy chain variable CDR2 from clone E16 2Asp
Ile Leu Cys Gly Thr Gly Arg Thr Arg Tyr Asn Glu Lys Leu Lys1 5 10
15Ala310PRTmus.Heavy chain variable CDR3 from clone E16 3Ser Ala
Ser Tyr Gly Asp Tyr Ala Asp Tyr1 5 10430PRTmus.Heavy chain variable
FR1 from clone E16 4Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met
Lys Pro Gly Ala1 5 10 15Ser Val Gln Ile Ser Cys Lys Ala Thr Gly Tyr
Thr Phe Ser 20 25 30514PRTmus.Heavy chain variable FR2 from clone
E16 5Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly1 5
10632PRTmus.Heavy chain variable FR3 from clone E16 6Met Ala Thr
Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Phe Met Gln1 5 10 15Leu Ser
Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg 20 25
30732PRThomo sapiensHeavy chain variable FR3 from VH1-18 7Arg Val
Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu1 5 10 15Leu
Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
30832PRTArtificial SequenceMutated Heavy chain variable FR3 from
VH1-18 8Arg Ala Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met
Glu1 5 10 15Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
Ala Arg 20 25 30932PRTArtificial SequenceMutated Heavy chain
variable FR3 from VH1-18 9Arg Val Thr Met Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr Met Glu1 5 10 15Leu Arg Ser Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys Ala Arg 20 25 301011PRTmus.Heavy chain variable
FR4 from clone E16 10Trp Gly His Gly Thr Thr Leu Thr Val Ser Ser1 5
101111PRTmus.Light chain variable CDR1 from clones E16, E24 and E34
11Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala1 5 10127PRTmus.Light
chain variable CDR2 from clones E16, E24 and E34 12Trp Ala Ser Thr
Arg His Thr1 5139PRTmus.Light chain variable CDR3 from clones E16,
E24 and E34 13Gln Gln His Tyr Thr Thr Pro Leu Thr1
51423PRTmus.Light chain variable FR1 from clones E16 and E24 14Asp
Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10
15Asp Arg Val Ser Ile Thr Cys 201515PRTmus.Light chain variable FR2
from clones E16 15Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu
Leu Ile Ser1 5 10 151615PRThomo sapiensLight chain variable FR2
from VK-B3 16Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile Tyr1 5 10 151715PRTArtificial SequenceMutated Light chain
variable FR2 from VK-B3 17Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
Lys Leu Leu Ile Ser1 5 10 151832PRTmus.Light chain variable FR3
from clones E16 and E24 18Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp Tyr Thr1 5 10 15Leu Thr Ile Ser Ser Val Gln Ala Glu
Asp Leu Ala Leu Tyr Tyr Cys 20 25 301910PRTmus.Light chain variable
FR4 from clones E16 and E24 19Phe Gly Ala Gly Thr Lys Leu Glu Leu
Lys1 5 1020119PRTmus.Heavy chain variable from clones E16 20Gln Val
Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Lys Pro Gly Ala1 5 10 15Ser
Val Gln Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asp Tyr 20 25
30Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45Gly Asp Ile Leu Cys Gly Thr Gly Arg Thr Arg Tyr Asn Glu Lys
Leu 50 55 60Lys Ala Met Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr
Ala Phe65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ala Ser Tyr Gly Asp Tyr Ala Asp
Tyr Trp Gly His Gly 100 105 110Thr Thr Leu Thr Val Ser Ser
11521119PRTArtificial Sequencehumanized E16 heavy chain version 1
21Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Leu Cys Gly Thr Gly Arg Thr Arg Tyr Asn
Glu Lys Leu 50 55 60Lys Ala Arg Val Thr Met Thr Thr Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ala Ser Tyr Gly Asp Tyr
Ala Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11522119PRTArtificial Sequencehumanized E16 heavy chain version 2
22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Leu Cys Gly Thr Gly Arg Thr Arg Tyr Asn
Glu Lys Leu 50 55 60Lys Ala Arg Ala Thr Phe Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ala Ser Tyr Gly Asp Tyr
Ala Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11523119PRTArtificial Sequencehumanized E16 heavy chain version 3
23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Leu Cys Gly Thr Gly Arg Thr Arg Tyr Asn
Glu Lys Leu 50 55 60Lys Ala Arg Val Thr Met Thr Ala Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ala Ser Tyr Gly Asp Tyr
Ala Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Val Thr Val Ser Ser
11524107PRTmus.Light chain variable from clone E16 24Asp Ile Val
Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln
Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr
Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
10525107PRTArtificial Sequencehumanized E16 light chain version 1
25Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1
5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ala65 70 75 80Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
His Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 10526107PRTArtificial Sequencehumanized E16 light chain
version 2 26Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser
Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val
Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
Lys Leu Leu Ile 35 40 45Ser Trp Ala Ser Thr Arg His Thr Gly Val Pro
Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Ala65 70 75 80Glu Asp Val Ala Val Tyr Tyr Cys
Gln Gln His Tyr Thr Thr Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105275PRTmus.Heavy chain variable CDR1 from
clone E24 27Ser Tyr Asp Ile Asn1 52817PRTmus.Heavy chain variable
CDR2 from clone E24 28Trp Ile Tyr Pro Gly Asp Gly Arg Ile Lys Tyr
Asn Glu Lys Phe Lys1 5 10 15Gly2910PRTmus.Heavy chain variable CDR3
from clone E24 29Gly Gly Ser Ser Gly Thr Tyr Phe Asp Tyr1 5
103030PRTmus.Heavy chain variable FR1 from clone E24 30Gln Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Leu Val
Lys Ile Ser Cys Lys Ala Ser Gly His Thr Phe Thr 20 25
303114PRTmus.Heavy chain variable FR2 from clone E24 and E34 31Trp
Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly1 5
103232PRTmus.Heavy chain variable FR3 from clone E24 32Lys Ala Ile
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln1 5 10 15Leu Ser
Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys Ala Arg 20 25
303311PRTmus.Heavy chain variable FR4 from clone E24 and E34 33Trp
Gly Gln Gly Thr Thr Leu Thr Val Ser Ser1 5 10349PRTmus.Light chain
variable CDR3 from clone E24 34Gln Gln His Tyr Ser Asn Pro Pro Thr1
53515PRTmus.Light chain variable FWR2 from clone E24 35Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr1 5 10
153610PRTmus.Light chain variable FWR4 from clone E24 36Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys1 5 1037119PRTmus.Heavy chain variable
from clone E24 37Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val
Lys Pro Gly Ala1 5 10 15Leu Val Lys Ile Ser Cys Lys Ala Ser Gly His
Thr Phe Thr Ser Tyr 20 25 30Asp Ile Asn Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Tyr Pro Gly Asp Gly Arg
Ile Lys Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Ile Leu Thr Ala
Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu
Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Gly Gly Ser
Ser Gly Thr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Leu
Thr Val Ser Ser 11538107PRTmus.Light chain variable from clone E24
38Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly1
5 10 15Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Val
Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg
Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Val Gln Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln
His Tyr Ser Asn Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 1053917PRTmus.Heavy chain variable CDR2 from clone E34
39Trp Ile Phe Pro Gly Asp Gly Arg Ile Lys Tyr Asn Glu Gln Ile Lys1
5 10 15Asp4010PRTmus.Heavy chain variable CDR3 from clone E34 40Ala
Ser Tyr Tyr Gly Ser Ile Phe Asp Tyr1 5 104130PRTmus.Heavy chain
variable FWR1 from clone E34 41Gln Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Thr1 5 10 15Leu Val Lys Ile Ser Cys Lys Thr
Ser Gly Tyr Thr Phe Thr 20 25 304232PRTmus.Heavy chain variable
FWR3 from clone E34 42Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala Tyr Met Glu1 5 10 15Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala
Val Tyr Phe Cys Ala Arg 20 25 304323PRTmus.Light chain variable
FWR1 from clone E34 43Asp Ile Val Met Thr Gln Ser His Lys Phe Met
Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Asn Ile Thr Cys
204415PRTmus.Light chain variable FWR2 from clone E34 44Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr1 5 10
154532PRTmus.Light chain variable FWR3 from clone E34 45Gly Val Pro
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Tyr Thr1 5 10 15Leu Thr
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys 20 25
3046119PRTmus.Heavy chain variable from clone E24 46Gln Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Leu Val Lys
Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asp Ile
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly
Trp Ile Phe Pro Gly Asp Gly Arg Ile Lys Tyr Asn Glu Gln Ile 50 55
60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe
Cys 85 90 95Ala Arg Ala Ser Tyr Tyr Gly Ser Ile Phe Asp Tyr Trp Gly
Gln Gly 100 105 110Thr Thr Leu Thr Val Ser Ser 11547107PRTmus.Light
chain variable from clone E24 47Asp Ile Val Met Thr Gln Ser His Lys
Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg Val Asn Ile Thr Cys Lys
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Trp Ala Ser Thr Arg
His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser Gly Thr
His Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu
Ala Leu Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Leu 85 90 95Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 1054830PRThomo sapiensHeavy
chain variable FR1 from VH1-18 48Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr 20 25 304914PRThomo sapiensHeavy chain
variable FR2 from VH1-18 49Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met Gly1 5 105011PRThomo sapiensHeavy chain variable FR4
from JH-6 50Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5
105123PRThomo sapiensLight chain variable FR1 from VK-B3 51Asp Ile
Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu
Arg Ala Thr Ile Asn Cys 205232PRThomo sapiensLight chain variable
FR3 from VK-B3 52Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val
Ala Val Tyr Tyr Cys 20 25 305310PRThomo sapiensLight chain variable
FR4 from VK-B3 53Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys1 5
1054296DNAhomo sapiensHeavy chain frame work from VH1-18
54caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc
60tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggatgg atcagcgctt acaatggtaa
cacaaactat
180gcacagaagc tccagggcag agtcaccatg accacagaca catccacgag
cacagcctac 240atggagctga ggagcctgag atctgacgac acggccgtgt
attactgtgc gagaga 2965563DNAhomo sapiensHeavy chain frame work from
JH-6 55attactacta ctactacggt atggacgtct gggggcaagg gaccacggtc
accgtctcct 60cag 6356305DNAhomo sapiensLight chain frame work from
VKB-3 56gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga
gagggccacc 60atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa
ctacttagct 120tggtaccagc agaaaccagg acagcctcct aagctgctca
tttactgggc atctacccgg 180gaatccgggg tccctgaccg attcagtggc
agcgggtctg ggacagattt cactctcacc 240atcagcagcc tgcaggctga
agatgtggca gtttattact gtcagcaata ttatagtact 300cctcc
3055722DNAArtificial SequenceSJ15R primer 57ggtcactgtc actggctcag
gg 225828DNAArtificial SequenceSJ16R primer 58aggcggatcc aggggccagt
ggatagac 285926DNAArtificial SequenceSJ17R primer 59gcacacgact
gaggcacctc cagatg 266034DNAArtificial SequenceSJ18R primer
60cggcggatcc gatggataca gttgtgtcag catc 34
* * * * *