Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR PREVENTING BIOFILM FORMATION
RELATED APPLICATION
This application claims priority to U.S. Provisional Application No.
61/643,650,
filed May 7, 2012. The content of this application is hereby incorporated by
reference in its
entirety.
BACKGROUND OF THE INVENTION
A biofilm is an aggregate of microorganisms in which the individual cells
adhere to
each other on a surface, usually embedded within a self-produced polymeric
matrix.
Biofilms have been found to be involved in a wide variety of microbial
infections in the
human body, including common conditions such as urinary tract infections,
catheter
infections, middle-ear infections, formation of dental plaque, gingivitis, as
well as less
common but more lethal conditions such as endocarditis, lung infections in
cystic fibrosis,
and infections of permanent indwelling devices, such as joint prostheses,
heart valves and
intrauterine devices. It has also been noted that bacterial biofilms may
impair cutaneous
wound healing and reduce topical antibacterial efficiency in healing or
treating infected skin
wounds.
In many bacteria, including staphylococci, the main molecule responsible for
intercellular adhesion is the glucosamine polymer, poly-N-acetylglucosamine
(PNAG).
PNAG is a beta-1-6-linked N-acetylglucosamine polymer, which together with
other
polymers such as teichoic acids and proteins forms the major part of the
extracellular matrix
of the bacterial biofilm.
Staphylococcus epidennidis and S. aureus are the most frequent causes of
nosocomial
infections (e.g., on indwelling medical devices), which characteristically
involve biofilms.
Biofilm formation by S. epidermidis and S. aureus is particularly problematic
in that it
protects the bacteria from attack by antibiotics and the host immune system,
thereby
rendering the infection difficult to treat.
Accordingly, there is a need in the art for alternative methods of treating or
preventing
S. epidennidis or S. aureus infections. Specifically, there is a need for
methods that act by
disrupting or inhibiting biofilm formation by these bacteria.
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SUMMARY OF THE INVENTION
The present invention provides methods for the treatment or prevention of
microbial
infections (e.g., nocosomial infection) in which the underlying pathology
involves a PNAG-
containing microbial biofilm. The methods of the invention generally involve
administering
to the subject an effective amount of an antibody that specifically binds to
PNAG and
disrupts or inhibits formation of PNAG-containing microbial biofilms. Such
methods are
particularly useful for the treatment of nosocomial staphylococcus (e.g.,
S.epidermidis and S.
aureus) infections.
Accordingly, in one aspect, the invention provides a method of preventing a
nocosomial infection comprising, the method generally comprising: identifying
a subject
(e.g., human subject) at risk of developing an poly-N-acetyl glucosamine
(PNAG)-containing
microbial biofilm from a medical procedure; and administering to the subject
an effective
amount of an antibody that specifically binds to PNAG and inhibits PNAG-
containing
microbial biofilm formation.
In certain embodiments, the nocosomial infection is a lung infection, joint
infection,
endocardial infection, skin infection, soft tissue infection, or septicemia.
In certain embodiments, the antibody is administered between about 0 and 240
hours
prior to the medical procedure.
In certain embodiments, the medical procedure is the installation of a
surgical implant
in the subject, e.g., a stent, catheter, cannula, prosthesis, or pace-maker.
In certain embodiments, the antibody is administered systemically. In other
embodiments, the antibody is administered locally to the site of implantation
of the surgical
implant.
In certain embodiments, the antibody is coated on the surgical implant or
adhered to
the surgical implant.
In certain embodiments, the antibody is administered in a controlled release
formulation.
In certain embodiments, the biofilm comprises Staphylococcus, e.g., S.
epidennidis
and S. aureus.
In certain embodiments, the antibody is a human antibody.
In certain embodiments, the antibody comprises a VH domain comprising the
HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID Nos: 1, 2,
and 3,
respectively.
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In certain embodiments, the antibody comprises a VL domain comprising the
LCDR1, LCDR2, and LCDR3 amino acid sequences set forth in SEQ ID Nos: 4, 5,
and 6,
respectively.
In certain embodiments, the antibody comprises the VH domain sequence set
forth in
SEQ ID No: 7.
The method of any one of the preceding claims, wherein the antibody comprises
the
VL domain sequence set forth in SEQ ID No: 8.
In another aspect, the invention provides a method of inhibiting formation of
a poly
N-acetyl glucosamine (PNAG)-containing microbial biofilm on a surgical implant
in a
subject (e.g., a human subject), the method generally comprising administering
to the subject
an effective amount of an antibody that specifically binds to PNAG prior to
implantation of
the surgical implant into the subject.
In certain embodiments, the surgical implant is a stent, catheter, cannula,
prosthesis,
or pace-maker.
In certain embodiments, the antibody is administered between about 0 and 240
hours
prior to the medical procedure.
In certain embodiments, the antibody is administered systemically. In other
embodiments, the antibody is administered locally to the site of implantation
of the surgical
implant.
In certain embodiments, the antibody is coated on the surgical implant or
adhered to
the surgical implant.
In certain embodiments, the antibody is administered in a controlled release
formulation.
In certain embodiments, the biofilm comprises Staphylococcus, e.g., S.
epidennidis
and S. aureus.
In certain embodiments, the antibody is a human antibody.
In certain embodiments, the antibody comprises a VH domain comprising the
HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID Nos: 1, 2,
and 3,
respectively.
In certain embodiments, the antibody comprises a VL domain comprising the
LCDR1, LCDR2, and LCDR3 amino acid sequences set forth in SEQ ID Nos: 4, 5,
and 6,
respectively.
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In certain embodiments, the antibody comprises the VH domain sequence set
forth in
SEQ ID No: 7.
The method of any one of the preceding claims, wherein the antibody comprises
the
VL domain sequence set forth in SEQ ID No: 8.
In a further aspect, the invention provides a method of inhibiting formation
of PNAG-
containing microbial biofilm on a substrate comprising contacting the
substrate with an
effective amount of an antibody that specifically binds to PNAG.
In certain embodiments, the biofilm comprises Staphylococcus, e.g., S.
epidennidis
and S. aureus.
In certain embodiments, the antibody is a human antibody.
In certain embodiments, the antibody comprises a VH domain comprising the
HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID Nos: 1, 2,
and 3,
respectively.
In certain embodiments, the antibody comprises a VL domain comprising the
LCDR1, LCDR2, and LCDR3 amino acid sequences set forth in SEQ ID Nos: 4, 5,
and 6,
respectively.
In certain embodiments, the antibody comprises the VH domain sequence set
forth in
SEQ ID No: 7.
The method of any one of the preceding claims, wherein the antibody comprises
the
VL domain sequence set forth in SEQ ID No: 8.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 depicts the results of in vitro assays measuring biofilm formation of
S.
aureus (ATCC 33592) in the presence and absence of F598.
Figure 2 depicts the results of in vitro assays measuring biofilm formation of
S.
epidermidis 1457 in the presence and absence of F598.
Figure 3 depicts the results of in vitro assays measuring biofilm formation of
S.
epidermidis RP62A in the presence and absence of F598.
DETAILED DESCRIPTION
The present invention provides methods for the treatment or prevention of
microbial
infections (e.g., nocosomial infection) in which the underlying pathology
involves a PNAG-
containing microbial biofilm. The methods the invention generally involve
administering to
the subject an effective amount of an antibody that specifically binds to PNAG
and disrupt or
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inhibit formation of PNAG-containing microbial biofilms. Such methods are
particularly
useful for the treatment of nosocomial staphylococcus (e.g., S.epidennidis and
S. aureus)
infections.
I. Definitions
In order that the present invention may be more readily understood, certain
terms are
first defined.
As used herein, the terms "poly-N-acetyl glucosamine" or "PNAG" refer to a
polymer
of N-acetyl glucosamine monomers linked via a beta 1-6 linkage. The terms also
encompass
partially or fully deacylated poly-N-acetyl glucosamine.
As used herein, the term "PNAG-containing microbial biofilm" refers to sessile
aggregate of microorganisms embedded in a PNAG matrix.
As used herein, the term "nosocomial infection" refers to infection acquired
in a
hospital or from a medical procedure performed inside or outside of a
hospital. Exemplary
nocosomial infections include sepsis (bloodstream infection), surgical site
infection, or
hospital-acquired pneuomia.
As used herein, the term "preventing a nocosomial infection" refers to an
inhibition or
reduction in the severity of an infection.
As used herein, the term "antibody" refers to immunoglobulin molecules
comprising
four polypeptide chains, two heavy (H) chains and two light (L) chains inter-
connected by
disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain
comprises a
heavy chain variable region (abbreviated VH) and a heavy chain constant
region. The heavy
chain constant region comprises three domains, CH1, CH2 and CH3. Each light
chain
comprises a light chain variable region (abbreviated VL) and a light chain
constant region.
The light chain constant region comprises one domain (CL1). The VH and VL
regions can be
further subdivided into regions of hypervariability, termed complementarity
determining
regions (CDRs), interspersed with regions that are more conserved, termed
framework
regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged
from
amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2,
CDR2, FR3,
CDR3, FR4.
As used herein, the term "antigen-binding portion" of an antibody include any
naturally occurring, enzymatically obtainable, synthetic, or genetically
engineered
polypeptide or glycoprotein that specifically binds an antigen to form a
complex. Antigen-
binding fragments of an antibody may be derived, e.g., from full antibody
molecules using
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any suitable standard techniques such as proteolytic digestion or recombinant
genetic
engineering techniques involving the manipulation and expression of DNA
encoding
antibody variable and optionally constant domains. Non-limiting examples of
antigen-
binding portions include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd
fragments; (iv) Fv
fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii)
minimal
recognition units consisting of the amino acid residues that mimic the
hypervariable region of
an antibody (e.g., an isolated complementarity determining region (CDR)).
Other engineered
molecules, such as diabodies, triabodies, tetrabodies and minibodies, are also
encompassed
within the expression "antigen-binding portion."
As used herein, the term "CDR" or "complementarity determining region" means
the
noncontiguous antigen combining sites found within the variable region of both
heavy and
light chain polypeptides. These particular regions have been described by
Kabat et al., J.
Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of
immunological
interest. (1991), and by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and
by MacCallum
et al., J. Mol. Biol. 262:732-745 (1996) where the definitions include
overlapping or subsets
of amino acid residues when compared against each other. The amino acid
residues which
encompass the CDRs as defined by each of the above cited references are set
forth for
comparison. Preferably, the term "CDR" is a CDR as defined by Kabat, based on
sequence
comparisons.
As used herein the term "framework (FR) amino acid residues" refers to those
amino
acids in the framework region of an Ig chain. The term "framework region" or
"FR region" as
used herein, includes the amino acid residues that are part of the variable
region, but are not
part of the CDRs (e.g., using the Kabat definition of CDRs). Therefore, a
variable region
framework is between about 100-120 amino acids in length but includes only
those amino
acids outside of the CDRs.
As used herein, the term "specifically binds to" refers to the ability of an
antibody or
an antigen-binding fragment thereof to bind to an antigen with an Kd of at
least about 1 x
10-6 M, 1 x 10-7M, 1 x 10-8M, 1 x 10-9M, 1 x 10-10 M, 1 x 10-11 M, 1 x 10-12
M, or more,
and/or bind to an antigen with an affinity that is at least two-fold greater
than its affinity for a
nonspecific antigen.
As used herein, the term "antigen" refers to the binding site or epitope
recognized by
an antibody or antigen binding portion thereof.
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As used herein, the term "effective amount" refers to that amount of an
antibody or an
antigen binding portion thereof that binds PNAG, which is sufficient to effect
treatment,
prognosis or diagnosis of a PNAG-containing microbial biofilm, as described
herein, when
administered to a subject. A therapeutically effective amount will vary
depending upon the
subject and disease condition being treated, the weight and age of the
subject, the severity of
the disease condition, the manner of administration and the like, which can
readily be
determined by one of ordinary skill in the art. The dosages for administration
can range from,
for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg,
about 10 ng to
about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg,
about 40 ng
to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000
mg, about
200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to
about 4,500 mg,
about 500 ng to about 4,000 mg, about 1 ug to about 3,500 mg, about 5 ug to
about 3,000 mg,
about 10 ug to about 2,600 mg, about 20 ug to about 2,575 mg, about 30 ug to
about 2,550
mg, about 40 ug to about 2,500 mg, about 50 ug to about 2,475 mg, about 100 ug
to about
2,450 mg, about 200 ug to about 2,425 mg, about 300 ug to about 2,000 mg,
about 400 ug to
about 1,175 mg, about 500 ug to about 1,150 mg, about 0.5 mg to about 1,125
mg, about 1
mg to about 1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about
1,050 mg,
about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg, about 3.0 mg
to about 975
mg, about 3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg
to about 900
mg, about 5 mg to about 875 mg, about 10 mg to about 850 mg, about 20 mg to
about 825
mg, about 30 mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to
about 750
mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg, about 300 mg
to about
675 mg, about 400 mg to about 650 mg, about 500 mg, or about 525 mg to about
625 mg, of
an antibody or antigen binding portion thereof, according to the invention.
Dosage regiments
may be adjusted to provide the optimum therapeutic response. An effective
amount is also
one in which any toxic or detrimental effects (i.e., side effects) of an
antibody or antigen
binding portion thereof are minimized and/or outweighed by the beneficial
effects.
As used herein, the term "subject" includes any human or non-human animal.
II. Bio film
The methods of the invention can be used to disrupt or inhibit the formation
of poly
N-acetyl glucosamine (PNAG)-containing microbial biofilms. PNAG-containing
biofilm can
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be produced by a variety of microbes, including bacteria and fungi. In
general, any PNAG-
containing microbial biofilm can be disrupted or inhibited using the methods
of the invention.
In the case of bacteria, PNAG-containing biofilms are commonly formed by, for
example, Staphylococci (e.g., S. epidermis, S. aureus (e.g., Multi Drug
Resistant S. aureus),
S. camosus, and S. haemolyticus). Known clinical isolates of Staphylococcus
that form
PNAG-containing biofilms include, without limitation, S. epidermis RP62A (ATCC
number
35984), S. epidermis RP12 (ATCC number 35983), S. epidermis M187, S. camosus
TM300
(pCN27), S. aureus RN4220 (pCN27), and S. aureus MN8 mucoid.
PNAG-containing biofilms are also formed by other bacteria including but not
limited
to Pseudomonas aeruginosa, E. coli (e.g., E. coli 0157:H7 and E. coli CFT073),
Yersinia
pestis, Yersinia entercolitica, Xanthomonas axonopodis, Pseudomonas
fluorescens,
Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae,
Ralstonia
solanacea rum, Bordetella pertussis, Bordetella parapertussis and Bordetella
bronchiseptica.
III. Nosocomial Infections
The invention provides methods for treating or preventing nosocomial
infections by
identifying a subject at risk of developing a poly-N-acetyl glucosamine (PNAG)-
containing
microbial biofilm from a medical procedure and administering to the subject an
effective
amount of an antibody that specifically binds to PNAG and inhibits PNAG-
containing
microbial biofilm formation.
Any medical procedure, whether performed inside or outside of a hospital, that
confers a risk to a patient of developing a PNAG-containing microbial biofilm
can be treated
or prevented using the methods of the invention. Examples of such medical
procedures
include, without limitation, surgery and implantation of a surgical device
(e.g., catheter,
cannula, prosthesis, respirator, replacement heart valve, and pace-maker).
Exemplary surgical
devices include, without limitation, central venous catheters; peritoneal
dialysis catheters;
orthopedic prostheses; orthopedic mesh; intracardiac devices such as
artificial valves,
pacemakers, and stents; cochlear implants; breast implants; endotracheal
tubes; voice
prostheses; intraocular lens
The skilled artisan will appreciate that in the case of an immunosupressed
subject, the
mere fact that the subject is in a hospital (or another environment that can
harbor bacteria
capable of PNAG-containing microbial biofilm) identifies this subject as being
at risk of
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developing a PNAG-containing microbial biofilm (e.g., in the lung, urinary
tract or in an
open wound) even if they do not receive a surgical procedure.
IV. Anti-PNAG Antibodies
Any antibody that binds to PNAG and inhibits formation of a PNAG-containing
bacterial biofilm can be used in the methods of the invention. Exemplary
antibody VH, VL
and CDR amino acid sequences suitable for use in the invention are set forth
in Table 1.
Table 1. VH, VL and CDR amino acid sequences of exemplary anti-PNAG
antibodies.
Antibody Sequence SEQ ID NO.
F598 HCDR3 DTYYYDSGDYEDAFDI 1
F598 HCDR2 YIHYSRSTNSNPALKS 2
F598 HCDR1 GYYWS 3
F598 LCDR3 QTWGAGIRV 4
F598 LCDR2 VNRDGSHIRGD 5
F598 LCDR1 TLSSGHSNYAIA 6
F598 VH QVQLQESGPGLVKPSETL SLTCTVSGGS I SGYY 7
WSWIRQPPGKGLEWIGYIHYSRS TNSNPALKSR
VT I SSDTSKNQLSLRLSSVTAADTAVYYCARDT
YYYDSGDYEDAFDIWGQGTMVTVSS
F598 VL QLVL TQSPSASAS LGASVKLTCT LS SGHSNYAI 8
AWHQQQPGKGPRYLMKVNRDGSHIRGDGIPDRF
SGS T SGAERYLT I SSLQSEDEADYYCQTWGAGI
RVFGGGTKLTVLG
F628 HCDR3 DTYYESSGHWFDGLDV 9
F628 HCDR2 YIHYSGSTNSNPSLKS 10
F628HCDR1 NYYWS 11
F628LCDR3 QTWGPGIRV 12
F628 LCDR2 VKSDGSHSKGD 13
F628 LCDR1 TLDSEHSRYT IA 14
F628VH QVQLQESGPGLVKPSETL SLTCTVSGGS I SNYY 15
WSWIRQSPGRGLEWIGYIHYSGS TNSNPSLKSR
VT I SVDTSKNQVSLKLGSVTAADTAIYYCARDT
YYESSGHWFDGLDVWGQGTSVTVSS
F628 VL QPVLTQSPSASASLGASVKLTCTLDSEHSRYT I 16
AWHQQQPEKGPRYLMKVKSDGSHSKGDGITDRF
SGS S SGAERYLT I SSLQSEDEADYYCQTWGPGI
RVFGGGTKLTVL
F630 HCDR3 DYYETSGYAYDDFAI 17
F630 HCDR2 WVSTYNGRTNYAQKFRG 18
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F630 HCDR1 NFGI S 19
F630 LCDR3 QTWGPGIRV 20
F630 LCDR2 VNSDGSHTKGD 21
F630LCDR1 TLSSGHSTYAIA 22
F630 VH QVQLVQSGAEMKRPGASVKVSCKASGYTFTNFG 23
I SWVRQAPGQGLEWI GWVS TYNGRTNYAQKFRG
RVTMTTDTSTNTAYMELRSLGSDDTAVFYCARD
YYETSGYAYDDFAIWGQGTLVTVSS
F630 VL QLVL TQSPSASAS LGASVKLTCT LS SGHSTYAI 24
AWHQQQPLRGPRFLMKVNSDGSHTKGDGIPDRF
SGS S SGAERYLS I SSLQSEDESDYYCQTWGPGI
RVFGGGTKLTVLG
In certain embodiments, the antibody, or antigen binding fragment thereof,
comprises
one or more CDR region amino acid sequence selected from the group consisting
of SEQ ID
NO: 1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, and 22.
In other embodiments, the antibody, or antigen binding fragment thereof,
comprises
HCDR3, HCDR2 and HCDR1 region amino acid sequences selected from the group
consisting of:
a) SEQ ID NO: 1, 2 and 3;
b) SEQ ID NO: 9, 10 and 11; and
c) SEQ ID NO: 17, 18 and 19, respectively.
In other embodiments, the antibody, or antigen binding fragment thereof,
comprises
the LCDR3, LCDR2 and LCDR1 region amino acid sequences selected from the group
consisting of:
a) SEQ ID NO: 4, 5 and 6;
b) SEQ ID NO: 12, 13 and 14; and
c) SEQ ID NO: 20, 21 and 22, respectively.
In other embodiments, the antibody, or antigen binding fragment thereof,
comprises
the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1 region amino acid sequences
selected from the group consisting of:
a) SEQ ID NO: 1, 2, 3, 4, 5 and 6;
b) SEQ ID NO: 9, 10, 21, 12, 13 and 14; and
c) SEQ ID NO: 17, 18, 21, 20, 21 and 22, respectively
In other embodiments, the antibody, or antigen binding fragment thereof,
comprises
the VH region amino acid sequences set forth in SEQ ID NO: 7, 15 and/or 23.
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In other embodiments, the antibody, or antigen binding fragment thereof,
comprises
the VL region amino acid sequences set forth in SEQ ID NO: 8, 16, and/or 24.
In other embodiments, the antibody, or antigen binding fragment thereof,
comprises
the VH and VL region amino acid sequences selected from the group consisting
of: SEQ ID
NO: 7 and 8; SEQ ID NO: 15 and 16; and SEQ ID NO: 23 and 24, respectively.
V. Modified Anti-PNAG antibodies
Anti-PNAG antibodies may comprise one or more modifications. Such modified
forms of anti-PNAG antibodies can be made using any techniques known in the
art.
i) Reducing Immunogenicity
In some embodiments, de-immunization can be used to decrease the
immunogenicity
of and antibody, or antigen binding portion thereof. As used herein, the term
"de-
immunization" includes alteration of an antibody, or antigen binding portion
thereof, to
modify T cell epitopes (see, e.g., W09852976A1, W00034317A2). For example, VH
and
VL sequences from the starting antibody may be analyzed and a human T cell
epitope "map"
may be generated from each V region showing the location of epitopes in
relation to
complementarity-determining regions (CDRs) and other key residues within the
sequence.
Individual T cell epitopes from the T cell epitope map are analyzed in order
to identify
alternative amino acid substitutions with a low risk of altering activity of
the final antibody.
A range of alternative VH and VL sequences are designed comprising
combinations of amino
acid substitutions and these sequences are subsequently incorporated into a
range of anti-
PNAG antibodies or fragments thereof for use in the methods disclosed herein,
which are
then tested for function. Complete heavy and light chain genes comprising
modified V and
human C regions are then cloned into expression vectors and the subsequent
plasmids
introduced into cell lines for the production of whole antibody. The
antibodies are then
compared in appropriate biochemical and biological assays, and the optimal
variant is
identified.
ii) Effector Functions and Fc Modifications
Anti-PNAG antibodies may comprise an antibody constant region (e.g. an IgG
constant region e.g., a human IgG constant region, e.g., a human IgG1 or IgG4
constant
region) which mediates one or more effector functions. For example, binding of
the Cl
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component of complement to an antibody constant region may activate the
complement
system. Activation of complement is important in the opsonisation and lysis of
cell
pathogens. The activation of complement also stimulates the inflammatory
response and may
also be involved in autoimmune hypersensitivity. Further, antibodies bind to
receptors on
various cells via the Fc region, with a Fc receptor binding site on the
antibody Fc region
binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors
which are
specific for different classes of antibody, including IgG (gamma receptors),
IgE (epsilon
receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody
to Fc receptors
on cell surfaces triggers a number of important and diverse biological
responses including
engulfment and destruction of antibody-coated particles, clearance of immune
complexes,
lysis of antibody-coated target cells by killer cells (called antibody-
dependent cell-mediated
cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer
and control of
immunoglobulin production. In preferred embodiments, the anti-PNAG antibodies,
or
fragments thereof, bind to an Fc.gamma. receptor. In alternative embodiments,
anti- PNAG
antibodies may comprise a constant region which is devoid of one or more
effector functions
(e.g., ADCC activity) and/or is unable to bind Fcy receptor.
Certain embodiments of the invention include anti-PNAG antibodies in which at
least
one amino acid in one or more of the constant region domains has been deleted
or otherwise
altered so as to provide desired biochemical characteristics such as reduced
or enhanced
effector functions, the ability to non-covalently dimerize, increased ability
to localize at the
site of a tumor, reduced serum half-life, or increased serum half-life when
compared with a
whole, unaltered antibody of approximately the same immunogenicity. For
example, certain
antibodies, or fragments thereof, for use in the diagnostic and treatment
methods described
herein are domain deleted antibodies which comprise a polypeptide chain
similar to an
immunoglobulin heavy chain, but which lack at least a portion of one or more
heavy chain
domains. For instance, in certain antibodies, one entire domain of the
constant region of the
modified antibody will be deleted, for example, all or part of the CH2 domain
will be deleted.
In certain other embodiments, anti-PNAG antibodies comprise constant regions
derived from different antibody isotypes (e.g., constant regions from two or
more of a human
IgGl, IgG2, IgG3, or IgG4). In other embodiments, anti-PNAG antibodies
comprises a
chimeric hinge (i.e., a hinge comprising hinge portions derived from hinge
domains of
different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule
and an IgG1
middle hinge domain). In one embodiment, an anti-PNAG antibodies comprises an
Fc region
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or portion thereof from a human IgG4 molecule and a Ser228Pro mutation (EU
numbering)
in the core hinge region of the molecule.
In certain anti-PNAG antibodies, the Fc portion may be mutated to increase or
decrease effector function using techniques known in the art. For example, the
deletion or
inactivation (through point mutations or other means) of a constant region
domain may
reduce Fc receptor binding of the circulating modified antibody thereby
increasing tumor
localization. In other cases it may be that constant region modifications
consistent with the
instant invention moderate complement binding and thus reduce the serum half
life and
nonspecific association of a conjugated cytotwdn. Yet other modifications of
the constant
region may be used to modify disulfide linkages or oligosaccharide moieties
that allow for
enhanced localization due to increased antigen specificity or flexibility. The
resulting
physiological profile, bioavailability and other biochemical effects of the
modifications, such
as tumor localization, biodistribution and serum half-life, may easily be
measured and
quantified using well know immunological techniques without undue
experimentation.
In certain embodiments, an Fc domain employed in an anti-PNAG antibody is an
Fc
variant. As used herein, the term "Fc variant" refers to an Fc domain having
at least one
amino acid substitution relative to the wild-type Fc domain from which said Fc
domain is
derived. For example, wherein the Fc domain is derived from a human IgG1
antibody, the Fc
variant of said human IgG1 Fc domain comprises at least one amino acid
substitution relative
to said Fc domain.
The amino acid substitution(s) of an Fc variant may be located at any position
(i.e.,
any EU convention amino acid position) within the Fc domain. In one
embodiment, the Fc
variant comprises a substitution at an amino acid position located in a hinge
domain or
portion thereof. In another embodiment, the Fc variant comprises a
substitution at an amino
acid position located in a CH2 domain or portion thereof. In another
embodiment, the Fc
variant comprises a substitution at an amino acid position located in a CH3
domain or portion
thereof. In another embodiment, the Fc variant comprises a substitution at an
amino acid
position located in a CH4 domain or portion thereof.
The antibodies may employ any art-recognized Fc variant which is known to
impart
an improvement (e.g., reduction or enhancement) in effector function and/or
FcR binding.
Said Fc variants may include, for example, any one of the amino acid
substitutions disclosed
in International PCT Publications W088/07089A1, W096/14339A1, W098/05787A1,
W098/23289A1, W099/51642A1, W099/5 8572A1, W000/09560A2, W000/32767A1,
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W000/42072A2, W002/44215A2, W002/060919A2, W003/074569A2, W004/016750A2,
W004/029207A2, W004/035752A2, W004/063351A2, W004/074455A2,
W004/099249A2, W005/040217A2, W005/070963A1, W005/077981A2,
W005/092925A2, W005/1 23780A2, W006/019447A1, W006/047350A2, and
W006/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046;
6,096,871;
6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056;
6,821,505;
6,998,253; and 7,083,784, each of which is incorporated by reference herein.
In one
exemplary embodiment, an anti-PNAG antibody may comprise an Fc variant
comprising an
amino acid substitution at EU position 268 (e.g., H268D or H268E). In another
exemplary
embodiment, an anti-PNAG antibody may comprise an amino acid substitution at
EU
position 239 (e.g., S239D or S239E) and/or EU position 332 (e.g., I332D or
I332Q).
In certain embodiments, an anti-PNAG antibody of may comprise an Fc variant
comprising an amino acid substitution which alters the antigen-independent
effector functions
of the antibody, in particular the circulating half-life of the antibody. Such
antibodies exhibit
either increased or decreased binding to FcRn when compared to antibodies
lacking these
substitutions, therefore, have an increased or decreased half-life in serum,
respectively. Fc
variants with improved affinity for FcRn are anticipated to have longer serum
half-lives, and
such molecules have useful applications in methods of treating mammals where
long half-life
of the administered antibody is desired, e.g., to treat a chronic disease or
disorder. In contrast,
Fc variants with decreased FcRn binding affinity are expected to have shorter
half-lives, and
such molecules are also useful, for example, for administration to a mammal
where a
shortened circulation time may be advantageous, e.g. for in vivo diagnostic
imaging or in
situations where the starting antibody has toxic side effects when present in
the circulation for
prolonged periods. Fc variants with decreased FcRn binding affinity are also
less likely to
cross the placenta and, thus, are also useful in the treatment of diseases or
disorders in
pregnant women. In addition, other applications in which reduced FcRn binding
affinity may
be desired include those applications in which localization the brain, kidney,
and/or liver is
desired. In one exemplary embodiment, the altered antibodies exhibit reduced
transport
across the epithelium of kidney glomeruli from the vasculature. In another
embodiment, the
altered antibodies exhibit reduced transport across the blood brain barrier
(BBB) from the
brain, into the vascular space. In one embodiment, an antibody with altered
FcRn binding
comprises an Fc domain having one or more amino acid substitutions within the
"FcRn
binding loop" of an Fc domain. The FcRn binding loop is comprised of amino
acid residues
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280-299 (according to EU numbering). Exemplary amino acid substitutions which
altered
FcRn binding activity are disclosed in International PCT Publication No.
W005/047327
which is incorporated by reference herein. In certain exemplary embodiments,
the antibodies,
or fragments thereof, comprise an Fc domain having one or more of the
following
substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).
In other embodiments, antibodies, for use in the diagnostic and treatment
methods
described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain
constant region,
which is altered to reduce or eliminate glycosylation. For example, an
antibody may also
comprise an Fc variant comprising an amino acid substitution which alters the
glycosylation
of the antibody. For example, said Fc variant may have reduced glycosylation
(e.g., N- or 0-
linked glycosylation). In exemplary embodiments, the Fc variant comprises
reduced
glycosylation of the N-linked glycan normally found at amino acid position 297
(EU
numbering). In another embodiment, the antibody has an amino acid substitution
near or
within a glycosylation motif, for example, an N-linked glycosylation motif
that contains the
amino acid sequence NXT or NXS. In a particular embodiment, the antibody
comprises an Fc
variant with an amino acid substitution at amino acid position 228 or 299 (EU
numbering). In
more particular embodiments, the antibody comprises an IgG1 or IgG4 constant
region
comprising an S228P and a T299A mutation (EU numbering).
Exemplary amino acid substitutions which confer reduce or altered
glycosylation are
disclosed in International PCT Publication No. W005/018572, which is
incorporated by
reference herein. In preferred embodiments, the antibodies, or fragments
thereof, are
modified to eliminate glycosylation. Such antibodies, or fragments thereof,
may be referred
to as "agly" antibodies, or fragments thereof, (e.g. "agly" antibodies). While
not being bound
by theory, it is believed that "agly" antibodies, or fragments thereof, may
have an improved
safety and stability profile in vivo. Exemplary agly antibodies, or fragments
thereof, comprise
an aglycosylated Fc region of an IgG4 antibody which is devoid of Fc-effector
function
thereby eliminating the potential for Fc mediated toxicity to the normal vital
organs. In yet
other embodiments, antibodies, or fragments thereof, comprise an altered
glycan. For
example, the antibody may have a reduced number of fucose residues on an N-
glycan at
Asn297 of the Fc region, i.e., is afucosylated. In another embodiment, the
antibody may have
an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc
region.
iii) Covalent Attachment
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Anti-PNAG antibodies may be modified, e.g., by the covalent attachment of a
molecule to the antibody such that covalent attachment does not prevent the
antibody from
specifically binding to its cognate epitope. For example, but not by way of
limitation, the
antibodies, or fragments thereof, may be modified by glycosylation,
acetylation, pegylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous
chemical
modifications may be carried out by known techniques, including, but not
limited to specific
chemical cleavage, acetylation, formylation, etc. Additionally, the derivative
may contain one
or more non-classical amino acids.
Antibodies, or fragments thereof, may further be recombinantly fused to a
heterologous polypeptide at the N- or C-terminus or chemically conjugated
(including
covalent and non-covalent conjugations) to polypeptides or other compositions.
For example,
anti-PNAG antibodies may be recombinantly fused or conjugated to molecules
useful as
labels in detection assays and effector molecules such as heterologous
polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO
91/14438; WO
89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
Anti-PNAG antibodies may be fused to heterologous polypeptides to increase the
in
vivo half life or for use in immunoassays using methods known in the art. For
example, in
one embodiment, PEG can be conjugated to the anti-PNAG antibodies to increase
their half-
life in vivo. Leong, S. R., et al., Cytokine 16:106 (2001); Adv. in Drug
Deliv. Rev. 54:531
(2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).
Moreover, anti-PNAG antibodies can be fused to marker sequences, such as a
peptide
to facilitate their purification or detection. 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., Proc. Natl. Acad. Sci.
USA 86:821-824
(1989), 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 "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson et al.,
Cell 37:767 (1984)) and the "flag" tag.
Anti-PNAG antibodies may be used in non-conjugated form or may be conjugated
to
at least one of a variety of molecules, e.g., to improve the therapeutic
properties of the
molecule, to facilitate target detection, or for imaging or therapy of the
patient. Anti-PNAG
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antibodies can be labeled or conjugated either before or after purification,
when purification
is performed. In particular, anti-PNAG antibodies may be conjugated to
therapeutic agents,
prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response
modifiers,
pharmaceutical agents, or PEG.
The present invention further encompasses anti-PNAG antibodies conjugated to a
diagnostic or therapeutic agent. The anti-PNAG antibodies can be used
diagnostically to, for
example, monitor the development or progression of a immune cell disorder
(e.g., CLL) as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given treatment and/or
prevention regimen. Detection can be facilitated by coupling the anti-PNAG
antibodies to a
detectable substance. Examples of detectable substances include various
enzymes, prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials, radioactive
materials, positron emitting metals using various positron emission
tomographies, and
nonradioactive paramagnetic metal ions. 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. Examples of suitable enzymes include horseradish peroxidase,
alkaline
phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of
suitable prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent
materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material include
1251, 1311, 111In or 99Tc.
Anti-PNAG antibodies for use in the diagnostic and treatment methods disclosed
herein may be conjugated to cytotwdns (such as radioisotopes, cytotoxic drugs,
or toxins)
therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides,
proteins, enzymes,
viruses, lipids, biological response modifiers, pharmaceutical agents,
immunologically active
ligands (e.g., lymphokines or other antibodies wherein the resulting molecule
binds to both
the neoplastic cell and an effector cell such as a T cell), or PEG.
In another embodiment, an anti-PNAG antibody for use in the diagnostic and
treatment methods disclosed herein can be conjugated to a molecule that
decreases tumor cell
growth. In other embodiments, the disclosed compositions may comprise
antibodies, or
fragments thereof, coupled to drugs or prodrugs. Still other embodiments of
the present
invention comprise the use of antibodies, or fragments thereof, conjugated to
specific
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biotoxins or their cytotoxic fragments such as ricin, gelonin, Pseudomonas
exotoxin or
diphtheria toxin. The selection of which conjugated or unconjugated antibodyto
use will
depend on the type and stage of cancer, use of adjunct treatment (e.g.,
chemotherapy or
external radiation) and patient condition. It will be appreciated that one
skilled in the art
could readily make such a selection in view of the teachings herein.
It will be appreciated that, in previous studies, anti-tumor antibodies
labeled with
isotopes have been used successfully to destroy tumor cells in animal models,
and in some
cases in humans. Exemplary radioisotopes include: 90Y, 1251, 1311, 1231,
111In, 105Rh,
153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. The radionuclides act by
producing
ionizing radiation which causes multiple strand breaks in nuclear DNA, leading
to cell death.
The isotopes used to produce therapeutic conjugates typically produce high
energy alpha- or
beta-particles which have a short path length. Such radionuclides kill cells
to which they are
in close proximity, for example neoplastic cells to which the conjugate has
attached or has
entered. They have little or no effect on non-localized cells. Radionuclides
are essentially
non-immunogenic.
VI. Pharmaceutical Formulations and Methods of Administration of Anti-
PNAG
Antibodies
Methods of preparing and administering anti-PNAG antibodies, or fragments
thereof,
to a subject are well known to or are readily determined by those skilled in
the art. The route
of administration of the antibodies, or fragments thereof, may be oral,
parenteral, by
inhalation or topical. The term parenteral as used herein includes
intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, rectal or vaginal
administration. The
intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral
administration
are generally preferred. While all these forms of administration are clearly
contemplated as
being within the scope, a form for administration would be a solution for
injection, in
particular for intravenous or intraarterial injection or drip. Usually, a
suitable pharmaceutical
composition for injection may comprise a buffer (e.g. acetate, phosphate or
citrate buffer), a
surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human
albumin), etc.
However, in other methods compatible with the teachings herein, the
polypeptides can be
delivered directly to the site of the adverse cellular population thereby
increasing the
exposure of the diseased tissue to the therapeutic agent.
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Preparations for parenteral administration include sterile aqueous or non-
aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous solvents are
propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters such
as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or
suspensions, including saline and buffered media. In the subject invention,
pharmaceutically
acceptable carriers include, but are not limited to, 0.01-0.1M and preferably
0.05M phosphate
buffer or 0.8% saline. Other common parenteral vehicles include sodium
phosphate solutions,
Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed
oils. Intravenous
vehicles include fluid and nutrient replenishers, electrolyte replenishers,
such as those based
on Ringer's dextrose, and the like. Preservatives and other additives may also
be present such
as for example, antimicrobials, antioxidants, chelating agents, and inert
gases and the like.
More particularly, pharmaceutical compositions suitable for injectable use
include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersions. In
such cases, the
composition must be sterile and should be fluid to the extent that easy
syringability exists. It
should be stable under the conditions of manufacture and storage and will
preferably be
preserved against the contaminating action of microorganisms, such as bacteria
and fungi.
The carrier can be a solvent or dispersion medium containing, for example,
water, ethanol,
polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and
the like), and
suitable mixtures thereof. The proper fluidity can be maintained, for example,
by the use of a
coating such as lecithin, by the maintenance of the required particle size in
the case of
dispersion and by the use of surfactants. Prevention of the action of
microorganisms can be
achieved by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars, polyalcohols, such
as mannitol,
sorbitol, or sodium chloride in the composition. Prolonged absorption of the
injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
In any case, sterile injectable solutions can be prepared by incorporating an
active
compound (e.g., an antibody by itself or in combination with other active
agents) in the
required amount in an appropriate solvent with one or a combination of
ingredients
enumerated herein, as required, followed by filtered sterilization. Generally,
dispersions are
prepared by incorporating the active compound into a sterile vehicle, which
contains a basic
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dispersion medium and the required other ingredients from those enumerated
above. In the
case of sterile powders for the preparation of sterile injectable solutions,
the preferred
methods of preparation are vacuum drying and freeze-drying, which yields a
powder of an
active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof. The preparations for injections are processed, filled into
containers such as
ampoules, bags, bottles, syringes or vials, and sealed under aseptic
conditions according to
methods known in the art. Further, the preparations may be packaged and sold
in the form of
a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S.
Ser. No.
09/259,338 each of which is incorporated herein by reference. Such articles of
manufacture
will preferably have labels or package inserts indicating that the associated
compositions are
useful for treating a subject suffering from, or at risk of obtaining a PNAG-
conatining
microbial biofilm.
Effective doses of the stabilized antibodies, or fragments thereof, of the
present
invention, for the treatment of the above described conditions vary depending
upon many
different factors, including means of administration, target site,
physiological state of the
patient, whether the patient is human or an animal, other medications
administered, and
whether treatment is prophylactic or therapeutic. Usually, the patient is a
human, but non-
human mammals including transgenic mammals can also be treated. Treatment
dosages may
be titrated using routine methods known to those of skill in the art to
optimize safety and
efficacy.
For passive immunization with an antibody, the dosage may range, e.g., from
about
0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25
mg/kg, 0.5
mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. For
example dosages
can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10
mg/kg,
preferably at least 1 mg/kg. Doses intermediate in the above ranges are also
intended to be
within the scope of the invention.
Subjects can be administered such doses daily, on alternative days, weekly or
according to any other schedule determined by empirical analysis. An exemplary
treatment
entails administration in multiple dosages over a prolonged period, for
example, of at least six
months. Additional exemplary treatment regimes entail administration once per
every two
weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules
include 1-
10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60
mg/kg weekly.
In some methods, two or more monoclonal antibodies with different binding
specificities are
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administered simultaneously, in which case the dosage of each antibody
administered may
fall within the ranges indicated.
Anti-PNAG antibodies, or fragments thereof, can be administered on multiple
occasions. Intervals between single dosages can be, e.g., daily, weekly,
monthly or yearly.
Intervals can also be irregular as indicated by measuring blood levels of
polypeptide or target
molecule in the patient. In some methods, dosage is adjusted to achieve a
certain plasma
antibody or toxin concentration, e.g., 1-1000 ug/ml or 25-300 ug/ml.
Alternatively,
antibodies, or fragments thereof, can be administered as a sustained release
formulation, in
which case less frequent administration is required. Dosage and frequency vary
depending on
the half-life of the antibody in the patient. In general, humanized antibodies
show the longest
half-life, followed by chimeric antibodies and nonhuman antibodies. In one
embodiment, the
antibodies, or fragments thereof, can be administered in unconjugated form. In
another
embodiment, the antibodies can be administered multiple times in conjugated
form. In still
another embodiment, the antibodies, or fragments thereof, can be administered
in
unconjugated form, then in conjugated form, or vise versa.
The dosage and frequency of administration can vary depending on whether the
treatment is prophylactic or therapeutic. In prophylactic applications,
compositions
containing the present antibodies or a cocktail thereof are administered to a
patient not
already in the disease state to enhance the patient's resistance. Such an
amount is defined to
be a "prophylactic effective dose." In this use, the precise amounts again
depend upon the
patient's state of health and general immunity, but generally range from 0.1
to 25 mg per
dose, especially 0.5 to 2.5 mg per dose. A relatively low dosage is
administered at relatively
infrequent intervals over a long period of time. Some patients continue to
receive treatment
for the rest of their lives.
In therapeutic applications, a relatively high dosage (e.g., from about 1 to
400 mg/kg
of antibody per dose, with dosages of from 5 to 25 mg being more commonly used
for
radioimmunoconjugates and higher doses for cytotwdn-drug conjugated molecules)
at
relatively short intervals is sometimes required until progression of the
disease is reduced or
terminated, and preferably until the patient shows partial or complete
amelioration of
symptoms of disease. Thereafter, the patent can be administered a prophylactic
regime.
In one embodiment, a subject can be treated with a nucleic acid molecule
encoding a
an anti-PNAG antibody (e.g., in a vector). Doses for nucleic acids encoding
polypeptides
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range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ug to 10 mg, or 30-300 ug
DNA per
patient. Doses for infectious viral vectors vary from 10-100, or more, virions
per dose.
Therapeutic agents can be administered by parenteral, topical, intravenous,
oral,
subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or
intramuscular means for
-- prophylactic and/or therapeutic treatment. Intramuscular injection or
intravenous infusion are
preferred for administration of an antibody. In some methods, therapeutic
antibodies, or
fragments thereof, are injected directly into the cranium. In some methods,
antibodies, or
fragments thereof, are administered as a sustained release composition or
device, such as a
MedipadTM device.
Anti-PNAG antibodies can optionally be administered in combination with other
agents that are effective in treating the disorder or condition in need of
treatment (e.g.,
prophylactic or therapeutic). Preferred additional agents are those which are
art recognized
and are standardly administered for a particular disorder.
Effective single treatment dosages (i.e., therapeutically effective amounts)
of 90Y-
-- labeled antibodies range from between about 5 and about 75 mCi, more
preferably between
about 10 and about 40 mCi. Effective single treatment non-marrow ablative
dosages of 1311-
labeled antibodies range from between about 5 and about 70 mCi, more
preferably between
about 5 and about 40 mCi. Effective single treatment ablative dosages (i.e.,
may require
autologous bone marrow transplantation) of 131I-labeled antibodies range from
between
-- about 30 and about 600 mCi, more preferably between about 50 and less than
about 500 mCi.
In conjunction with a chimeric modified antibody, owing to the longer
circulating half life
vis-a-vis murine antibodies, an effective single treatment non-marrow ablative
dosages of
iodine-131 labeled chimeric antibodies range from between about 5 and about 40
mCi, more
preferably less than about 30 mCi. Imaging criteria for, e.g., the 111In
label, are typically less
-- than about 5 mCi.
While a great deal of clinical experience has been gained with 1311 and .90Y,
other
radiolabels are known in the art and have been used for similar purposes.
Still other
radioisotopes are used for imaging. For example, additional radioisotopes
which are
compatible with the scope of the instant invention include, but are not
limited to, 1231, 1251,
-- 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 875r, 113In, 127Cs, 129Cs, 1321,
197Hg, 203Pb,
206Bi, 177Lu, 186Re, 212Pb, 212Bi, 475c, 105Rh, 109Pd, 1535m, 188Re, 199Au,
225Ac,
211A 213Bi. In this respect alpha, gamma and beta emitters are all compatible
with in the
instant invention. Further, in view of the instant disclosure it is submitted
that one skilled in
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the art could readily determine which radionuclides are compatible with a
selected course of
treatment without undue experimentation. To this end, additional radionuclides
which have
already been used in clinical diagnosis include 1251, 1231, 99Tc, 43K, 52Fe,
67Ga, 68Ga, as
well as 111In. Antibodies have also been labeled with a variety of
radionuclides for potential
use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111-125
(1987)).
These radionuclides include 188Re and 186Re as well as 199Au and 67Cu to a
lesser extent.
U.S. Pat. No. 5,460,785 provides additional data regarding such radioisotopes
and is
incorporated herein by reference.
As previously discussed, the antibodies, or fragments thereof, can be
administered in
a pharmaceutically effective amount for the in vivo treatment of mammalian
disorders. In this
regard, it will be appreciated that the disclosed antibodies, or fragments
thereof, will be
formulated so as to facilitate administration and promote stability of the
active agent.
Preferably, pharmaceutical compositions in accordance with the present
invention comprise a
pharmaceutically acceptable, non-toxic, sterile carrier such as physiological
saline, non-toxic
buffers, preservatives and the like. For the purposes of the instant
application, a
pharmaceutically effective amount of an antibody, conjugated or unconjugated
to a
therapeutic agent, shall be held to mean an amount sufficient to achieve
effective binding to a
target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or
disorder or to
detect a substance or a cell. In the case of tumor cells, the polypeptide will
be preferably be
capable of interacting with selected immunoreactive antigens on neoplastic or
immunoreactive cells and provide for an increase in the death of those cells.
Of course, the
pharmaceutical compositions of the present invention may be administered in
single or
multiple doses to provide for a pharmaceutically effective amount of the
polypeptide.
In keeping with the scope of the present disclosure, anti-PNAG antibodies may
be
administered to a human or other animal in accordance with the aforementioned
methods of
treatment in an amount sufficient to produce a therapeutic or prophylactic
effect. The anti-
PNAG antibodies can be administered to such human or other animal in a
conventional
dosage form prepared by combining the antibody with a conventional
pharmaceutically
acceptable carrier or diluent according to known techniques. It will be
recognized by one of
skill in the art that the form and character of the pharmaceutically
acceptable carrier or
diluent is dictated by the amount of active ingredient with which it is to be
combined, the
route of administration and other well-known variables. Those skilled in the
art will further
appreciate that a cocktail comprising one or more species of polypeptides
according to the
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present invention may prove to be particularly effective.
VII. Methods of Treating or Preventing Infections Involving PNAG-Containing
Microbial Biofilm
The invention provides methods for treating or preventing PNAG-containing
bacterial
biofilm by administering to a subject in need of thereof a pharmaceutical
composition
comprising one or more anti-PNAG antibody, or antigen binding fragment
thereof.
In certain embodiments, a pharmaceutical composition comprising one or more
anti-
PNAG antibodies, or antigen binding fragments thereof, are administered to a
subject in
combination with one or more additional therapeutic agents. In a particular
embodiments, the
one or more additional therapeutic agents is administered concurrently with
the
pharmaceutical composition comprising one or more anti-PNAG antibodies.
Suitable
additional therapeutic agents include antibacterial agents (e.g.,
antibiotics). Suitable anti-
bacterial agents include, without limitation, penicillin G, penicillin V,
ampicillin, amwdcillin,
bacampicillin, cyclacillin, epicillin, hetacillin, pivampicillin, methicillin,
nafcillin, oxacillin,
cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, ticarcillin,
avlocillin, mezlocillin,
piperacillin, amdinocillin, cephalexin, cephradine, cefadoxil, cefaclor,
cefazolin, cefuroxime
axetil, cefamando le, cefonicid, cefoxitin, cefotaxime, ceftizoxime,
cefinenoxine, ceftriaxone,
moxalactam, cefotetan, cefoperazone, ceftazidme, imipenem, clavulanate,
timentin,
sulbactam, neomycin, erythromycin, metronidazole, chloramphenicol,
clindamycin,
lincomycin, vancomycin, trimethoprim-sulfamethoxazole, aminoglycosides,
quinolones,
tetracyclines and rifampin. (See e.g., Goodman and Gilman's, Pharmacological
Basics of
Therapeutics, 8th Ed., 1993, McGraw Hill Inc.)
One skilled in the art would be able, by routine experimentation, to determine
what an
effective, non-toxic amount of antibody (or additional therapeutic agent)
would be for the
purpose of treating a PNAG -associated disease or disorder. For example, a
therapeutically
active amount of a polypeptide may vary according to factors such as age, sex,
medical
complications (e.g., immunosuppressed conditions or diseases) and weight of
the subject, and
the ability of the antibody to elicit a desired response in the subject. The
dosage regimen may
be adjusted to provide the optimum therapeutic response. For example, several
divided doses
may be administered daily, or the dose may be proportionally reduced as
indicated by the
exigencies of the therapeutic situation. Generally, however, an effective
dosage is expected
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to be in the range of about 0.05 to 100 milligrams per kilogram body weight
per day and
more preferably from about 0.5 to 10, milligrams per kilogram body weight per
day.
The precise time of administration of the anti-PNAG antibody can be adjusted
according to the patients needs. In the case of prophylactic treatment to
inhibit the formation
of a PNAG-containing microbial biofilm in a subject, the anti-PNAG antibody
can be given
at any time prior to exposure to the biofilm-forming microbe (e,g, upon entry
into hospital or
at the start of a medical procedure). For example, the anti-PNAG antibody can
be
administered between 0 and 240 hours prior to exposure to the biofilm-forming
microbe, e.g.,
about 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23 24, 30,
40, 60, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
2010, 220, 230,
and/or 240 hours.
VIII. Exemplification
The present invention is further illustrated by the following examples which
should
not be construed as further limiting. The contents of Sequence Listing,
figures and all
references, patents and published patent applications cited throughout this
application are
expressly incorporated herein by reference.
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Example 1. Inhibition of S. aureus biofilm by F598
S. aureus (ATCC 33592) was grown overnight in TSB medium, adjusted at 1E+07
CFU/ml. The bacteria were then aliquoted into MBEC plates (MBEC AssaysTM,
Innovotech)
containing 5, or 20 ug/ml of concentration of F598 or human IgG control and
incubated for
5.5h. Following the incubation period, the pegs of the MBEC plates were
removed, washed
and the amount of formed biofilm quantified using by luminescence (BacTiter-
Glo TM
Microbial Cell Viability Assay, Promega). In this experiment F598 demonstrated
a clear
protective effect against S. aureus in vitro biofilm development.
Specifically, F598 showed
more than 50% inhibition of S. aureus biofilm development (43 +/- 8 % and 37+/-
3%, at 5
and 20 g/ml, respectively) (see Figure 1).
Example 2. Inhibition of S. epidermidis (clinical isolate 1457) biofilm by
F598
S. epidermidis (clinical isolate 1457) was grown in multiwell plates in TSB
medium
for 8 h in the presence of 25, 50, 100, or 200 11g/m1 of F598 or human IgG1
control. Wells
were then washed and stained with crystal violet. Biofilms were quantified by
measuring the
sample absorbance at a wavelength of 490 nm. In this experiment F598
demonstrated a clear,
dose-dependent protective effect against S. epidennidis 1457 in vitro biofilm
development.
Specifically, at 25 lag/m1 biofilm formation was 40% of the control (0.2 vs
0.5 OD), and at
200 iug/m1 biofilm formation was 20% of the control (0.1 vs 0.5 OD) (see
Figure 2).
Example 3. Inhibition of S. epidermidis (clinical isolate RP62A) biofilm by
F598
S. epidermidis (clinical isolate RP62A) was grown in multiwell plates in TSB
medium
for 8 h in the presence of 19, 38, or 76 iug/m1 of F598 or PBS buffer control.
Wells were then
washed and stained with crystal violet. Biofilms were quantified by measuring
the sample
absorbance at a wavelength of 490 nm. In this experiment F598 demonstrated a
clear, dose-
dependent protective effect against S. epidermidis RP62A in vitro biofilm
development.
Specifically, 19, 38, and 76 ug/m1 of F598 reduced biofilm formation 36, 60
and 76%,
respectively (relative to the corresponding PBS control) (see Figure 3).
26
