Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN ANTIBODIES TO STAPHYLOCOCCUS AUREUS
Background of the Invention
Staphylococcus aureus is a human pathogen capable of causing symptoms
ranging from skin boils to septicemia and death (1). Staphylococci are among
the most
common nosocomial pathogens in hospitals and long-term-care facilities. In
particular,
S. aureus is an important cause of infections associated with indwelling
medical devices
such as heart valves and joint prostheses. Examples of particularly
susceptible class of
patients include the elderly and immunocompromised hospital patients. In
addition to
being a major cause of nosocomial infections, S. aureus can also mediate
diseases
through community-acquired infections.
Antibiotic resistant strains of S. aureus have emerged since the widespread
use
of antibiotics. In addition, hospital strains are ofen resistant to multiple
antibiotics.
Methicillin resistance has been reported soon after this antibiotic was first
introduced for
treating penicillin-resistant S. aureus in 1959, and has gradually become a
serious
problem in many countries (2,3). Methicillin-resistant S. aureus (MRSA)
strains are
generally resistant to all (3-lactam antibiotics by expression of a mutated
penicillin
binding protein (PBP 2a) that has a low binding affinity for these antibiotics
(4). At
present, MRSA is treated with the glycopeptide antibiotic vancomycin. Although
"true"
vancomycin resistance has not been seen in clinical cases of S. aureus, the
genes for
acquiring resistance to vancomycin are known and vancomycin resistant S.
aureus
strains have been artificially generated (5). Thus, the emergence of S. aureus
resistant
strains in clinical settings may be only a matter of time.
In spite of the fact that vancomycin treatment remains widely effective for
MRSA infections, this treatment regimen induces significant renal toxicities
(6). Few
effective alternatives to glycopeptides have been developed, which include the
use of the
drugs Synercid and the oxazolidinones (7). These new types of antibiotics,
although
effective, may generate resistant organisms after their widespread use.
Moreover, some
patients are unable to take antibiotics because of allergic reactions.
The polysaccharide capsule (CP) of S. aureus has been a major focus for groups
developing immunotherapy for S aureus infections ( 13,14). CP vaccines have
been
investigated in animal models (14) and given to human volunteers to raise
hyperimmune
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serum (11). Hyperimmunized serum from human volunteers immunized with CP
vaccines is one alternative for passive immunization of individuals at risk of
S. aureus
infections that is currently being investigated in clinical trials (11).
However, this
approach has several disadvantages, including the fact that 1 ) human
immunizations are
typically restricted to certain well characterized antigens, 2) human serum
must be
extensively screened and still may contain infectious agents, and 3) the
relative activity
of different batches of serum may be difficult to control. These drawbacks may
result in
a potentially infectious, expensive treatment that targets a single antigen
and is difficult
to standardize.
Accordingly, the need exists for developing improved strategies for providing
safer, less toxic alternatives to the current therapy for MRSA, and which are
less likely
to cause resistant organisms after their widespread use.
Summary of the Invention
The present invention provides isolated human monoclonal antibodies which
specifically bind to Staphylococcus aureus by binding to an antigen of S.
aureus, as well
as compositions containing one or a combination of such antibodies.
Preferably, the
human antibodies cross-react with an epitope present on multiple (i.e., two or
more) S.
aureus clinical isolates. In certain embodiments, the human antibodies are
also
characterized by binding to S. aureus or an S. aureus-antigen with high
affinity, and by
inhibiting S. aureus growth and/or mediating phagocytosis and cell killing of
S. aureus
(in vitro and in vivo) in the presence of human effector cells. Accordingly,
the human
monoclonal antibodies of the invention can be used as diagnostic or
therapeutic agents
in vivo and in vitro.
Isolated human antibodies of the invention encompass various antibody
isotypes,
such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, and IgE.
Typically,
they include IgGI (e.g., IgGlK) and IgM isotypes. The antibodies can be full-
length
(e.g., an IgGl or IgG4 antibody) or can include only an antigen-binding
portion (e.g., a
Fab, F(ab')2, Fv or a single chain Fv fragment). In one embodiment, the human
antibodies are recombinant human antibodies. In another embodiment, the human
antibodies are produced by a hybridoma which includes a B cell obtained from a
transgenic non-human animal, e.g., a transgenic mouse, having a genome
comprising a
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human heavy chain transgene and a human light chain transgene, fused to an
immortalized cell. In particular embodiments, the antibodies are produced by
hybridomas referred to herein as 2GD 12, 2H 12, 8.1 E5, 8.2C 1, 7F 1, 6D 12
and SH 10.
In another embodiment, human anti- S. aureus antibodies of the present
invention can be characterized by one or more of the following properties:
a) crossreactivity with at least one, preferably multiple, S. aureus isolates,
e.g., S.
aureus clinical isolates;
b) a binding affinity to S. aureus or an S. aureus-antigen with an affinity
constant
of at least about 10' M-l, preferably about 108 M-~, and more preferably,
about 109 M-1
to 10' ° M-~ or higher;
c) an association constant (K~SO~) with S. aureus or an S. aureus-antigen of
at
least about 103, more preferably about 104 and most preferably about 105 M~~S-
1;
d) a dissociation constant (Kd;s) from S. aureus or an S. aureus-antigen of
about
10-3 s', preferably about 10-4 s', more preferably, 10-5 s-~, and most
preferably, 10-6 s-~;
e) the ability to opsonize S. aureus; or
f) the ability to inhibit growth and colonization of S. aureus and/or mediate
phagocytosis and killing of S. aureus in the presence of human effector cells
at a
concentration of about 10 ~g/ml or less (e.g., in vitro).
Examples of S. aureus clinical isolates which can be targeted by the human
antibodies of the invention include, but are not limited to: IBERIAN, EMRSA,
ONTARIO and BRAZILIAN strains. In a particular embodiment, the antibodies bind
to
at least one methicillin-resistant S. aureus (MRSA) strain.
Isolated human antibodies of the invention can bind any S. aureus antigen
including for example, antigens secreted by, or present on the surface of,
S. aureus strains. Examples of such antigens include capsular polysaccharides
(e.g., CP
type 5 or CP type 8), fibronectin binding proteins, protein A, toxins (a-, (3-
, 8, and y-
toxin, enterotoxins, epidemolytic toxin), toxic shock syndrome toxin
superantigens,
coagulase, staphylokinase, penicillin binding protein 2a (PBP 2a) and
adhesins, among
others. In one embodiment, the human antibodies bind to S. aureus or an S.
aureus
antigen with an affinity constant of at least about 10' M-1, preferably about
10g M-1, and
more preferably, about 109 M-~ to 10'° M-~ or stronger, and are capable
of mediating
phagocytosis and killing of S aureus by human effector cells, e.g.,
polymorphonuclear
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cells (PMNs), with an IC50 of about 1 x 10-7 M or less, or at a concentration
of about 10
pg/ml or less in vitro.
In another aspect, the invention provides nucleic acid molecules encoding the
antibodies, or antigen-binding portions, of the invention. Accordingly,
recombinant
expression vectors which include the antibody-encoding nucleic acids of the
invention,
and host cells transfected with such vectors, are also encompassed by the
invention, as
are methods of making the antibodies of the invention by culturing these host
cells.
In yet another aspect, the invention provides isolated B-cells from a
transgenic
non-human animal, e.g., a transgenic mouse, which are capable of expressing
various
isotypes (e.g., IgG, IgA and/or IgM) of human monoclonal antibodies that
specifically
bind to S. aureus or an S. aureus-antigen. Preferably, the isolated B cells
are obtained
from a transgenic non-human animal, e.g., a transgenic mouse, which has been
immunized with whole S. aureus or a purified or enriched preparation of an S.
aureus-
antigen. Preferably, the transgenic non-human animal, e.g., a transgenic
mouse, has a
genome comprising a human heavy chain transgene and a human light chain
transgene.
The isolated B-cells are then immortalized to provide a source (e.g., a
hybridoma) of
human monoclonal antibodies to S. aureus or an S. aureus-antigen.
Accordingly, the present invention also provides a hybridoma capable of
producing human monoclonal antibodies that specifically bind to S. aureus or
an S.
aureus-antigen. In one embodiment, the hybridoma includes a B cell obtained
from a
transgenic non-human animal, e.g., a transgenic mouse, having a genome
comprising a
human heavy chain transgene and a human light chain transgene, fused to an
immortalized cell. The transgenic non-human animal can be immunized with whole
S.
aureus or a purified or enriched preparation of an S. aureus-antigen to
generate
antibody-producing hybridomas. Particular hybridomas of the invention include
2GD12, 2H12, 8.1E5, 8.2C1, 7F1, 6D12, and SH10.
In yet another aspect, the invention provides a transgenic non-human animal,
such as a transgenic mouse (also referred to herein as "HuMab"), which express
human
monoclonal antibodies that specifically bind to S. aureus or an S. aureus-
antigen. In a
particular embodiment, the transgenic non-human animal is a transgenic mouse
having a
genome comprising a human heavy chain transgene and a human light chain
transgene.
The transgenic non-human animal can be immunized with whole S. aureus or a
purified
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or enriched preparation of an S. aureus-antigen. Preferably, the transgenic
non-human
animal, e.g., the transgenic mouse, is capable of producing multiple isotypes
of human
monoclonal antibodies to S aureus or an S. aureus antigen (e.g., IgG, IgA
and/or IgM)
by undergoing V-D-J recombination and isotype switching. Isotype switching may
occur by, e.g., classical or non-classical isotype switching.
In another aspect, the present invention provides methods for producing human
monoclonal antibodies which specifically react with S. aureus or an S. aureus-
antigen.
In one embodiment, the method includes immunizing a transgenic non-human
animal,
e.g., a transgenic mouse, having a genome comprising a human heavy chain
transgene
and a human light chain transgene, with whole S. aureus or a purified or
enriched
preparation of an S. aureus-antigen. B cells (e.g., splenic B cells) of the
animal are then
obtained and fused with myeloma cells to form immortal, hybridoma cells that
secrete
human monoclonal antibodies against S. aureus or an S. aureus-antigen.
Isolated anti-S. aureus human monoclonal antibodies of the invention, or
antigen
binding portions thereof, can be derivatized or linked to another functional
molecule,
e.g., another peptide or protein (e.g., an Fab' fragment). For example, an
antibody or
antigen-binding portion of the invention can be functionally linked (e.g., by
chemical
coupling, genetic fusion, noncovalent association or otherwise) to one or more
other
molecular entities, such as another antibody (e.g., a bispecific or a
multispecific
antibody). Accordingly, in another aspect, the present invention features a
bispecific or
multispecific molecule comprising at least one first binding specificity for
S. aureus or
an S. aureus-antigen and a second binding specificity for an Fc receptor,
e.g., human Fcy
RI or a human Fca receptor.
Multispecific molecules of the invention also include trispecific,
tetraspecific
and other multispecific molecules. In one embodiment the multispecific
molecule
includes an anti-enhancement factor (EF) portion, e.g., a molecule which binds
to a
surface protein involved in cytotoxic activity.
In a particular embodiment, bispecific and multispecific molecules of the
invention comprise at least one antibody, or fragment thereof (e.g., an Fab,
Fab', F(ab')2,
Fv, or a single chain Fv). In a particular embodiment, the antibody or
fragment thereof
is a completely human antibody or a portion thereof, or a "chimeric" or a
"humanized"
antibody or a portion thereof (e.g., has a variable region, or at least a
complementarity
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determining region (CDR), derived from a non-human antibody (e.g., murine)
with the
remaining portions) being human in origin).
In one embodiment, the at least one antibody or fragment thereof of the
bispecific or multispecific molecule binds to an Fc receptor, such as a human
IgG
receptor, e.g., an Fc-gamma receptor (FcyR), such as FcyRI (CD64),
FcyRII(CD32), and
FcyRIII (CD 16). A preferred Fcy receptor is the high affinity Fcy receptor,
FcyRI.
However, other Fc receptors, such as human IgA receptors (e.g. FcaRI) also can
be
targeted. The Fc receptor is preferably located on the surface of an effector
cell, e.g., a
monocyte, macrophage or an activated polymorphonuclear cell. In a preferred
embodiment, the bispecific and multispecific molecules bind to an Fc receptor
at a site
which is distinct from the immunoglobulin (e.g., IgG or IgA) binding site of
the
receptor. Therefore, the binding of the bispecific and multispecific molecules
is not
blocked by physiological levels of immunoglobulins.
In another aspect, the present invention provides target-specific effector
cells
which comprise an effector cell expressing an Fc receptor, e.g., a macrophage
or an
activated PMN cell, and a bispecific or multispecific molecule of the
invention.
In another aspect, the present invention provides compositions, e.g.,
pharmaceutical and diagnostic compositions, comprising a pharmaceutically
acceptable
carrier and at least one human monoclonal antibody of the invention, or an
antigen-
binding portion thereof, which specifically binds to S. aureus or an S. aureus-
antigen.
In one embodiment, the composition comprises a combination of the human
antibodies
or antigen-binding portions thereof, preferably each of which binds to a
distinct epitope.
For example, a pharmaceutical composition comprising a human monoclonal
antibody
that exhibits limited crossreactivity to S. aureus isolates, but that mediates
highly
effective phagocytosis of S. aureus, can be combined with another human
monoclonal
antibody that exhibits broad crossreactivity to S. aureus strains. Thus, the
combination
provides multiple therapies tailored to provide the maximum therapeutic
benefit.
Compositions, e.g., pharmaceutical compositions, comprising a combination of
at least
one human monoclonal antibody of the invention, or antigen-binding portions
thereof,
and at least one bispecific or multispecific molecule of the invention, are
also within the
scope of the invention.
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In yet another aspect, the invention provides a method for inhibiting
infectivity
of S. aureus by inhibiting growth and/or colonization, and/or by inducing
phagocytosis
and/or killing, of S. aureus bacteria by human effector cells, such as human
polymorphonuclear cells (PMNs), using an antibody, or antigen-binding portion
thereof
(or a bispecific or multispecific antibody) of the invention. In one
embodiment, the
method comprises contacting S. aureus either in vitro or in vivo with one or a
combination of human monoclonal antibodies of the invention, or an antigen-
binding
portion thereof, in the presence of a human effector cell. The method can be
employed
in culture, e.g. in vitro or ex vivo (e.g., cultures comprising S. aureus and
effector cells).
For example, a sample containing S. aureus and effector cells can be cultured
in vitro,
and combined with an antibody of the invention, or an antigen-binding portion
thereof
(or a bispecific or multispecific antibody of the invention). Alternatively,
the method
can be performed in a subject, e.g., as part of an in vivo (e.g., therapeutic
or
prophylactic) protocol.
For in vivo methods, the antibody, or antigen-binding portion thereof (or a
bispecific or multispecific antibody of the invention), can be administered to
a human
subject suffering from an S. aureus-mediated disease such that growth
inhibition,
phagocytosis and/or killing of S. aureus is induced. In one embodiment, the
subject can
be additionally treated with an agent that modulates, e.g., enhances or
inhibits, the
expression or activity of Fc receptor, e.g., an Fca, receptor or an Fcy
receptor, by for
example, treating the subject with a cytokine. Preferred cytokines for
administration
during treatment with the bispecific and multispecific molecules include
granulocyte
colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating
factor
(GM-CSF), interferon-y (IFN-y), and tumor necrosis factor (TNF).
Isolated human monoclonal antibody compositions of the invention also can be
administered in combination with other known anti-bacterial therapies.
Exemplary diseases that can be treated (e.g., ameliorated) or prevented using
the
methods and compositions of the invention include, but are not limited to,
invasive or
toxigenic infectious diseases. Such invasive diseases include Bacteremia,
osteomyelitis,
septic arthritis, septic thrombophlebitis and acute bacterial endocarditis.
Such toxigenic
diseases include Staphylococcol food poisoning, scalded skin syndrome and
toxic shock
syndrome. Additional examples of diseases that can be treated (e.g., prevented
or
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ameliorated) using the methods of the invention include infections of the
upper and/or
lower respiratory, heart, gastrointestinal tract, CNS, eye, kidney and urinary
tract, skin,
and bone and joint.
In yet another aspect, the present invention provides a method for detecting
in
vitro or in vivo the presence of S. aureus in a sample, e.g., for diagnosing
an S. aureus-
mediated disease. In one embodiment, this is achieved by contacting a sample
to be
tested, along with a control sample, with a human monoclonal antibody of the
invention,
or an antigen-binding portion thereof ( or a bispecific or multispecific
molecule), under
conditions that allow for formation of a complex between the antibody and S.
aureus.
Complex formation is then detected (e.g., using an ELISA) in both samples, and
any
statistically significant difference in the formation of complexes between the
samples is
indicative the presence of S. aureus in the test sample.
Other features and advantages of the instant invention be apparent from the
following detailed description and claims.
Brief Description of the Drawings
Figure 1 is a bar graph depicting the levels of anti-S. aureus human
antibodies present in the plasma of HuMAb mice immunized with heat killed S.
aureus,
as compared to non-immunized controls. Shown are results of optical density
with
respect to the indicated dilution of pooled plasma from S. aureus immunized
mice
(hatched bar) and non-immunized mice (solid bar) analyzed by ELISA.
Figure 2 is a bar graph depicting the binding of the supernatants from six
mixed
hybridoma cultures, 2GD 12, 2H 12, 8.1 E5, 8.2C l, 7F 1 and 6D 12, to S.
aureus strain
209, compared to human IgG controls. The anti-FcyRI antibody H22 was used as a
control.
Figure 3 is a bar graph depicting the binding of the supernatants from mixed
hybridoma cultures (2GD 12, 2H 12, 8.1 E5, 8.2C 1, 7F l and 6D 12) to
Methicillin
resistant S. aureus strain BK2058 compared to media alone (bkd), as measured
by
ELISA.
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Figure 4 is a bar graph depicting the binding of the supernatants from the
mixed
hybridoma cultures (2GD 12, 2H 12, 8.1 E5, 8.2C 1, 7F 1 and 6D 12) to mixed
bacteria
(Escherichia coli, Pseudomonas aeroginosa, or Micrococcus luteus) as measured
by
ELISA.
Figure 5 is a bar graph comparing the binding of the supernatants from the
mixed hybridoma cultures (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to S.
aureus
(speckled bar) or E. coli (solid bar), as measured by flow cytometry.
Figure 6 is a bar graph depicting the binding of the supernatants from the
indicated subcloned hybridoma cultures to S. aureus FDA 209, as measured by
ELISA.
Figure 7 is a bar graph showing binding of supernatants from subcloned
hybridoma cultures to S. aureus ATCC 27661, as measured by ELISA.
Figures 8A-8C are histograms showing neutrophil-mediated phagocytosis of S.
aureus upon incubation of polymorphonuclear cells (PMNs) with mixed hybridoma
supernatants. FACScan analyses of control PMNs alone (Figure 8A), PMNs and S.
aureus, with or without supernatant from mixed hybridoma cultures (Figure 8B),
and
PMNs and control E. coli, with or without supernatant from mixed hybridoma
cultures
(Figure 8C) are shown.
Figures 9A-9B show binding of purified human monoclonal antibody 6D 12 to S.
aureus FDA 209 compared to IgG controls. Figure 9A is a bar graph depicting
the
binding of two concentrations ( 1 and 10 ~g/ml) of purified human monoclonal
antibody
6D 12 (open bars) to S. aureus FDA 209, compared to IgG controls (speckled
bars) as
measured using ELISA. Figure 9B is a histogram showing direct binding of FITC-
labeled 6D12 monoclonal antibodies to S. aureus FDA 209 compared to FITC-
labeled
humanized anti-EGF receptor antibody control (H425) as detected by flow
cytometry.
Figures l0A-IOB show binding of purified human monoclonal antibody 6D 12 to
MRSA strain 2058 compared to IgG controls. Figure 10A is a linear graph
depicting the
binding of two concentrations (1 and 10 ~g/ml) of purified human monoclonal
antibody
6D12 to MRSA strain 2058, compared to IgG controls. Figure IOB is a histogram
showing direct binding of FITC-labeled 6D I 2 monoclonal antibodies to MRSA
strain
2058 compared to FITC-labeled humanized anti-EGF receptor antibody control
(H425)
as detected by flow cytometry.
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Figure 11 is a bar graph showing killing of S. aureus after antibody
opsonization
and PMN phagocytosis using human monoclonal antibodies against
S. aureus.
Detailed Description of the Invention
The present invention provides novel antibody-based therapeutics for treating
and diagnosing S. aureus infections. Therapeutic and diagnostic reagents of
the
invention include isolated human monoclonal antibodies, or antigen-binding
portions
thereof, which bind to an epitope present on at least one, and preferably
multiple, strains
of S. aureus clinical isolates, including MRSA strains. In one embodiment, the
human
antibodies are produced in a non-human transgenic animal, e.g., a transgenic
mouse,
capable of producing multiple isotypes of human monoclonal antibodies to S.
aureus or
an S. aureus antigen (e.g., IgG, IgA and/or IgE) by undergoing V-D-J
recombination
and isotype switching. Accordingly, various aspects of the invention include
antibodies
and antibody fragments, and pharmaceutical compositions thereof, as well as
non-
human transgenic animals, and B-cells and hybridomas for making such
monoclonal
antibodies. Methods of using the antibodies of the invention to detect S.
aureus or to
inhibit S. aureus infectivity, either in vitro or in vivo, are also
encompassed by the
invention.
In order that the present invention may be more readily understood, certain
terms
are first defined. Additional definitions are set forth throughout the
detailed description.
The term "Staphylococcus aureus" (abbreviated herein as "S. aureus"), as used
herein, refers to any strain, genotype or isolate of S. aureus, a Gram-
positive bacteria
which is among the most common of nosocomial pathogens in hospitals and long-
term
care facilities (1) today. These organisms are capable of causing symptoms
ranging
from skin boils to septicemia and death. The term "S. aureus" also includes
antibiotic-
resistant strains of S. aureus, e.g., penicillin- and methicillin-resistant S.
aureus
(abbreviated herein as "MRSA"), including, for example, IBERIAN, EMRSA,
ONTARIO, BRAZILIAN and COL strains (2,3). MRSA strains are generally resistant
to (3-lactam antibiotics by expression of a mutated penicillin binding protein
(PBP 2a)
that has a low binding capacity for these antibiotics (4).
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As used herein, the term "S. aureus-antigen" includes any S. aureus antigen,
including secreted S. aureus antigens and antigens which naturally occur on
the surface
of S. aureus. In preferred embodiments, the S. aureus-antigen is present in a
large
percentage of clinical isolates to provide for broad crossreactivity of the
antibody that
binds to the antigen. In other preferred embodiments, binding of an antibody
of the
invention to the S. aureus-antigen mediates effector cell phagocytosis and
killing of S.
aureus. Preferably, the S. aureus antigen can elicit a protective immune
response upon
immunization of a non-human transgenic animal. Examples of such antigens
include
capsular polysaccharides (e.g., CP type 5 or CP type 8), fibronectin binding
proteins,
protein A, toxins (oc-, (3-, 8, and y-toxin, enterotoxins, epidemolytic
toxin), toxic shock
syndrome toxin superantigens, coagulases, staphylokinases, penicillin binding
protein 2a
(PBP 2a) and adhesins, among others (28). For example, the CP antigens (CPS
and
CP8) are present on about 70-80% of clinical isolates.
As used herein, the term "antibody" refers to a glycoprotein comprising at
least
two heavy (H) chains and two light (L) chains inter-connected by disulfide
bonds. Each
heavy chain is comprised of a heavy chain variable region (abbreviated herein
as HCVR
or VH) and a heavy chain constant region. The heavy chain constant region is
comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of
a
light chain variable region (abbreviated herein as LCVR or VL) and a light
chain
constant region. The light chain constant region is comprised of one domain,
CL. The
VH and VL regions can be further subdivided into regions of hypervariability,
termed
complementarity determining regions (CDR), interspersed with regions that are
more
conserved, termed framework regions (FR). Each 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. The variable regions of
the heavy and light chains contain a binding domain that interacts with an
antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to
host tissues or factors, including various cells of the immune system (e.g.,
effector cells)
and the first component (Clq) of the classical complement system.
The term "antigen-binding portion" of an antibody (or simply "antibody
portion"), as used herein, refers to one or more fragments of an antibody that
retain the
ability to specifically bind to an antigen (e.g., S. aureus or an S. aureus
antigen). It has
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been shown that the antigen-binding function of an antibody can be performed
by
fragments of a full-length antibody. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include (i) a Fab
fragment, a
monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab')2
fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide
bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains;
(iv) a Fv fragment consisting of the VL and VH domains of a single arm of an
antibody,
(v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of
a VH
domain; and (vi) an isolated complementarity determining region (CDR).
Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded for by
separate
genes, they can be joined, using recombinant methods, by a synthetic linker
that enables
them to be made as a single protein chain in which the VL and VH regions pair
to form
monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988)
Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. U.SA
85:5879-
5883). Such single chain antibodies are also intended to be encompassed within
the
term "antigen-binding portion" of an antibody. These antibody fragments are
obtained
using conventional techniques known to those with skill in the art, and the
fragments are
screened for utility in the same manner as are intact antibodies.
The term "bispecific molecule" is intended to include any agent, e.g., a
protein,
peptide, or protein or peptide complex, which has two different binding
specificities
which bind to, or interact with (a) a cell surface antigen and (b) an Fc
receptor on the
surface of an effector cell. The term "multispecific molecule" or
"heterospecific
molecule" is intended to include any agent, e.g., a protein, peptide, or
protein or peptide
complex, which has more than two different binding specificities which bind
to, or
interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of
an effector
cell, and (c) at least one other component. Accordingly, the invention
includes, but is
not limited to, bispecific, trispecific, tetraspecific, and other
multispecific molecules
which are directed to cell surface antigens, such as S. aureus or an S. aureus
antigen,
and to Fc receptors on effector cells. The term "bispecific antibodies"
further includes
diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and
VL
domains are expressed on a single polypeptide chain, but using a linker that
is too short
to allow for pairing between the two domains on the same chain, thereby
forcing the
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domains to pair with complementary domains of another chain and creating two
antigen
binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.
USA 90:6444-
6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).
As used herein, the term "heteroantibodies" refers to two or more antibodies,
antibody binding fragments (e.g., Fab), derivatives therefrom, or antigen
binding regions
linked together, at least two of which have different specificities. These
different
specificities include a binding specificity for an Fc receptor on an effector
cell, and a
binding specificity for an antigen or epitope on a target cell, e.g., S.
aureus.
The term "human antibody", as used herein, is intended to include antibodies
having variable and constant regions derived from human germline
immunoglobulin
sequences. The human antibodies of the invention may include amino acid
residues not
encoded by human germline immunoglobulin sequences (e. g., mutations
introduced by
random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
However,
the term "human antibody", as used herein, is not intended to include
antibodies in
which CDR sequences derived from the germline of another mammalian species,
such
as a mouse, have been grafted onto human framework sequences.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used
herein refer to a preparation of antibody molecules of single molecular
composition. A
monoclonal antibody composition displays a single binding specificity and
affinity for a
particular epitope. Accordingly, the term "human monoclonal antibody" refers
to
antibodies displaying a single binding specificity which have variable and
constant
regions derived from human germline immunoglobulin sequences. In one
embodiment,
the human monoclonal antibodies are produced by a hybridoma which includes a B
cell
obtained from a transgenic non-human animal, e.g., a transgenic mouse, having
a
genome comprising a human heavy chain transgene and a light chain transgene,
fused to
an immortalized cell.
The term "recombinant human antibody", as used herein, is intended to include
all human antibodies that are prepared, expressed, created or isolated by
recombinant
means, such as antibodies isolated from an animal (e.g., a mouse) that is
transgenic for
human immunoglobulin genes (described further in Section I, below); antibodies
expressed using a recombinant expression vector transfected into a host cell,
antibodies
isolated from a recombinant, combinatorial human antibody library, or
antibodies
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prepared, expressed, created or isolated by any other means that involves
splicing of
human immunoglobulin gene sequences to other DNA sequences. Such recombinant
human antibodies have variable and constant regions derived from human
germline
immunoglobulin sequences. In certain embodiments, however, such recombinant
human antibodies are subjected to in vitro mutagenesis (or, when an animal
transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and thus the
amino acid
sequences of the VH and VL regions of the recombinant antibodies are sequences
that,
while derived from and related to human germline VH and VL sequences, may not
naturally exist within the human antibody germline repertoire in vivo.
As used herein, a "heterologous antibody" is defined in relation to the
transgenic
non-human organism producing such an antibody. This term refers to an antibody
having an amino acid sequence or an encoding nucleic acid sequence
corresponding to
that found in an organism not consisting of the transgenic non-human animal,
and
generally from a species other than that of the transgenic non-human animal.
As used herein, a "heterohybrid antibody" refers to an antibody having a light
and heavy chains of different organismal origins. For example, an antibody
having a
human heavy chain associated with a murine light chain is a heterohybrid
antibody.
Examples of heterohybrid antibodies include chimeric and humanized antibodies,
discussed supra.
An "isolated antibody", as used herein, is intended to refer to an antibody
which
is substantially free of other antibodies having different antigenic
specificities (e.g., an
isolated antibody that specifically binds to S. aureus or an S. aureus antigen
is
substantially free of antibodies that specifically bind antigens other than S.
aureus or an
S. aureus antigen). An isolated antibody that specifically binds to one strain
or one
antigen of S. aureus may, however, have cross-reactivity to other S. aureus
strains or
antigens. Moreover, an isolated antibody may be substantially free of other
cellular
material and/or chemicals. In one embodiment of the invention, a combination
of
"isolated" monoclonal antibodies having different specificities are combined
in a well
defined composition.
As used herein, "specific binding" refers to antibody binding to a
predetermined
antigen. Typically, the antibody binds with an affinity of at least about 1 x
10' M~~, and
binds to the predetermined antigen with an affinity that is at least two-fold
greater than
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its affinity for binding to a non-specific antigen (e.g., BSA, casein) other
than the
predetermined antigen or a closely-related antigen. The phrases "an antibody
recognizing an antigen" and " an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds specifically to
an
antigen".
As used herein, the term "high affinity" for an IgG antibody refers to a
binding affinity
of at least about 1 x 109M-1, typically at least about 5 x 109M-1, frequently
more than
about 1 x 101°M-1, and sometimes 5 x 101°M-~ to 1 x 1011M-1 or
greater. "High affinity"
binding can vary for other antibody isotypes. For example, "high affinity"
binding for
an IgM isotype is at least about 1 x 107M-1.
The term "Kassoc~~~ as used herein, is intended to refer to the association
constant
of a particular antibody-antigen interaction.
The term "Kdis", as used herein, is intended to refer to the dissociation
constant
of a particular antibody-antigen interaction.
As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGI)
that is
encoded by heavy chain constant region genes.
As used herein, "isotype switching" refers to the phenomenon by which the
class, or isotype, of an antibody changes from one Ig class to one of the
other Ig classes.
As used herein, "nonswitched isotype" refers to the isotypic class of heavy
chain
that is produced when no isotype switching has taken place; the CH gene
encoding the
nonswitched isotype is typically the first CH gene immediately downstream from
the
functionally rearranged VDJ gene. Isotype switching has been classified as
classical or
non-classical isotype switching. Classical isotype switching occurs by
recombination
events which involve at least one switch sequence region in the transgene. Non-
classical isotype switching may occur by, for example, homologous
recombination
between human 6w and human ~u (8-associated deletion). Alternative non-
classical
switching mechanisms, such as intertransgene and/or interchromosomal
recombination,
among others, may occur and effectuate isotype switching.
As used herein, the term "switch sequence" refers to those DNA sequences
responsible for switch recombination. A "switch donor" sequence, typically a ~
switch
region, will be 5' (i.e., upstream) of the construct region to be deleted
during the switch
recombination. The "switch acceptor" region will be between the construct
region to be
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deleted and the replacement constant region (e.g., y, s, etc.). As there is no
specific site
where recombination always occurs, the final gene sequence will typically not
be
predictable from the construct.
As used herein, "glycosylation pattern" is defined as the pattern of
carbohydrate
units that are covalently attached to a protein, more specifically to an
immunoglobulin
protein. A glycosylation pattern of a heterologous antibody can be
characterized as
being substantially similar to glycosylation patterns which occur naturally on
antibodies
produced by the species of the nonhuman transgenic animal, when one of
ordinary skill
in the art would recognize the glycosylation pattern of the heterologous
antibody as
being more similar to said pattern of glycosylation in the species of the
nonhuman
transgenic animal than to the species from which the CH genes of the transgene
were
derived.
The term "naturally-occurring" as used herein as applied to an object refers
to the
fact that an object can be found in nature. For example, a polypeptide or
polynucleotide
sequence that is present in an organism (including viruses) that can be
isolated from a
source in nature and which has not been intentionally modified by man in the
laboratory
is naturally-occurring.
The term "rearranged" as used herein refers to a configuration of a heavy
chain
or light chain immunoglobulin locus wherein a V segment is positioned
immediately
adjacent to a D-J or J segment in a conformation encoding essentially a
complete VH or
VL domain, respectively. A rearranged immunoglobulin gene locus can be
identified by
comparison to germline DNA; a rearranged locus will have at least one
recombined
heptamer/nonamer homology element.
The term "unrearranged" or "germline configuration" as used herein in
reference
to a V segment refers to the configuration wherein the V segment is not
recombined so
as to be immediately adjacent to a D or J segment.
The term "nucleic acid molecule", as used herein, is intended to include DNA
molecules and RNA molecules. A nucleic acid molecule may be single-stranded or
double-stranded, but preferably is double-stranded DNA.
The term "isolated nucleic acid molecule", as used herein in reference to
nucleic
acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind
to S.
aureus or an S. aureus antigen, is intended to refer to a nucleic acid
molecule in which
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the nucleotide sequences encoding the antibody or antibody portion are free of
other
nucleotide sequences encoding antibodies or antibody portions that bind
antigens other
than S. aureus or an S. aureus antigen, which other sequences may naturally
flank the
nucleic acid in human genomic DNA.
For nucleic acids, the term "substantial homology" indicates that two nucleic
acids, or designated sequences thereof, when optimally aligned and compared,
are
identical, with appropriate nucleotide insertions or deletions, in at least
about 80% of the
nucleotides, usually at least about 90% to 95%, and more preferably at least
about 98%
to 99.5% of the nucleotides. Alternatively, substantial homology exists when
the
segments will hybridize under selective hybridization conditions, to the
complement of
the strand.
The percent identity between two sequences is a function of the number
of identical positions shared by the sequences (i.e., % homology = # of
identical
positions/total # of positions x 100), taking into account the number of gaps,
and the
length of each gap, which need to be introduced for optimal alignment of the
two
sequences. The comparison of sequences and determination of percent identity
between
two sequences can be accomplished using a mathematical algorithm, as described
in the
non-limiting examples below.
The percent identity between two nucleotide sequences can be
determined using the GAP program in the GCG software package (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,
60,
70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity
between two
nucleotide or amino acid sequences can also determined using the algorithm of
E.
Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120 weight
residue
table, a gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity
between two amino acid sequences can be determined using the Needleman and
Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been
incorporated into
the GAP program in the GCG software package (available at http://www.gcg.com),
using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,
14, 12,
10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
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The nucleic acid and protein sequences of the present invention can
further be used as a "query sequence" to perform a search against public
databases to,
for example, identify related sequences. Such searches can be performed using
the
NBLAST and XBLAST programs (version 2.0) of Altschul, et al. ( 1990) J. Mol.
Biol.
215:403-10. BLAST nucleotide searches can be performed with the NBLAST
program,
score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the
nucleic
acid molecules of the invention. BLAST protein searches can be performed with
the
XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences
homologous to the protein molecules of the invention. To obtain gapped
alignments for
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al.,
(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped
BLAST programs, the default parameters of the respective programs (e.g.,
XBLAST
and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
The nucleic acids may be present in whole cells, in a cell lysate, or in a
partially
purified or substantially pure form. A nucleic acid is "isolated" or "rendered
substantially pure" when purified away from other cellular components or other
contaminants, e.g., other cellular nucleic acids or proteins, by standard
techniques,
including alkaline/SDS treatment, CsCI banding, column chromatography, agarose
gel
electrophoresis and others well known in the art. See, F. Ausubel, et al., ed.
Current
Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New
York
( 1987).
The nucleic acid compositions of the present invention, while often in a
native
sequence (except for modified restriction sites and the like), from either
cDNA, genomic
DNA or mixtures thereof, may be mutated in accordance with standard techniques
to
provide gene sequences. For coding sequences, these mutations, may affect the
amino
acid sequence as desired. In particular, DNA sequences substantially
homologous to or
derived from native V, D, J, constant, switches and other such sequences
described
herein are contemplated (where "derived" indicates that a sequence is
identical or
modified from another sequence).
A nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid sequence. For instance, a promoter or
enhancer is
operably linked to a coding sequence if it affects the transcription of the
sequence. With
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respect to transcription regulatory sequences, operably linked means that the
DNA
sequences being linked are contiguous and, where necessary to join two protein
coding
regions, contiguous and in reading frame. For switch sequences, operably
linked
indicates that the sequences are capable of effecting switch recombination.
The term "vector", as used herein, is intended to refer to a nucleic acid
molecule
capable of transporting another nucleic acid to which it has been linked. One
type of
vector is a "plasmid", which refers to a circular double stranded DNA loop
into which
additional DNA segments may be ligated. Another type of vector is a viral
vector,
wherein additional DNA segments may be ligated into the viral genome. Certain
vectors are capable of autonomous replication in a host cell into which they
are
introduced (e.g., bacterial vectors having a bacterial origin of replication
and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can
be
integrated into the genome of a host cell upon introduction into the host
cell, and thereby
are replicated along with the host genome. Moreover, certain vectors are
capable of
directing the expression of genes to which they are operatively linked. Such
vectors are
referred to herein as "recombinant expression vectors" (or simply, "expression
vectors")
In general, expression vectors of utility in recombinant DNA techniques are
often in the
form of plasmids. In the present specification, "plasmid" and "vector" may be
used
interchangeably as the plasmid is the most commonly used form of vector.
However,
the invention is intended to include such other forms of expression vectors,
such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and adeno-
associated
viruses), which serve equivalent functions.
The term "recombinant host cell" (or simply "host cell"), as used herein, is
intended to refer to a cell into which a recombinant expression vector has
been
introduced. It should be understood that such terms are intended to refer not
only to the
particular subject cell but to the progeny of such a cell. Because certain
modifications
may occur in succeeding generations due to either mutation or environmental
influences,
such progeny may not, in fact, be identical to the parent cell, but are still
included within
the scope of the term "host cell" as used herein.
Various aspects of the invention are described in further detail in the
following
subsections.
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I. Production of Human Antibodies to S. aureus
Human monoclonal antibodies (mAbs) of the invention can be produced by a
variety of techniques, including conventional monoclonal antibody methodology
e.g.,
the standard somatic cell hybridization technique of Kohler and Milstein,
Nature 256:
495 (1975). Although somatic cell hybridization procedures are preferred, in
principle,
other techniques for producing monoclonal antibody can be employed e.g., viral
or
oncogenic transformation of B lymphocytes.
The preferred animal system for preparing hybridomas is the murine system.
Hybridoma production in the mouse is a very well-established procedure.
Immunization
protocols and techniques for isolation of immunized splenocytes for fusion are
known in
the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures
are also
known.
In a preferred embodiment, human monoclonal antibodies directed against S.
aureus are generated using transgenic mice carrying the complete human immune
system rather than the mouse system, referred to herein as "HuMab" transgenic
mice.
For example, HuMAb transgenic mice can be used which contain a human
immunoblobulin gene miniloci that encodes unrearranged human heavy (~ and y)
and K
light chain immunoglobulin sequences, together with targeted mutations that
inactivate
the endogenous ~ and K chain loci (Lonberg, N. et al. (1994) Nature 368(6474):
856-
859). Accordingly, these mice show reduced expression of mouse IgM or K, and
in
response to immunization, the introduced human heavy and light chain
transgenes
undergo class switching and somatic mutation to generate high affinity human
IgGK
monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994)
Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D.
(1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N.
(1995)
Ann. N. Y. Acad. Sci 764:536-546). The preparation of HuMab mice is described
in
detail Section II below and in Taylor, L. et al. (1992) Nucleic Acids Research
20:6287-
6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et
al. (1993)
Proc. Natl. Acad Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics
4:117-123;
Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.
152:2912-2920; Lonberg et al., (1994) Nature 368(6474): 856-859; Lonberg, N.
(1994)
Handbook of Experimental Pharmacology 113 :49-1 O 1; Taylor, L. et al. ( 1994)
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International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern.
Rev.
Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. ( 1995) Ann. N. Y. Acad.
Sci
764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the
contents
of all of which are hereby incorporated by reference in their entirety. See
further, U.S.
Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;
5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, and
GenPharm
International; U.S. Patent No. 5,545,807 to Surani et al.; International
Publication Nos.
WO 98/24884, published on June 11, 1998; WO 94/25585, published November 10,
1994; WO 93/1227, published June 24, 1993; WO 92/22645, published December 23,
1992; WO 92/03918, published March 19, 1992, the disclosures of all of which
are
hereby incorporated by reference in their entity.
HuMab Immunizations
To generate fully human monoclonal antibodies to S. aureus, mice, preferably
HuMab transgenic mice, can be immunized, for example, with heat killed whole
S.
aureus (e.g., purified or enriched immunogen containing S - 20 pg S. aureus
antigen)
using, for example, the immunization protocol described in Lonberg, N. et al.
( 1994)
Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:
845-
851 and WO 98/24884. Preferably, the mice are 6-16 weeks of age upon the first
infusion.
For example, heat killed whole S. aureus from a single clinical isolate that
expresses the CP antigen, CP type 5 (CPS), can be used to immunize the HuMab
mice
intraperitoneally. CP type 5 is a suitable S. aureus antigen for use in the
invention
because it is a common CP type shared by MRSA isolates. Furthermore, previous
studies have demonstrated that antibodies which bind type 5 capsule can
mediate
protection against experimental S. aureus infections (14, 28). In the event
that
immunizations using a single S. aureus strain do not result in antibodies
cross-reactive
with two or more S. aureus strains, mice can be immunized with alternating
strains of S.
aureus to promote immune responses against shared antigens. Prior to
immunization,
the bacteria is generally typed, and can be grown either on agar plates to
promote
expression of capsule (29), or in broth to minimize capsule expression.
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Cumulative experience with various antigens has shown that the HuMAb
transgenic mice respond best when initially immunized intraperitoneally (IP)
with
antigen in complete Freund's adjuvant, followed by every other week IP
immunizations
(up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune
response
can be monitored over the course of the immunization protocol with plasma
samples
being obtained by retroorbital bleeds. The plasma can be screened by ELISA (as
described below), and mice with sufficient titers of anti-S. aureus human
immunoglobulin can be used for fusions. Mice can be boosted intravenously with
antigen 3 days before sacrifice and removal of the spleen. It is expected that
2-3 fusions
for both the high capsule and the low-capsule immunizations may need to be
performed.
Six mice will be immunized for each antigen. For example, a total of twelve
HuMAb
mice of the HC07 and HC012 strains can be immunized.
Generation of Hybridomas Producing Monoclonal Antibodies to S. aureus
The mouse splenocytes can be isolated and fused with PEG to a mouse myeloma
cell line based upon standard protocols (21, 30). The resulting hybridomas are
then
screened for the production of antigen-specific antibodies. For example,
single cell
suspensions of splenic lymphocytes from immunized mice are fused to one-sixth
the
number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with
50% PEG. Cells are plated at approximately 2 x 1 OS in flat bottom microtiter
plate,
followed by a two week incubation in selective medium containing 20% fetal
Clone
Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM
L~glutamine, 1 mM sodium pyruvate, SmM HEPES, 0.055 mM 2-mercaptoethanol, 50
units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1X HAT
(Sigma;
the HAT is added 24 hours after the fusion). After two weeks, cells are
cultured in
medium in which the HAT is replaced with HT. Individual wells are then
screened by
ELISA for human anti- S aureus monoclonal IgM and IgG antibodies. Once
extensive
hybridoma growth occurs, medium is observed usually after 10-14 days. The
antibody
secreting hybridomas are replated, screened again, and if still positive for
human IgG,
anti-S. aureus monoclonal antibodies, can be subcloned at least twice by
limiting
dilution. The stable subclones are then cultured in vitro to generate small
amounts of
antibody in tissue culture medium for characterization.
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Characterization of Binding of Human Monoclonal Antibodies to S. aureus
To characterize binding of human monoclonal anti- S. aureus antibodies of the
invention, sera from immunized mice can be tested, for example, by ELISA.
Briefly, an
S. aureus immunizing strain can be grown overnight in Columbia broth medium or
on
Columbia agar plates. Bacteria can be recovered from plates by gently
resuspending the
cells in sterile saline. The bacteria from broth or plates can be washed by
centrifugation
and diluted to an OD59o of 0.2. Bacterial suspensions (50 ~l) can be added to
96-well
flat bottom plates, and plates are allowed to dry in fume hood. The bacteria
can be fixed
to the plate with 1 % glutaraldehyde for 5 minutes. The plates are blocked
with 20%
mouse serum (which blocks IgG binding to protein A). These plates can be
stored at -80
°C until needed. Plates can then be washed with PBS-tween buffer
(Dulbeccos PBS, .OS
Tween 20, 1 mM EDTA, .25% BSA, .OS % NaN3), and dilutions of plasma from S.
aureus-immunized mice and irrelevant antigen-immunized mice are added, and
incubated at 37 °C for 1 hr. Plates are washed and then reacted with
anti-human IgG Fc
specific alkaline phosphatase and incubated at 37 °C for 1 hour. Plates
can be washed
again, developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650.
Preferably, mice which develop the highest titers will be used for fusions.
To screen hybridomas using an ELISA assay, microtiter plates can be prepared
as described above using several strains of S. aureus in addition to strain
used for
immunizations. Exemplary S. aureus strains that can be used include the
IBERIAN
(BK2058 and BK2709), EMRSA, ONTARIO, and BRAZILIAN isolates and/or purified
antigens, e.g., capsular polysaccharides 5 and 8, fibronectin binding protein
penicillin
binding protein and protein A, which have been demonstrated to spread rapidly
in
hospital settings. Hybridomas that show positive reactivity with the
immunizing strain
of S. aureus (by, e.g., ELISA assays as described above) can be tested for
cross-
reactivity on plates coated with the other strains. Irrelevant human IgG will
be used as a
negative control. Hybridomas that bind with high avidity to most S. aureus
strains will
be subcloned and further characterized. One clone from each hybridoma, which
retains
the reactivity of the parent cells (by ELISA), can be chosen for making a S-10
vial cell
bank stored at -140 °C, and for antibody purification.
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To purify human anti-S. aureus antibodies, selected hybridomas can be grown in
two-liter spinner-flasks for monoclonal antibody purification. Supernatants
can be
filtered and concentrated before affinity chromatography with protein A-
sepharose
(Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis
and
high performance liquid chromatography to ensure purity. The buffer solution
can be
exchanged into PBS, and the concentration can be determined by OD28o using
1.43
extinction coefficient. The monoclonal antibodies can be aliquoted and stored
at -80 °C.
To determine if the selected human anti-S. aureus monoclonal antibodies bind
to
unique epitopes, each antibody can be biotinylated using commercially
available
reagents (Pierce, Rockford, IL). Competition studies using unlabeled
monoclonal
antibodies and biotinylated monoclonal antibodies can be performed using S.
aureus
coated-ELISA plates as described above. Biotinylated mAb binding can be
detected
with a strep-avidin-alkaline phosphatase probe.
To determine the isotype of purified antibodies, isotype ELISAs can be
performed. Wells of microtiter plates can be coated with 10 ~,g/ml of anti-
human Ig
overnight at 4°C. After blocking with 5% BSA, the plates are reacted
with 10 pg/ml of
monoclonal antibodies or purified isotype controls, at ambient temperature for
two
hours. The wells can then be reacted with either human IgGI or human IgM-
specific
alkaline phosphatase-conjugated probes. Plates are developed and analyzed as
described
above.
In order to demonstrate binding of monoclonal antibodies to live S. aureus,
flow
cytometry (e.g., as described by Poutrel et at. (31)) can be used. Briefly,
bacteria
(grown as above, at 10' cells/ml) are mixed with various concentrations of
monoclonal
antibodies in PBS containing 0.1% Tween 80 and 20% mouse serum, and incubated
at
37°C for 1 hour. After washing, the bacteria are reacted with
Fluorescein-labeled anti-
human IgG antibody under the same conditions as the primary antibody staining.
The
samples can be analyzed by FACScan instrument using light and side scatter
properties
to gate on single bacteria. Short sonication steps (which have been shown not
to
significantly affect cell viability) may be included to reduce clumping of
bacteria. An
alternative assay using fluorescence microscopy may be used (in addition to or
instead
of) the flow cytometry assay. Bacteria can be stained exactly as described
above and
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examined by fluorescence microscopy. This method allows visualization of
individual
cells, but may have diminished sensitivity depending on the density of the
antigen.
Anti-S. aureus human IgGs can be further tested for reactivity with specific
S.
aureus antigens by Western blotting. Briefly, cell wall extracts from selected
S. aureus
isolates can be prepared and subjected to sodium dodecyl sulfate
polyacrylamide gel
electrophoresis. After electrophoresis, the separated antigens will be
transferred to
nitrocellulose membranes, blocked with 20% mouse serum, and probed with the
monoclonal antibodies to be tested. Human IgG binding can be detected using
anti-
human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets
(Sigma Chem. Co., St. Louis, MO). The antigens that are specifically bound by
the
monoclonal antibodies tested can be identified by direct sequencing or through
the use
of mutants known to lack or produce very low amounts of certain cell wall
components.
Phagocytic and Cell Killing Activities of Human Anti-S. aureus Antibodies
In addition to demonstrating the ability of selected monoclonal antibodies to
bind
specifically to S. aureus, the ability of these antibodies to mediate
phagocytosis and
killing of S. aureus in vitro (i.e., their therapeutic utility) can also be
determined. The
testing of monoclonal antibody activity in vitro will provide an initial
screening prior to
testing in vivo models. Briefly, polymorphonuclear cells (PMN) from healthy
donors
can be purified by Ficoll Hypaque density centrifugation, followed by lysis of
contaminating erythrocytes. Washed PMNs, can be suspended in RPMI supplemented
with 10% heat-inactivated fetal calf serum and mixed with S. aureus, grown as
described above, at various ratios of PMN to bacteria (PMN:bacteria). Purified
human
anti-S. aureus IgGs can then be added at various concentrations. Irrelevant
human IgG
can be used as negative control. Assays can be carried out for 0-120 minutes
at 37°C.
Samples can be diluted in water and then plated on tryptic soy agar plates for
overnight
incubation before determining colony counts. anti-S. aureus monoclonal can
also be
tested in combinations with each other to determine whether phagocytosis is
enhanced
with multiple monoclonal antibodies.
Human monoclonal antibodies which show, e.g., high affinity binding and cross-
reactivity with one or more of the S. aureus clinical isolates, can be tested
in an in vivo
challenge model in mice to determine the efficacy against S. aureus infection.
These
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antibodies can be selected, for example, based on the following criteria which
are not
intended to be exclusive:
1. binding to live S. aureus cells grown on agar or in broth;
2. high affinity of binding to S. aureus;
3. cross-reactivity with a variety of S. aureus clinical isolates by ELISA
and/or
flow cytometry;
4. binding to a unique epitope on S. aureus (to eliminate the possibility that
monoclonal antibodies with complimentary activities when used in
combination would compete for binding to the same epitope);
5. opsonization of S. aureus;
4. mediation of growth inhibition, phagocytosis and/or killing of S. aureus in
the
presence of human effector cells.
Preferred monoclonal antibodies meet one or more, and preferably all, of these
criteria.
In a particular embodiment, the human monoclonal antibodies of the present
invention are used in combination, e.g., as a pharmaceutical composition
comprising
two or more anti-S. aureus monoclonal antibodies, or fragments thereof. For
example.
human anti-S. aureus monoclonal antibodies having different, but complementary
activities can be combined in a single therapy to achieve a desired
therapeutic or
diagnostic effect. In one embodiment, this is achieved by combining a
monoclonal
antibody having limited cross-reactivity, but mediating efficient
phagocytosis, with one
or more other human monoclonal antibodies exhibiting broader cross-reactivity,
or
which exhibit effective inhibition of S. aureus growth and/or colonization.
II. Production of Trans~enic Nonhuman Animals Which Generate Human Monoclonal
Anti-S. aureus Antibodies
In yet another aspect, the invention provides transgenic non-human animals,
e.g.,
a transgenic mice, which are capable of expressing the aforesaid human
monoclonal
antibodies that specifically bind to S. aureus or an S. aureus-antigen. In a
preferred
embodiment, the transgenic non-human animals, e.g., the transgenic mice, have
a
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genome comprising a human heavy chain transgene and a light chain transgene.
In one
embodiment, the transgenic non-human animals, e.g., the transgenic mice, have
been
immunized with whole S. aureus or a purified or enriched preparation of an S.
aureus-
antigen. Preferably, the transgenic non-human animals, e.g., the transgenic
mice, are
capable of producing multiple isotypes of human monoclonal antibodies to S.
aureus or
an S aureus antigen (e.g., IgG, IgA and/or IgE) by undergoing V-D-J
recombination
and isotype switching. Isotype switching may occur by, e.g., classical or non-
classical
isotype switching.
The design of a transgenic non-human animal that responds to foreign antigen
stimulation with a heterologous antibody repertoire, requires that the
heterologous
immunoglobulin transgenes contain within the transgenic animal function
correctly
throughout the pathway of B-cell development. In a preferred embodiment,
correct
function of a heterologous heavy chain transgene includes isotype switching.
Accordingly, the transgenes of the invention are constructed so as to produce
isotype
switching and one or more of the following: ( 1 ) high level and cell-type
specific
expression, (2) functional gene rearrangement, (3) activation of and response
to allelic
exclusion, (4) expression of a sufficient primary repertoire, (5) signal
transduction, (6)
somatic hypermutation, and (7) domination of the transgene antibody locus
during the
immune response.
Not all of the foregoing criteria need be met. For example, in those
embodiments wherein the endogenous immunoglobulin loci of the transgenic
animal are
functionally disrupted, the transgene need not activate allelic exclusion.
Further, in
those embodiments wherein the transgene comprises a functionally rearranged
heavy
and/or light chain immunoglobulin gene, the second criteria of functional gene
rearrangement is unnecessary, at least for that transgene which is already
rearranged.
For background on molecular immunology, see, Fundamental Immunology, 2nd
edition
(1989), Paul William E., ed. Raven Press, N.Y., which is incorporated herein
by
reference.
In certain embodiments, the transgenic non-human animals used to generate the
human monoclonal antibodies of the invention contain rearranged, unrearranged
or a
combination of rearranged and unrearranged heterologous immunoglobulin heavy
and
light chain transgenes in the germline of the transgenic animal. Each of the
heavy chain
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transgenes comprises at least one CH gene. In addition, the heavy chain
transgene may
contain functional isotype switch sequences, which are capable of supporting
isotype
switching of a heterologous transgene encoding multiple CH genes in the B-
cells of the
transgenic animal. Such switch sequences may be those which occur naturally in
the
germline immunoglobulin locus from the species that serves as the source of
the
transgene CH genes, or such switch sequences may be derived from those which
occur in
the species that is to receive the transgene construct (the transgenic
animal). For
example, a human transgene construct that is used to produce a transgenic
mouse may
produce a higher frequency of isotype switching events if it incorporates
switch
sequences similar to those that occur naturally in the mouse heavy chain
locus, as
presumably the mouse switch sequences are optimized to function with the mouse
switch recombinase enzyme system, whereas the human switch sequences are not.
Switch sequences may be isolated and cloned by conventional cloning methods,
or may
be synthesized de novo from overlapping synthetic oligonucleotides designed on
the
basis of published sequence information relating to immunoglobulin switch
region
sequences (Mills et al., Nucl. Acids Res. 15:7305-7316 (1991); Sideras et al.,
Intl.
Immunol. 1:631-642 (1989), which are incorporated herein by reference).
For each of the foregoing transgenic animals, functionally rearranged
heterologous
heavy and light chain immunoglobulin transgenes are found in a significant
fraction of
the B-cells of the transgenic animal (at least 10 percent).
The transgenes used to generate the transgenic animals of the invention
include a
heavy chain transgene comprising DNA encoding at least one variable gene
segment,
one diversity gene segment, one joining gene segment and at least one constant
region
gene segment. The immunoglobulin light chain transgene comprises DNA encoding
at
least one variable gene segment, one joining gene segment and at least one
constant
region gene segment. The gene segments encoding the light and heavy chain gene
segments are heterologous to the transgenic non-human animal in that they are
derived
from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene
segments from a species not consisting of the transgenic non-human animal. In
one
aspect of the invention, the transgene is constructed such that the individual
gene
segments are unrearranged, i.e., not rearranged so as to encode a functional
immunoglobulin light or heavy chain. Such unrearranged transgenes support
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recombination of the V, D, and J gene segments (functional rearrangement) and
preferably support incorporation of all or a portion of a D region gene
segment in the
resultant rearranged immunoglobulin heavy chain within the transgenic non-
human
animal when exposed to an S. aureus antigen.
In an alternate embodiment, the transgenes comprise an unrearranged "mini-
locus". Such transgenes typically comprise a substantial portion of the C, D,
and J
segments as well as a subset of the V gene segments. In such transgene
constructs, the
various regulatory sequences, e.g. promoters, enhancers, class switch regions,
splice-
donor and splice-acceptor sequences for RNA processing, recombination signals
and the
like, comprise corresponding sequences derived from the heterologous DNA. Such
regulatory sequences may be incorporated into the transgene from the same or a
related
species of the non-human animal used in the invention. For example, human
immunoglobulin gene segments may be combined in a transgene with a rodent
immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively,
synthetic regulatory sequences may be incorporated into the transgene, wherein
such
synthetic regulatory sequences are not homologous to a functional DNA sequence
that is
known to occur naturally in the genomes of mammals. Synthetic regulatory
sequences
are designed according to consensus rules, such as, for example, those
specifying the
permissible sequences of a splice-acceptor site or a promoter/enhancer motif.
For
example, a minilocus comprises a portion of the genomic immunoglobulin locus
having
at least one internal (i.e., not at a terminus of the portion) deletion of a
non-essential
DNA portion (e.g., intervening sequence; intron or portion thereof) as
compared to the
naturally-occurring germline Ig locus.
In a preferred embodiment of the invention, the transgenic animal used to
generate human antibodies to S. aureus contains at least one, typically 2-10,
and
sometimes 25-50 or more copies of the transgene described in Example 12 of WO
98/24884 (e.g., pHCl or pHC2) bred with an animal containing a single copy of
a light
chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the
offspring
bred with the JH deleted animal described in Example 10 of WO 98/24884, the
contents
of which are hereby expressly incorporated by reference. Animals are bred to
homozygosity for each of these three traits. Such animals have the following
genotype:
a single copy (per haploid set of chromosomes) of a human heavy chain
unrearranged
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mini-locus (described in Example 12 of WO 98/24884), a single copy (per
haploid set of
chromosomes) of a rearranged human K light chain construct (described in
Example 14
of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus
that
removes all of the functional JH segments (described in Example 10 of WO
98/24884).
Such animals are bred with mice that are homozygous for the deletion of the JH
segments (Examples 10 of WO 98/24884) to produce offspring that are homozygous
for
the JH deletion and hemizygous for the human heavy and light chain constructs.
The
resultant animals are injected with antigens and used for production of human
monoclonal antibodies against these antigens.
B cells isolated from such an animal are monospecific with regard to the human
heavy and light chains because they contain only a single copy of each gene.
Furthermore, they will be monospecific with regards to human or mouse heavy
chains
because both endogenous mouse heavy chain gene copies are nonfunctional by
virtue of
the deletion spanning the JH region introduced as described in Example 9 and
12 of WO
98/24884. Furthermore, a substantial fraction of the B cells will be
monospecific with
regards to the human or mouse light chains because expression of the single
copy of the
rearranged human K light chain gene will allelically and isotypically exclude
the
rearrangement of the endogenous mouse K and lambda chain genes in a
significant
fraction of B-cells.
The transgenic mouse of the preferred embodiment will exhibit immunoglobulin
production with a significant repertoire, ideally substantially similar to
that of a native
mouse. Thus, for example, in embodiments where the endogenous Ig genes have
been
inactivated, the total immunoglobulin levels will range from about 0.1 to 10
mg/ml of
serum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mglml. When a
transgene
capable of effecting a switch to IgG from IgM has been introduced into the
transgenic
mouse, the adult mouse ratio of serum IgG to IgM is preferably about 10:1. The
IgG to
IgM ratio will be much lower in the immature mouse. In general, greater than
about
I 0%, preferably 40 to 80% of the spleen and lymph node B cells express
exclusively
human IgG protein.
The repertoire will ideally approximate that shown in a non-transgenic mouse,
usually at least about 10% as high, preferably 25 to 50% or more. Generally,
at least
about a thousand different immunoglobulins (ideally IgG), preferably 104 to
106 or
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more, will be produced, depending primarily on the number of different V, J
and D
regions introduced into the mouse genome. These immunoglobulins will typically
recognize about one-half or more of highly antigenic proteins, e.g.,
staphylococcus
protein A. Some of the immunoglobulins will exhibit an affinity for
preselected
antigens of at least about 107M-1, preferably l O8M-1 to 109M-1 or greater.
In some embodiments, it may be preferable to generate mice with predetermined
repertoires to limit the selection of V genes represented in the antibody
response to a
predetermined antigen type. A heavy chain transgene having a predetermined
repertoire
may comprise, for example, human VH genes which are preferentially used in
antibody
responses to the predetermined antigen type in humans. Alternatively, some VH
genes
may be excluded from a defined repertoire for various reasons (e.g., have a
low
likelihood of encoding high affinity V regions for the predetermined antigen;
have a low
propensity to undergo somatic mutation and affinity sharpening; or are
immunogenic to
certain humans). Thus, prior to rearrangement of a transgene containing
various heavy
or light chain gene segments, such gene segments may be readily identified,
e.g. by
hybridization or DNA sequencing, as being from a species of organism other
than the
transgenic animal.
The transgenic mice of the present invention can be immunized with whole S.
aureus or an S. aureus antigen as described in Section I, supra. The mice will
produce B
cells which undergo class-switching via intratransgene switch recombination
(cis-
switching) and express immunoglobulins reactive with whole S. aureus or an S.
aureus
antigen. The immunoglobulins can be human sequence antibodies, wherein the
heavy
and light chain polypeptides are encoded by human transgene sequences, which
may
include sequences derived by somatic mutation and V region recombinatorial
joints, as
well as germline-encoded sequences; these human sequence immunoglobulins can
be
referred to as being substantially identical to a polypeptide sequence encoded
by a
human VL or VH gene segment and a human JL or JL segment, even though other
non-
germline sequences may be present as a result of somatic mutation and
differential V-J
and V-D-J recombination joints. With respect to such human sequence
antibodies, the
variable regions of each chain are typically at least 80 percent encoded by
human
germline V, J, and, in the case of heavy chains, D, gene segments; frequently
at least 85
percent of the variable regions are encoded by human germline sequences
present on the
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transgene; often 90 or 95 percent or more of the variable region sequences are
encoded
by human germline sequences present on the transgene. However, since non-
germline
sequences are introduced by somatic mutation and VJ and VDJ joining, the human
sequence antibodies will frequently have some variable region sequences (and
less
frequently constant region sequences) which are not encoded by human V, D, or
J gene
segments as found in the human transgene(s) in the germline of the mice.
Typically,
such non-germline sequences (or individual nucleotide positions) will cluster
in or near
CDRs, or in regions where somatic mutations are known to cluster.
The human sequence antibodies which bind to the predetermined antigen can
result from isotype switching, such that human antibodies comprising a human
sequence
y chain (such as y1, y2a, y2B, or y3) and a human sequence light chain (such
as K) are
produced. Such isotype-switched human sequence antibodies often contain one or
more
somatic mutation(s), typically in the variable region and often in or within
about 10
residues of a CDR) as a result of affinity maturation and selection of B cells
by antigen,
particularly subsequent to secondary (or subsequent) antigen challenge. These
high
affinity human sequence antibodies may have binding affinities of at least 1 x
109 M-~,
typically at least 5 x 109 M-~, frequently more than 1 x 101° M-~, and
sometimes 5 x l0io
M-~ to 1 x 10" M-~ or greater.
Another aspect of the invention pertains to the B cells from such mice which
can
be used to generate hybridomas expressing monoclonal high affinity (greater
than 2 x
109 M-~) human sequence antibodies against whole S. aureus or an S. aureus
antigen.
These hybridomas can be used to generate a composition comprising an
immunoglobulin having an affinity constant (Ka) of at least 2 x 109 M-~ for
binding
whole S. aureus or an S. aureus antigen, wherein said immunoglobulin
comprises:
a human sequence light chain composed of ( 1 ) a light chain variable region
having a polypeptide sequence which is substantially identical to a
polypeptide sequence
encoded by a human VL gene segment and a human J~ segment, and (2) a light
chain
constant region having a polypeptide sequence which is substantially identical
to a
polypeptide sequence encoded by a human CL gene segment; and
a human sequence heavy chain composed of a (1) a heavy chain variable region
having a polypeptide sequence which is substantially identical to a
polypeptide sequence
encoded by a human VH gene segment, optionally a D region, and a human JH
segment,
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and (2) a constant region having a polypeptide sequence which is substantially
identical
to a polypeptide sequence encoded by a human CH gene segment.
The development of high affinity human sequence antibodies against whole S.
aureus or an S. aureus antigen is facilitated by a method for expanding the
repertoire of
human variable region gene segments in a transgenic mouse having a genome
comprising an integrated human immunoglobulin transgene, said method
comprising
introducing into the genome a V gene transgene comprising V region gene
segments
which are not present in said integrated human immunoglobulin transgene.
Often, the V
region transgene is a yeast artificial chromosome comprising a portion of a
human V,-~ or
VL (VK) gene segment array, as may naturally occur in a human genome or as may
be
spliced together separately by recombinant methods, which may include out-of
order or
omitted V gene segments. Often at least five or more functional V gene
segments are
contained on the YAC. In this variation, it is possible to make a transgenic
mouse
produced by the V repertoire expansion method, wherein the mouse expresses an
immunoglobulin chain comprising a variable region sequence encoded by a V
region
gene segment present on the V region transgene and a C region encoded on the
human
Ig transgene. By means of the V repertoire expansion method, transgenic mice
having
at least 5 distinct V genes can be generated; as can mice containing at least
about 24 V
genes or more. Some V gene segments may be non-functional (e.g., pseudogenes
and
the like); these segments may be retained or may be selectively deleted by
recombinant
methods available to the skilled artisan, if desired.
Once the mouse germline has been engineered to contain a functional YAC
having an expanded V segment repertoire, substantially not present in the
human Ig
transgene containing the J and C gene segments, the trait can be propagated
and bred
into other genetic backgrounds, including backgrounds where the functional YAC
having an expanded V segment repertoire is bred into a mouse germline having a
different human Ig transgene. Multiple functional YACs having an expanded V
segment repertoire may be bred into a germline to work with a human Ig
transgene (or
multiple human Ig transgenes). Although referred to herein as YAC transgenes,
such
transgenes when integrated into the genome may substantially lack yeast
sequences,
such as sequences required for autonomous replication in yeast; such sequences
may
optionally be removed by genetic engineering (e.g., restriction digestion and
pulsed-field
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gel electrophoresis or other suitable method) after replication in yeast in no
longer
necessary (i.e., prior to introduction into a mouse ES cell or mouse
prozygote). Methods
of propagating the trait of human sequence immunoglobulin expression, include
breeding a transgenic mouse having the human Ig transgene(s), and optionally
also
having a functional YAC having an expanded V segment repertoire. Both VH and
VL
gene segments may be present on the YAC. The transgenic mouse may be bred into
any
background desired by the practitioner, including backgrounds harboring other
human
transgenes, including human Ig transgenes and/or transgenes encoding other
human
lymphocyte proteins. The invention also provides a high affinity human
sequence
immunoglobulin produced by a transgenic mouse having an expanded V region
repertoire YAC transgene. Although the foregoing describes a preferred
embodiment of
the transgenic animal of the invention, other embodiments are contemplated
which have
been classified in four categories:
I. Transgenic animals containing an unrearranged heavy and rearranged light
immunoglobulin transgene;
II. Transgenic animals containing an unrearranged heavy and unrearranged light
immunoglobulin transgene;
III. Transgenic animal containing rearranged heavy and an unrearranged light
immunoglobulin transgene; and
IV. Transgenic animals containing rearranged heavy and rearranged light
immunoglobulin transgenes.
Of these categories of transgenic animal, the preferred order of preference is
as
follows II > I > III > IV where the endogenous light chain genes (or at least
the K gene)
have been knocked out by homologous recombination (or other method) and I > II
> III
>IV where the endogenous light chain genes have not been knocked out and must
be
dominated by allelic exclusion.
III. Production of Bispecific/ Multispecific Molecules Which Bind to S. aureus
In yet another embodiment of the invention, human monoclonal antibodies to S.
aureus or an S. aureus-antigen, or antigen-binding portions thereof, of the
invention are
derivatized or linked to another functional molecule, e.g., another peptide or
protein
(e.g., an Fab' fragment). For example, an antibody or antigen-binding portion
of the
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invention can be functionally linked (e.g., by chemical coupling, genetic
fusion,
noncovalent association or otherwise) to one or more other binding molecules,
such as
another antibody (e.g., a bispecific or a multispecific antibody), antibody
fragment,
peptide, or binding mimetic.
Accordingly, in another aspect, the present invention features bispecific and
multispecific molecules comprising at least one first binding specificity for
S. aureus or
an S. aureus-antigen and a second binding specificity an Fc receptor, e.g.,
human FcyRI
or a human Fca receptor. These bispecific and multispecific molecules are
capable of
binding both to FcyRl-or FcaR-expressing effector cells (e.g., monocytes,
macrophages
or polymorphonuclear cells (PMNs)), and to target cells, including S. aureus
or an S.
aureus antigen. When binding in this manner, the bispecific and multispecific
molecules trigger Fc receptor-mediated effector cell activities, such as
phagocytosis of
an S. aureus cell, antibody dependent cellular cytoxicity (ADCC), cytokine
release, or
generation of superoxide anion.
Multispecific molecules of the invention can further include a third binding
specificity, in addition to an anti-Fc binding specificity and an anti-target
cell antigen
binding specificity, such as S. aureus or an S. aureus antigen. In one
embodiment, the
third binding specificity is an anti-enhancement factor (EF) portion, e.g., a
molecule
which binds to a surface protein involved in cytotoxic activity and thereby
increases the
immune response against the target cell. The "anti-enhancement factor portion"
can be
an antibody, functional antibody fragment or a ligand that binds to a given
molecule,
e.g., an antigen or a receptor, and thereby results in an enhancement of the
effect of the
binding determinants for the Fc receptor or target cell antigen. The "anti-
enhancement
factor portion" can bind an Fc receptor or a target cell antigen.
Alternatively, the anti-
enhancement factor portion can bind to an entity that is different from the
entity to
which the first and second binding specificities bind. For example, the anti-
enhancement factor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3,
CDB, CD28,
CD4, CD40, ICAM-1 or other immune cell that results in an increased immune
response
against the target cell).
In one embodiment, the bispecific and multispecific molecules of the invention
comprise as a binding specificity at least one antibody, or an antibody
fragment thereof,
including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody
may also be
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a light chain or heavy chain dimer, or any minimal fragment thereof such as a
Fv or a
single chain construct as described in Ladner et al. U.S. Patent No.
4,946,778, issued
August 7, 1990, the contents of which is expressly incorporated by reference.
In one embodiment bispecific and multispecific molecules of the invention
comprise a binding specificity for an FcyR or an FcaR present on the surface
of an
effector cell, and a second binding specificity for a target cell antigen,
e.g., an S. aureus
or an S. aureus antigen.
In one embodiment, the binding specificity for an Fc receptor is provided by a
human monoclonal antibody, the binding of which is not blocked by human
immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any
of the
eight y-chain genes located on chromosome 1. These genes encode a total of
twelve
transmembrane or soluble receptor isoforms which are grouped into three Fcy
receptor
classes: FcyRI (CD64), FcyRII(CD32), and FcyRIII (CD16). In one preferred
embodiment, the Fcy receptor a human high affinity FcyRI. The human FcyRI is a
72
kDa molecule, which shows high affinity for monomeric IgG (108 - 109 M-').
The production and characterization of these preferred monoclonal antibodies
are
described by Fanger et al. in PCT application WO 88/00052 and in U.S. Patent
No.
4,954,617, the teachings of which are fully incorporated by reference herein.
These
antibodies bind to an epitope of FcyRI, FcyRII or FcyRIII at a site which is
distinct from
the Fcy binding site of the receptor and, thus, their binding is not blocked
substantially
by physiological levels of IgG. Specific anti-FcyRI antibodies useful in this
invention
are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb
32 is available from the American Type Culture Collection, ATCC Accession No.
HB9469. Anti-FcyRI mAb 22, F(ab')2 fragments of mAb 22, and can be obtained
from
Medarex, Inc. (Annandale, N.J.). In other embodiments, the anti-Fcy receptor
antibody
is a humanized form of monoclonal antibody 22 (H22). The production and
characterization of the H22 antibody is described in Graziano, R.F. et al.
(1995) J.
Immunol 155 (10): 4996-5002 and PCT/US93/10384. The H22 antibody producing
cell
line was deposited at the American Type Culture Collection on November 4, 1992
under
the designation HA022CL1 and has the accession no. CRL 11177.
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In still other preferred embodiments, the binding specificity for an Fc
receptor is
provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha
receptor
(FcaR (CD89)). Preferably, the antibody binds to a human IgA receptor at a
site that is
not blocked by endogenous IgA. The term "IgA receptor" is intended to include
the
gene product of one a-gene (FcaRI) located on chromosome 19. This gene is
known to
encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa.
FcaRI
(CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and
neutrophilic granulocytes, but not on non-effector cell populations. Fca,RI
has medium
affinity (~ 5 X 10~ M-~) for both IgAl and IgA2, which is increased upon
exposure to
cytokines such as G-CSF or GM-CSF (Morton, H.C. et al. (1996) Critical Reviews
in
Immunology 16:423-440). Four FcaRI-specific monoclonal antibodies, identified
as
A3, A59, A62 and A77, which bind FcocRI outside the IgA ligand binding domain,
have
been described (Monteiro, R.C. et al., 1992, J. Immunol. 148:1764).
FcaRI and FcyRI are preferred trigger receptors for use in the invention
because
they are (1) expressed primarily on immune effector cells, e.g., monocytes,
PMNs,
macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-
100,000 per
cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4)
mediate
enhanced antigen presentation of antigens, including self antigens, targeted
to them.
In other embodiments, bispecific and multispecific molecules of the invention
further comprise a binding specificity which recognizes, e.g., binds to, a
target cell
antigen, e.g., S. aureus antigen. In a preferred embodiment, the binding
specificity is
provided by a human monoclonal antibody of the present invention.
An "effector cell specific antibody" as used herein refers to an antibody or
functional antibody fragment that binds the Fc receptor of effector cells.
Preferred
antibodies for use in the subject invention bind the Fc receptor of effector
cells at a site
which is not bound by endogenous immunoglobulin.
As used herein, the term "effector cell" refers to an immune cell which is
involved in the effector phase of an immune response, as opposed to the
cognitive and
activation phases of an immune response. Exemplary immune cells include a cell
of a
myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells
including
cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages,
monocytes,
eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells,
and
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basophils. Effector cells express specific Fc receptors and carry out specific
immune
functions. In preferred embodiments, an effector cell is capable of inducing
antibody-
dependent cellular toxicity (ADCC), e.g., a neutrophil capable of inducing
ADCC. For
example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes
which
express FcaR are involved in specific killing of target cells and presenting
antigens to
other components of the immune system, or binding to cells that present
antigens. In
other embodiments, an effector cell can phagocytose a target antigen, target
cell, or
microorganism. The expression of a particular FcR on an effector cell can be
regulated
by humoral factors such as cytokines. For example, expression of FcyRl has
been found
to be up-regulated by interferon gamma (IFN-y). This enhanced expression
increases
the cytotoxic activity of FcyRI-bearing cells against targets. An effector
cell can
phagocytose or lyse a target antigen or a target cell.
"Target cell" shall mean any undesirable cell in a subject (e.g., a human or
animal) that can be targeted by a composition (e.g., a human monoclonal
antibody, a
bispecific or a multispecific molecule) of the invention. In preferred
embodiments, the
target cell is an S. aureus cell. For example, an S. aureus cell can be an
antibiotic -
resistant strains of S. aureus, e.g., penicillin- and methicillin-resistant S.
aureus,
including, for example, IBERIAN, EMRSA, ONTARIO, BRAZILIAN and COL strains
(2,3). MRSA strains are generally resistant to (3-lactam antibiotics by
expression of a
mutated penicillin binding protein (PBP 2a) that has a low binding capacity
for these
antibiotics (4).
In certain embodiments, the antibodies used in the bispecific or multispecific
molecules of the invention can be human, chimeric or humanized antibody to an
Fc
receptor.
Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) can be
produced by recombinant DNA techniques known in the art. For example, a gene
encoding the Fc constant region of a murine (or other species) monoclonal
antibody
molecule is digested with restriction enzymes to remove the region encoding
the murine
Fc, and the equivalent portion of a gene encoding a human Fc constant region
is
substituted. (see Robinson et al., International Patent Publication
PCT/LJS86/02269;
Akira, et al., European Patent Application 184,187; Taniguchi, M., European
Patent
Application 171,496; Morrison et al., European Patent Application 173,494;
Neuberger
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et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No.
4,816,567;
Cabilly et al., European Patent Application 125,023; Better et al. (1988
Science
240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J.
Immunol.
139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987,
Canc. Res.
47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J.
Natl
Cancer Inst. 80:1553-1559).
The chimeric antibody can be further humanized by replacing sequences of the
Fv variable region which are not directly involved in antigen binding with
equivalent
sequences from human Fv variable regions. General reviews of humanized
chimeric
antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207 and by
Oi et
al., 1986, BioTechniques 4:214. Those methods include isolating, manipulating,
and
expressing the nucleic acid sequences that encode all or part of
immunoglobulin Fv
variable regions from at least one of a heavy or light chain. Sources of such
nucleic acid
are well known to those skilled in the art and, for example, may be obtained
from 7E3,
an anti-GPIIbIIIa antibody producing hybridoma. The recombinant DNA encoding
the
chimeric antibody, or fragment thereof, can then be cloned into an appropriate
expression vector. Suitable humanized antibodies can alternatively be produced
by
CDR substitution U.S. Patent 5,225,539; Jones et al. 1986 Nature 321:552-525;
Verhoeyan et al. 1988 Science 239:1534; and Beidler et al. 1988 J. Immunol.
141:4053-
4060.
All of the CDRs of a particular human antibody may be replaced with at least a
portion of a non-human CDR or only some of the CDRs may be replaced with non-
human CDRs. It is only necessary to replace the number of CDRs required for
binding
of the humanized antibody to the Fc receptor.
An antibody can be humanized by any method, which is capable of replacing at
least a portion of a CDR of a human antibody with a CDR derived from a non-
human
antibody. Winter describes a method which may be used to prepare the humanized
antibodies of the present invention (UK Patent Application GB 2188638A, filed
on
March 26, 1987), the contents of which is expressly incorporated by reference.
The
human CDRs may be replaced with non-human CDRs using oligonucleotide site-
directed mutagenesis as described in International Application WO 94/10332
entitled,
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Humanized Antibodies to Fc Receptors for Immunoglobulin G on Human Mononuclear
Phagocytes.
Also within the scope of the invention are chimeric and humanized antibodies
in
which specific amino acids have been substituted, deleted or added. In
particular,
preferred humanized antibodies have amino acid substitutions in the framework
region,
such as to improve binding to the antigen. For example, in a humanized
antibody
having mouse CDRs, amino acids located in the human framework region can be
replaced with the amino acids located at the corresponding positions in the
mouse
antibody. Such substitutions are known to improve binding of humanized
antibodies to
the antigen in some instances. Antibodies in which amino acids have been
added,
deleted, or substituted are referred to herein as modified antibodies or
altered antibodies.
The term modified antibody is also intended to include antibodies, such as
monoclonal antibodies, chimeric antibodies, and humanized antibodies which
have been
modified by, e.g., deleting, adding, or substituting portions of the antibody.
For
example, an antibody can be modified by deleting the constant region and
replacing it
with a constant region meant to increase half life, e.g., serum half life,
stability or
affinity of the antibody. Any modification is within the scope of the
invention so long
as the bispecific and multispecific molecule has at least one antigen binding
region
specific for an FcyR and triggers at least one effector function.
Bispecific and multispecific molecules of the present invention can be made
using chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Natl.
Acad. Sci.
USA 78:5807), "polydoma" techniques (See U.S. Patent 4,474,893, to Reading),
or
recombinant DNA techniques.
In particular, bispecific and multispecific molecules of the present invention
can
be prepared by conjugating the constituent binding specificities, e.g., the
anti-FcR and
anti-S. aureus binding specificities, using methods known in the art and
described in the
examples provided herein. For example, each binding specificity of the
bispecific and
multispecific molecule can be generated separately and then conjugated to one
another.
When the binding specificities are proteins or peptides, a variety of coupling
or cross-
linking agents can be used for covalent conjugation. Examples of cross-linking
agents
include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-
succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-
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maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky
et al.
(1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described by Paulus (Behring Ins. Mitt.
(1985)
No. 78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et
al. (J.
Immunol. (1987) 139: 2367-2375). Preferred conjugating agents are SATA and
sulfo-
SMCC, both available from Pierce Chemical Co. (Rockford, IL).
When the binding specificities are antibodies (e.g., two humanized
antibodies), they can
be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the
two heavy
chains. In a particularly preferred embodiment, the hinge region is modified
to contain
an odd number of sulfhydryl residues, preferably one, prior to conjugation.
Alternatively, both binding specificities can be encoded in the same vector
and
expressed and assembled in the same host cell. This method is particularly
useful where
the bispecific and multispecific molecule is a mAb x mAb, mAb x Fab, Fab x
F(ab')2 or
ligand x Fab fusion protein. A bispecific and multispecific molecule of the
invention,
e.g., a bispecific molecule can be a single chain molecule, such as a single
chain
bispecific antibody, a single chain bispecific molecule comprising one single
chain
antibody and a binding determinant, or a single chain bispecific molecule
comprising
two binding determinants. Bispecific and multispecific molecules can also be
single
chain molecules or may comprise at least two single chain molecules. Methods
for
preparing bi- and multispecific molecules are described for example in U.S.
Patent
Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175;
U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number
5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and
U.S.
Patent Number 5,482,858.
Binding of the bispecific and multispecific molecules to their specific
targets can
be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay
(RIA), or a Western Blot Assay. Each of these assays generally detects the
presence of
protein-antibody complexes of particular interest by employing a labeled
reagent (e.g.,
an antibody) specific for the complex of interest. For example, the FcR-
antibody
complexes can be detected using e.g., an enzyme-linked antibody or antibody
fragment
which recognizes and specifically binds to the antibody-FcR complexes.
Alternatively,
the complexes can be detected using any of a variety of other immunoassays.
For
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example, the antibody can be radioactively labeled and used in a
radioimmunoassay
(RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays,
Seventh
Training Course on Radioligand Assay Techniques, The Endocrine Society, March,
1986, which is incorporated by reference herein). The radioactive isotope can
be
detected by such means as the use of a y counter or a scintillation counter or
by
autoradiography.
IV. Antibody Conjugates/Immunotoxins
In another aspect, the present invention features a human anti-PSMA monoclonal
antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a
cytotoxin,
a drug or a radioisotope. When conjugated to a cytotoxin, these antibody
conjugates are
referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any
agent that is
detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B,
gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),
anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). An
antibody of the
present invention can be conjugated to a radioisotope, e.g., radioactive
iodine, to
generate cytotoxic radiopharmaceuticals for treating a PSMA-related disorder,
such as a
cancer.
The antibody conjugates of the invention can be used to modify a given
biological response, and the drug moiety is not to be construed as limited to
classical
chemical therapeutic agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins may
include, for
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example, an enzymatically active toxin, or active fragment thereof, such as
abrin, ricin
A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor or
interferon-y; or, biological response modifiers such as, for example,
lymphokines,
interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte
macrophage colony stimulating factor ("GM-CSF"), granulocyte colony
stimulating
factor ("G-CSF"), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs
In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al.
(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug
Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
623-53
(Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In
Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results,
And Future
Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer
Therapy", in
Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp.
303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And
Cytotoxic
Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).
V. Pharmaceutical Compositions
In another aspect, the present invention provides a composition, e.g., a
pharmaceutical composition, containing one or a combination of human
monoclonal
antibodies, or antigen-binding portions) thereof, of the present invention,
formulated
together with a pharmaceutically acceptable carrier. In a preferred
embodiment, the
compositions include a combination of multiple (e.g., two or more) isolated
human
antibodies or antigen-binding portions thereof of the invention. Preferably,
each of the
antibodies or antigen-binding portions thereof of the composition binds to a
distinct, pre-
selected epitope of an S. aureus or S. aureus-antigen.
In one embodiment, human anti-S. aureus monoclonal antibodies having
complementary activities are used in combination, e.g., as a pharmaceutical
composition, comprising two or more human anti-S. aureus monoclonal
antibodies. For
example, a human monoclonal antibody having limited cross-reactivity among S.
aureus
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strains, but mediating very efficient phagocytosis can be used in combination
with other
human monoclonal antibodies to S. aureus exhibiting broader specificity.
In another embodiment, the composition comprises one or a combination of
bispecific or multispecific molecules of the invention (e.g., which contains
at least one
S binding specificity for an Fc receptor and at least one binding specificity
for an S.
aureus or an S. aureus antigen).
Pharmaceutical compositions of the invention also can be administered in
combination therapy, i.e., combined with other agents. For example, the
combination
therapy can include a composition of the present invention with at least one
anti-
infectious agent (e.g., an antibiotic), or other conventional therapy.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents, and the like that are physiologically compatible.
Preferably,
the carrier is suitable for intravenous, intramuscular, subcutaneous,
parenteral, spinal or
epidermal administration (e.g., by injection or infusion). Depending on the
route of
administration, the active compound, i.e., antibody, bispecific and
multispecific
molecule, may be coated in a material to protect the compound from the action
of acids
and other natural conditions that may inactivate the compound.
A "pharmaceutically acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any undesired
toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci.
66:1-19).
Examples of such salts include acid addition salts and base addition salts.
Acid addition
salts include those derived from nontoxic inorganic acids, such as
hydrochloric, nitric,
phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as
well as
from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-
substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic
and
aromatic sulfonic acids and the like. Base addition salts include those
derived from
alkaline earth metals, such as sodium, potassium, magnesium, calcium and the
like, as
well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-
methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine,
procaine
and the like.
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A composition of the present invention can be administered by a variety of
methods known in the art. As will be appreciated by the skilled artisan, the
route and/or
mode of administration will vary depending upon the desired results. The
active
compounds can be prepared with carriers that will protect the compound against
rapid
release, such as a controlled release formulation, including implants,
transdermal
patches, and microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for the
preparation of
such formulations are patented or generally known to those skilled in the art.
See, e.g.,
Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed.,
Marcel
Dekker, Inc., New York, 1978.
To administer a compound of the invention by certain routes of administration,
it
may be necessary to coat the compound with, or co-administer the compound
with, a
material to prevent its inactivation. For example, the compound may be
administered to
a subject in an appropriate carrier, for example, liposomes, or a diluent.
Pharmaceutically acceptable diluents include saline and aqueous buffer
solutions.
Liposomes include water-in-oil-in-water CGF emulsions as well as conventional
liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).
Pharmaceutically acceptable carriers include sterile aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable
solutions or dispersion. The use of such media and agents for pharmaceutically
active
substances is known in the art. Except insofar as any conventional media or
agent is
incompatible with the active compound, use thereof in the pharmaceutical
compositions
of the invention is contemplated. Supplementary active compounds can also be
incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the
conditions of manufacture and storage. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure suitable to high
drug
concentration. The carrier can be a solvent or dispersion medium containing,
for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyethylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity can
be maintained, for example, by the use of a coating such as lecithin, by the
maintenance
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of the required particle size in the case of dispersion and by the use of
surfactants. In
many cases, it will be preferable to include isotonic agents, for example,
sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition.
Prolonged absorption of the injectable compositions can be brought about by
including
in the composition an agent that delays absorption, for example, monostearate
salts and
gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound in the required amount in an appropriate solvent with one or a
combination of
ingredients enumerated above, as required, followed by sterilization
microfiltration.
Generally, dispersions are prepared by incorporating the active compound into
a sterile
vehicle that contains a basic dispersion medium and the required other
ingredients from
those enumerated above. In the case of sterile powders for the preparation of
sterile
injectable solutions, the preferred methods of preparation are vacuum drying
and freeze-
drying (lyophilization) that yield a powder of the active ingredient plus any
additional
desired ingredient from a previously sterile-filtered solution thereof.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be administered,
several divided
doses may be administered over time or the dose may be proportionally reduced
or
increased as indicated by the exigencies of the therapeutic situation. It is
especially
advantageous to formulate parenteral compositions in dosage unit form for ease
of
administration and uniformity of dosage. Dosage unit form as used herein
refers to
physically discrete units suited as unitary dosages for the subjects to be
treated; each
unit contains a predetermined quantity of active compound calculated to
produce the
desired therapeutic effect in association with the required pharmaceutical
carrier. The
specification for the dosage unit forms of the invention are dictated by and
directly
dependent on (a) the unique characteristics of the active compound and the
particular
therapeutic effect to be achieved, and (b) the limitations inherent in the art
of
compounding such an active compound for the treatment of sensitivity in
individuals.
Examples of pharmaceutically-acceptable antioxidants include: ( 1 ) water
soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate,
sodium
metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such
as ascorbyl
palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
lecithin,
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propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating
agents, such as
citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric
acid, and the like.
For the therapeutic compositions, formulations of the present invention
include
those suitable for oral, nasal, topical (including buccal and sublingual),
rectal, vaginal
and/or parenteral administration. The formulations may conveniently be
presented in
unit dosage form and may be prepared by any methods known in the art of
pharmacy.
The amount of active ingredient which can be combined with a carrier material
to
produce a single dosage form will vary depending upon the subject being
treated, and
the particular mode of administration. The amount of active ingredient which
can be
combined with a carrier material to produce a single dosage form will
generally be that
amount of the composition which produces a therapeutic effect. Generally, out
of one
hundred per cent, this amount will range from about 0.01 per cent to about
ninety-nine
percent of active ingredient, preferably from about 0.1 per cent to about 70
per cent,
most preferably from about 1 per cent to about 30 per cent.
Formulations of the present invention which are suitable for vaginal
administration also include pessaries, tampons, creams, gels, pastes, foams or
spray
formulations containing such carriers as are known in the art to be
appropriate. Dosage
forms for the topical or transdermal administration of compositions of this
invention
include powders, sprays, ointments, pastes, creams, lotions, gels, solutions,
patches and
inhalants. The active compound may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives, buffers, or
propellants
which may be required.
The phrases "parenteral administration" and "administered parenterally" as
used
herein means modes of administration other than enteral and topical
administration,
usually by injection, and includes, without limitation, intravenous,
intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,
intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular,
subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Examples of suitable aqueous and nonaqueous carriers which may be employed
in the pharmaceutical compositions of the invention include water, ethanol,
polyols
(such as glycerol, propylene glycol, polyethylene glycol, and the like), and
suitable
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mixtures thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as
ethyl oleate. Proper fluidity can be maintained, for example, by the use of
coating
materials, such as lecithin, by the maintenance of the required particle size
in the case of
dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of presence of
microorganisms may be ensured both by sterilization procedures, supra, and by
the
inclusion of various antibacterial and antifungal agents, for example,
paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to
include
isotonic agents, such as sugars, sodium chloride, and the like into the
compositions. In
addition, prolonged absorption of the injectable pharmaceutical form may be
brought
about by the inclusion of agents which delay absorption such as aluminum
monostearate
and gelatin.
When the compounds of the present invention are administered as
pharmaceuticals, to humans and animals, they can be given alone or as a
pharmaceutical
composition containing, for example, 0.01 to 99.5% (more preferably. 0.1 to
90%) of
active ingredient in combination with a pharmaceutically acceptable carrier.
Regardless of the route of administration selected, the compounds of the
present
invention, which may be used in a suitable hydrated form, and/or the
pharmaceutical
compositions of the present invention, are formulated into pharmaceutically
acceptable
dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical
compositions of the present invention may be varied so as to obtain an amount
of the
active ingredient which is effective to achieve the desired therapeutic
response for a
particular patient, composition, and mode of administration, without being
toxic to the
patient. The selected dosage level will depend upon a variety of
pharmacokinetic
factors including the activity of the particular compositions of the present
invention
employed, or the ester, salt or amide thereof, the route of administration,
the time of
administration, the rate of excretion of the particular compound being
employed, the
duration of the treatment, other drugs, compounds and/or materials used in
combination
with the particular compositions employed, the age, sex, weight, condition,
general
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health and prior medical history of the patient being treated, and like
factors well known
in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily
determine
and prescribe the effective amount of the pharmaceutical composition required.
For
example, the physician or veterinarian could start doses of the compounds of
the
invention employed in the pharmaceutical composition at levels lower than that
required
in order to achieve the desired therapeutic effect and gradually increase the
dosage until
the desired effect is achieved. In general, a suitable daily dose of a
compositions of the
invention will be that amount of the compound which is the lowest dose
effective to
produce a therapeutic effect. Such an effective dose will generally depend
upon the
factors described above. It is preferred that administration be intravenous,
intramuscular, intraperitoneal, or subcutaneous, preferably administered
proximal to the
site of the target. If desired, the effective daily dose of a therapeutic
compositions may
be administered as two, three, four, five, six or more sub-doses administered
separately
at appropriate intervals throughout the day, optionally, in unit dosage forms.
While it is
possible for a compound of the present invention to be administered alone, it
is
preferable to administer the compound as a pharmaceutical formulation
(composition).
Therapeutic compositions can be administered with medical devices known in
the art. For example, in a preferred embodiment, a therapeutic composition of
the
invention can be administered with a needleless hypodermic injection device,
such as
the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335,
5,064,413,
4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and
modules
useful in the present invention include: U.S. Patent No. 4,487,603, which
discloses an
implantable micro-infusion pump for dispensing medication at a controlled
rate;
U.S. Patent No. 4.,486,194, which discloses a therapeutic device for
administering
medicants through the skin; U.S. Patent No. 4,447,233, which discloses a
medication
infusion pump for delivering medication at a precise infusion rate; U.S.
Patent
No. 4,447,224, which discloses a variable flow implantable infusion apparatus
for
continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an
osmotic drug
delivery system having mufti-chamber compartments; and U.S. Patent No.
4,475,196,
which discloses an osmotic drug delivery system. These patents are
incorporated herein
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by reference. Many other such implants, delivery systems, and modules are
known to
those skilled in the art.
In certain embodiments, the human monoclonal antibodies of the invention can
be formulated to ensure proper distribution in vivo. For example, the blood-
brain barrier
(BBB) excludes many highly hydrophilic compounds. To ensure that the
therapeutic
compounds of the invention cross the BBB (if desired), they can be formulated,
for
example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S.
Patents
4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more
moieties which are selectively transported into specific cells or organs, thus
enhance
targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol.
29:685).
Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent
5,416,016 to
Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.
153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais
et al.
(1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor
(Briscoe
et al. (1995) Am. J. Physiol. 1233:134), different species of which may
comprise the
formulations of the inventions, as well as components of the invented
molecules; p1 20
(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L.
Laukkanen
( 1994) FEBS Lett. 346:123; J.J. Killion; LJ. Fidler ( 1994) Immunomethods
4:273. In
one embodiment of the invention, the therapeutic compounds of the invention
are
formulated in liposomes; in a more preferred embodiment, the liposomes include
a
targeting moiety. In a most preferred embodiment, the therapeutic compounds in
the
liposornes are delivered by bolus injection to a site proximal to the tumor or
infection.
The composition must be fluid to the extent that easy syringability exists. It
must be
stable under the conditions of manufacture and storage and must be preserved
against
the contaminating action of microorganisms such as bacteria and fungi.
A "therapeutically effective dosage" preferably inhibits tumor growth by at
least
about 20%, more preferably by at least about 40%, even more preferably by at
least
about 60%, and still more preferably by at least about 80% relative to
untreated subjects.
The ability of a compound to inhibit cancer can be evaluated in an animal
model system
predictive of efficacy in human tumors. Alternatively, this property of a
composition
can be evaluated by examining the ability of the compound to inhibit, such
inhibition in
vitro by assays known to the skilled practitioner. A therapeutically effective
amount of
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a therapeutic compound can decrease tumor size, or otherwise ameliorate
symptoms in a
subject. One of ordinary skill in the art would be able to determine such
amounts based
on such factors as the subject's size, the severity of the subject's symptoms,
and the
particular composition or route of administration selected.
The composition must be sterile and fluid to the extent that the composition
is
deliverable by syringe. In addition to water, the carrier can be an isotonic
buffered
saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. Proper
fluidity can be
maintained, for example, by use of coating such as lecithin, by maintenance of
required
particle size in the case of dispersion and by use of surfactants. In many
cases, it is
preferable to include isotonic agents, for example, sugars, polyalcohols such
as manitol
or sorbitol, and sodium chloride in the composition. Long-term absorption of
the
injectable compositions can be brought about by including in the composition
an agent
which delays absorption, for example, aluminum monostearate or gelatin.
When the active compound is suitably protected, as described above, the
compound may be orally administered, for example, with an inert diluent or an
assimilable edible carrier.
VI. Uses and Methods of the Invention
The compositions (e.g., human antibodies and derivatives thereof) of the
present
invention have in vitro and in vivo diagnostic and therapeutic utilities. For
example,
these molecules can be administered to cells in culture, e.g. in vitro or ex
vivo, or in a
subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders.
As used herein,
the term "subject" is intended to include human and non-human animals.
Preferred
human animals include a human patient having an S. aureus-mediated disorder.
For
example, the methods and compositions of the present invention can be used to
treat a
subject at risk of acquiring a nosocomial infection. Examples of particularly
susceptible
classes of subjects include the elderly and immunocompromised hospital
patients. The
term "non-human animals" of the invention includes all vertebrates, e.g.,
mammals and
non-mammals, such as non-human primates, sheep, dog, cow, chickens,
amphibians,
reptiles, etc.
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The compositions (e.g., human antibodies, multispecific and bispecific
molecules) of the invention can be initially tested for binding activity
associated with
therapeutic or diagnostic use in vitro. For example, compositions of the
invention can
be tested using the ELISA and flow cytometric assays described in the Examples
below.
Moreover, the activity of these molecules in triggering at least one effector-
mediated
effector cell activity, including phagocytosis of S. aureus can be assayed.
Protocols for
assaying for effector cell-mediated phagocytosis are described in the Examples
below.
The compositions (e.g., human antibodies, multispecific and bispecific
molecules) of the invention have additional utility in therapy and diagnosis
of S. aureus-
mediated diseases. For example, the human monoclonal antibodies, the
multispecific or
bispecific molecules can be used, for example, to elicit in vivo or in vitro
one or more of
the following biological activities: to opsonize S. aureus; to mediate
phagocytosis or to
inhibit growth of S. aureus in the presence of human effector cells; or to
inhibit S.
aureus growth.
For example, human antibodies and derivatives thereof of the invention can be
used in vivo to treat, prevent or diagnose a variety of S. aureus-mediated
diseases.
Examples of S. aureus-mediated diseases include, for example, invasive and
toxigenic
infectious diseases. Exemplary invasive diseases include: Bacteremia,
osteomyelitis,
septic arthritis, septic thrombophlebitis and acute bacterial endocarditis.
Exemplary
toxigenic diseases include: Staphylococcol food poisoning, scalded skin
syndrome and
toxic shock syndrome. Additional examples of S. aureus-mediated diseases that
can be
treated using the methods and compositions of the invention include infections
of the
upper respiratory tract (e.g., Otis media, bacterial trachetis, acute
epiglottitis,
thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,
infective
endocarditis), gastrointestinal (e.g., secretory diarrhea, splenic abscess ,
retroperitoneal
abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis
, keratitis,
endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney
and urinary
tract (e.g., epididymitis, intrarenal and perinephric abscess , toxic shock
system), skin
(e.g., impetigo, folloculitis, cutaneuos abscesses, cellulitis, wound
infection, bacterial
myositis), bone and joint (e.g., septic arthritis, osteomyelitis).
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Methods of administering the compositions (e.g., human antibodies,
multispecific and bispecific molecules) of the invention are known in the art.
Suitable
dosages of the molecules used will depend on the age and weight of the subject
and the
particular drug used. The molecules can be coupled to radionuclides, such as
131I, 90Y,
lOSRh, etc., as described in Goldenberg, D.M. et al. (1981) Cancer Res. 41:
4354-4360,
and in EP 0365 997. The compositions (e.g., human antibodies, multispecific
and
bispecific molecules) of the invention can also be coupled to anti-infectious
agents.
Target-specific effector cells, e.g., effector cells linked to compositions
(e.g.,
human antibodies, multispecific and bispecific molecules) of the invention can
also be
used as therapeutic agents. Effector cells for targeting can be human
leukocytes such as
macrophages, neutrophils or monocytes. Other cells include and other IgG- or
IgA-
receptor bearing cells. If desired, effector cells can be obtained from the
subject to be
treated. The target-specific effector cells, can be administered as a
suspension of cells in
a physiologically acceptable solution. The number of cells administered can be
in the
1 ~ order of 108-109 but will vary depending on the therapeutic purpose. In
general, the
amount will be sufficient to obtain localization at the target cell, e.g., an
S. aureus cell,
and to effect cell killing by, e.g., phagocytosis. Routes of administration
can also vary.
Therapy with target-specific effector cells can be performed in conjunction
with
other techniques for removal of targeted cells. For example, anti-bacterial
therapy using
the compositions (e.g., human antibodies, multispecific and bispecific
molecules) of the
invention and/or effector cells armed with these compositions can be used in
conjunction with antibiotic therapy. Additionally, combination immunotherapy
may be
used to direct two distinct cytotoxic effector populations toward tumor cell
rejection.
For example, anti-S. aureus antibodies linked to anti-Fc-gammaRI or anti-T3
may be
used in conjunction with IgG- or IgA-receptor specific binding agents.
Bispecific and multispecific molecules of the invention can also be used to
modulate FcyR or FcocR levels on effector cells, such as by capping and
elimination of
receptors on the cell surface. Mixtures of anti-Fc receptors can also be used
for this
purpose.
The compositions (e.g., human antibodies, multispecific and bispecific
molecules) of the invention which have complement binding sites, such as
portions from
IgGI, -2, or -3 or IgM which bind complement can also be used in the presence
of
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complement. In one embodiment, ex vivo treatment of a population of cells
comprising
target cells with a binding agent of the invention and appropriate effector
cells can be
supplemented by the addition of complement or serum containing complement.
Phagocytosis of target cells coated with a binding agent of the invention can
be
improved by binding of complement proteins. In another embodiment target cells
coated with the compositions (e.g., human antibodies, multispecific and
bispecific
molecules) of the invention can also be lysed by complement.
The compositions (e.g., human antibodies, multispecific and bispecific
molecules) of the invention can also be administered together with complement.
Accordingly, within the scope of the invention are compositions comprising
human
antibodies, multispecific or bispecific molecules and serum or complement.
These
compositions are advantageous in that the complement is located in close
proximity to
the human antibodies, multispecific or bispecific molecules. Alternatively,
the human
antibodies, multispecific or bispecific molecules of the invention and the
complement or
serum can be administered separately.
Also within the scope of the invention are kits comprising the compositions
(e.g.,
human antibodies, multispecific and bispecific molecules) of the invention and
instructions for use. The kit can further contain a least one additional
reagent, such as
complement, or one or more additional human antibodies of the invention (e.g.,
a human
antibody having a complementary activity which binds to an epitope in S.
aureus or an
S. aureus antigen distinct from the first human antibody).
In other embodiments, the subj ect can be additionally treated with an agent
that
modulates, e.g., enhances or inhibits, the expression or activity of Fcy or
Fca receptors,
by for example, treating the subject with a cytokine. Preferred cytokines for
administration during treatment with the multispecific molecule include of
granulocyte
colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating
factor
(GM-CSF), interferon-y (IFN-y), and tumor necrosis factor (TNF)
The compositions (e.g., human antibodies, multispecific and bispecific
molecules) of the invention can also be used to target cells expressing FcyR
or S.
aureus-antigens, for example for labeling such cells. For such use, the
binding agent
can be linked to a molecule that can be detected. Thus, the invention provides
methods
for localizing ex vivo or in vitro cells expressing FcyR or S. aureus-
antigens. The
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detectable label can be, e.g., a radioisotope, a fluorescent compound, an
enzyme, or an
enzyme co-factor. In one embodiment, the invention provides methods for
detecting the
presence of S. aureus in a sample, comprising:
contacting the sample, and a control sample, with a human monoclonal antibody,
or an antigen binding portion thereof, which specifically binds to S. aureus
or an
S. aureus-antigen, under conditions that allow for formation of a complex
between the antibody or portion thereof and the S. aureus or an S. aureus-
antigen, and
detecting the formation of a complex,
wherein a difference complex formation between the sample compared to the
control
sample is indicative the presence of S. aureus in the sample.
In still another embodiment, the invention provides a method for detecting the
presence of an Fc-expressing cell in vivo or in vitro. The method comprises
(i)
administering to a subject a composition (e.g., a mufti- or bispecific
molecule) of the
invention or a fragment thereof, conjugated to a detectable marker; (ii)
exposing the
subject to a means for detecting said detectable marker to identify areas
containing Fc-
expressing cells.
The present invention is further illustrated by the following examples, which
should not be construed as further limiting.
FXAMP1,FR
Example 1 Production of Human Monoclonal Antibodies Against S. aureus
Human anti-S. aureus monoclonal antibodies were generated by immunizing two
strains of HC07 HuMAb mice with heat killed S. aureus strain FDA 209. The HC07
HuMAb mice used in the study were generated as described in U.S. Patent Nos.
5,545,806, 5,625,825, and 5,545,807, the entire disclosures of which are
hereby
incorporated by reference.
In particular, HC07 mice were immunized for 12 weeks using heat killed FDA
209 grown in broth and emulsified in CFA. Immunized mice were boosted twice at
two
week intervals with the same bacteria in IFA. At 8 and 10 weeks, serum was
collected
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from these mice. S. aureus-specific titers were detected in the immunized
animals after
the third immunization. Figure 1 shows the optical density (OD (405-650)) for
the
indicated dilution of pooled plasma from S. aureus immunized mice (hatched
bar) and
non-immunized mice (solid bar) as analyzed by ELISA using S. aureus-coated
microtiter plates and detected with an alkaline phosphatase-conjugated anti-
human IgG
probe. When the mice showed a titer against S. aureus 209 as measured by ELISA
assay, they were boosted with an i.v. injection of heat killed S. aureus 209.
Three days
later the mice were sacrificed and spleens were harvested.
To generate hybridomas producing anti-S. aureus antibodies, splenocytes from
mice showing plasma containing anti-S. aureus antibodies were fused with
P3X63-Ag8.653 cells (deposited with the ATCC under designation ATCC CRL 1580
nonsecreting mouse myeloma cells) and PEG. Approximately 800 mixed hybridoma
cultures were generated. Supernatants from these hybridoma cultures were
screened for
production of human IgGI and for binding to S. aureus 209 strain. Figure 2
depicts the
binding of six supernatants from the hybridoma cultures termed 2612, 2H12,
8.1E5,
8.2C 1, 7F 1 and 6D 12 to S. aureus 209 strain as measured by ELISA. Briefly,
bacteria
were dried onto a 96-well plate and fixed with .25% glutaraldehyde.
Supernatants were
added were added and incubated for 1 hour. Human IgG was detected using goat
anti-
human IgG alkaline phosphatase and pNPP. Binding to human IgG of the anti-
FcyRI
(H22 antibody) was used as a control. Significant binding was detected with
the six
hybridoma supernatants shown as compared to the H22 control.
Example 2 Characterization of Human Monoclonal Antibodies Against S.
aureus
The mixed hybridoma supernatants generated in Example 1 were tested for
binding to Methicillin resistant S. aureus (MRSA) strains as follows.
Binding of the supernatants to the MRSA strains BK2058 and BK2709, were
tested using an ELISA assay. These two strains are Iberian MRSA clones
isolated from
a hospital outbreak and they both overexpress a Penicillin binding protein.
BK2058 also
overexpresses a fibronectin binding protein.
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BK2058 and BK2709 strains were fixed to wells of a 96 well plate and the
antibody in the supernatant was detected using the goat anti-human IgG
alkaline
phosphatase described in Example 1. Media alone was used as a control. Figure
3
shows the binding of the mixed hybridoma supernatants 2G 12, 2H 12, 8.1 E5,
8.2C 1, 7F 1
and 6D12 to S. aureus MRSA strain 2058. No significant binding of the
supernatant
from the mixed hybridoma cultures (2G 12, 2H 12, 8.1 E5, 8.2C 1, 7F 1 and 6D
12) to
mixed bacteria (Escherichia coli, Pseudomonas aeroginosa, or Micrococcus
luteus) was
detected, as measured by ELISA (Figure 4).
The specificity of the human anti-S. aureus monoclonal antibodies was
confirmed by comparing the binding of the supernatants from mixed hybridoma
cultures
(2612, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to S. aureus (speckled bar) or E.
coli (solid
bar), as measured by flow cytometry (Figure 5). As shown in Figure 5,
significant
levels of supernatant binding were detected only upon incubation with S.
aureus.
Figure 6 is a bar graph showing the binding of the supernatants from subcloned
hybridoma cultures (2G 12, 2H 12, 8.1 E5, 8.2C 1, 7F 1 and 6D 12) to S. aureus
FDA 209,
as measured be ELISA. As before, S. aureus cells were fixed to wells of a 96-
well plate
and the monoclonal antibody present on the supernatant was detected using anti-
human
IgG (kappa chain) alkaline phosphatase. The second antibody confirmed that the
supernatants contain a human antibody that binds to S. aureus. Binding to
human IgG
of the anti-FcyRI (H22 antibody) was used as a control.
In addition, supernatants from subclones of the hybridoma cultures 6D 12 and
SH10 were assayed by ELISA for binding to S. aureus strain ATCC 27661.
Briefly, S.
aureus bacteria were grown overnight in trypticase soy agar broth, resuspended
in 1
gelatin in PBS, dried onto a 96-well plate at 40°C overnight, and fixed
with 0.1%
glutaraldehyde in PBS. The S. aureus coated plates were blocked with 20% mouse
sera,
0.5% EDTA, 0.25% BSA, 0.5% NaN3, washed, and hybridoma supernatants (or
antibodies (25 ~g/ml)) were added at 100 ~.l/well and incubated for 2 hours at
37°C or
overnight at 4°C. Human antibody was detected by incubation with anti-
human IgG
Fab'2 conjugated to alkaline phosphatase for 1 hour at 37°C, followed
by the addition of
pNPP. The 6D12 and SH10 subcloned hybridoma supernatants demonstrated
significant
binding to S. aureus as compared to the isotype control antibody (Figure 7).
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Selected hybridoma supernatants that showed significant, specific binding to
S.
aureus (e.g., 2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) were then tested for the
ability
to mediate S. aureus phagocytosis (Figures 8A-8C). Briefly, PMNs were
incubated for
30 minutes at 37°C with FITC labeled S. aureus, with and without
supernatant from
mixed hybridoma cultures. Changes in fluorescence were detected using a
FACSscan.
Figures 8A-8C are histograms showing neutrophil-mediated phagocytosis of S.
aureus
upon incubation of polymorphonuclear cells (PMNs) with a mixed hybridoma
supernatant. Figures 8A-8C show the results from one representative
supernatant of 6
supernatants that had previously been shown to contain anti-S. aureus human
IgG.
Figures 8A-8C show FACScan analyses of control PMNs alone (Figure 8A), PMNs
and
S. aureus, with or without supernatant from mixed hybridoma cultures (Figure
8B), and
PMNs and control E coli, with or without supernatant from mixed hybridoma
cultures
(Figure 8C). A shift in the fluorescence was detected when PMNs and S. aureus
were
incubated with the supernatant from the mixed hybridoma cultures, which
indicates that
there was phagocytosis of FITC-labeled S. aureus (Figure 8B). No significant
changes
in bacterial phagocytosis were detected in the following conditions: PMNs
alone
(Figure 8A); upon incubation of PMNs with S. aureus without supernatant from
the
mixed hybridoma cultures (Figure 8B); and upon incubation of PMNs with E. coli
with
supernatant from the mixed hybridoma cultures (Figure 8C).
Selected hybridomas that showed specific binding and phagocytotic activities
(e. g., , 2G 12, 2H 12, 8.1 E5, 8.2C 1, 7F 1 and 6D 12) were also subcloned to
develop
purified cultures that produced human monoclonal antibodies against S. aureus.
The
resulting purified antibodies were again tested for binding to S. aureus FDA
209
(Figures 9A and 9B) and MRSA strain 2058 (Figures 10A and l OB) and for their
ability
to mediate S. aureus phagocytosis as described above.
Figures 9A-9B show binding of purified human monoclonal antibody 6D 12 to S.
aureus FDA 209 compared to IgG controls. In particular, Figure 9A is a bar
graph
depicting the binding of two concentrations (1 and 10 pg/ml) of purified human
monoclonal antibody 6D 12 (open bars) to S. aureus FDA 209, compared to IgG
controls
(speckled bars) as measured using ELISA. The purified 6D12 human monoclonal
antibody was compared with a control human IgG (anti-FcyRI monoclonal antibody
H22) for binding to S aureus FDA 209. Bacteria dried onto microtiter plates
were
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blocked with 20% mouse serum, and then reacted with monoclonal antibody
preparations. Monoclonal antibody binding was detected with an alkaline
phosphatase-
conjugated anti-human IgG Fc specific probe. Figure 9B is a histogram showing
direct
binding of FITC-labeled 6D 12 monoclonal antibody to S. aureus FDA 209
compared to
FITC-labeled humanized anti-EGF receptor antibody control (H425) as detected
by flow
cytometry. Briefly, 25 ~g/ml of FITC-labeled 6D 12 monoclonal antibody and
FITC-
labeled H425 control were incubated with S. aureus FDA 209 and 2.8 mg/ml of
irrelevant human IgG to block protein A. S. aureus cells were fixed with
paraformaldehyde and analyzed on a BD flow cytometer.
Figures l0A-lOB show binding of purified human monoclonal antibody 6D12 to
MRSA strain 2058 compared to IgG controls. In particular, Figure 10A is a
linear graph
depicting the binding of two concentrations (1 and 10 ~g/ml) of purified human
monoclonal antibody 6D12 to MRSA strain 2058, compared to IgG controls. Figure
1 OB is a histogram showing direct binding of FITC-labeled 6D 12 monoclonal
antibodies
to MRSA strain 2058 compared to FITC-labeled humanized anti-EGF receptor
antibody
control (H425) as detected by flow cytometry. Briefly, 25 ~.g/ml of FITC-
labeled 6D12
monoclonal antibody and FITC-labeled H425 control were incubated with MRSA
strain
2058 and 2.8 mg/ml of irrelevant human IgG to block protein A. S. aureus cells
were
fixed with paraformaldehyde and analyzed on a BD flow cytometer.
The survival of S. aureus strain FDA-209 after PMN phagocytosis was then
assessed as follows. Bacteria were grown overnight in trypicase soy agar
broth,
harvested and resuspended in PBS at a concentration of 106 cfu/ml. PMNs were
collected from whole blood from a healthy volunteer. Briefly, 120 ml of whole
blood
was diluted 1:1 with RPMI cell media, layered over Ficoll-paque, and
centrifuged at 500
x g for 30 minutes. The PMN layer was collected and contaminating red blood
cells
were lysed with KC03 lysing solution. PMNs were washed twice, counted , and
adjusted to a concentration of 106 cells/ml. All reagents were prepared in 2%
human
sera which was preabsorbed to live S. aureus. Bacteria (200 g.1) were
opsonized with 10
~l of the 6D12 antibody and an isotype control antibody (100 g.g/ml) for 10
minutes at
room temperature, and then incubated with human PMNs (200 g1) for 90 minutes
at
37°C with constant rotation. The bacteria and PMNs were then microfuged
for 5
minutes and the cell pellet was resuspended in 1 ml of distilled water. Serial
dilutions
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were made in distilled water and 50 ~.1 of each dilution was plated on tryptic
soy agar
plates and surviving S. aureus was counted.
As shown in Figure 11, the human anti-S. aureus antibody 6D12 increased the
PMN mediated killing of S. aureus by 60% as compared to a human isotype
control
antibody against CD30.
rnnr~neinn
The foregoing Examples demonstrate the generation of human monoclonal
antibodies that specifically react with high affinity to at least two strains
of S. aureus
(FDA 209 and MRSA strain 2058). In addition, the human monoclonal anti-S.
aureus
antibodies effectively mediate phagocytic activity against at least two
strains of S.
aureus (FDA 209 and MRSA strain 2058) using PMNs as effector cells. Thus, the
phagocytic activities of these human antibodies against Methicillin-resistant
S. aureus
was established. These results support the conclusion that the fully human
monoclonal
antibodies against S. aureus of the present invention are useful for the
treatment of S.
aureus nosocomial infections.
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Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents of the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
Incorporation by Reference
The entire contents of all references, pending patent applications and issued
patents cited herein are hereby expressly incorporated by reference into the
present
disclosure.
