U.S. patent application number 11/642053 was filed with the patent office on 2008-01-17 for multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria.
This patent application is currently assigned to Biosynexus Incorporated. Invention is credited to Gerald Walter Fischer, Simon J. Foster, John Fitzgerald Kokai-Kun, Richard F. Schuman, Jeffrey R. Stinson.
Application Number | 20080014202 11/642053 |
Document ID | / |
Family ID | 29714989 |
Filed Date | 2008-01-17 |
United States Patent
Application |
20080014202 |
Kind Code |
A1 |
Schuman; Richard F. ; et
al. |
January 17, 2008 |
Multifunctional monoclonal antibodies directed to peptidoglycan of
gram-positive bacteria
Abstract
The present invention encompasses protective monoclonal
antibodies that bind to peptidoglycan of Gram-positive bacteria.
The antibodies also bind to whole bacteria and enhance phagocytosis
and killing of the bacteria in vitro and block nasal colonization
by Gram-positive bacteria in vivo. The invention also provides
human, humanized and chimeric antibodies. The invention also sets
forth the heavy chain and light chain variable regions of an
antibody within the invention.
Inventors: |
Schuman; Richard F.;
(Gaithersburg, MD) ; Kokai-Kun; John Fitzgerald;
(Frederick, MD) ; Foster; Simon J.; (Sheffield,
GB) ; Stinson; Jeffrey R.; (Brookville, MD) ;
Fischer; Gerald Walter; (Bethesda, MD) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP
ONE POST OFFICE SQUARE
BOSTON
MA
02109-2127
US
|
Assignee: |
Biosynexus Incorporated
Gaithersburg
MD
|
Family ID: |
29714989 |
Appl. No.: |
11/642053 |
Filed: |
December 19, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10323903 |
Dec 20, 2002 |
7169903 |
|
|
11642053 |
Dec 19, 2006 |
|
|
|
60343444 |
Dec 21, 2001 |
|
|
|
Current U.S.
Class: |
424/150.1 ;
424/190.1; 424/243.1; 424/244.1; 424/246.1; 435/346; 530/387.3;
530/388.2 |
Current CPC
Class: |
C07K 16/1278 20130101;
C07K 2317/24 20130101; A61K 2039/505 20130101; C07K 16/1275
20130101; A61K 39/00 20130101; C07K 2317/77 20130101; C07K 16/1271
20130101; C07K 2319/30 20130101 |
Class at
Publication: |
424/150.1 ;
424/190.1; 424/243.1; 424/244.1; 424/246.1; 435/346; 530/387.3;
530/388.2 |
International
Class: |
A61K 39/40 20060101
A61K039/40; A61K 39/02 20060101 A61K039/02; A61K 39/07 20060101
A61K039/07; C07K 16/12 20060101 C07K016/12; A61K 39/085 20060101
A61K039/085; A61K 39/09 20060101 A61K039/09 |
Claims
1. A method for treating a patient comprising, administering a
therapeutically effective amount of a composition comprising at
least one MAb that binds to peptidoglycan (PepG) of Gram-positive
bacteria; wherein said administration results in therapeutically
beneficial outcome.
2. The method of claim 1, wherein the therapeutically beneficial
outcome comprises a discernable reduction in the number of
Gram-positive bacteria in the nares of a colonized patient.
3. The method of claim 1, wherein the therapeutically beneficial
outcome comprises a discernable reduction in the likelihood of
future nasal colonization.
4. The method of claim 1, wherein the therapeutically beneficial
outcome comprises a discernable reduction in the number of
Gram-positive bacteria in an infected patient.
5. The method of claim 1, wherein the therapeutically beneficial
outcome comprises a discernable reduction in the likelihood of
future infection.
6. The method of claim 1, wherein the therapeutically beneficial
outcome comprises a discernable reduction in the likelihood of
nosocomial infection.
7. The method of claim 1, wherein the composition comprises at
least one MAb that enhances the opsonophagocytic killing of
Gram-positive bacteria by at least 50%.
8. The method of claim 1, wherein the composition comprises at
least one MAb that binds PepG of Gram-positive bacteria at a level
at least two-fold greater than background in an ELISA.
9. The method of claim 1, wherein the composition comprises a
pharmaceutically acceptable carrier.
10. The method of claim 1, wherein the composition comprises at
least one MAb that binds to lipoteichoic acid (LTA) of
Gram-positive bacteria.
11. The method of claim 1, wherein the composition comprises at
least one MAb that blocks colonization by Gram-positive bacteria
upon instillation into the nares of a patient.
12. The method of claim 1, wherein the composition comprises at
least one MAb that specifically binds PepG of a Gram-positive
bacteria selected from Staphylococcus aureus, Staphylococcus
epidermidis, Streptococcus mutans, Bacillus subtilis, Bacillus
megaterium, Enterococcus faecalis, and Listeria monocytogenes.
13. The method of claim 1, wherein the composition comprises at
least one MAb that specifically binds PepG of Staphylococcus
aureus.
14. The method of claim 1, wherein the composition comprises at
least one MAb that specifically binds PepG of Staphylococcus
epidermidis.
15. The method of claim 1, wherein the composition comprises at
least one MAb that specifically binds PepG of Staphylococcus aureus
and Staphylococcus epidermidis.
16. The method of claim 1, wherein the composition comprises at
least one MAb that specifically binds PepG of Bacillus
subtilis.
17. The method of claim 1, wherein the composition comprises at
least one MAb selected from MAb-11-232.3, MAb-11-248.2,
MAb-11-569.3, MAb-11-232.3 IE9, MAb-99-110FC12 IE4, A130, and
M130.
18. The method of claim 1, wherein the composition comprises at
least one MAb comprising a light chain variable region comprising
the amino acid sequence set forth in SEQ ID NO: 1.
19. The method of claim 1, wherein the composition comprises at
least one MAb comprising a light chain variable region having at
least 80% identity with the amino acid sequence set forth in SEQ ID
NO: 1.
20. The method of claim 1, wherein the composition comprises at
least one MAb comprising a heavy chain variable region comprising
the amino acid sequence set forth in SEQ ID NO: 3.
21. The method of claim 1, wherein the composition comprises at
least one MAb comprising a heavy chain variable region having at
least 80% identity with the amino acid sequence set forth in SEQ ID
NO: 3.
22. The method of claim 1, wherein the composition comprises at
least one MAb selected from chimeric, humanized, and human
MAbs.
23. The method of claim 1, wherein the composition comprises at
least one MAb comprising a modified Fc portion, wherein said
modification reduces nonspecific binding of the MAb via the Fc
portion.
24. The method of claim 1, wherein the composition comprises at
least one MAb selected from a Fab, Fab', F(ab').sub.2, Fv, SFv, and
scFv.
25. The method of claim 1, wherein the composition further
comprises at least one antistaphylococcal drug.
26. The method of claim 1, wherein the patient is selected from a
hospitalized infant, a premature infant, a burn victim, an elderly
patient, an immunocompromised patient, an immununosuppressed
patient, a patient undergoing an invasive procedure, and a health
care worker.
27. The method of claim 1, wherein the protective monoclonal
antibody is administered by a route selected from intravenous,
intraperitoneal, intracorporeal injection, intra-articular,
intraventricular, intrathecal, intramuscular, subcutaneous,
intranasal, intravaginal, and oral.
28. A composition comprising, a therapeutically effective amount of
at least one MAb that binds to peptidoglycan (PepG) of
Gram-positive bacteria; wherein said MAb provides a therapeutically
beneficial outcome upon administration to a patient.
29. The composition of claim 28, wherein administration reduces the
number of Gram-positive bacteria in a patient.
30. The composition of claim 28, wherein at least one MAb binds
PepG of Gram-positive bacteria at a level at least two-fold greater
than background in an ELISA.
31. The composition of claim 28, wherein at least one MAb enhances
opsonophagocytic of Gram-positive bacteria by at least 50%.
32. The composition of claim 28, further comprising a
pharmaceutically acceptable carrier.
33. The composition of claim 28, further comprising at least one
MAb that binds to lipoteichoic acid (LTA) of Gram-positive
bacteria.
34. The composition of claim 28, wherein at least one MAb blocks
colonization by Gram-positive bacteria upon instillation into the
nares of a patient.
35. The composition of claim 28, wherein at least one MAb
specifically binds PepG of a Gram-positive bacteria selected from
Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus
mutans, Bacillus subtilis, Bacillus megaterium, Enterococcus
faecalis, and Listeria monocytogenes.
36. The composition of claim 28, wherein at least one MAb
specifically binds PepG of Staphylococcus aureus.
37. The composition of claim 28, wherein at least one MAb
specifically binds PepG of Staphylococcus epidermidis.
38. The composition of claim 28, wherein at least one MAb
specifically binds PepG of Staphylococcus aureus and Staphylococcus
epidermidis.
39. The composition of claim 28, wherein at least one MAb
specifically binds PepG of Bacillus subtilis.
40. The composition of claim 28, wherein at least one MAb is
selected from MAb-11-232.3, MAb-11-248.2, MAb-11-569.3,
MAb-11-232.3 IE9, MAb-99-110FC12 IE4, A130, and M130.
41. The composition of claim 28, wherein at least one MAb comprises
a light chain variable region comprising the amino acid sequence
set forth in (SEQ ID NO: 1).
42. The composition of claim 28, wherein at least one MAb comprises
a light chain variable region having at least 80% identity with the
amino acid sequence set forth in (SEQ ID NO: 1).
43. The composition of claim 28, wherein at least one MAb comprises
a heavy chain variable region comprising the amino acid sequence
set forth in SEQ ID NO: 3.
44. The composition of claim 28, wherein at least one MAb comprises
a heavy chain variable region having at least 80% identity with the
amino acid sequence set forth in (SEQ ID NO: 3).
45. The composition of claim 28, wherein at least one MAb is
selected from chimeric, humanized, and human MAbs.
46. The composition of claim 28, wherein at least one MAb comprises
a modified Fc portion, wherein said modification reduces
nonspecific binding of the MAb via the Fc portion.
47. The composition of claim 28, wherein at least one MAb is
selected from a Fab, Fab', F(ab').sub.2, Fv, SFv, and scFv.
48. The composition of claim 28, further comprising at least one
antistaphylococcal drug.
49. A hybridoma cell line deposited at the ATCC under accession no.
PTA-2492.
50. A hybridoma cell line deposited at the ATCC under accession no.
PTA-3659.
51. A vaccine comprising at least one purified PepG, peptides,
fragments and epitopes thereof, in a pharmaceutically acceptable
carrier.
52. A method for treating a patient comprising, administering a
therapeutically effective amount of the vaccine of claim 51.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 10/323,903, filed Dec. 20, 2002; which is a
non-provisional application of U.S. Provisional Application No.
60/343,444, filed Dec. 21, 2001.
[0002] This application is related to U.S. patent application Ser.
No. 09/097,055, filed Jun. 15, 1998, and to U.S. Patent Application
No. 60/341,806, and the application entitled, Methods for Blocking
or Alleviating Staphylococcal Nasal Colonization by Intranasal
Application of Monoclonal Antibodies filed on Dec. 20, 2001, and to
U.S. Pat. Nos. 5,571,511 and 5,955,074. The entire contents of each
of the above mentioned applications and patents are hereby
incorporated by reference in their entirety.
DESCRIPTION OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention in the fields of immunology and infectious
diseases relates to protective antibodies that are specific for
Gram-positive bacteria, particularly to bacteria bearing exposed
peptidoglycan on the surface. The invention includes monoclonal
antibodies, as well as fragments, regions and derivatives
thereof.
[0005] 2. Introduction
[0006] Man has long battled infections caused by bacteria,
particularly Gram-positive bacteria. The surface structures and
cell wall of Gram-positive bacteria form a complex matrix that
performs functions essential in bacteria and host interactions. The
cell wall consists of a peptidoglycan macromolecule (repeating
units of N-acetylglucosamine and N-acetylmuramic acid) and attached
accessory molecules including teichoic acids, lipoteichoic acids,
and carbohydrates (see, e.g., (9) and (24)). In addition, there are
many surface proteins anchored to the bacterial cell wall (see,
e.g., (17)).
[0007] To protect itself against such bacteria, the body employs a
variety of means. Mechanical barriers, such as the skin and mucous
membranes, are the body's first line of defense. If a pathogen is
able to circumvent these barriers and begin multiplying, then white
blood cells called polymorphonuclear leukocytes, or PMNs, are the
next mechanism that the body uses to respond to an infection.
Finally, acquired immune mechanisms step in, wherein circulating
antibodies and complement (soluble plasma proteins that can bind
foreign targets non-specifically) bind to the invading pathogen and
attract phagocytic cells, which in turn engulf and digest the
pathogen. This latter mechanism is called phagocytosis, and the
antibodies and complement that bind to the pathogen and promote
phagocytosis are called opsonins. The enhancement of phagocytosis
by opsonins is in turn called opsonization. Opsonization may rely
on a combination of antibodies and complement (the "classical
pathway"), or just on complement (the "alternative pathway"). The
systems for opsonization and phagocytosis are significant because
defective phagocytosis and killing of staphylococci (and other
Gram-positive bacteria) leads to host invasion, infection and
occasionally death.
[0008] Because of the prevalence of these bacteria on the skin and
other surfaces, most mammals are exposed to Gram-positive bacteria.
Thus, the polyclonal serum from any mammal, including humans, is
likely to contain IgG that will bind to many different cell wall
and surface components of Gram-positive bacteria. Such a collection
of IgGs may serve to protect against Gram-positive bacteria because
polyclonal IgG binding to many epitopes on surface antigens or cell
wall molecules (such as peptidoglycan, teichoic acid, lipoteichoic
acid, proteins and carbohydrates) may collectively be opsonic and
promote phagocytosis of Gram-positive bacteria. Thus, the composite
function of the antibodies in polyclonal serum may account for the
serum's functional activity: However, such polyclonal IgG is
clearly not always protective, as evidenced by the continued
presence of infections due to such bacteria. To augment the level
of antibodies against Gram-positive bacteria, clinicians administer
vaccines based on these bacteria. However, many bacterial cell
extracts that are used for immunization are not pure for one
epitope or antigen, so the activity of the resulting antibodies may
represent activities against several different cell wall
components. This is particularly problematic if the cell wall is
the antibody target, and the purity of the cell wall preparation
cannot be verified.
[0009] Moreover, although perhaps protective in some individuals,
polyclonal serum cannot be used to elucidate the functional role of
an antibody to a single epitope because, by definition, a
polyclonal serum contains many different antibodies, which bind to
multiple antigens and epitopes. Each antibody may contribute to the
composite functional activity. Consequently, the ability of
antibodies directed against specific epitopes on the cell wall to
act as opsonic factors for Gram-positive bacteria is not
defined.
[0010] In addition, it is likely that some antibodies to an epitope
will promote phagocytosis, while others will have different
functions, such as blocking adherence of the bacteria to a cell.
Thus, only monoclonal antibodies to specific epitopes can elucidate
the potential functions of specific antibodies, such as enhancing
phagocytosis, blocking bacterial adherence, or neutralizing toxic
activities, and thereby form the basis of predictably protective
treatments.
[0011] Moreover, until recently, determining the role of
peptidoglycan or of antibodies to peptidoglycan was complicated by
the impurity of peptidoglycan preparations. Teichoic acids and
lipoteichoic acids are closely associated with cell wall
peptidoglycan. In addition, for some bacteria, such as S.
epidermidis, teichoic acid and lipoteichoic acid have the same
glycerol phosphate backbone. These teichoic acid moieties can
easily contaminate peptidoglycan preparations, which are prepared
from cell wall extracts. Thus, the activity of serum raised against
these preparations may not result from the activity of antibodies
to peptidoglycan, but instead from the activity of antibodies to
contaminates (see, e.g., (36)). Recently, we have developed
monoclonal antibodies to LTA that have multiple functional
activities, including opsonic activity, against Gram-positive
bacteria. These antibodies can be used to confirm that
peptidoglycan preparations are free of LTA contamination.
[0012] Furthermore, since peptidoglycan is ubiquitous in the
bacterial world, highly specific opsonic or protective antibodies
to peptidoglycan seem unlikely. In addition, the question about the
role of protective antibody remained. Peterson and colleagues
showed that normal human serum opsonized cell wall extracts and
peptidoglycan (20). However, there were clearly many different
antibodies to many different epitopes in the serum. At least three
different antigenic sites have been distinguished on the
peptidoglycan matrix. When Peterson and colleagues cleaved the
peptidoglycan into small, soluble fragments with lysostaphin, the
fragments were no longer opsonized in the presence of normal human
serum. One explanation is that the smaller fragments could not
support binding of a sufficient number of different antibodies, and
that antibodies to a single epitope on peptidoglycan are not
opsonic. Consequently, while peptidoglycan can activate the
alternative pathway, which promotes opsonization and phagocytosis
of S. aureus by complement alone, the role of antibodies and the
classical pathway in opsonization and phagocytosis remained
difficult to understand.
[0013] The role of antibodies in these processes was further in
doubt when IgG deficient serum was found to be fully opsonic in
studies by the same group. This result was consistent with studies
by others that showed a normal level of killing by PMNs using serum
that had been depleted of antibodies, and after blocking neutrophil
IgG Fc receptors. An additional complication lies in the fact that
many cell wall epitopes are deep under the surface and may be
covered by proteins and capsular polysaccharide in live growing
bacteria (18, 19).
[0014] Thus it was not known whether or not a monoclonal antibody
to peptidoglycan that binds to a specific epitope could have
functional activity without working in concert with other
antibodies having other antigen or epitope specificities. It was
also not known if an antibody with a single specificity could
perform several functions important for host immunity and
protection. Such monoclonal antibodies would be useful to prevent
or treat Gram-positive infections, and the epitopes or antigens to
which they bind would be useful as vaccines to induce protective
immunity in a host.
SUMMARY OF THE INVENTION
[0015] This invention relates to therapeutic compositions
comprising protective monoclonal antibodies (MAbs) to peptidoglycan
(PepG) that enhance phagocytosis, block colonization and/or inhibit
PepG induced- or facilitated-toxicity. As noted above, phagocytosis
is important for effective immunity against Gram-positive bacteria.
This invention provides protective opsonic MAbs to PepG that
enhance phagocytosis and killing of Gram-positive bacteria and thus
can block or treat systemic infections. Nasal colonization has been
shown to be a primary reservoir for staphylococci, and a strong
correlation has been demonstrated between staphylococcal nasal
colonization and subsequent staphylococcal infections. This
invention provides protective anti-PepG MAbs that block and/or
alleviate nasal colonization by Gram-positive bacteria, such as
staphylococci, and thereby reduce the incidence and/or severity of
associated infections. Given intravenously, subcutaneously,
intramuscularly, or through any other route of administration,
protective anti-PepG MAbs may reduce the toxic effects of cell wall
components. Thus, these therapeutic compositions both prevent and
treat infections by Gram-positive bacteria.
[0016] The protective monoclonal antibodies of the invention
include both IgG and IgM anti-PepG MAbs specific for PepG and
include mouse, mouse/human chimeric, humanized or fully human MAbs
specific for PepG. The protective monoclonal antibodies of this
invention are directed to any of the multiple epitopes on PepG.
They exhibit multiple binding characteristics and functional
activities.
[0017] These protective monoclonal antibodies can be administered
singly or in combination into the nares of normal or colonized
human subjects or other mammals to block or alleviate bacterial
colonization of the nasal mucosa and to thereby preclude systemic
infections or reduce the spread of Gram-positive bacteria.
[0018] The invention also includes methods of using both single
protective anti-PepG MAbs and combinations of MAbs to enhance
phagocytosis, inhibit bacterial infection, which may result from
colonization of the nasal mucosa and reduce toxic effects of PepG
and other cell wall components or toxins.
[0019] In addition, PepG epitopes or antigens and peptides that
mimic those epitopes and antigens would be useful as vaccines to
elicit opsonic antibodies to Gram-positive bacteria.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows the cDNA cloning strategy for the heavy chain
and light chain variable regions of M130.
[0021] FIG. 2 shows the polypeptide and nucleic acid sequences of
(A) the M130 antibody light chain variable region (SEQ ID NO: 1 and
SEQ ID NO.: 2) and (B) the M130 antibody heavy chain variable
region (SEQ ID NO: 3 and SEQ ID NO: 4).
[0022] FIG. 3 is shows the pJSB22 heavy chain expression
plasmid.
[0023] FIG. 4 shows the pJSB6 light chain expression plasmid.
[0024] FIG. 5 shows the pLG1 bi-cistronic expression plasmid.
[0025] FIG. 6 shows the binding of anti-human IgG to the
mouse/human chimeric antibody A130.
[0026] FIG. 7 shows the binding activity of the mouse/human
chimeric antibody, A130, to S. aureus peptidoglycan.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0027] The term "antibody", as used herein, includes full-length
antibodies and portions thereof. A full-length antibody has one
pair or, more commonly, two pairs of polypeptide chains, each pair
comprising a light and a heavy chain. Each heavy or light chain is
divided into two regions, the variable region (which confers
antigen recognition and binding) and the constant region
(associated with localization and cellular interactions). Thus, a
full-length antibody commonly contains two heavy chain constant
regions (HC or CH), two heavy chain variable regions (HV or VH),
two light chain constant regions (LC or CL), and two light chain
variable regions (LV or VL) (FIG. 2). The light chains or chain may
be either a lambda or a kappa chain. Thus, in an embodiment of the
invention, the antibodies include at least one heavy chain variable
region and one light chain variable region, such that the antibody
binds antigen.
[0028] Another aspect of the invention involves the variable region
that comprises alternating complementarity determining regions, or
CDRs, and framework regions, or FRs. The CDRs are the sequences
within the variable region that generally confer antigen
specificity.
[0029] The invention also encompasses portions of antibodies which
comprise sufficient variable region sequence to confer antigen
binding. Portions of antibodies include, but are not limited to
Fab, Fab', F(ab').sub.2, Fv, SFv, scFv (single-chain Fv), whether
produced by proteolytic cleavage of intact antibodies, such as
papain or pepsin cleavage, or by recombinant methods, in which the
cDNAs for the intact heavy and light chains are manipulated to
produce fragments of the heavy and light chains, either separately,
or as part of the same polypeptide.
[0030] MAbs of the present invention encompass antibody sequence
corresponding to human and non-human animal antibodies, and hybrids
thereof. The term "chimeric antibody," as used herein, includes
antibodies that have variable regions derived from an animal
antibody, such as rat or mouse antibody, fused to another molecule,
for example, a constant region domain derived from a human IgG,
IgA, or IgM antibody.
[0031] One type of chimeric antibody, a "Humanized antibody" has
the variable regions altered (through mutagenesis or CDR grafting)
to match (as much as possible) the known sequence of human variable
regions. CDR grafting involves grafting the CDRs from an antibody
with desired specificity onto the FRs of a human antibody, thereby
replacing much of the non-human sequence with human sequence.
Humanized antibodies, therefore, more closely match (in amino acid
sequence) the sequence of known human antibodies. By humanizing
mouse monoclonal antibodies, the severity of the human anti-mouse
antibody, or HAMA, response is diminished. The invention also
includes fully human antibodies which would avoid the HAMA respose
as much as possible.
[0032] The invention also encompasses "modified antibodies", which
include, for example, the proteins or peptides encoded by truncated
or modified antibody-encoding genes. Such proteins or peptides may
function similarly to the antibodies of the invention. Other
modifications, such as the addition of other sequences that may
enhance an effector function, which includes the ability to block
or alleviate nasal colonization by staphylococci, are also within
the present invention. Such modifications include, for example, the
addition of amino acids to the antibody's amino acid sequence,
deletion of amino acids in the antibody's amino acid sequence,
substitution of one or more amino acids in the antibody amino acid
sequence with alternate amino acids, isotype switching, and class
switching.
[0033] In certain embodiments, an antibody may be modified in its
Fc region to prevent binding to bacterial proteins. The Fc region
normally provides binding sites for accessory cells of the immune
system. As the antibodies bind to bacteria, and coat them, these
accessory cells recognize the coated bacteria and respond to
infection. When a bacterial protein binds to the Fc region near the
places where accessory cells bind, the normal function of these
cells is inhibited. For example, Protein A, a bacterial protein
found in the cell membrane of S. aureus, binds to the Fc region of
IgG near accessory cell binding sites. In doing so, Protein A
inhibits the function of these accessory cells, thus interfering
with clearance of the bacterium. To circumvent this interference
with the antibacterial immune response, the Fc portion of the
antibody of the invention may be modified to prevent nonspecific
binding of Protein A while retaining binding to accessory cells
(see, e.g., (10)).
[0034] In light of these various forms, the antibodies of the
invention include full-length antibodies, antibody portions,
chimeric antibodies, humanized antibodies, fully human antibodies,
and modified antibodies and will be referred to collectively as
"MAbs" unless otherwise indicated.
[0035] The term "epitope", as used herein, refers to a region, or
regions, of PepG that is bound by an antibody to PepG. The regions
that are bound may or may not represent a contiguous portion of the
molecule.
[0036] The term "antigen", as used herein, refers to a polypeptide
sequence, a non-proteinaceous molecule, or any molecule that can be
recognized by the immune system. An antigen may be a full-sized
staphylococcal protein or molecule, or a fragment thereof, wherein
the fragment is either produced from a recombinant cDNA encoding
less than the full-length protein, or a fragment derived from the
full-sized molecule or protein or a fragment thereof. Such
fragments may be made by proteolysis. An antigen may also be a
polypeptide sequence that encompasses an epitope of a
staphylococcal protein, wherein the epitope may not be contiguous
with the linear polypeptide sequence of the protein. The DNA
sequence encoding an antigen may be identified, isolated, cloned,
and transferred to a prokaryotic or eukaryotic cell for expression
by procedures well-known in the art (25). An antigen may be 100%
identical to a region of the staphylococcal molecule or protein
amino acid sequence, or it may be at least 95% identical, or at
least 90% identical, or at least 85% identical. An antigen may also
have less than 100%, 95%, 90% or 85% identity with the
staphylococcal molecule or protein amino acid sequence, provided
that it still is able to elicit antibodies that bind to a native
staphylococcal molecule or protein.
[0037] The percent identity of a peptide antigen can be determined,
for example, by comparing the sequence of the target antigen or
epitope to the analagous portion of staphylococcal sequence using
the GAP computer program, version 6.0 described by Devereux et al.
(Nucl. Acids Res. 12:387, 1984) and available from the University
of Wisconsin Genetics Computer Group (UWGCG) (40). The GAP program
utilizes the alignment method of Needleman and Wunsch (J. Mol.
Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl.
Math 2:482, 1981), and is applicable to determining the percent
identity of protein or nucleotide sequences referenced herein (41,
42). The preferred default parameters for the GAP program include:
(1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) for nucleotides, and the
weighted comparison matrix of Gribskov and Burgess, Nucl. Acids.
Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds.,
Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for
each gap and an additional 0.10 penalty for each symbol in each
gap; and (3) no penalty for end gaps (43, 44).
[0038] Alternatively, for simple comparisons over short regions up
to 10 or 20 units, or regions of relatively high homology, for
example between antibody sequences, or homologous portions thereof,
the percent identity over a defined region of peptide or nucleotide
sequence may by determined by dividing the number of matching amino
acids or nucleotides by the total length of the aligned sequences,
multiplied by 100%. Where an insertion or gap of one, two, or three
amino acids occurs in a MAb chain, for example in or abutting a
CDR, the insertion or gap is counted as single amino acid
mismatch.
[0039] Antigens may be bacterial surface antigens and/or virulence
and/or adherence antigens. Surface antigens are antigens that are
accessible to an antibody when the antigen is in the configuration
of the whole intact bacterium, i.e., the antigen is not inside the
cell cytoplasm. Virulence antigens are antigens that are involved
in the pathogenic process, causing disease in a host. Virulence
antigens include, for example, LTA, peptidoglycan, toxins, fimbria,
flagella, and adherence antigens. Adherence antigens mediate the
ability of a staphylococcal bacterium to adhere to the surface of
the nares. An antigen may be a non-proteinaceous component of
staphylococci such as a carbohydrate or lipid. For example,
peptidoglycan and lipoteichoic acid are two non-proteinaceous
antigens found in the cell wall of staphylococci. Antigens may
comprise or include fragments of non-proteinaceous molecules as
long as they elicit an immune response.
[0040] As used herein, antigens include molecules that can elicit
an antibody response to PepG. An antigen may be a PepG molecule, or
a fragment thereof, wherein the fragment may be enzymatically, or
otherwise, derived from the entire molecule or a fragment thereof.
An antigen may also be, a fragment of PepG that encompasses an
epitope of PepG, wherein the epitope may not be contiguous with the
macromolecular structure of the molecule. An antigen may be 100%
identical to a region of PepG, or it may be 95% identical, or 90%
identical, or 85% identical. An antigen may also have less identity
with the PepG molecule, provided that it is able to elicit
antibodies that bind to PepG. An antigen may also be an unrelated
molecule, which, through some structural similarity, is able to
elicit antibodies that bind to PepG. In certain embodiments of the
invention, an antigen elicits antibodies that bind to PepG on the
surface of bacteria. In certain embodiments, an antigen is a
peptide that elicits antibodies that bind to PepG, and can be
encoded by a cDNA. Procedures are generally described in Molecular
Cloning: A Laboratory Manual, 2.sup.nd Ed., which is herein
incorporated by reference for any purpose (25).
[0041] Particular antigens of the invention include antigens that
bind to any of the monoclonal antibodies produced by hybridomas
11-232.3, 11-248.2, 11-569.3, 11-232.3 IE9, 99-110FC12 IE4 (also
referred to as MAb-11-232.3, MAb-11-248.2, MAb-11-569.3,
MAb-11-232.3 IE9, and MAb-99-110FC12 IE4), A130, or M130, described
herein.
[0042] An antibody is said to specifically bind to an antigen,
epitope, or protein, if the antibody gives a signal by protein
ELISA or other assay that is at least two fold, at least three
fold, at least five fold, and at least ten fold greater than the
background signal, i.e., at least two fold, at least three fold, at
least five fold, or at least ten fold greater than the signal
ascribed to non-specific binding. An antibody is said to
specifically bind to a bacterium if the antibody gives a signal by,
for example, methanol-fixed bacteria ELISA or live bacteria ELISA
that is at least 1.5 fold, 2 fold, or 3 fold greater than the
background signal.
[0043] "Enhanced phagocytosis", as used herein, means an increase
in phagocytosis over a background level as assayed by the methods
in this application, or another comparable assay. The level deemed
valuable may well vary depending on the specific circumstances of
the infection, including the type of bacteria and the severity of
the infection. For example, enhanced phagocytic activity may be
equal to or greater than 75%, 80%, 85%, 90%, 95%, or 100% over
background phagocytosis. Enhanced phagocytosis may also be equal to
or greater than 50%, 55%, 60%, 65%, or 70% over background
phagocytosis. As used herein, opsonic activity may also be assessed
by assays that measure neutrophil mediated opsonophagocytotic
bactericidal activity.
[0044] The MAb's of the invention are useful for the prophylaxis
and other treatment of systemic and local staphylococcal
infections. In this respect, a MAb of the invention is said to
"alleviate" staphylococcal nasal colonization if it is able to
decrease the number of colonies in the nares of a mammal when the
MAb is administered before, concurrently with, or after exposure to
staphylococci, whether that exposure results from the intentional
instillation of staphylococcus or from general exposure. For
instance, in the nasal colonization animal model described below, a
MAb or collection of MAbs is considered to alleviate colonization
if the extent of colonization, or the number of bacterial colonies
that can be grown from a sample of nasal tissue, is decreased after
administering the MAb or collection of MAbs. A MAb or collection of
MAbs alleviates colonization in the nasal colonization assays
described herein when it reduces the number of colonies by at least
25%, at least 50%, at least 60%, at least 75%, at least 80%, or at
least 90%. 100% alleviation may also be referred to as
eradication.
[0045] A MAb is said to "block" staphylococcal colonization if it
is able to prevent the nasal colonization of a human or non-human
mammal when it is administered prior to, or concurrently with,
exposure to staphylococci, whether by intentional instillation or
otherwise into the nares. A MAb blocks colonization, as in the
nasal colonization assay described herein, if no staphylococcal
colonies can be grown from a sample of nasal tissue taken from a
mammal treated with the MAb of the invention for an extended period
such as 1.2 hours or longer or 24 hours or longer compared to
control mammals. A MAb also blocks colonization in the nasal
colonization assay described herein if it causes a reduction in the
number of animals that are colonized relative to control animals.
For instance, a MAb is considered to block colonization if the
number of animals that are colonized after administering the
material and the Gram-positive bacteria is reduced by at least 25%,
at least 50%, and at least 75%, relative to control animals or if
no colonies can be grown from a sample taken from a treated
individual for an extended period such as 12 hours or 24 hours or
longer.
[0046] In a clinical setting, the presence or absence of nasal
colonization in a human patient is determined by culturing nasal
swabs on an appropriate bacterial medium. These cultures are scored
for the presence or absence of staphylococcal colonies. In this
type of qualitative assay system, it may be difficult to
distinguish between blocking and alleviation of staphylococcal
colonization. Thus, for the purposes of qualitative assays, such as
nasal swabs, a MAb "blocks" colonization if it prevents future
colonization in human patients who show no signs of prior
colonization for an extended period such as 12 hours or 24 hours or
longer. A MAb "alleviates" colonization if it causes a discernable
decrease in the number of positive cultures taken from a human
patient who is already positive for staphylococci before the MAbs
of the invention are administered.
[0047] Thus, the MAbs of the invention may be administered
intranasally to block and/or alleviate staphylococcal nasal
colonization. Administration (instillation) of an "effective
amount" of the MAb results in a mammal that exhibits any of: 1) no
nasal colonization by staphylococci for at least 12 hours after
administration, 2) discernable, medically meaningful, or
statistically significant decrease in the number of Gram-positive
or staphylococcal colonies in the nares, or 3) a discernable,
medically meaningful, or statistically significant decrease in the
frequency of Gram-positive or staphylococcal cultures taken from
the nares, or 4) a discernable, medically meaningful, or
statistically significant decrease in the frequency of
Gram-positive or staphylococcal infections.
[0048] "Instillation" encompasses any delivery system capable of
providing a effective amount of a MAb to the mammalian nares.
[0049] A goal of the invention is to reduce the frequency of
staphylococcal infections, including nosocomial infections. The
administration of an effective amount includes that sufficient to
demonstrate a discernable, medically meaningful, or statistically
significant of decrease in the likelihood of staphylococcal
infection involving a body site other than the nares, for example
systemic infection, or infections at the site of trauma or surgery.
Such demonstrations may encompass, for example, animal studies or
clinical trials of patients at risk of infection by Gram-positive
bacteria, including, but not limited to: premature infants, persons
undergoing inpatient or outpatient surgery, burn victims, patients
receiving indwelling catheters, stents, joint replacements and the
like, geriatric patients, and those with genetically, chemically or
virally suppressed immune systems.
[0050] As used herein, a "treatment" of a patient encompasses any
administration of a composition of the invention that results in a
"therapeutically beneficial outcome," hereby defined as: 1) any
discernable, medically meaningful, or statistically significant
reduction, amelioration, or alleviation of existing Gram-positive
bacterial infection or colonization, or 2) any discernable,
medically meaningful, or statistically significant blocking or
prophylaxis against future bacterial challenge, infection, or
colonization, or 3) any discernable, medically meaningful, or
statistically significant reduction in the likelihood of nosocomial
infection. Treatment thus encompasses a discernable, medically
meaningful, or statistically significant reduction in the number of
Gram-positive bacteria in a colonized or infected patient as well
as a reduction in likelihood of future colonization or infection.
As used herein, "colonized" refers to the subclinical presence of
Gram-positive bacteria in patient, most particularly in the nares,
whereas "infected" refers to clinical infection in any body
site.
[0051] As used herein, "medically meaningful" encompasses any
treatment that improves the condition of a patient; improves the
prognosis for a patient; reduces morbidity or mortality of a
patient; or reduces the incidence of morbidity or rates of
mortality from the bacterial infections addressed herein, among a
population of patients. The specific determination or
identification of a "statistically significant" result will depend
on the exact statistical test used. One of ordinary skill in the
art can readily recognize a statistically significant result in the
context of any statistical test employed, as determined by the
parameters of the test itself. Examples of these well-known
statistical tests include, but are not limited to, X.sup.2 Test
(Chi-Squared Test), Students t Test, F Test, M test, Fisher Exact
Text, Binomial Exact Test, Poisson Exact Test, one way or two way
repeated measures analysis of variance, and calculation of
correlation efficient (Pearson and Spearman).
[0052] MAbs of the invention include "protective Mabs." Protective
MAbs 1) exhibit strong binding to PepG, 2) enhance the opsonization
and killing of Gram-positive bacteria (opsonophagocytic killing),
and 3) reduce bacterial colonization. Such MAbs may also inhibit
the toxicity that is induced or facilitated by PepG. In another
embodiment, these protective MAbs encompass therapeutic
compositions for the treatment of Gram-positive infections.
[0053] A vaccine is considered to confer a protective immune
response if it stimulates the production of protective opsonic
antibodies to Gram-positive bacteria. Production of protective
opsonic antibodies may be measured by the presence of such
antibodies in the serum of a test subject that has been
administered the vaccine, relative to a control that has not
received the vaccine. The presence of protective opsonic antibodies
in the serum may be measured by the activity assays described
herein, or by other equivalent assays. If an opsonophagocytic
bactericidal assay is used, then killing by the test serum of at
least 50% more bacteria, 75% more bacteria, and at least 100% more
bacteria, relative to the control serum, is considered to be
enhanced immunity.
EMBODIMENTS OF THE INVENTION
[0054] One aspect of the invention relates to protective anti-PepG
MAbs that bind to whole bacteria. Bacteria include all
Gram-positive bacteria, and in particular, staphylococci and
streptococci. Since many epitopes of PepG may be unavailable on the
surface of Gram-positive bacteria, this invention provides
protective MAbs that bind to whole bacteria as well as to isolated
PepG. By binding PepG, these protective MAbs may neutralize the
toxic effects of these molecules.
[0055] Another aspect of the invention relates to protective MAbs
that function as opsonins, binding in a manner that allows
interaction with phagocytes, thereby promoting phagocytosis. Such
protective MAbs may block or alleviate Gram-positive bacterial
infections. These protective anti-PepG MAbs may be used either
alone or in combination with MAbs of different specificity, for
example, MAbs specific for LTA, to treat diseases caused by
Gram-positive bacteria and/or other organisms. A further aspect of
the invention is protective anti-PepG MAbs that may block or
alleviate bacterial nasal colonization.
[0056] Particular embodiments of the invention include protective
MAbs comprising the antigen-binding domains of the monoclonal
antibodies MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, MAb-11-232.3
IE9, MAb-99-110FC12 IE4, A130, or M130, described herein.
[0057] The invention also includes protective chimeric anti-PepG
MAbs, in which the variable regions from a mouse monoclonal
antibody are fused to human constant regions, and the chimeric
antibody is produced in mammalian cell culture.
[0058] For example, a chimeric heavy chain may comprise the antigen
binding region of the heavy chain variable region of a protective
mouse anti-PepG MAb of the invention linked to at least a portion
of a human heavy chain constant region. This chimeric heavy chain
may be combined with a chimeric light chain that comprises the
antigen binding region of the light chain variable region of a
protective mouse anti-PepG MAb linked to at least a portion of a
human light chain constant region.
[0059] In certain embodiments of the invention, a protective
chimeric antibody is the human/mouse chimeric A130 antibody
described herein. In another embodiment, a protective chimeric
antibody comprises the antigen-binding domains of any of the
monoclonal antibodies MAb-11-232.3, MAb-11-248.2, MAb-11-569.3,
MAb-11-232.3 IE9, MAb-99-110FC12 IE4, A130, or M130, described
herein.
[0060] Epitopes and antigens that are bound by protective anti-PepG
monoclonal antibodies are also aspects of the invention. Further
aspects of the invention include epitopes and antigens that elicit
opsonic antibodies that bind to PepG of Gram-positive bacteria in
vertebrates. These epitopes and antigens elicit protective opsonic
antibodies when introduced into a human, a mouse, a rat, a rabbit,
a dog, a cat, a cow, a sheep, a pig, a goat, or a chicken. Peptides
that mimic those epitopes and antigens, and which can elicit
opsonic antibodies to PepG of Gram-positive bacteria are also
encompassed by the invention. These epitopes, antigens, peptides,
and fragments of PepG may be used as vaccines to protect against,
or alleviate, infections caused by Gram-positive bacteria.
[0061] The present invention also discloses therapeutic
compositions comprising the protective anti-PepG MAbs of the
invention, whether chimeric, humanized, or fully human, as well as
fragments, regions, and derivatives thereof. These compositions may
also include a pharmaceutically acceptable carrier. The therapeutic
compositions of the invention may alternatively comprise the
isolated antigen, epitope, or portions thereof, together with a
pharmaceutically acceptable carrier.
[0062] In certain embodiments, a therapeutic composition of the
invention includes, but is not limited to, a protective antibody
comprising the antigen-binding domains of any of the monoclonal
antibodies MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, MAb-11-232.3
IE9, MAb-99-110FC12 IE4, A130, or M130, described herein.
[0063] Pharmaceutically acceptable carriers include, but are not
limited to, sterile liquids, such as water, oils, including
petroleum oil, animal oil, vegetable oil, peanut oil, soybean oil,
mineral oil, sesame oil, and the like. Saline solutions, aqueous
dextrose, and glycerol solutions can also be employed as liquid
carriers. Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, 18.sup.th Edition (8), which
is herein incorporated by reference for any purpose.
[0064] Additionally, the invention may be practiced with various
delivery vehicles and/or carriers. Such vehicles may increase the
half-life of the Mabs in storage and upon administration including,
but not limited to, application to mucus membranes, for example,
upon inhalation or instillation into the nares. These carriers
comprise natural polymers, semi-synthetic polymers, synthetic
polymers, liposomes, and semi-solid dosage forms (8, 16, 22, 26,
29, 30, 37). Natural polymers include, for example, proteins and
polysaccharides. Semi-synthetic polymers are modified natural
polymers such as chitosan, which is the deacetylated form of the
natural polysaccharide, chitin. Synthetic polymers include, for
example, polyphosphoesters, polyethylene glycol, poly (lactic
acid), polystyrene sulfonate, and poly (lactide coglycolide).
Semi-solid dosage forms include, for example, dendrimers, creams,
ointments, gels, and lotions. These carriers can also be used to
microencapsulate the MAbs or be covalently linked to the MAbs.
[0065] In one embodiment, the MAbs of the invention comprise, or
are covalently or non-covalently bound to the outside of a carrier
particle, which may be formulated as a powder, spray, aerosol,
cream, gel, etc for application to the nares or infected area. In
one embodiment, the MAbs are coated onto a carrier particle core in
a dissolvable film, which may comprise a mucoadhesive. The carrier
particle core may be inert, or dissolvable.
[0066] The invention further comprises any delivery system capable
of providing a effective amount of a MAb to the mammalian nares or
other infected area. Representative and non-limiting formats
include drops, sprays, powders, aerosols, mists, catheters, tubes,
syringes, applicators for creams, particulates, pellets, and the
like. Also encompassed within the invention are kits comprising a
composition containing one or more MAbs of the invention, in
connection with an appropriate delivery device or applicator for
the composition, for example: catheters, tubes, sprayers, syringes,
atomizers, or other applicator for creams, particulates, pellets,
powders, liquids, gels and the like.
[0067] Finally, the present invention provides methods for treating
a patient infected with, or suspected of being infected with, a
Gram-positive bacteria. The method comprises administering to a
patient a therapeutically effective amount of a therapeutic
composition comprising one or more of the protective anti-PepG MAbs
(including monoclonal, chimeric, humanized, fully human, fragments,
regions, and derivatives thereof) and a pharmaceutically acceptable
carrier. A patient can be any human or non-human mammal in need of
prophylaxis or other treatment. Representative patients include any
mammal subject to S. aureus, staphylococcal, or Gram-positive
infection or carriage, including humans and non-human animals such
as mice, rats, rabbits, dogs, cats, pigs, sheep, goats, horses,
primates, ruminants including beef and milk cattle, buffalo,
camels, as well as fur-bearing animals, herd animals, laboratory,
zoo, and farm animals, kenneled and stabled animals, domestic pets,
and veterinary animals.
[0068] A therapeutically effective amount is an amount reasonably
believed to provide measurable relief, assistance, prophylactive or
preventive effect in the treatment of the infection. Such therapy
as above or as described below may be primary or supplemental to
additional treatment, such as antibiotic therapy for a
Gram-positive bacterial infection, an infection caused by a
different agent, or an unrelated disease. Combination therapy with
other antibodies is expressly contemplated within the
invention.
[0069] A further embodiment of the present invention is a method of
blocking or alleviating such infections, comprising administering
an effective amount of a therapeutic composition comprising the
protective anti-PepG MAb (whether monoclonal or chimeric,
humanized, or fully human, including fragments, regions, and
derivatives thereof) and a pharmaceutically acceptable carrier.
[0070] An effective amount may be reasonably believed to provide
some measure of blocking or alleviating infection by Gram-positive
bacteria. Such therapy as above or as described below may be
primary or supplemental to additional treatment, such as antibiotic
therapy, for a staphylococcal infection, an infection caused by a
different Gram-positive bacterial agent, or an unrelated disease.
Indeed, combination therapy with other antibodies is expressly
contemplated within the invention.
[0071] In another embodiment, a peptide that mimics any of the PepG
epitopes would be useful to block binding of Gram-positive bacteria
to epithelial cells and thereby inhibit colonization. For example,
a therapeutic composition containing one or more such peptides may
be administered intranasally to block or inhibit colonization, and
therefore prevent or alleviate further infection.
[0072] Yet another embodiment of the present invention is a vaccine
comprising one or more of the epitopes of the PepG antigen or one
or more of the peptides that mimic a PepG epitope in a
pharmaceutically acceptable carrier. Upon introduction into a host,
the vaccine elicits an antibody broadly protective and opsonic
against infection by Gram-positive bacteria. The vaccine may
include the epitope, a peptide that mimics an epitope, any mixture
of epitopes and peptides that mimic an epitope, the antigen,
different antigens, or any combination of epitopes, peptides that
mimic epitopes, and antigens. Standard techniques for immunization
and analysis of the subsequent antibody response are found in:
Antibodies: A Laboratory Manual, (Harlow & Lane eds., 1988),
Cold Spring Harbor Laboratory Press; Conjugate Vaccines, (J. M.
Cruse, R. E. Lewis, Jr. eds., 1989), Karger, Basel; U.S. Pat. Nos.
5,955,079, and 6,432,679 each of which is incorporated by
reference.
[0073] The protective anti-PepG MAbs, vaccines, and therapeutic
compositions of the invention are particularly beneficial for
individuals known to be or suspected of being at risk of infection
by Gram-positive bacteria, such as infant and elderly patients,
immunocompromised patients, patients undergoing invasive
procedures, patients undergoing chemotherapy, patients undergoing
radiation therapy, and health care workers. This includes infants
with immature immune systems, patients receiving body implants,
such as valves, patients with indwelling catheters, patients
preparing to undergo surgery involving breakage or damage of skin
or mucosal tissue, certain health care workers, and patients
expected to develop impaired immune systems from some form of
therapy, such as chemotherapy or radiation therapy. Among non-human
patients, those at risk include zoo animals, herd animals, and
animals maintained in close quarters.
[0074] The MAbs of the invention may be administered in conjunction
with other antibiotic anti-staphylococcal drugs including
antibiotics like mupirocin and bacitracin; anti-staphylococcal
agents like lysostaphin, lysozyme, mutanolysin, and cellozyl
muramidase; anti-bacterial peptides like nisin; and lantibiotics,
or any other lanthione-containing molecule, such as nisin or
subtilin.
[0075] In view of the disclosure provided, the administration of
the MAbs of the invention is within the know-how and experience of
one of skill in the art in light of the particular formulation and
delivery method selected. In particular, the amount of MAbs
required, combinations with appropriate carriers, the dosage
schedule and amount may be varied within a wide range based on
standard knowledge in the field without departing from the claimed
invention. In one example, the MAbs of the invention may be given
by intravenous drip or in discrete doses, doses may range from 1 to
4 or more times daily giving 0.1 to 20 mg per dose. In one
embodiment, the amount of MAb administered would be 2-4 times per
day at 0.1-3 mg per dose, a dose known to be effective with an
inoculum of 10.sup.8 S. aureus bacteria, an amount of bacteria
known to ensure 100% colonization in an animal model. Such a dosing
regimen would be effective on patients either admitted to the
hospital for surgical procedures, patients suffering from various
conditions that predispose them to staphylococcal infections,
convalescing patients, infants with immature immune systems, or
prior to a patients' release from hospitals.
[0076] The protective anti-PepG antibodies, vaccines, and the
therapeutic compositions of the invention may be administered by
intravenous, intraperitoneal, intracorporeal injection,
intra-articular, intraventricular, intrathecal, intramuscular,
subcutaneous, intranasally, dermally, intradermally,
intravaginally, orally, or by any other effective method of
administration. The composition may also be given locally, such as
by injection to the particular area infected, either
intramuscularly or subcutaneously. Administration can comprise
administering the therapeutic composition by swabbing, immersing,
soaking, or wiping directly to a patient. The treatment can also be
applied to objects to be placed within a patient, such as
indwelling catheters, cardiac values, cerebrospinal fluid shunts,
joint prostheses, other implants into the body, or any other
objects, instruments, or appliances at risk of becoming infected
with a Gram-positive bacteria, or at risk of introducing such an
infection into a patient.
[0077] Particular aspects of the invention are now presented in the
form of the following "Materials and Methods" as well as the
specific Examples. Of course, these are included only for purposes
of illustration and are not intended to limit the present
invention.
Materials and Methods
Bacteria
[0078] S. aureus, type 5, is deposited at the ATCC under Accession
No. 49521.
[0079] S. aureus type 8, is deposited at the ATCC under Accession
No. 12605.
[0080] S. epidermidis strain Hay, was deposited at the ATCC on Dec.
19, 1990 under Accession No. 55133.
[0081] S. hemolyticus is deposited at the ATCC under Accession No.
43252.
Hybridomas
[0082] Hybridoma 96-105CE11 IF6 (M110) was deposited at the ATCC on
Jun. 13, 1997 under Accession No. HB-12368.
[0083] Hybridoma 99-110 FC12 IE4 was deposited at the ATCC on Sep.
21, 2000 under Patent Deposit PTA-2492.
[0084] Hybridoma 11-232.3 IE9 (M130) was deposited at the ATCC on
Aug. 21, 2001 under PTA-3659.
Isotype Determination Assay
[0085] Isotype was determined using a mouse immunoglobulin isotype
kit obtained from Zymed Laboratories (Cat. No. 90-6550).
Binding Assays
[0086] In the binding assays of the invention, immunoglobulin is
incubated with a preparation of whole cell staphylococci or with a
preparation of bacterial cell wall components such as LTA or PepG.
The binding assay may be an agglutination assay, a coagulation
assay, a colorimetric assay, a fluorescent binding assay, or any
other suitable binding assay that is known in the art. A
particularly suitable assay is either an enzyme-linked
immunosorbent assay (ELISA) or a radio-immunoassay (RIA). Binding
is detected directly and can also be detected indirectly by using
competitive or noncompetitive binding procedures known in the
art.
[0087] The whole cell staphylococcus preparation, LTA preparation,
PepG preparation, or a combination of those preparations, may be
fixed using standard techniques to a suitable solid support,
including, but not limited to, a plate, a well, a bead, a
micro-bead, a paddle, a propeller, or a stick. Solid supports may
be comprised of, for example, glass or plastic. In certain
embodiments of the invention, the solid support is a microtiter
plate.
[0088] Generally, a binding assay requires the following steps.
First, the fixed preparation is incubated with an immunoglobulin
source. In one embodiment of the assay, the immunoglobulin source
is, for example, tissue culture supernatant or a biological sample
such as ascites, plasma, serum, whole blood, or body tissue. In
another embodiment, the immunoglobulin may be further isolated or
purified from its source by means known in the art, and the
purified or isolated immunoglobulin is subsequently used in the
assay. The amount of binding is determined by comparing the binding
in a test sample to the binding in a negative control. A negative
control is defined as any sample that does not contain
antigen-specific immunoglobulin. In the binding assay, a positive
binding reaction results when the amount of binding observed for
the test sample is greater than the amount of binding for a
negative control. Positive binding may be determined from a single
positive/negative binding reaction or from the average of a series
of binding reactions. The series of binding reactions may include
samples containing a measured amount of immunoglobulin that
specifically binds to the fixed antigen, thereby creating a
standard curve. This standard curve may be used to quantitate the
amount of antigen-specific immunoglobulin in an unknown sample.
[0089] In an alternate embodiment of the assay, antibodies are
fixed to a solid support and an unknown immunoglobulin sample is
characterized by its ability to bind a bacterial preparation. The
other aspects of the assays discussed above apply where
appropriate.
[0090] The specific binding assays used in the Examples are set
forth below:
[0091] Live Bacteria ELISA (LBE): The LBE assay was performed to
measure the ability of antibodies to bind to live bacteria. Various
types of bacteria may be used in this assay, including S. aureus
type 5, type 5-USU, type 8, S. epidermidis strain Hay, and S.
hemolyticus. Bacteria from an overnight plate culture was
transferred to 35 ml of Tryptic Soy Broth (TSB) and grown with
gentle shaking for 1.5-2.0 hours at 37.degree. C. The bacteria were
then pelleted by centrifugation at 1800-2000.times.g for 15 minutes
at room temperature. The supernatant was removed and the bacteria
were resuspended in 35-45 ml of phosphate buffered saline
containing 0.1% bovine serum albumin (PBS/BSA). The bacteria were
again pelleted by centrifugation, the supernatant discarded and the
bacteria resuspended in PBS/BSA to a percent transmittance (% T) of
65%-70% at 650 nm. From this suspension the bacteria were further
diluted 15-fold in sterile 0.9% sodium chloride (Sigma cat. no.
S8776, or equivalent), and 100 .mu.l of this suspension was added
to replicate wells of a flat-bottomed, sterile 96-well plate.
[0092] Each antibody to be tested was diluted to the desired
concentration in PBS/BSA containing 0.05% Tween-20 and horse radish
peroxidase-conjugated Protein A (Protein A-HRP, Zymed Laboratories)
at a 1:10000 dilution (PBS/BSA/Tween/Prot A-HRP). The Protein A-HRP
was allowed to bind to the antibodies for 30-60 minutes at room
temperature before use, thereby generating an antibody-Protein
A-HRP complex to minimize the potential non-specific binding of the
antibodies to the Protein A found on the surface of S. aureus.
Generally, several dilutions of test antibody were used in each
assay. From each antibody dilution, 50 .mu.l of the
antibody-Protein A-HRP complex was added to replicate wells and the
mixture of bacteria and antibody-Protein A-HRP complex was
incubated at 37.degree. C. for 30-60 minutes with gentle rotation
(50-75 rpm) on an orbital shaker.
[0093] Following the incubation, the bacteria were pelleted in the
plate by centrifugation at 1800-2000.times.g. The supernatant was
carefully removed from the wells and 200 .mu.l of PBS/BSA
containing 0.05% Tween-20 (PBS/BSA/Tween) was added to all wells to
dilute unbound reagents. The bacteria were again pelleted by
centrifugation and the supernatant was removed. One hundred
microliters of TMB substrate (BioFx, Inc. cat. no. TMBW-0100-01)
was added to each well and the reactions were allowed to proceed
for 15 minutes at room temperature. The reactions were stopped by
adding 100 .mu.l of TMB stop reagent (450 nm Stop Reagent; BioFx,
Inc. catalog no. STPR-0100-01). The absorbance of each well was
determined using a microplate reader fitted with a 450 nm
filter.
[0094] In this assay, the intensity of the color development was
directly proportional to the binding of the antibodies to the
bacteria. Control wells contained bacteria and Protein A-HRP
without antibody.
[0095] Immunoassay on Methanol-Fixed Bacteria: Heat-killed bacteria
were suspended in sterile 0.9% sodium chloride (Sigma cat. no.
S8776, or equivalent) at a % transmittance (% T) of 70-75% at 650
nm. Ten milliliters the bacterial suspension was diluted 15-fold in
sterile 0.9% sodium chloride and then pelleted by centrifugation at
1800.times.g for 15 minutes at 10-15.degree. C. The supernatant was
discarded and the pellet was resuspended in 1500 ml of methanol
(MeOH). One hundred microliters of the bacteria-MeOH suspension was
distributed into each well of Nunc Maxisorp Stripwells (Nunc
catalog no. 469949). The MeOH was allowed to evaporate, fixing the
bacteria to the plastic wells. The bacteria-coated stripwells were
stored in plastic bags in the dark at room temperature and used
within 2 months of preparation.
[0096] For evaluation of antibodies, the bacteria-coated plates w
ere washed four times with phosphate buffered saline containing
0.05% Tween-20 (PBS-T) as follows. Approximately 250 .mu.l of PBS-T
was added to each well. The buffer was removed by flicking the
plate over the sink and the remaining buffer removed by inverting
the plate and tapping it on absorbent paper. The antibody was
diluted in PBS-T and then added to the wells. Supernatants,
ascites, or purified antibodies were tested at the dilutions
indicated in the Examples. Control wells received PBS-T alone.
After addition of the antibody, the wells were incubated at room
temperature for 30-60 minutes in a draft-free environment. The
wells were again washed four times with PBS-T. Ninety-five
microliters of detection antibody was then added to each well. The
detection antibody was one of the following: rabbit anti-mouse
IgG.sub.3, rabbit anti-mouse IgM, or goat anti-human IgG
(gamma-specific), all conjugated to horse radish peroxidase (HRP)
and diluted 1:6000 in PBS-T (Zymed catalog numbers 61-0420, 61-6820
and 62-8420, respectively).
[0097] Following another 30-60 minute incubation at room
temperature, the wells were washed four times with PBS-T and each
well received 100 .mu.l of TMB substrate solution (BioFx
#TMBW-0100-01). Plates were incubated in the dark at room
temperature for 15 minutes and the reactions were stopped by the
addition of 100 .mu.l of TMB stop solution (BioFx #STPR-0100-01).
The absorbance of each well was measured at 450 nm using a
Molecular Devices Vmax plate reader.
[0098] Immunoassay with Protein A: In order to evaluate the binding
of the MAbs to S. aureus, the immunoassay procedure was modified
for methanol-fixed bacteria, described above. Because S. aureus
expresses Protein A on its surface, and Protein A binds strongly to
the constant region of the heavy chains of gamma-globulins, it is
possible that false positive results may be obtained from
non-specific binding of the antibodies to Protein A. To overcome
this difficulty, the immunoassay wells were coated with bacteria as
described above, but prior to the addition of the antibodies to the
bacteria-coated wells, the MAbs were incubated with a solution of
recombinant Protein A conjugated to HRP (Zymed Laboratories Cat.
No. 10-1123), diluted 1:8000 in PBS-T. The binding reaction was
allowed to proceed for 30 minutes at room temperature. The wells
were washed four times with PBS-T and 100 .mu.l of the solution of
each Protein A-HRP-MAb combination was added to the wells. The
presence of the Protein A-HRP from the pretreatment blocked the
MAbs from binding to the Protein A on the S. aureus. Furthermore,
the binding of the Protein A-HRP to the constant region of the
heavy chain did not interfere with the antibody binding site on the
MAbs, thereby allowing evaluation of the MAbs on S. aureus and
other bacteria.
[0099] The Protein A-HRP-MAb solutions were allowed to bind in the
coated wells for 30-60 minutes at room temperature. The wells were
then washed with PBS-T and TMB substrate solution was added and the
assay completed as described above.
[0100] Immunoassay on LTA and PepG: The binding of the MAbs to LTA
was measured by immunoassay on wells coated with S. aureus LTA
(Sigma Cat. No. 2515). One hundred microliters of a 1 .mu.g/ml LTA
solution in PBS was distributed into replicate Nunc Maxisorp
Stripwells and incubated overnight at room temperature. The unbound
material was removed from the wells by washing four times with
PBS-T. Antibody, diluted in PBS-T, was then added to the wells and
the assay continued as described above for the Immunoassay on
Methanol-Fixed Bacteria.
[0101] For immunoassays on PepG, Nunc Maxisorp Stripwell plates
were coated with 100 .mu.l of a 5 .mu.g/ml solution of PepG (S.
Foster; also can be prepared by the procedure set forth in Example
2) in 0.1 M carbonate buffer (pH 9.2-9.6) overnight at room
temperature. Unbound antigen was removed from the plate by washing
four times with PBS-T. Sample supernatants, ascites, or antibodies,
diluted in PBS-T, were added to replicate wells. The plate was
covered with a plate sealer and incubated for 30-60 minutes at room
temperature in a draft-free environment. The plate was again washed
with PBS-T, and 95 .mu.l of gamma-specific Rabbit anti-Mouse IgG,
conjugated to horseradish peroxidase (HRP) (Zymed Laboratories) was
added to all wells. The plate was again covered and incubated in a
draft-free environment for 30-60 minutes at room temperature. The
plate was washed with PBS-T and 100 .mu.l of TMB substrate solution
was added to each well. After a 15 minute incubation at room
temperature in the dark, 100 .mu.l of TMB stop solution was added
to all wells and the absorbance of each well was measured using a
Molecular Devices V.sub.max plate reader with a 450 nm filter.
Activity Assays
[0102] Antibodies that bind to an antigen may not necessarily
enhance opsonization or enhance protection from infection.
Therefore, an opsonization assay was used to determine the
functional activities of the antibodies.
[0103] An opsonization assay can be a calorimetric assay, a
chemiluminescent assay, a fluorescent or radiolabel uptake assay, a
cell-mediated bactericidal assay, or any other appropriate assay
known in the art which measures the opsonic potential of a
substance and thereby identifies reactive immunoglobulin. In an
opsonization assay, an infectious agent, a eukaryotic cell, and the
opsonizing substance to be tested, or an opsonizing substance plus
a purported opsonizing enhancing substance, are incubated
together.
[0104] In certain embodiments, the opsonization assay is a
cell-mediated bactericidal assay. In this in vitro assay, an
infectious agent such as a bacterium, a phagocytic cell, and an
opsonizing substance, such as immunoglobulin, are incubated
together. Any eukaryotic cell with phagocytic or binding ability
may be used in a cell-mediated bactericidal assay. In certain
embodiments, phagocytic cells are macrophages, monocytes,
neutrophils, or any combination of these cells. Complement proteins
may be included to promote opsonization by both the classical and
alternate pathways.
[0105] The amount or number of infectious agents remaining after
incubation determines the opsonic ability of an antibody. The fewer
the number of infectious agents that remain after incubation, the
greater the opsonic activity of the antibody tested. In a
cell-mediated bactericidal assay, opsonic activity is measured by
comparing the number of surviving bacteria between two similar
assays, only one of which contains the antibody being tested.
Alternatively, opsonic activity is determined by measuring the
number of viable organisms before and after incubation with a
sample antibody. A reduced number of bacteria after incubation in
the presence of antibody indicates a positive opsonizing activity.
In the cell-mediated bactericidal assay, positive opsonization is
determined by culturing the incubation mixture under appropriate
bacterial growth conditions. Any reduction in the number of viable
bacteria comparing pre-incubation and post-incubation samples, or
between samples that contain immunoglobulin and those that do not,
is a positive reaction.
[0106] Neutraphil-Mediated Opsonophagocytic Bactericidal Assay: The
assay was performed using neutrophils isolated from adult venous
blood by sedimentation using PMN Separation Medium (Robbins
Scientific catalog no. 1068-00-0). Forty microliters of antibody,
serum, or other immunoglobulin source, was added at various
dilutions to replicate wells of a round-bottom microtiter plate.
Forty microliters of neutrophils (approximately 10.sup.6 cells per
well) was then added to each well, followed immediately by
approximately 3.times.10.sup.4 mid-log phase bacteria (S.
epidermidis strain Hay, ATCC 55133 or S. aureus type 5, ATCC 49521)
in 10 .mu.l Tryptic Soy Broth (Difco cat. no. 9063-74, or
equivalent). Finally, 10 .mu.l of immunoglobulin-depleted human
serum was added as a source of active complement. (Immunoglobulins
were removed from human serum complement by preincubating the serum
with Protein G-agarose and Protein L-agarose before use in the
assay. This depletion of immunoglobulins minimized the
concentrations of anti-staphylococcal antibodies in the complement,
thereby reducing bacterial killing caused by inherent antibodies in
the complement solution.)
[0107] The plates were incubated at 37.degree. C. with constant,
vigorous shaking. Aliquots of 10 .mu.l were taken from each well at
zero time, when the sample antibody was first added, and after 2
hours of incubation. To determine the number of viable bacteria in
each aliquot harvested from each sample well, each aliquot was
diluted 20-fold in a solution of 0.1% BSA in water (to lyse the
PMNs), mixed vigorously by rapid pipetting, and cultured on blood
agar plates (Remel, cat. no. 01-202, or equivalent) overnight at
37.degree. C. The opsonic activity was measured by comparing the
number of bacterial colonies observed from the sample taken at two
hours with the number of bacterial colonies observed from the
sample taken at time zero. Colonies were enumerated using an IPI
Minicount Colony Counter.
[0108] This cell-mediated bactericidal assay has been correlated
with in vivo efficacy, as set forth in Examples 11 and 12 of U.S.
Pat. No. 5,571,511.
[0109] Nasal Colonization Assay: The mouse nasal colonization model
for S. aureus was based on the work of Kiser et al. (11). Briefly,
streptomycin resistant S. aureus type 5 is grown on high salt
Columbia agar (Difco) to promote capsule formation. The bacteria
are washed with sterile saline (0.9% NaCl in water) to remove media
components and resuspended at .about.10.sup.8 bacteria/animal dose
in saline (0.9% NaCl in water) containing various concentrations
and combinations of anti-staphylococcal or irrelevant control MAbs.
Following one hour preincubation, the bacteria are pelleted and
resuspended in a final volume of 10 .mu.l per animal dose in either
saline or saline containing antibody. Mice that have been
maintained on streptomycin-containing water for 24 hours are
sedated with anesthesia. Staphylococci are instilled into the nares
of the mice by pipetting without contacting the nose.
[0110] After four to seven days, during which the animals are
maintained on streptomycin-containing water, the animals are
sacrificed and the noses removed surgically and dissected. Nasal
tissue is vortexed vigorously in saline (0.9% NaCl in water) plus
0.5% Tween-20 to release adherent bacteria and the saline is plated
on Columbia blood agar (Remel) and tryptic soy agar (Difco)
containing streptomycin to determine colonization.
[0111] The invention, having been described above, may be better
understood by reference to examples. The following examples are
intended for illustration purposes only, and should not be
construed as limiting the scope of the invention in any way.
EXAMPLES
Example 1
The Production of Hybridomas and Monoclonal Antibodies to S. aureus
PepG Immunization of Mice
[0112] To produce monoclonal antibodies directed against S. aureus
PepG, immunizations were carried out using 5-6 week old female
BALB/c mice, obtained from Harlan Sprague Dawley (Indianapolis,
Ind.). The immunogen for the primary immunization was S. aureus
PepG (gift from Roman Dziarski; PepG can also prepared as described
in Example 2). Five microliters of PepG (7 mg/ml suspension) was
mixed with 345 .mu.l of PBS and 350 .mu.l of RIBI adjuvant (RIBI
Immunochemicals, Hamilton, N.H.). The resulting suspension
contained 50 .mu.g/ml of PepG. Each mouse was immunized with a
subcutaneous (sc) dose of 0.1 ml (5 .mu.g per mouse).
[0113] Approximately four weeks following the initial immunization,
a booster immunization was given. PBS (873 .mu.l) was mixed with
7.1 .mu.l of PepG (7 mg/ml suspension) and 120 .mu.l of Alhydrogel
(Accurate Chemical and Scientific, Co., Westbury, N.Y.). Each mouse
received an sc dose of 0.1 ml (5 .mu.g of PepG per mouse).
[0114] After an additional eight weeks, the mice were immunized
with a 50 .mu.g/ml solution containing 50% Alum adjuvant (Pierce
Cat. No. 77161) in PBS (0.2 ml/mouse). Sera from the immunized mice
were tested by ELISA as described above. As shown in Table 1, serum
from mouse 8813 bound most strongly to PepG. This mouse was given a
final, pre-fusion, intraperitoneal boost of 10 .mu.g PepG in PBS
three days prior to the generation of hybridomas. TABLE-US-00001
TABLE 1 PepG ELISA of Sera from Mice Immunized with PepG Mouse ID
Serum Dilution Serum Sample #1.sup.a Serum Sample #2.sup.b Buffer
100 0.107 0.125 8810 100 0.341 0.339 8811 100 0.219 0.215 8812 100
0.267 0.249 8813 100 0.308 2.143 8814 100 0.223 0.296 .sup.aSample
was taken 9 weeks after the first immunization. .sup.bSample was
taken 18 weeks after the first immunization.
Generation of Hybridomas
[0115] Hybridomas were prepared by the general methods of Shulman,
Wilde and Kohler and Bartal, A. H. and Hirshaut (2, 28). Spleen
cells from mouse 8813 were mixed with SP2/0 mouse myeloma cells
(ATCC No. CRL1581) at a ratio of 10 spleen cells per SP2/0 cell,
pelleted by centrifugation (400.times.g, 10 minutes at room
temperature) and washed in serum free DMEM (Hyclone cat. no.
SH30081.01, or equivalent). The supernatant was removed and fusion
of the cell mixture was accomplished in a sterile 50 ml centrifuge
conical by the addition of 1 ml of a 50% w/v solution of
polyethylene glycol (PEG; mw 1500; Boehringer Mannheim cat. no.
783641) over a period of 60-90 seconds. Serum free medium was then
added slowly in successive volumes of 1, 2, 4, 8, 16 and 19 ml. The
hybridoma cell suspension was gently resuspended into the medium
and the cells pelleted by centrifugation (500.times.g, 10 minutes
at room temperature). The supernatant was removed and the cells
resuspended in RPMI 1640, supplemented with 10% heat-inactivated
fetal bovine serum, 0.05 mM hypoxanthine and 16 .mu.M thymidine (HT
medium; Life Technologies cat. no. 11067-030, or equivalent). One
hundred microliters of the hybridoma suspension cells were plated
into 96-well tissue culture plates. Eight wells (column 1 of plate
A) received approximately 2.5.times.10.sup.4 SP2/0 cells in 100
.mu.l. The SP2/0 cells served as a control for killing by the
selection medium added 24 hours later.
[0116] Twenty-four hours after preparation of the hybridomas, 100
.mu.l of RPMI 1640, supplemented with 10% heat-inactivated fetal
bovine serum, 0.1 mM hypoxanthine, 0.8 .mu.M aminopterin and 32
.mu.M thymidine (HAT medium, Life Technologies cat. no. 11067-030,
or equivalent) was added to each well.
[0117] Ninety-six hours after the preparation of the hybridomas,
the SP2/0 cells in plate A, column 1 were dead, indicating that the
HAT selection medium had successfully killed the unfused SP2/0
cells. Twelve days after the preparation of the hybridomas,
supernatants from all wells were tested by ELISA for the presence
of antibodies that bind to PepG.
[0118] Based on the results of this preliminary assay, cells from
28 of the original 760 wells were transferred to a 24-well culture
dishes. Four days later, supernatant from these cultures were
retested by ELISA for the presence of antibodies that bind to PepG,
using isotype-specific testing reagents. Briefly, supernatants from
the cultures were added to 96-well plates coated with PepG and
allowed to bind. To simultaneously detect binding of the antibodies
to PepG, and the isotype of the antibodies, replicate wells were
incubated with HRP-conjugated rabbit anti-mouse IgA, HRP-conjugated
rabbit anti-mouse IgG, and HRP-conjugated rabbit anti-mouse IgM.
The wells were then washed and developed by standard methods. As
shown in Table 2, one of the cultures (99-110CF10) produced an IgG
antibody and was passaged further. In addition, fifteen other
cultures produced IgM antibodies and were also passaged further.
TABLE-US-00002 TABLE 2 PepG ELISA Assay of 99-110 Supernatants
Supernatant Supernatant ID Dilution Isotype Absorbance Buffer 2 M
0.077 99-110AD2 2 M 2.020 99-110AF5 2 M 1.741 99-110AA10 2 M 2.924
99-110BG8 2 M 3.702 99-110BA11 2 M 2.168 99-110CB2 2 M 3.747
99-110CG2 2 M 2.465 99-110DA4 2 M 2.606 99-110DF6 2 M 3.211
99-110DA10 2 M 3.570 99-110DA11 2 M 3.333 99-110EE4 2 M 1.131
99-110FC12 2 M 4.000 99-110GH4 2 M 4.000 99-110GC8 2 M 3.732
99-110CF10 2 G 0.102
[0119] Cultures 99-110CF10 and 99-110FC12 were subcloned by
limiting dilution, as follows. Hybridomas were enumerated using a
hemocytometer and adjusted to a concentration of 225 cells/ml. One
ml of the cell solution was then mixed with 36 ml of RPMI 1640
medium, 7.5 ml of heat-inactivated fetal bovine serum, 0.5 ml of 10
mg/ml kanamycin solution (GIBCO BRL cat #15160-054), and 5 ml of
Hybridoma Serum Free Medium (Life Technologies Cat. No. 12045-084).
The resulting suspension contained 4.5 cells/ml. Two hundred
microliters of this suspension was then added to each well of two
96-well culture dishes. As shown in Table 2 and Table 3, culture
99-110CF10 did not produce antibodies that bound to PepG.
Subsequent subclones of culture 99-110CF10 likewise did not elicit
PepG-specific antibodies. TABLE-US-00003 TABLE 3 PepG ELISA of
99-110CF10 Clones Clone Supernatant ID Dilution Absorbance Buffer
0.067 CF10IC1 2 0.170 CF10IE1 2 0.161 CF10IG1 2 0.161 CF10IH2 2
0.153 CF10ID3 2 0.135 CF10IC7 2 0.129 CF10IF10 2 0.129
[0120] The clones from culture 99-110FC12, shown in Table 4,
continued to produce IgM antibodies that bound to PepG. Thirty-two
clones from 99-110FC12 were tested by ELISA on plates coated with
PepG. Of these, 31 were strongly positive, producing absorbance
values of 3.159 or greater. Four clones, designated 99-110FC12 IE4,
ID3, IIH5 and IIC6 were expanded and cryopreserved. Clone IE4 was
selected for additional analysis. TABLE-US-00004 TABLE 4 PepG ELISA
99-110FC12 Clones Clone Supernatant ID Dilution Absorbance Buffer
0.039 FC121F1 2 3.495 FC12IE2 2 2.651 FC12ID3 2 4.000 FC12IF3 2
3.159 FC12IG3 2 3.811 FC12IE4 2 4.000 FC12ID6 2 3.937 FC12IE6 2
4.000 FC12IC7 2 0.074 FC12IH7 2 4.000 FC12IF8 2 4.000 FC12IG8 2
3.533 FC12IF11 2 4.000 FC12IG11 2 4.000 FC12IA12 2 4.000 FC12IIB2 2
3.456 FC12IID2 2 4.000 FC12IIG2 2 4.000 FC12IIF3 2 4.000 FC12IIG3 2
3.379 FC12IIB4 2 4.000 FC12IIG4 2 4.000 FC12IIA5 2 3.887 FC12IIH5 2
4.000 FC12IIC6 2 4.000 FC12IIE6 2 3.756 FC12IIG6 2 4.000 FC12IIA10
2 3.844 FC12IIC10 2 3.450 FC12IIH10 2 3.980 FC12IIH11 2 4.000
[0121] Clone 99-110FC12 IE4 was grown in an Integra Biosystems
Culture system, designed to produce high quantities of
immunoglobulin in culture supernatants. Supernatant from the IE4
clone was tested by ELISA on wells coated with methanol-fixed S.
epidermidis strain Hay, PepG, and LTA. As shown in Table 5, the
antibody bound strongly to S. aureus PepG, but not to the
methanol-fixed bacteria, or to S. aureus LTA. TABLE-US-00005 TABLE
5 Binding of 99-110FC12 IE4 supernatant by ELISA Supernatant On S.
aureus On S. aureus On methanol-Fixed Dilution PepG LTA S. epi.
Strain Hay 10 3.789 0.051 0.078 30 3.983 0.052 0.075 90 3.974 0.048
0.073 270 4.00 0.047 0.069 810 3.858 0.044 0.065 PBS-T 0.044 0.045
0.056
Example 2
Production of Hybridomas and Monoclonal Antibodies to B. subtilis
PepG Purification of Peptidoglycan
[0122] Bacillus subtilis HR (gift of Howard Roger, University of
Kent, UK) vegetative cell walls were made as previously described
under stringent conditions, using lipopolysaccharide-free materials
(6, 38), which are herein incorporated for any purpose). Proteins
were removed from the peptidoglycan by treatment with pronase, and
teichoic acid and other attached polymers were removed by treatment
with HF (48% v/v) for 24 h at 4.degree. C. The insoluble
peptidoglycan was pelleted by centrifugation (13,000 g, 5 min,
4.degree. C.) and resuspended in distilled water to 2 mg/ml PepG.
This step was repeated once. The peptidoglycan was then pelleted
and resuspended in 50 mM Tris HCl pH7.5 to 2 mg/ml PepG, and this
step was repeated once. Finally, the peptidoglycan was pelleted and
resuspended in distilled water to 2 mg/ml PepG three more times.
The peptidoglycan was resuspended at about 10 mg/ml in distilled
water and stored at -20.degree. C.
[0123] PepG preparations were analyzed as previously described by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), which revealed no evidence of contaminating protein
(39). The purity of the peptidoglycan was further verified by amino
acid analysis of hydrolyzed PepG, which gave only the expected
amino acids, and by reverse phase chromatography analysis of
enzymatically digested material, both assays performed as
previously described (1). This level of purity had previously not
been ensured during the production of anti-peptidoglycan
antibodies.
Preparation of Muropeptide Conjugate for Immunization
[0124] Peptidoglycan conjugate was made as per the manufacturers
protocol for the Imject SuperCarrier EDC System for Peptides
(Pierce, cat. no. 77152).
[0125] Muropeptides were made by Cellosyl digestion. Cellosyl is a
muramidase that cleaves the bond between N-acetylmuramic acid and
N-acetylglucosamine in the glycan backbone of PepG. Complete
cellosyl digestion of PepG results in the production of small,
soluble muropeptides. One milliliter of 11.5 mg/ml purified
peptidoglycan was harvested by centrifugation (13,000 g, 5 min,
4.degree. C.) and resuspended in 1 ml conjugation buffer, supplied
with the Imject SuperCarrier EDC System for Peptides (Pierce, cat.
no. 77152). Twenty-five microliters Cellosyl (2 mg/ml; Hoechst) was
added to the PepG suspension and incubated at 37.degree. C. with
rotary mixing for 7 hours. The sample was then boiled for 10 min
and any insoluble material removed by centrifugation as above.
Three hundred microliters of cellosyl-digested PepG was added to
200 .mu.l of conjugation buffer.
[0126] Preparation of SuperCarrier (Pierce, cat. no. 77152),
conjugation and purification of conjugate were performed as per
manufacturers protocol. The PepG conjugate was stored at
-20.degree. C.
Immunization of Mice
[0127] To produce monoclonal antibodies directed against B.
subtilis PepG, immunizations were carried out using four 8-12 week
old BALB/C mice, obtained from Sheffield University Field
Laboratories. The immunogen for the primary immunization was B.
subtilis PepG, prepared as muropeptide conjugate (described above).
For each mouse, 50 .mu.g of conjugate in 50 .mu.L of PBS was mixed
with 50 .mu.l of Freund's complete adjuvant, and this mixture was
injected subcutaneously.
[0128] At about day 14, 29, 113, and 232 following the primary
immunization, each mouse was injected intraperitoneally with 50
.mu.g of conjugate in 50 .mu.L of PBS, which had been mixed with 50
.mu.l of Freund's incomplete adjuvant. Hybridomas were generated
four days after the final boost of PepG conjugate.
Generation of Hybridomas
[0129] Hybridomas were prepared by the general methods of Shulman,
Wilde and Kohler and Bartal and Hirshaut (2, 28). Spleen cells from
immunized mice 1 and 2 were pooled and were mixed with SP2/0 mouse
myeloma cells at a ratio of 5 spleen cells per SP2/0 cell, pelleted
by centrifugation (770.times.g, 5 minutes at 30.degree. C.) and
washed in serum-free RPMI. One milliliter of PEG 1500 (50% in 75 mM
HEPES, Boehringer cat. no. 783 641) was added over 1 minute,
followed by 1 ml of RPMI over 1 minute, and then 9 ml of RPMI over
2 minutes. Cells were then pelleted by centrifugation at
430.times.g for 15 minutes at 30.degree. C. Cells were resuspended
in RPMI-1640/HAT (1 ml of 50.times.HAT concentrate (Invitrogen cat
no. 21060-017) in 50 ml RPMI-1640) containing 20% FCS at
approximately 1.times.10.sup.6 cells/ml. One hundred microliters of
cell suspension was added to each well of ten 96-well plates and
grown at 37.degree. C. Unfused SP2/0 cells were used as a selection
control and died 5 days after plating.
[0130] Thirteen days after preparation of the hybridomas,
supernatants from all wells were assayed by ELISA for the presence
of antibodies that bind PepG. Thirteen of 829 wells tested were
positive.
[0131] Based on the results of the ELISA, positive cells were
expanded in a 24-well culture plate and retested for stable
antibody secretion by ELISA. Six lines were found to secrete
antibodies to PepG. Line BB4 was found to produce antibodies with
the highest affinity to B. subtilis cell walls. At about 75%
confluence, BB4 was cloned by limiting dilution and the subclones
retested for anti-PepG antibody secretion. Two clones, BB4/A4 and
BB4/A5, were found to secrete antibodies to B. subtilis PepG.
Isotype determination, using Isotype Strips (Roche Diagnostics,
cat. no. 1493027), showed that both antibodies were IgG.
Example 3
Substrate Affinities of PepG Antibodies
Cellosyl Digestion of PepG
[0132] PepG from Bacillus subtilis, Staphylococcus aureus,
Streptococcus mutans, Bacillus megaterium, Enterococcus faecalis,
Staphylococcus epidermidis, and Listeria monocytogenes was purified
as described in Example 2. One milliliter of each PepG (10 mg/ml)
in 25 mM sodium phosphate buffer pH5.6 was digested with 250
.mu.g/ml cellosyl (Hoechst AG) for 15 hours at 37.degree. C. The
samples were boiled for 3 minutes to stop the reaction and
insoluble material was removed by centrifugation (14,000.times.g
for 8 minutes at room temperature). The soluble cellosyl-digested
PepG was stored at -20.degree. C.
[0133] Staphylococcal PepG has a unique pentaglycine crossbridge,
which can be cleaved by lysostaphin, a glycine-glycine
endopeptidase. Lysostaphin (25 .mu.g/ml; Sigma Cat No. L0761) was
added to the cellosyl digestion of S. aureus and S. epidermidis
PepG to cleave this peptide cross bridge. S. aureus was also
digested without lysostaphin.
ELISA to Determine Antibody Affinities for PepG and
Cellosyl-Digested PepG
[0134] Three hybridoma lines, identified as 11-232.3, 11-248.2, and
11-569.3 (QED Biosciences), were produced by immunizing mice with
UV-inactivated whole S. aureus, and the MAbs they produce were
subsequently shown to bind to PepG. The affinities of the MAbs
produced by 11-232.3 (when purified, the MAb is referred to as
702PG), 11-248.2, and 11-569.3, BB4/A4, and BB4/A5 for PepG from
various bacteria, and cellosyl-digested PepG from various bacteria,
were compared by ELISA as follows. 96-well immunoassay plates (NUNC
Immunoplate Maxisorp) were coated with 100 .mu.g/ml poly-L-lysine
(Sigma Chemicals, cat. no. P6407) in 0.05 M carbonate/bicarbonate
buffer pH 9.6 (0.015 M Na.sub.2CO.sub.3, 0.035 M NaHCO.sub.3, pH
9.6, hereafter referred to as carbonate buffer) for 1 hour at room
temperature. The carbonate buffer was removed, and the wells were
washed once with carbonate buffer. The wells were then coated with
100 .mu.l 5 .mu.g/ml purified PepG substrate, or cellosyl-digested
PepG substrate, in carbonate buffer overnight at 4.degree. C. The
substrate solution was removed, and the wells were washed twice
with PBS-T. The wells were then blocked with 150 .mu.l PBS-T
containing 0.2% w/v bovine gelatin (Sigma cat. no. A7030; blocking
buffer) for 2 hours at 37.degree. C. The blocking buffer was
removed and the wells were washed four times with PBS-T.
[0135] Fifty microliters of one of the MAbs listed above (diluted
appropriately in blocking buffer) was added to each well and the
binding reaction was incubated for 2 hours at 37.degree. C. The
monoclonal antibody was removed and the wells were washed three
times in PBS-T. Fifty microliters of HRP-conjugated goat anti-mouse
IgG (Biorad), diluted 1:20,000 in blocking buffer, was added to
each well and the binding reaction was incubated for 1 hour at
37.degree. C. The secondary antibody was removed and the wells were
washed three times with PBS-T. Fifty microliters of TMB enzyme
substrate (Biorad cat. no. 172-1068) was added to each well and the
color was developed for fifteen minutes at room temperature. The
reaction was stopped by addition of 50 .mu.l of 2M H.sub.2SO.sub.4
to each well, and the absorbance was read on a VICTOR plate reader
(Wallac) at 450 nm. The results of the ELISA are shown in Table
6.
[0136] Table 6 demonstrates that the PepG antibodies described are
not identical, as each shows a different range of specificity and
affinity for the different PepG substrates. Specifically, 702PG,
MAb-11-232.3, and MAb-11-248.2 show high affinity for S. aureus
PepG, and low affinity for B. subtilis PepG, while the antibodies
produced by BB4/A4 and BB4/A5 (also called MAb-BB4/A4 and
MAb-BB4/A5) show the reverse specificity. MAb-BB4/A4 and MAb-BB4/A5
also show high affinity for S. epidermidis, while the others do
not. TABLE-US-00006 TABLE 6 Binding of PepG antibodies to bacterial
substrates Conc. of MAb (.mu.g/ml) that gave reading >0.1 at 450
nm. PepG from: 702 PG * 11-232.3 11-248.2 11-569.3 BB4/A4 BB4/A5
Bacillus subtilis 168 HR 1000 1000 >1000 100 <1 <1
Staphylococcus aureus <1 <1 <1 100 100 100 8325/4
Streptococcus mutans LTII >1000 1000 >1000 100 100 100
Bacillus megaterium KM >1000 >1000 >1000 1000 1000 1000
spore cortex Enterococcus faecalis >1000 >1000 >1000 100
100 1000 NCTC 775 Staphylococcus epidermidis 100 100 100 100 <1
<1 138 Listeria monocytogenes >1000 >1000 >1000 1000
1000 1000 EGD Bacillus subtilis 168 HR >1000 1000 >1000 100
100 100 Cellosyl digested Staphylococcus aureus >1000 1000 100
100 100 100 8325/4 Cellosyl digested Streptococcus mutans LTII
>1000 1000 >1000 100 100 10 Cellosyl digested Bacillus
megaterium KM >1000 >1000 >1000 100 100 1000 spore cortex
Cellosyl digested Enterococcus faecalis >1000 >1000 >1000
100 100 100 NCTC 775 Cellosyl digested Staphylococcus 100 >1000
100 1000 100 100 epidermidis 138 Cellosyl and lysostaphin digested
Listeria monocytogenes >1000 >1000 >1000 100 100 1000 EGD
Cellosyl digested Staphylococcus aureus 100 >1000 >1000 1000
1000 1000 8325/4 Cellosyl and lysostaphin digested Bacillus
subtilis 168 HR was a gift from Howard Roger, University of Kent,
U.K. Staphylococcus aureus 8325/4 was a gift from Richard Novick,
Skirball Institute, NY, U.S.A. Streptococcus mutans LTII was a gift
from Roy Russell, University of Newcastle, U.K. Bacillus megaterium
KM was a gift from Keith Johnstone, University of Cambridge, U.K.
Staphylococcus epidermidis 138 was a gift from Paul Williams,
University of Nottingham, U.K. Listeria monocytogenes EGD was a
gift from W. Goebel, University of Wurzburg, Germany
[0137] Cellosyl digestion, which cleaves glycan strands, of any of
the PepG substrates abrogates binding of antibodies 702PG,
MAb-11-232.3, MAb-11-248.2, MAb-BB4/A4, and MAb-BB4/A5. Thus, these
antibodies may interact with an epitope that requires an intact
glycan strand. The affinity of MAb-11-569.3, on the other hand, is
unaffected by cellosyl digestion, indicating that it may interact
with an epitope that is not associated with the bond that is
cleaved. The cellosyl/lysostaphin results further confirm the
single digest results.
[0138] Finally, S. aureus PepG has a higher level of O-acetylation
on glucosamine residues than PepG from B. subtilis, suggesting that
this O-acetylation may be important for the binding of antibodies
702PG, MAb-11-232.3, and MAb-11-248.2, and may negatively affect
the binding of MAb-BB4/A4 and MAb-BB4/A5.
Example 4
Binding of the Monoclonal Antibodies to LTA, PepG and
Staphylococci
[0139] MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, and MAb-99-110FC12
IE4 were assayed for binding to S. aureus PepG, S. aureus LTA, and
to methanol-fixed S. aureus and methanol-fixed S. epidermidis. In
addition to those MAbs, a human/mouse chimeric anti-LTA antibody,
referred to as A110, was included in the assays as a positive
control for LTA and S. epidermidis binding. A description of the
production and chimerization of A110 can be found in U.S. patent
application Ser. No. 09/097,055, filed Jun. 15, 1998.
[0140] As shown in Table 7, MAb-11-232.3, 11-248.2 ascites, and
99-110FC12 IE4 supernatant all bound strongly to S. aureus PepG. As
expected, A110, the anti-LTA antibody, does not bind to PepG.
TABLE-US-00007 TABLE 7 Binding of MAbs on Wells Coated with S.
aureus Peptidoglycan Purified 11-232.3 11-569.3 *A110 11-248.2
99-110FC12 Antibody Purified Purified Purified Ascites Ascites
Supernatant IE4 sup. (.mu.g/ml) Ms IgG.sub.3 Ms IgG.sub.3 Hu
IgG.sub.1 Dilution Ms IgM Dilution Ms IgM 3 3.674 0.449 0.086 100
3.903 10 3.789 1 3.642 0.311 0.077 300 4.000 30 3.983 0.33 3.659
0.160 0.069 900 4.000 90 3.974 0.11 3.085 0.104 0.066 2700 4.000
270 4.000 0.037 2.113 0.086 0.068 8100 3.902 810 3.858 None 0.074
0.069 0.103 None 0.059 None 0.070 *Negative anti-LTA Control
[0141] When tested on LTA, as shown in Table 8, only A110 showed
strong binding. No binding was obtained with MAb-11-232.3,
MAb-11-569.3, and 99-110FC12 IE4 supernatant. Slight
cross-reactivity was obtained with 11-248.2 ascites at a 1:100
dilution, which may be due to high concentrations of non-specific
immunoglobulins in the ascites. TABLE-US-00008 TABLE 8 Binding of
MAbs on Wells Coated with S. aureus LTA Purified 11-232.3 11-569.3
*A110 11-248.2 99-110FC12 Antibody Purified Purified Purified
Ascites Ascites Supernatant IE4 sup. (.mu.g/ml) Ms IgG.sub.3 Ms
IgG.sub.3 Hu IgG.sub.1 Dilution Ms IgM Dilution Ms IgM 3 0.055
0.087 3.357 100 0.404 10 0.051 1 0.046 0.071 3.083 300 0.206 30
0.052 0.33 0.052 0.061 1.996 900 0.131 90 0.048 0.11 0.043 0.049
1.077 2700 0.092 270 0.047 0.037 0.045 0.045 0.411 8100 0.067 810
0.044 None 0.048 0.045 0.081 None 0.054 None 0.045 *Positive
control for LTA
[0142] These data indicated that MAb-11-232.3 and MAb-11-248.2,
which were raised to whole UV-killed S. aureus, are specific for
PepG on the surface of the bacteria. MAb-11-569.3, which was also
raised to UV-killed S. aureus, shows much weaker binding to PepG,
and no binding to LTA, indicating that it may be specific for PepG,
although it may also be specific for another surface antigen, but
cross-react with PepG. As expected, MAb-99-110FC12 IE4, which was
raised to purified S. aureus PepG, binds to PepG, but not LTA,
quite strongly.
[0143] Each of the MAbs was also tested on plates coated with
methanol-fixed S. epidermidis strain Hay and S. aureus, as shown in
Tables 9 and 10, respectively.
[0144] All of the antibodies, except MAb-99-110FC12 IE4, bound to
S. epidermidis strain Hay. Interestingly, MAb-11-569.3 bound to S.
epidermidis strain Hay more strongly than did MAb-11-232.3,
although MAb-11-569.3 bound less strongly to PepG and S. aureus
than did the MAb-11-232.3. This result indicates that the antigen
on the surface of S. aureus to which MAb-11-569.3 was raised, which
may or may not be PepG, is likely conserved between S. aureus and
S. epidermidis, resulting in strong binding by MAb-11-569.3 to both
bacteria. The IgM antibodies (from hybridomas 11-248.2 and
99-110FC12 IE4) were not tested against S. aureus, because the
immunoassay Protein A method used for the S. aureus-coated plates
does not work with IgM antibodies, which do not bind to protein.
TABLE-US-00009 TABLE 9 Binding of MAbs on Wells Coated with
methanol-Fixed S. epidermidis Strain Hay Purified 11-232.3 11-569.3
*A110 11-248.2 99-110FC12 Antibody Purified Purified Purified
Ascites Ascites Supernatant IE4 sup. (.mu.g/ml) Ms IgG.sub.3 Ms
IgG.sub.3 Hu IgG.sub.1 Dilution Ms IgM Dilution Ms IgM 3 0.761
3.044 1.412 100 2.905 10 0.078 1 0.518 2.672 1.324 300 2.809 30
0.075 0.33 0.301 1.733 1.058 900 2.749 90 0.073 0.11 0.150 0.476
0.664 2700 2.699 270 0.069 0.037 0.087 0.147 0.324 8100 2.288 810
0.065 None 0.054 0.052 0.089 None 0.056 None 0.056 *Anti-LTA
[0145] TABLE-US-00010 TABLE 10 Binding of MAbs on Wells Coated with
methanol-Fixed S. aureus Type 5 Purified 11-232.3 11-569.3 *A110
11-248.2 99-110FC12 Antibody Purified Purified Purified Ascites
Ascites Supernatant IE4 sup. (.mu.g/ml) Ms IgG.sub.3 Ms IgG.sub.3
Hu IgG.sub.1 Dilution Ms IgM Dilution Ms IgM 3 2.687 2.233 4.000 ND
ND ND ND 1 2.371 1.083 4.000 ND ND ND ND 0.33 1.541 0.330 4.000 ND
ND ND ND 0.11 0.596 0.144 3.671 ND ND ND ND 0.037 0.201 0.087 1.095
ND ND ND ND None 0.052 0.052 0.049 ND ND ND ND ND = not determined
*Anti-LTA
[0146] As noted above, peptidoglycan is a cell wall component found
in Gram-positive bacteria. These assays show MAb-11-232.3,
MAb-11-248.2, and MAb-99-110FC12 IE4 bind PepG strongly and do not
bind LTA, another cell wall component common to Gram-positive
bacteria. MAb-11-569.3 binds PepG less strongly in an ELISA (Table
7) than it binds S. aureus type 5 in a methanol-fixed ELISA (Table
10). Differences observed in the binding of the MAbs may be due to
the specific epitope bound by the MAbs and the presentation of that
epitope in protein and whole-bacteria ELISAs. Alternatively,
MAb-11-569.3 may bind to a different antigen, but cross-react with
PepG. MAb-11-232.3, 11-248.2 ascites, and MAb-11-569.3 also bind in
an ELISA assay to S. epidermidis strain Hay. Furthermore,
MAb-11-232.3 and MAb-11-569.3 also bind in an ELISA assay to S.
aureus (binding of 11-248.2 ascites in the ELISA to S. aureus could
not be determined). The lack of binding of 99-110FC12 IE4
supernatant to S. epidermidis strain Hay in the ELISA suggests that
this antibody binds an epitope found on S. aureus PepG, but not
expressed or available for binding in S. epidermidis strain
Hay.
Example 5
The Opsonophagocytic Activity of the Monoclonal Antibodies
[0147] Antibodies that bind to an antigen may not necessarily
enhance opsonization or enhance protection from infection.
Therefore, a neutrophil mediated bactericidal assay was used to
determine the functional activity of anti-PepG MAb against S.
aureus and S. epidermidis strain Hay. Neutrophils (PMNs) were
isolated from adult venous blood by using PMN separation medium
(Robbins Scientific Cat. No. 1068-00-0). Forty microliters of PMNs
were added to round-bottomed wells of micro titer plates
(approximately 2.times.10.sup.6 cells per well) with approximately
3.times.10.sup.4 mid-log phase bacteria. Human serum, treated with
Protein G and Protein L to remove antibodies that bind to S. aureus
and S. epidermidis strain Hay, was used as a source of active
complement. Forty microliters of antibody was added to the wells at
various dilutions and the plates were incubated at 37.degree. C.
with constant, vigorous shaking. Samples of 10 .mu.l were taken
from each well at zero time and after 2 hours of incubation. Each
was diluted, vigorously vortexed to disperse the bacteria, and
cultured on blood agar plates overnight at 37.degree. C. to
quantitate the number of viable bacteria.
[0148] Results are presented as percent reduction in numbers of
bacterial colonies observed compared to control samples. In an
opsonophagocytic bactericidal assay, 99-110FC12 IE4 supernatant was
active against S. aureus type 5, but not against S. epidermidis
strain Hay as shown in Table 11. TABLE-US-00011 TABLE 11
Opsonophagocytic Activity of 99-110FC12 IE4 supernatant Percent
Killed Antibody S. aur. S. epi. Dilution type 5 Hay neat + PMN + C
84 0 1:2 + PMN + C 80 N.D. 1:4 + PMN + C 68 0 PMN + C 30 9 MAb
alone 0 0 N.D. = not determined
[0149] Hybridoma 99-110FC12 IE4 was produced by immunization of
mice with PepG, while hybridomas 11-232.3, 11-248.2, and 11-569.3,
were produced by immunizing mice with UV-inactivated whole S.
aureus. Each of the anti-PepG MAbs from the hybridoma lines was
tested for activity in the opsonophagocytic bactericidal assay. In
addition, A110, which binds LTA, was also included in the assay.
The MAbs produced by 11-232.3 and 11-569.3 are mouse IgG.sub.3,
kappa light chain antibodies, and were purified before use. A110,
which is a human/mouse chimeric antibody with a human IgG.sub.1 and
a kappa light chain, was also purified before use. MAb-99-110FC12
IE4 and MAb-11-248.2 are mouse IgM, kappa light chain antibodies
and were used as either cell culture supernatant (99-110FC12 IE4)
or as ascites (11-248.2). Opsonic studies were performed to
determine if the MAbs enhanced phagocytosis and killing of both
groups of staphylococci.
[0150] As shown in Table 12A, each of the anti-PepG antibodies
demonstrated enhanced killing of S. aureus. When PMNs were mixed
with complement but without antibody, killing of the S. aureus was
less than 20%. However, addition of MAb-11-232.3 or MAb-11-569.3 at
100 .mu.g/ml resulted in killing of 76% and 82%, respectively. The
use of undiluted ascites from 11-248.2 (a mouse IgM) resulted in
killing of 89%, while 75% killing was obtained with neat
supernatant from 99-110FC12 IE4 (also a mouse IgM). Surprisingly,
although A110 binds strongly to S. aureus LTA (Table 8), and to
methanol-fixed S. aureus (Table 10), it shows very weak
opsonization of S. aureus in this assay. TABLE-US-00012 TABLE 12A
Opsonophagocytic Killing of S. aureus Type 5 By Monoclonal
Antibodies Antibody or Conc. Hybridoma Target (.mu.g/ml) % Killed
ID Isotype Antigen or Dilution S. aureus A110 Human IgG.sub.1, LTA
300 9 kappa 100 23 33.3 20 MAb-11-232.3 Mouse IgG.sub.3, Peptido-
100 76 kappa glycan 33.3 63 MAb-11-569.3 Mouse IgG.sub.3, Peptido-
100 82 kappa glycan 33.3 53 11-248.2 Mouse IgM, Peptido- Neat 89
ascites kappa glycan 1:4 49 1:16 49 99-110FC12IE4 Mouse IgM,
Peptido- Neat 75 supernatant kappa glycan 1:2 51 Background killing
(PMNs and complement without antibody) was less than 20% for all
assays
[0151] Previous assays have demonstrated stronger opsonization of
S. aureus by M110, the mouse monoclonal antibody from which A110 is
derived (Table 12B and U.S. patent application Ser. No.
09/097,055). We believe that the difference in activity between
A110 and M110 is due to dosage effects in the assays, rather than
activity differences between the chimeric and nonchimeric
antibodies. As demonstrated in Table 13, A110 retains its activity
against S. epidermidis. TABLE-US-00013 TABLE 12B Opsonophagocytic
Killing of S. aureus Type 5 by M110 Group Ab % Killed % Killed
Description Dilution S. epidermidis S. aureus C' only 0.0 0.0 PMN
only 0.0 0.0 PMN + C' No Ab 49.5 53.7 PMN + Ab + C' 10 -- 83.3 40
-- 78.9 80 100.0 61.0
[0152] When S. epidermidis strain Hay was used as the target
organism, the results for MAb-11-232.3, 11-248.2 ascites, and
MAb-11-569.3 were similar to those obtained with S. aureus type 5,
as shown in Table 13. At 300 .mu.g/ml, 66% and 83% killing was
obtained with MAb-11-232.3 and MAb-11-569.3, respectively. 95%
killing was obtained with neat ascites from hybridoma 11-248.2.
However, no killing was obtained with supernatant from 99-110FC12
IE4, which is consistent with its very poor binding to
methanol-fixed S. epidermidis in Table 9. Finally, strong killing
(>98%) was obtained with A110 at all doses tested (11.1
.mu.g/ml-300 .mu.g/ml), and background killing, obtained by mixing
PMNs with complement, but without antibody, was 22%. TABLE-US-00014
TABLE 13 Opsonophagocytic Killing of S. epidermidis Strain Hay By
Monoclonal Antibodies Antibody or Conc. % Killed Hybridoma Target
(.mu.g/ml) Strain ID Isotype Antigen or Dilution Hay A110 Human
IgG.sub.1, LTA 300 100 kappa (S. epi. 100 99 strain 33.3 98 Hay)
11.1 100 MAb-11-232.3 Mouse IgG.sub.3, Peptido- 300 66 kappa glycan
100 41 33.3 41 11.1 51 MAb-11-569.3 Mouse IgG.sub.3, Peptido- 300
83 kappa glycan 100 74 33.3 61 11.1 59 11-248.2 Mouse IgM, Peptido-
neat 95 ascites kappa glycan 1:2 31 1:4 24 99-110FC12IE4 Mouse IgM,
Peptido- neat 0 supernatant kappa glycan 1:4 1 Background killing
(PMNs and complement without antibody) was less than 22% for all
assays.
[0153] These data show that the MAb-11-232.3, 11-248.2 ascites, and
MAb-11-569.3 can enhance phagocytosis and killing of S. aureus type
5 and S. epidermidis strain Hay. The data also show that A110 is
less effective against S. aureus than MAb-11-232.3, 11-248.2
ascites, and MAb-11-569.3, but is highly active against S.
epidermidis strain Hay. The 99-110FC12 IE4 supernatant is active
against S. aureus, but not S. epidermidis strain Hay. These data
demonstrate a strong correlation between binding to methanol-fixed
bacteria and the ability to enhance opsonization of those bacteria,
with the notable exception of A110, which, although it binds
strongly to methanol-fixed S. aureus, is only weakly opsonic
against live S. aureus.
Example 6
Nasal Colonization Assay
[0154] Using a staphylococcal nasal colonization model in mice, we
demonstrated that intranasal instillation of the MAb-11-232.3
significantly reduces nasal colonization.
[0155] To ensure that the blocking of nasal colonization obtained
with the test MAbs was specific for anti-staphylococcal antibodies,
we examined the capacity of an irrelevant control chimeric IgG to
block staphylococcal nasal colonization. The control was Medi 493,
a chimeric IgG, MAb against RSV (MedImmune, Inc.). In the same
experiment, we also tested MAb-11-232.3 for its capacity to block
colonization.
[0156] Streptomycin resistant S. aureus type 5 (SA5, 1 to
3.times.10.sup.8 bacteria/mouse) was preincubated for 1 hour in
saline (0.9% NaCl in water), saline containing MAb-11-232.3 (2-3 mg
purified IgG per mouse dose of 1-3.times.10.sup.8 bacteria) or
saline containing Medi 493 (2-3 mg purified IgG/mouse dose of
1-3.times.10.sup.8 bacteria). Following preincubation, the bacteria
were pelleted and resuspended in saline (10 .mu.l/mouse dose), in
saline containing MAb-11-232.3 (10 .mu.l/mouse dose), or in saline
containing Medi 493 (10 .mu.l/mouse dose). Eight or nine mice each
were intranasally instilled with SA5 in saline, SA5 in
MAb-11-232.3, or SA5 in Medi 493. After seven days, the mice were
sacrificed and the nasal tissue dissected and plated on Columbia
blood agar and tryptic soy agar containing streptomycin to
determine colonization. Table 14 shows that MAb-11-232.3 reduced
staphylococcal nasal colonization in mice, but that an anti-RSV
MAb, Medi 493, had no effect. TABLE-US-00015 TABLE 14 Nasal
Colonization Assay against S. aureus Type 5 2 .times. 10.sup.8 SA5
Number of mice Average number of instilled with: colonized colonies
recovered Sterile Saline 9/9 70 MAb-11-232.3 3/8 8 (2 mg/mouse
dose) Medi 493 9/9 137 (2 mg/mouse dose)
[0157] Specifically, Table 14 shows that both the number of mice
colonized, and the number of colonies, are reduced in an
antibody-specific manner by anti-S. aureus surface antigen-specific
MAb-11-232.3. All of the mice in the saline and the irrelevant
chimeric IgG control groups were colonized with S. aureus, but only
three out of eight mice were colonized in the MAb-11-232.3 group.
This reduction in the number of mice colonized demonstrates that
the administered MAb 11-232.3 is protective because five of
the--eight mice are free from bacterial colonization. The number of
colonies recovered per mouse in the MAb-11-232.3 group was also
dramatically reduced as compared with the other two groups. The
saline control group exhibited an average of 70 colonies in the
nine mice colonized and the irrelevant antibody control group
exhibited even greater number of average colonies, 187, in the nine
mice colonized. In contrast, only three of the eight animals in the
treated group exhibited any sign of colonization and that level of
colonization, an average of 8 colonies per mouse nose, was greatly
reduced. Such a reduction in colonies recovered is similarly
profylactically beneficial in vivo. Therefore, the administered
anti-PepG MAb is protective from S. aureus nasal colonization.
These data also demonstrate that the effect is specific for
anti-staphylococcal surface antigen MAbs, and is not just a general
consequence of antibody binding through the Fc portion of the
antibody to surface Protein A on the staphylococci. Additional MAbs
against S. aureus peptidoglycan, MAb-11-248.2 and MAb-11-569.3, may
demonstrate similar inhibitory effects on S. aureus colonization as
described above. Studies are in progress to affirm the
effectiveness of MAb-11-248.2 and MAb-11-569.3 in the in vivo mouse
model described above.
Example 7
Subcloning of Hybridoma 11-232.3 to Produce Hybridoma 11-232.3
IE9
[0158] QED cell culture 11-232.3 was cloned by limiting dilution.
Briefly, the cells were diluted to a concentration of 225 viable
cells per ml. One ml of this suspension was added to 36 ml of RPMI
1640. The cell suspension was further diluted by the addition of
7.5 ml of FBS, 0.5 ml of 10 mg/ml kanamycin solution (Gibco BRL Cat
#15160-054) and 5 ml of Hybridoma SFM medium (Gibco BRL Cat
#12045-084). The final volume of the suspension was 50 ml,
resulting in a cell concentration of 4.5 cells/ml. Two hundred
microliters of the cell suspension was added to each well of two
96-well tissue culture dishes. The cultures were incubated for 10
days at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 in
air. The presence of clones was verified by microscopic observation
of single foci of cells in individual wells. Approximately 40% of
all wells had growing clones of 11-232.3. When tested by ELISA, all
supernatants bound peptidoglycan. Four cultures, 11-232.3-IG9,
-IE9, -IH7 and -IB6 were expanded and cryopreserved. The binding of
these four clones to peptidoglycan and LTA is shown in Table 15.
The MAb produced by hybridoma 11-232.3 IE9 was subsequently
designated M130. TABLE-US-00016 TABLE 15 Binding of 11-232.3
subclones to LTA and PepG Supernatant Absorbance Absorbance Culture
ID Dilution of PepG on LTA 232.3 uncloned 2 4.000 0.090 232.3
uncloned 2 4.000 0.084 232.3IG9 2 3.384 0.084 232.3IE9 2 3.141
0.100 232.3IH7 2 2.863 0.092 232.3IB6 2 3.570 0.086 Buffer Only
0.090 0.075
[0159] As shown in Table 16, the monoclonal antibody produced by
hybridoma 11-232.3 IE9, M130, bound S. aureus in the LBE assay.
Surprisingly, M130 did not bind to S. epidermidis strain Hay in
this assay, although it shows opsonic activity against S.
epidermidis strain Hay (Table 7). The opsonic assay uses antibody
at a concentration of up to 300 .mu.g/ml, while the LBE assay uses
concentrations of up to 3 .mu.g/ml, so the difference in the
activity of M130 in the two assays may result from the large
difference in concentrations used. TABLE-US-00017 TABLE 16 Binding
of MAb M130 by LBE Assay SA5 Antibody SA5 ATCC SA8 S. hemo S. epi
.mu.g/ml USU 49521 ATCC 12605 ATCC 43252 Hay 3 2.981 2.319 2.365
0.133 0.101 1 1.765 1.457 1.313 0.120 0.082 0.33 0.633 0.641 0.441
0.120 0.072 0.11 0.252 0.248 0.155 0.112 0.076 Buffer 0.100 0.808
0.110 0.120 0.072
Example 8
Cloning and Sequencing of the M130 Variable Regions
[0160] Total RNA was isolated from 2.times.10.sup.6 frozen IE9
(232-3) hybridoma cells using the Midi RNA Isolation kit (Qiagen)
following the manufacturer's procedure. The RNA was dissolved in 10
mM Tris, 0.1 mM EDTA (pH 8.4) containing of 0.25 .mu.g/.mu.l Prime
RNase Inhibitor (0.03 U/.mu.g; Sigma).
[0161] FIG. 1 shows the strategy for cloning the variable region
genes. Table 17 shows the sequences of the oligonucleotide primers
used for the procedures (SEQ ID NOS: 5-12). The total RNA (2 .mu.g)
was converted to cDNA by using Superscript II-MMLV Reverse
Transcriptase (Life Technologies) and mouse Kappa-specific primer
(JSBX-18; SEQ ID NO: 8; Sigma-Genosys) and a mouse heavy
chain-specific primer (JSBX-25A; SEQ ID NO: 9; Sigma-Genosys)
according to the manufacturer's procedures (see Table 12 for primer
sequences). The first strand cDNA synthesis products were purified
using a Centricon-30 concentrator device (Amicon). Of the 40 .mu.l
of cDNA recovered, 5 .mu.l was used as template DNA for PCR. PCR
amplification reactions (50 .mu.l) contained template DNA, 30
pmoles of the appropriate primers (JSBX-11A, -12A and -18 for light
chains; SEQ ID NOS: 6-8; JSBX-5 and -25A for heavy chains; SEQ ID
NO: 5 and SEQ ID NO: 9), 2.5 units of ExTaq polymerase (PanVera),
1.times.ExTaq reaction buffer, 200 .mu.M each dNTP, 2 mM
MgCl.sub.2. The template was denatured by an initial incubation at
96.degree. C. for 3 min. The products were amplified by 30 thermal
cycles of 96.degree. C. for 1 min., 60.degree. C. for 30 sec.,
72.degree. C. for 30 sec. The PCR products from the successful
reactions were purified using the Nucleospin PCR Purification
system (Clontech) as per manufacturer's procedure. TABLE-US-00018
TABLE 17 Oligonucleotide primers used SEQ ID Name Length
Description Oligo Sequence NO: JSBX-5 40 mouse HCV
TGTTTTCGTACGTCTTGTC 5 front primer CCAGGTBCARCTKMARSAR for TC
MegaVector JSBX- 32 mLCV Front TACCGTACCGGTGAYATYM 6 11A for
AGATGACMCAGWC MegaVector JSBX- 32 mLCV Front TACCGTACCGGTSAAATTG 7
12A for WKCTSACYCAGTC MegaVector JSBX-18 23 Mouse Kappa
GCACCTCCAGATGTTAACT 8 Constant GCTC reverse primer JSBX- 22 Mouse
IgG CTGGACAGGGMTCCAKAGT 9 25a reverse TCC primer (123-144) JSBX-27
38 Mouse Back ATAGGATTCGAAAAGTGTA 10 Primer for CTTMCGTTTCAGYTCCARC
MegaVector JSBX-44 23 M130 HCV TGTTTTCGTACGTCTTGTC 11 front primer
CCAG for MegaVector JSBX-45 35 M130 HCV TTTTCTGAATTCTGCAGAG 12 back
primer ACAGTGACCAGAGTCC for MegaVector Note: each of the following
letters is used to denote an equal mixture of nucleotides in that
position: B = C, G, or T; D = A, G, or T; K = G or T; M = A or C; R
= A or G; S = C or G; V = A, C, or G; W = A or T; Y = C or T.
[0162] The PCR products (approximately 400 base pairs) were then
cloned into bacterial vector pGEM T (Promega), a T/A style cloning
vector, following the manufacturer's procedures using a 3:1 insert
to vector molar ratio. One half (5 .mu.l) of the ligation reactions
were used to transform Ultracompetent XL1Blue cells (Stratagene) as
per the manufacturer's procedure. Bacterial clones containing
plasmids with DNA inserts were identified using diagnostic
restriction enzyme digestions with DraIII and BsiWI (for heavy
chain clones) or DraIII and EcoRV (for light chain clones) (New
England Biolabs). Plasmids containing inserts of the appropriate
size (.about.400 bp) were then sequenced. The pGEM vector
containing the M130 heavy chain variable region is referred to as
pJSB18-6, and the pGEM vector containing the M130 light chain
variable region is referred to as pJSB4-4. The final consensus DNA
sequence of the light and heavy chain variable regions is shown in
FIG. 2 (SEQ ID NO: 2 and SEQ ID NO: 4, respectively).
Example 9
Cloning of Mouse/Human Chimeric Antibody A130
[0163] The heavy and light chain variable regions of M130 were then
subcloned into a mammalian expression plasmid vector for production
of recombinant chimeric mouse/human antibody molecules under the
control of CMV transcription promoters. The variable region of M130
is fused directly to the human IgG, constant domain. The light
chain of M130, on the other hand, has a mouse Kappa intron domain
3' of the variable region coding sequence. After splicing, the
variable region becomes fused to a human Kappa constant region
exon. The selectable marker for both vectors in mammalian cells is
Neomycin (G418).
[0164] The variable region gene fragments were re-amplified by PCR
using primers that adapted the fragments for cloning into the
expression vector (FIG. 1, Table 17). The heavy chain front primer
(JSBX-44; SEQ ID NO: 11) includes a 5' tail that encodes the
C-terminus of the heavy chain leader and a BsiWI restriction site
for cloning, while the heavy chain reverse primer (JSBX-45; SEQ ID
NO: 12) adds a 3' EcoRI restriction site for cloning. This results
in the addition of two amino acids, glutamine (E) and phenylalanine
(F) between the heavy chain variable region and the human IgG.sub.1
constant region.
[0165] The light chain front primer (JSBX-11A; SEQ ID NO: 6)
introduces a 5' tail that encodes the two C-terminal amino acids of
the light chain leader and an AgeI restriction site for cloning
purposes. The light chain reverse primer (JSBX-27; SEQ ID NO: 10)
adds a 3' DNA sequence for the joining region-Kappa exon splice
junction followed by a BstBI restriction site for cloning. PCRs
were performed as described above, using pJSB18-6 as a template for
the heavy chain and pJSB4-4 as a template for the light chain.
Following a three minute incubation at 96.degree. C. the PCR
perimeters were 30 thermal cycles of 58.degree. C. for 30 sec.,
70.degree. C. for 30 sec., and 96.degree. C. for 1 min.
[0166] The heavy chain PCR product was digested with BsiWI and
EcoRI (New England Biolabs), purified using a Nucleospin PCR
Purification column (Clontech), per the manufacturer's
instructions, and ligated into BsiWI/EcoRI/PfiMI digested and
gel-purified pJRS383, using the Takara Ligation Kit (Panvera), per
the manufacturer's procedure. The ligation mix was then transformed
into XL1Blue cells (Stratagene); clones were selected and screened
for the correct insert, resulting in mammalian expression vector
pJSB22 (FIG. 3).
[0167] The light chain PCR product (approximately 350 base pairs)
was digested with AgeI and BstBI (New England Biolabs), purified
using a Nucleospin PCR Purification column (Clontech), as described
by the manufacturer. This fragment was then ligated into pJRS384
that had been AgeI/BstBI/XcmI digested and gel-purified, using the
Takara Ligation Kit (Panvera), per the manufacturer's procedure.
The ligation mix was then transformed into XL1Blue cells
(Stratagene); clones were selected and screened for the correct
insert, resulting in mammalian expression plasmid pJSB6.1 (see FIG.
4).
[0168] Combining the two individual plasmids then made a single
expression construct containing both the light and heavy chain
expression cassettes. The plasmid pJSB6.1 was digested with BamHI
and NheI (New England Biolabs) and purified using a Nucleospin PCR
Purification column (Clontech), as described by the manufacturer.
The plasmid pJSB22 was digested with BglII and NheI (New England
Biolabs), separated on an agarose gel and the fragment containing
the heavy chain expression domain was isolated using a Nucleospin
Gel Fragment DNA Purification column (Clontech). These fragments
were ligated together using the Takara Ligation Kit (Panvera)
following the manufacturer's procedure and transformed into XL1Blue
cells (Stratagene). The resulting bi-cistronic expression vector,
pLG1-MEGA, was then transfected into mammalian cells for A130
chimeric antibody production, after sequence confirmation of the
variable regions (FIG. 5).
Example 10
Transient Production of Recombinant Chimeric Mouse/Human A130
Antibody
[0169] The plasmid pLG1-MEGA was transfected into COS-7 (ATCC no.
CRL 1651) cells using Superfect (Qiagen) in 6 well tissue culture
dishes as described by the manufacturer. After two days the
supernatant was assayed for the production of chimeric antibody and
for the capability for the expressed antibody to bind to S. aureus
peptidoglycan antigen as follows.
[0170] 8-well strips (Maxisorp F8; Nunc, Inc.) were coated with a
1:500 dilution in PBS of goat anti-human Fc (Pierce). The plates
were then covered with pressure sensitive film and incubated
overnight at 4.degree. C. Plates were then washed once with
1.times. Wash solution (KPL cat. no. 50-63-01). One hundred
microliters of culture supernatant dilutions were then applied to
duplicate wells and allowed to incubate for 60 minutes on a plate
rotator at room temperature. The plates were washed seven times
with Wash solution. A goat anti-human kappa-HRP (Zymed) conjugate
was diluted 1:800 in the sample/conjugate diluent (0.02 M Tris pH
7.4, 0.25 M NaCl.sub.2, 2% gelatin, 0.1% Tween-20). One hundred
microliters was added to the samples, and then incubated on a plate
rotator for 60 minutes at room temperature. The samples were washed
seven times, as above and then incubated with 100 .mu.L/well of TMB
substrate (BioFx, cat. no. TMBW-0100-01) for <1 minute at room
temperature. The reaction was stopped with 100 .mu.L/well of TMB
Stop reagent (BioFx, cat. no. STPR-0100-01) and the absorbance
value at 450 nm was determined using an automated microtiter plate
ELISA reader (Spetramax Plus; Molecular Devices, Inc.). FIG. 6
shows that the mouse/human chimeric A130 antibody is bound by a
goat anti-human IgG kappa antibody, indicating that transfection of
cells with the pLG1-MEGA plasmid results in the cells producing a
molecule containing both human IgG and Kappa domains.
[0171] The supernatants were then assayed for the ability of the
expressed antibodies to bind to peptidoglycan. 8-well strips
(Maxisorp F8; Nunc, Inc.) were coated with a 5 .mu.g/ml solution of
S. aureus peptidoglycan (prepared by the method set forth in
Example 2) in carbonate coating buffer, pH 9.5 (0.1M sodium
bicarbonate) overnight at 4.degree. C. Plates were washed once with
PBS. One hundred microliters of culture supernatant dilutions were
applied to duplicate wells and allowed to incubate for 60 minutes
on a plate rotator at room temperature. The plates were washed
seven times with Wash solution. One hundred microliters of Goat
anti-Human IgG H+L-HRP (Zymed) diluted 1:4000 in sample/conjugate
diluent was added to the samples, and the plates were incubated on
a plate rotator for 60 minutes at room temperature. The samples
were washed seven times with Wash buffer and then incubated with
100 .mu.L/well of TMB substrate (BioFx) for 10-15 minutes on a
plate rotator at room temperature. The reaction was stopped with
100 .mu.L/well of TMB Stop reagent (BioFx) and the absorbance value
at 450 nm was determined using an automated microtiter plate ELISA
reader (Spectramax Plus, Molecular Devices).
[0172] As a positive control, the original mouse monoclonal
antibody M130 was used, and assayed with a 1:2000 dilution of Goat
anti-Mouse Fc-HRP conjugate. FIG. 7 shows that the transfection of
cells with the pLG1-MEGA plasmid results in the cells producing a
molecule that binds to S. aureus peptidoglycan. These results
suggest that the mouse/human chimeric antibody, A130, retains the
peptidoglycan binding ability of the mouse monoclonal antibody
M130, from which it was derived.
Example 11
Specific Binding of the Monoclonal Antibodies to PepG
[0173] To confirm that the monoclonal antibodies are specific for
PepG, and do not bind to a contaminant in the PepG preparation used
for the ELISA assay shown in Table 7, sandwich assays were
conducted. In brief, multiwell plates were coated with "capture"
antibodies specific for PepG, LTA, or an unknown antigen; PepG was
then added to the wells and bound by the antibodies; and then a
"detection" antibody was added to measure its affinity for the PepG
captured by the capture antibody.
[0174] Specifically, monoclonal antibodies M110, M130,
MAb-11-230.3, MAb-11-232.3, MAb-11-391.4, MAb-11-557.3,
MAb-11-564.4, MAb-11-580.5, and MAb-11-586.3 were diluted to 3
.mu.g/ml in PBS. Hybridomas 11-230.3, 11-232.3, 11-391.4, 11-557.3,
11-564.4, 11-580.5, and 11-586.3 were derived from mice that had
been immunized with whole UV-killed S. aureus. As set forth above,
MAb-11-230.3, MAb-11-232.3, MAb-11-557.3, MAb-11-564.4, and
MAb-11-586.3 have been previously shown to bind to PepG, while
MAb-11-391.4 binds to LTA, and MAb-11-580.5 binds to an unknown
epitope on S. aureus. These MAbs were used to coat four columns of
Nunc Maxisorp Stripwells (Nunc Cat # 469949), and are referred to
as "capture" antibodies. Coating of each capture antibody was
accomplished by adding 100 .mu.l of the 3 .mu.g/ml solution to the
appropriate wells and incubating overnight (18-26 hours) at room
temperature. The unbound material was then removed from the wells
by washing four times with PBS-T. For each capture antibody, S.
aureus peptidoglycan (prepared by S. Foster as described in Example
2), diluted to 10 .mu.g/ml in PBS-T, was added to the wells of two
of the four columns, and PBS-T without peptidoglycan was added to
the wells of the other two columns.
[0175] The wells were incubated for 30-60 minutes at room
temperature and washed four times with PBS-T. The wells then
received 100 .mu.l of A130 at the concentrations indicated in Table
17 (2-fold dilutions from 5 .mu.g/ml to 0.078 .mu.g/ml). An
anti-PepG monoclonal antibody, A130, which is described above, was
the "detection" antibody and was diluted in PBS-T with 0.1% human
serum without IgG, IgA and IgM (Axell Cat BYA20341, Lot H2415). The
detection antibody was expected to react with any of the PepG bound
to the capture MAbs used for coating. Thus, it was expected that
PepG would not be captured in wells coated with MAbs M110 and
391.4, which are specific for LTA, and in wells coated with
MAb-11-580.5, a MAb of unknown specificity.
[0176] Following another 30-60 minute incubation, the wells were
washed with PBS-T and each well received 95 .mu.l of HRP-conjugated
gamma-specific goat anti-human IgG (Zymed Cat 62-8420), diluted
1:6000 in PBS-T. After 30-60 minutes, the wells were washed with
PBS-T and 100 .mu.l of TMB substrate solution was added to each
well (BioFX Cat TMBW-0100-01). The reaction was allowed to proceed
for 15 minutes at room temperature in the dark and was stopped by
addition of 100 .mu.l TMB Stop Reagent to each well (BioFX Cat
STPR-0100-01). The absorbance of each well was measured using a
Molecular Devices Vmax microplate reader with a 450 nm filter.
[0177] Table 18 shows the results of the capture assay using a
battery of capture MAbs, followed by PepG, and then A130 as the
detection MAb. After coating a well with MAb-11-230.2,
MAb-11-232.3, MAb-11-557.3, MAb-11-564.4, or MAb-11-586.3, and
incubating with PepG, the detection antibody, A130, binds to the
captured PepG on the plate. If an anti-LTA antibody, such as that
produced by hybridoma 391.4, or M110, is used to capture, A130 does
not bind, presumably because the capture antibody failed to bind
PepG. Furthermore, MAb-11-580.5, which is of unknown specificity,
also fails to capture significant PepG, as evidenced by the lack of
binding by A130. As a positive control, if M130, which has
identical variable regions as A130, is to capture PepG, A130 binds
strongly to the plate. These results suggest that A130 binds to
PepG, and not to a contaminant in the PepG preparation, because
capturing PepG with a battery of a different antibodies that are
believed to bind to PepG results in binding of A130 to the captured
material. TABLE-US-00019 TABLE 18 PepG Sandwich Assay: Capture with
anti-S. aureus MAbs and Detection with A130 capture MAb
MAb-11-230.3 MAb-11-232.3 MAb-11-391.4 detection MAb (anti-PepG)
(anti-PepG) (anti-LTA) A130 (.mu.g/ml) +PepG -PepG +PepG -PepG
+PepG -PepG 5 0.513 0.096 0.529 0.077 0.085 0.072 2.5 0.576 0.090
0.529 0.073 0.088 0.069 1.25 0.703 0.091 0.557 0.074 0.087 0.068
0.625 0.561 0.091 0.526 0.072 0.085 0.074 0.3125 0.493 0.093 0.450
0.072 0.084 0.072 0.156 0.420 0.091 0.499 0.066 0.085 0.072 0.078
0.322 0.090 0.303 0.071 0.078 0.068 Buffer 0.090 0.090 0.071 0.081
0.063 0.076 capture MAb MAb-11-557.3 MAb-11-564.4 MAb-11-580.5
detection MAb (anti-PepG) (anti-PepG) (unknown) A130 (.mu.g/ml)
+PepG -PepG +PepG -PepG +PepG -PepG 5 0.599 0.079 0.530 0.095 0.151
0.079 2.5 0.610 0.076 0.564 0.093 0.159 0.082 1.25 0.588 0.079
0.560 0.095 0.158 0.086 0.625 0.612 0.079 0.522 0.093 0.163 0.086
0.3125 0.593 0.079 0.498 0.091 0.158 0.081 0.156 0.525 0.078 0.432
0.091 0.151 0.085 0.078 0.434 0.080 0.387 0.095 0.139 0.083 Buffer
0.081 0.078 0.093 0.090 0.080 0.088 capture MAb MAb-11-586.3 M110
M130 detection MAb (anti-PepG) (anti-LTA) (anti-PepG) A130
(.mu.g/ml) +PepG -PepG +PepG -PepG +PepG -PepG 5 0.635 0.088 0.182
0.172 0.723 0.072 2.5 0.661 0.085 0.172 0.161 0.666 0.072 1.25
0.636 0.096 0.172 0.163 0.633 0.071 0.625 0.524 0.085 0.169 0.156
0.568 0.070 0.3125 0.599 0.085 0.169 0.153 0.467 0.083 0.156 0.526
0.084 0.167 0.151 0.351 0.070 0.078 0.411 0.084 0.163 0.149 0.269
0.070 Buffer 0.083 0.084 0.146 0.146 0.067 0.068
[0178] To confirm that the anti-PepG antibodies do not bind to
possible LTA contamination in the PepG preparation, a similar
sandwich assay was conducted in which A110, which binds to LTA, was
used as the capture antibody, LTA was bound to the capture
antibody, and detection antibodies were then added to measure their
binding to the captured LTA.
[0179] Nunc Maxisorp Stripwells (Nunc Cat #469949) were coated with
100 .mu.l of 3 .mu.g/ml A110 in PBS and incubated overnight (18-26
hours) at room temperature. After overnight incubation, unbound
material was removed from the wells by washing four times with
PBS-T. Replicate wells then received 100 .mu.l of LTA solution
(Sigma Cat #2515 diluted to 1 .mu.g/ml in PBS-T) or PBS-T alone.
The wells were then incubated for 30-60 minutes at room temperature
and washed four times with PBS-T.
[0180] The wells received 100 .mu.l of an antibody selected from
monoclonal antibodies M110, M130, MAb-11-230.3, MAb-11-232.3,
MAb-11-391.4, MAb-11-557.3, MAb-11-564.4, MAb-11-580.5, and
MAb-11-586.3, titrated in 2-fold dilutions from 5 .mu.g/ml to 0.078
.mu.g/ml in PBS-T with 0.1% human serum without IgG, IgA and IgM
(Axell Cat BYA20341, Lot H2415). Each MAb was titrated in four
columns, two of which had received LTA and two of which had
received PBS-T alone. These mouse MAbs served as detection
antibodies, analogous to the A130 detection antibody used in Table
18.
[0181] Following another 30-60 minute incubation, the wells were
again washed with PBS-T and each well received 95 .mu.l of
HRP-conjugated gamma-specific anti-mouse IgG (Jackson
Immunoresearch Cat 115-035-164), diluted 1:10,000 in PBS-T. After
30-60 minutes, the wells were washed with PBS-T and 100 .mu.l of
TMB substrate solution was added to each well (BioFX Cat
TMBW-0100-01). The reaction was allowed to proceed for 15 minutes
at room temperature in the dark and stopped by addition of 100
.mu.l TMB Stop Reagent to each well (BioFX Cat STPR-0100-01). The
absorbance of each well was, measured using a Molecular Devices
Vmax microplate reader with a 450 nm filter.
[0182] Table 19 shows the results of the sandwich assay using A110
as the capture antibody, followed by LTA, and then a battery of
detection MAbs. This assay confirms that the antibodies that are
believed to bind to PepG do not in fact bind to possible LTA
contaminants in the PepG preparation. A110 was used as the capture
antibody to capture LTA on the plate. MAb-11-230.2, MAb-11-232.3,
MAb-11-557.3, MAb-11-564.4, MAb-11-586.3, and M130, all of which
bind to PepG, fail to bind to the captured LTA on the plate. As
would be expected, the anti-LTA antibodies, including those
produced by hybridoma 391.4, and M110, show strong binding to the
captured LTA. Finally, MAb-11-580.5, which is of unknown
specificity, does not bind to the captured LTA. These results
further confirm that the anti-PepG monoclonal antibodies, including
M130, do not bind to LTA. TABLE-US-00020 TABLE 19 LTA Sandwich
Assay: Capture with A110, Detection with anti-S. aureus MAbs
detection MAb MAb-11-230.3 MAb-11-232.3 MAb-11-391.4 detection MAb
(anti-PepG) (anti-PepG) (anti-LTA) conc. (.mu.g/ml) +PepG -PepG
+PepG -PepG +PepG -PepG 5 0.063 0.063 0.066 0.066 1.751 0.118 2.5
0.059 0.080 0.073 0.065 0.930 0.090 1.25 0.059 0.061 0.064 0.067
0.479 0.082 0.625 0.057 0.058 0.063 0.065 0.277 0.072 0.3125 0.056
0.059 0.060 0.063 0.160 0.067 0.156 0.059 0.061 0.061 0.066 0.113
0.066 0.078 0.057 0.058 0.061 0.062 0.086 0.061 Buffer 0.055 0.058
0.066 0.064 0.060 0.063 detection MAb MAb-11-557.3 MAb-11-564.4
MAb-11-580.5 detection MAb (anti-PepG) (anti-PepG) (unknown) conc.
(.mu.g/ml) +PepG -PepG +PepG -PepG +PepG -PepG 5 0.064 0.058 0.072
0.074 0.066 0.066 2.5 0.056 0.056 0.065 0.067 0.064 0.063 1.25
0.058 0.057 0.086 0.066 0.062 0.065 0.625 0.056 0.061 0.063 0.063
0.059 0.061 0.3125 0.057 0.055 0.062 0.061 0.064 0.063 0.156 0.058
0.059 0.060 0.062 0.059 0.062 0.078 0.056 0.059 0.061 0.060 0.057
0.059 Buffer 0.054 0.056 0.084 0.061 0.054 0.059 detection MAb
MAb-11-586.3 M110 M130 detection MAb (anti-PepG) (anti-LTA)
(anti-PepG) conc. (.mu.g/ml) +PepG -PepG +PepG -PepG +PepG -PepG 5
0.060 0.062 0.743 0.059 0.068 0.070 2.5 0.059 0.060 0.509 0.059
0.061 0.063 1.25 0.061 0.062 0.362 0.062 0.060 0.063 0.625 0.061
0.062 0.257 0.060 0.059 0.060 0.3125 0.060 0.059 0.179 0.060 0.055
0.058 0.156 0.061 0.061 0.133 0.060 0.057 0.057 0.078 0.058 0.057
0.100 0.058 0.054 0.057 Buffer 0.060 0.057 0.055 0.059 0.055
0.058
[0183] Finally, to further confirm that M110 does not capture a
contaminant in the PepG preparation that is recognized by M130, the
following sandwich assay was performed. M110 was bound to plates as
the capture antibody, LTA or PepG was then bound to the plates,
followed by either A110 or A130 as the detection antibody.
[0184] Nunc Maxisorp Stripwells (Nunc Cat #469949) were coated with
100 .mu.l of M10 at 3 .mu.g/ml and incubated overnight (18-26
hours) at room temperature. The unbound material was then removed
from the wells by washing four times with PBS-T. Four columns of
wells then received 100 .mu.l/well of LTA solution (Sigma Cat #2515
diluted to 1 .mu.g/ml in PBS-T), four columns received 100
.mu.l/well of PepG solution (10 .mu.g/ml in PBS-T), and four
columns received PBS-T alone. The wells were incubated for 30-60
minutes at room temperature and then washed four times with PBS-T.
Two columns of each LTA-bound, PepG-bound, and PBS-t received 100
.mu.l of A130, titrated in 2-fold dilutions from 5 .mu.g/ml to
0.078 .mu.g/ml in PBS-T with 0.1% human serum without IgG, IgA and
IgM (Axell Cat BYA20341, Lot H2415). Two columns of each LTA-bound,
PepG-bound, and PBS-t received A110, similarly diluted and
titrated. These chimeric antibodies served as "detection"
antibodies, analogous to the A130 detection antibody used in Table
18.
[0185] Following a 30-60 minute incubation, the wells were washed
with PBS-T and each well received 95 .mu.l of HRP-conjugated
gamma-specific goat anti-human IgG (Zymed Cat 62-8420), diluted
1:6000 in PBS-T. After 30-60 minutes, the wells were washed with
PBS-T and 100 .mu.l of TMB substrate solution was added to each
well (BioFX Cat TMBW-0100-01). The reaction was allowed to proceed
for 15 minutes at room temperature in the dark and stopped by the
addition of 100 .mu.l TMB Stop Reagent to each well (BioFX Cat
STPR-0100-01). The absorbance of each well was measured using a
Molecular Devices Vmax microplate reader with a 450 nm filter.
[0186] Table 20 shows the results of the sandwich assay using M110
as the capture antibody, followed by PepG or LTA, and then A110 or
A130 as the detection antibody. These results demonstrate again
that M110 does not capture an antigen that can be bound by A130.
Furthermore, this result demonstrates that the PepG preparation
used in these assays does not contain appreciable levels of LTA, as
capture of the PepG with anti-LTA M110 antibody does not result in
sufficient captured LTA to show binding of A110 above background
levels. TABLE-US-00021 TABLE 20 Capture with M110, Binding of LTA
or PepG, Detection with A110 or A130 detection with -LTA detection
with -LTA A130 (.mu.g/ml) +LTA +PepG -PepG A110 (.mu.g/ml) +LTA
+PepG -PepG 5 0.186 0.197 0.179 5 1.237 0.177 0.174 2.5 0.181 0.197
0.180 2.5 1.119 0.172 0.168 1.25 0.209 0.213 0.190 1.25 1.036 0.175
0.172 0.625 0.198 0.223 0.200 0.625 0.961 0.189 0.180 0.3125 0.192
0.224 0.200 0.3125 0.829 0.190 0.185 0.156 0.192 0.229 0.199 0.156
0.688 0.200 0.197 0.078 0.188 0.217 0.194 0.078 0.523 0.187 0.187
Buffer 0.192 0.196 0.190 Buffer 0.180 0.179 0.182
Example 12
Human Antibodies that Bind PepG
[0187] Rather than humanizing a mouse antibody to minimize the HAMA
response during treatment as described above, a skilled artisan can
isolate a protective anti-PepG antibody that is fully human. There
are a number of well-known alternative strategies one of ordinary
skill in the art may use to produce completely human recombinant
antibodies. One is the generation of antibodies using phage display
technologies (50, 54). Specifically, human RNA is used to produce a
cDNA library of antibody heavy and light chain fragments expressed
on the surface of bacteriophage. These libraries can be used to
probe against the antigen of interest (i.e., PepG) and the phage
that bind, because of the antibody expressed on the surface, are
then isolated. The DNA encoding the variable regions is sequenced
and cloned for antibody expression.
[0188] Another method of producing human antibodies employs
"humanized" mice. These transgenic mice have had their own antibody
genes replaced with a portion of the human antibody gene complex so
that upon inoculation with antigen, they produce human antibodies
(48, 50, 51, 52, 54). The antibody producing cells that result can
then be incorporated into the standard hybridoma technology for the
establishment of specific monoclonal antibody producing cell
lines.
[0189] Recombinant human antibodies are also produced by isolating
antibody-producing B cells from human volunteers that have a robust
anti-PepG response. Using fluorescence activated cell sorting
(FACS) and fluorescently labeled PepG, cells producing the
anti-PepG antibodies can be separated from the other cells. The RNA
can then be extracted and the sequence of the reactive antibody
variable regions determined (49, 53). The DNA sequence of the
functional variable regions can be synthesized or cloned into
mammalian expression vectors for large-scale human recombinant
antibody production.
CONCLUSION
[0190] Monoclonal antibodies were raised in mice against S. aureus
PepG, an abundant cell surface molecule on Gram-positive bacteria.
One hybridoma clone, 99-110FC12 IE4, produces an IgM antibody that
bound strongly in ELISA assays to PepG, but not to S. epidermidis
strain Hay, or to LTA, another surface molecule common to
Gram-positive bacteria (Example 1, Table 5).
[0191] Monoclonal antibodies were also raised against B. subtilis
PepG. Hybridomas BB4/A4 and BB4/A5 produce IgG antibodies that bind
to B. subtilis PepG (Example 2). The affinity of the monoclonal
antibodies produced by BB4/A4, BB4/A5, 11-232.3, 11-248.2,
11-569.3, and antibody 702 PG, which is purified from hybridoma
11-232.3, were tested for binding to PepG from a number of
different bacteria in an ELISA assay. MAb-BB4/A4 and MAb-BB4/A5,
which were produced from the same mouse, bound strongly to PepG
from B. subtilis and S. epidermidis, while MAb-11-232.3 and
MAb-11-248.2 bound strongly to PepG S. aureus (Example 3, Table 6).
These results demonstrate that monoclonal antibodies raised against
PepG from one Gram positive bacteria may bind PepG from another
Gram-positive bacteria, which may indicate binding to a conserved
epitope on PepG.
[0192] A110, MAb-11-232.3, MAb-11-248.2, MAb-99-110FC12 IE4, and
MAb-11-569.3 were tested for binding to S. aureus PepG in an ELISA
assay. MAb-11-232.3, MAb-11-248.2, and MAb-99-110FC12 IE4, which
were raised to either whole UV-killed S. aureus or to S. aureus
PepG, bound strongly to S. aureus PepG. MAb-11-569.3 bound weakly
to PepG, although it was also raised to whole UV-killed S. aureus,
indicating that it may bind to an epitope other than PepG on the
surface of the bacteria. The anti-LTA antibody, A110, did not bind
to S. aureus PepG (Example 4, Table 7). In a similar assay to
measure binding of the antibodies to S. aureus LTA, only A110
showed appreciable binding (Example 4, Table 8). The results
demonstrate that antibodies raised to whole UV-killed S. aureus may
in fact be specific for PepG on the surface of the bacteria.
[0193] Each of the antibodies was then tested for binding to
methanol-fixed S. epidermidis strain Hay. A110, which was raised to
S. epidermidis LTA, bound most strongly to methanol-fixed S.
epidermidis. MAb-11-569.3 and MAb-11-248.2 also bound strongly to
the methanol-fixed S. epidermidis, in spite of the fact that they
were raised to UV-killed S. aureus. This suggests that these
antibodies may bind to a conserved epitope on the surface of the
two bacterial strains. MAb-11-232.3, which was also raised to
UV-killed S. aureus bound less strongly to methanol-fixed S.
epidermidis. Similarly, MAb-99-110FC12 IE4, which was raised
against S. aureus PepG, did not bind methanol-fixed S. epidermidis
(Example 4, Table 9).
[0194] Finally, A110, MAb-11-232.3, and MAb-11-569.3 were tested
for binding to methanol-fixed S. aureus. Not surprisingly,
MAb-11-232.3 and MAb-11-569.3 bound strongly, as they were raised
to whole UV-killed S. aureus. A110 also bound to methanol-fixed S.
aureus, although it was raised to heat-killed S. epidermidis,
indicating that it may bind to an epitope that is conserved between
the two bacteria (Example 4, Table 10).
[0195] Hybridoma 99-110FC12 IE4 supernatant was tested for opsonic
activity against S. aureus and S. epidermidis in the presence of
PMNs and complement, which was derived from human serum that had
been depleted of antibodies to S. aureus and S. epidermidis.
MAb-99-110FC12 IE4 was opsonic against S. aureus, but not against
S. epidermidis (Example 5, Table 11). As discussed in the preceding
paragraphs, MAb-99-110FC12 IE4 binds to S. aureus PepG, but not to
whole S. epidermidis, suggesting a correlation between binding
ability and opsonic activity.
[0196] MAb-11-232.2, MAb-11-569.3, MAb-11-248.2, each of which was
raised to whole UV-killed S. aureus, as well as MAb-99-110FC12 IE4,
which was raised to purified S. aureus PepG, and A110 were tested
in a similar opsonophagocytic assay against S. aureus type 5. The
antibodies that were raised against UV-killed S. aureus,
MAb-11-232.2, MAb-11-569.3, and MAb-11-248.2, as well as
MAb-110FC12 IE4, which was raised against S. aureus PepG, showed at
least 75% killing of S. aureus in the assay. The anti-LTA antibody,
A110, which was raised against LTA from S. epidermidis, showed only
23% killing (Example 5, Table 12). Surprisingly, although A110
bound strongly to both S. aureus LTA and whole methanol-fixed S.
aureus, it was not strongly opsonic against S. aureus. The
non-chimerized version of A110, M110, was somewhat more opsonic
against S. aureus in a previous assay (Example 5, Table 12B). This
variation, however, is likely due to assay to assay variations and
dosage effects, rather than differences in activity between the
chimerized and non-chimerized antibodies, because A110 retains its
activity against S. epidermidis (Example 5, Table 13). The
antibodies that were raised against S. aureus PepG, and against
whole UV-killed S. aureus, on the other hand, showed strong opsonic
activity against the bacteria, as was expected.
[0197] In a similar assay against S. epidermidis strain Hay,
MAb-11-232.2, MAb-11-569.3, and MAb-11-248.2 showed at least 66%
killing, while MAb-99-110FC12 IE4 showed little or no killing.
A110, in contrast with the previous assay, showed at least 98%
killing of S. epidermidis strain Hay (Example 5, Table 13). These
results, and those discussed above, show a strong correlation
between the ability to bind to methanol-fixed bacteria and opsonic
activity against that bacteria. A110 is a notable exception,
however, because it was able to bind to methanol-fixed S. aureus,
but was not opsonic against live S. aureus. Furthermore, these
results demonstrate that monoclonal antibodies that have been
raised against UV-killed S. aureus can have opsonic activity
against S. epidermidis, suggesting a conserved determinant between
the two bacteria that allows a MAb raised against one to be opsonic
for the other. These results also indicate that weak binding may be
sufficient for opsonic activity, because MAb-11.232.3 was still
somewhat opsonic against S. epidermidis, in spite of its poor
binding to those bacteria in the methanol-fixed bacteria ELISA.
[0198] MAb-11-232.3 was tested for its ability to block nasal
colonization in mice. After preincubation of S. aureus type 5 with
MAb-11-232.3, only 3 out of 8 mice were colonized by S. aureus, as
compared to 9 out of 9 mice in the control groups. Furthermore, of
the mice that were colonized, the mice that received S. aureus that
had been preincubated with MAb-11-232.3 had one-tenth the number of
bacterial colonies as control mice (Example 6, Table 14). These
results suggest that MAb-11-232.3, which binds to and is opsonic
against S. aureus, is able to block nasal colonization by the
bacteria, and is also able to reduce the number of bacteria in mice
that are colonized.
[0199] Hybridoma 11-232.3 was subcloned, and the antibody produced
by subclone 11-232.3 IE9, M130, was further analyzed. M130 was
tested for binding to a number of different bacteria in a live
bacteria ELISA assay. M130 bound to three different strains of live
S. aureus, but did not bind to S. hemolyticus or S. epidermidis
(Example 7, Table 16). These results are consistent with the
methanol-fixed bacteria ELISAs, in which MAb-11-232.3 bound
strongly to S. aureus, but weakly to S. epidermidis. Therefore,
although MAb-11.232.3 and M130 are specific for S. aureus, they are
broadly reactive against different strains of bacteria, as
MAb-11-232.3 was still somewhat opsonic against S. epidermidis, in
spite of its weaker binding.
[0200] The variable regions of M130 were cloned and human/mouse
chimeric antibodies were produced that have the M130 variable
regions and human constant regions (Examples 8 and 9). These
chimeric antibodies, referred to as A130, retained the ability to
bind to S. aureus PepG (Example 10, FIG. 7). These human/mouse
chimeric antibodies are expected to have a reduced HAMA response in
humans, which may be therapeutically advantageous.
[0201] Finally, sandwich assays were conducted to confirm that the
anti-PepG antibodies of the invention are in fact specific for
PepG, and do not bind to contaminants in PepG preparations. In the
first assay, a battery of antibodies were used to capture PepG on a
plate, and then A130 was used as a detection antibody to detect the
captured PepG (Example 11, Table 18). As expected, antibodies that
are known to bind to PepG were able to capture PepG, as detected by
binding of A130 antibody. Antibodies that bind to LTA did not
capture an antigen that could be detected by A130, indicating that
A130 does not bind to LTA. In the second assay, anti-LTA A110
antibody was used to capture LTA on a plate, which was then
detected with the same battery of antibodies as was used for
capture in the first assay (Example 11, Table 19). As expected, the
anti-LTA antibodies were able to detect the LTA that was captured
by A110. The anti-PepG antibodies, on the other hand, were unable
to detect an antigen captured by A110, suggesting that they do not
bind to LTA. Finally, M110 was used to capture either LTA or PepG
on a plate, and the captured antigen was then detected with either
A110 or A130 (Example 11, Table 20). As expected, M110 was not able
to capture an antigen from either the PepG or LTA preparations that
was detectable by A130. M110 was able to capture LTA that was
detectable by A110, however. Significantly, M110 was not able to
capture sufficient LTA from the PepG preparation to allow
measurable detection by A110, suggesting that the PepG preparation
is substantially free of LTA. This level of purity in a PepG
preparation has not previously been demonstrated.
[0202] Previously, it was unclear whether a monoclonal antibody
could enhance phagocytosis of Gram-positive bacteria, because the
polyclonal sera that were used contained many different antibodies
that bound to many different epitopes on the surface of the
bacteria, and the sum of this collective binding and activities may
have accounted for the overall activity of the serum. Here, we
demonstrate that a monoclonal antibody, which binds to a single
epitope on the surface of bacteria, can be opsonic against that
bacteria. We have also demonstrated that monoclonal antibodies
raised against PepG can have that activity, and that those
antibodies may be opsonic for a number of different types of
Gram-positive bacteria.
[0203] The antibodies of the invention can block or alleviate nasal
colonization. These antibodies may therefore be useful protective
molecules in the fight against antibiotic-resistant Gram-positive
bacterial infections.
REFERENCES
[0204] 1. Atrih, Abdelmadjid; Bacher, Gerold; Allmaier, Gunter;
Williamson, Michael P.; and Foster, Simon J. 1999. Analysis of
Peptidoglycan Structure from Vegetative Cells of Bacillus subtilis
168 and Role PBP 5 in Peptidoglycan Maturation, Journal of
Bacteriology 181: 3956-3966. [0205] 2. Bartal, Arie H.; Hirshaut,
Yashar. 1987. Current Methods in Hybridoma Formation Bartal, A. H.
et al. (ed.) Methods of Hybridoma Formation, Humana Press, Clifton,
N.J. [0206] 3. Espersen, F.; Hertz, J. B.; and Hoiby, N. 1981.
Cross-Reactions Between Staphylococcus epidermis and 23 Other
Bacterial Species, Acta Path. Microbial. Scand., Sect. B. 89:
253-260. [0207] 4. Fischer, Gerald W. Broadly reactive opsonic
antibodies that react with common staphylococcal antigens, U.S.
Pat. No. 5,571,511, issued Nov. 5, 1996. [0208] 5. Fleer, A.;
Senders R. C.; Visser M. R.; Bijlmer R. P.; Gerards L. J.;
Kraaijeveld C. A.; Verhoef J. 1983. Septicemia due to
coagulase-negative staphylococci in a neonatal intensive care unit:
clinical and bacteriological features and contaminated parenteral
fluids as a source of sepsis, Pediatr. Infect. Dis. 2: 426-431.
[0209] 6. Foster, Simon J. 1992. Analysis of the Autolysins of
Bacillus subtilis 168 during Vegetative Growth and Differentiation
by Using Renaturin Polyacrylamide Gel Electrophoresis, Journal of
Bacteriology 174: 464-470. [0210] 7. Fournier, Jean-Michel. 1991.
Staphylococcus Aureus, Vaccines and Immunotherapy, Ch. 13, pp.
166-171. [0211] 8. Genarro, A. (ed.) 1990. Remington's
Pharmaceutical Sciences, 18.sup.th Edition, Mack Publishing,
Easton, Pa. [0212] 9. Hancock, I. C. 1997. Bacterial Cell Surface
Carbohydrates: Structure and Assembly, Biochem. Soc. Trans. 25:
183-187. [0213] 10. Jendeberg, Lena; Nilsson, Peter; Larsson,
Antonella; Denker, Per; Uhlen, Mathias; Nilsson, Bjorn; Nygren,
Per-Ake. 1997. Engineering of Fc1 and Fc3 from Human Immunoglobulin
G to Analyse Subclass Specificity for Staphylococcal Protein A, J.
Immunol. Methods 201: 25-34. [0214] 11. Kiser, Kevin B.;
Cantey-Kiser, Jean M.; Lee, Jean C. 1999. Development and
Characterization of a Staphylococcus Aureus Nasal Colonization
Model in Mice, Infection and Immunity 67: 5001-5006. [0215] 12.
Krieger, Monty; Joiner, Keith A. Method for Treating Gram Positive
Septicemia, U.S. Pat. No. 5,624,904, issued Apr. 29, 1997. [0216]
13. Lee, J. C. 1996. The prospects for developing a vaccine against
Staphylococcus aureus, Trends in Micro. 4: 162-66. [0217] 14.
LoBuglio A. F.; Wheeler R. H.; Trang J.; Haynes A.; Rogers K.;
Harvey E. B.; Sun L.; Ghrayeb J.; Khazaeli M. B. 1989. Mouse/human
chimeric monoclonal antibody in man: kinetics and immune response,
P.N.A.S. 86: 4220-4224. [0218] 15. Nakamura, K. et al. 1999. Uptake
and Release of Budesonide from Mucoadhesive, pH-sensitive
Copolymers and Their Application to Nasal Delivery. J. Control.
Release 61:329-335. [0219] 16. Natsume, H., S. Iwata, K. Ohtak, M.
Miyamoto, M. Yamaguchi, K. Hosoya, and D. Kobayashi. 1999.
Screening of cationic compounds as an absorption enhancer for nasal
drug delivery. Int. J. Pharma. 185:1-12. [0220] 17. Navarre,
William Wiley and Schneewind, Olaf. 1999. Surface Proteins of
Gram-Positive Bacteria and Mechanisms of Their Targeting to the
Cell Wall Envelope, Microbiology and Molecular Biology Reviews 63:
174-229. [0221] 18. Peterson, Phillip K.; Verhoef, Jan; Sabath, L.
D.; and Quie, Paul G. 1997. Effect of Protein A on Staphylococcal
Opsonization, Infection and Immunity 15: 760-764. [0222] 19.
Peterson, Phillip K.; Wilkinson, Brian J.; Kim, Youngki; Schmeling,
David; and Quie, Paul G. 1978. Influence of Encapsulation on
Staphylococcal Opsonization and Phagocytosis by Human
Polymorphonuclear Leukocytes, Infection and Immunity 19: 943-949.
[0223] 20. Peterson, Phillip K.; Wilkinson, Brian J.; Kim, Youngki;
Schmeling, David; Douglas, Steven D.; Quie, Paul G.; and Verhoef,
January 1978. The Key Role of Peptidoglycan in the Opsonization of
Staphylococcus Aureus, The Journal of Clinical Investigation 61:
597-609. [0224] 21. Quie, Paul G.; Hill, Harry R.; and Davis, Todd
A. 1974. Defective Phagocytosis of Staphylococci, Annals New York
Academy of Sciences, pp. 233-243. [0225] 22. Ramkissoon-Ganorkar,
C. et al. 1999. Modulating insulin-release profile from
pH/thermosensivite polymeric beads through polymer molecular
weight. J. Contr. Release 59:287-298. [0226] 23. Romero-Vivas J.;
Rubio M.; Fernandez C.; Picazo J. J. 1995. Mortality associated
with nosocomial bacteremia due to methicillin-resistant
Staphylococcus aureus, Clin. Infect. Dis. 21: 1417-23. [0227] 24.
Salton, M. R. J. 1994. The Bacterial Cell Envelope--A Historical
Perspective, in J.-M. Ghuyson and R. Hakenbeck (ed.), Bacterial
Cell Wall, Elsevier Science BV, Amsterdam, pp. 1-22. [0228] 25.
Sambrook, Joseph; Russell, David W. 1989. Molecular Cloning: A
Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. [0229] 26. Schwab, U. E., A. E. Wold, J. L.
Carson, M. W. Leigh, P.-W. Cheng, P. H. Gilligan and T. F. Boat.
1993. Increased adherence of Staphylococcus aureus from cystic
fibrosis lungs to airway epithelial cells. Am. Rev. Respir. Dis.
148:365-369. [0230] 27. Shockman, Gerald D.; Jackson, Dianne E.;
Wong, William. Monoclonal antibodies to Peptidoglycan and Methods
of Preparing Same, U.S. Pat. No. 4,596,769, issued Jun. 24, 1986.
[0231] 28. Shulman, M.; Wilde, C. D.; Kohler, G. 1978. A Better
Cell Line for Making Hybridomas Secreting Specific Antibodies,
Nature 276: 269-270. [0232] 29. Soto, N., A. Vaghjimal, A.
Stahl-Avicolli, J. Protic, L. Lutwick and E. Chapnick. 1999.
Bacitracin versus mupirocin for Staphylococcus aureus nasal
colonization. Infect. Cont. Hosp. Epidem. 20: 351-353. [0233] 30.
Suzuki, Y. and Y. Makino. 1999. Mucosal drug delivery using
cellulose derivative as a functional polymer. J. Control. Release.
62:101-107. [0234] 31. Timmerman C. P.; Besnier J. M.; De Graaf L.;
Torensma R.; Verkley A. J.; Fleer A.; Verhoef J. 1991.
Characterisation and functional aspects of monoclonal antibodies
specific for surface proteins of coagulase-negative staphylococci,
J. Med. Micro. 35: 65-71. [0235] 32. Tomasz, Alexander. 2000. The
Staphylococcal Cell Wall, in V. A. Fischetti et al. (ed.)
Gram-Positive Pathogens, Ch. 36, pp. 351-355. [0236] 33. Verbrugh,
Henri A.; Peters, Roel; Rozenberg-Arska, Maja; Peterson, Phillip
K.; and Verhoef, January 1981. Antibodies to Cell Wall
Peptidoglycan of Staphylococcus aureus in Patients with Serious
Staphylococcal Infections, The Journal of Infectious Disease 144:
1-9. [0237] 34. Verbrugh, Henri A.; Van Dijk, Willemien C.; Peters,
Roel; Van Erne, Marijke E.; Daha, Mohamed R.; Peterson, Phillip K.
and Verhoeff, January 1980. Opsonic Recognition of Staphylococci
Mediated by Cell Wall Peptidoglycan: Antibody-Independent
Activation of Human Complement and Opsonic Activity of
Peptidoglycan Antibodies, The Journal of Immunology 124: 1167-1173.
[0238] 35. Waldvogel, Francis A. 1990. Staphylococcus Aureus
(Including toxic Shock Syndrome), Mandell, G. L. et al. (ed.),
Principles and Practices of Infectious Diseases, Third Edition,
Churchill Livingstone, Philadelphia, Pa., pp. 1489-1510. [0239] 36.
Waldvogel, Francis A. 2000. Staphylococcus Aureus (Including toxic
Shock Syndrome), Mandell, G. L. et al. (ed.) Principles and
Practice of Infectious Diseases, Fifth Edition, Churchill
Livingstone, Philadelphia, Pa., pp. 1760-1775. [0240] 37. Merkus,
F. W., J. C. Verhoef, N. G. Schipper, and E. Marttin. 1999.
Cyclodextrins in nasal drug delivery. Advan. Drug Deliv. Rev. 36:
41-57. [0241] 38. Kengatharan, K. M., De Kimpe, S., Robson, C.,
Foster, S. J. & Thiemermann, C. 1998. Mechanism of
Gram-positive shock: Identification of peptidoglycan and
lipoteichoic acid moieties essential in the induction of nitric
oxide synthase, shock and multiple organ failure. Journal of
Experimental Medicine 188: 305-315. [0242] 39. Foster, S. J. 1993.
Molecular analysis of three major wall-associated proteins of
Bacillus subtilis 168: evidence for the processing the product of a
gene encoding a 258 kDa precursor two-domain ligand-binding
protein. Molecular Microbiology 8: 299-310. [0243] 40. Devereux,
J., Haeberli, P. & Smithies, O. 1984. A comprehensive set of
sequence analysis programs for the VAX. Nucl. Acids Res. 12:
387-395. [0244] 41. Needleman, S. B. & Wunsch, C. D. 1970. A
general method applicable to the search for similarities in the
amino acid sequence of two proteins. J. Mol. Biol. 48: 443-453.
[0245] 42. Smith, T. F. & Waterman, M. S. 1981. Comparison of
biosequences. Adv. Appl. Math 2: 482-489. [0246] 43. Gribskov, M.
& Burgess, R. R. 1986. Sigma factors from E. coli, B. subtilis,
phage SP01, and phage T4 are homologous proteins. 14: 6745-6763.
[0247] 44. Schwartz, R. M. & Dayhoff, M. O. 1979. "Matrices for
detecting distant relationships", pp. 353-358 in: Atlas of Protein
Sequence and Structure. National Biomedical Research Foundation.
[0248] 45. Ausubel et al. (ed.) 1989. Current Protocols in
Molecular Biology, John Wiley & Sons. [0249] 46. Borrebaeck,
Carl A. K. 1995. Antibody Engineering, 2nd Ed., Oxford University
Press, NY. [0250] 47. Harlow, Ed; Lane, David. 1988. Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. [0251] 48. Green, L. L., M. C. Hardy, et al. (1994).
"Antigen-specific human monoclonal antibodies from mice engineered
with human Ig heavy and light chain YACs." Nat Genet. 7(1): 13-21.
[0252] 49. Kantor, A. B., C. E. Merrill, et al. (1995).
"Development of the antibody repertoire as revealed by single-cell
PCR of FACS-sorted B-cell subsets." Ann N Y Acad Sci 764: 224-7.
[0253] 50. Low, N. M., P. H. Holliger, et al. (1996). "Mimicking
somatic hypermutation: affinity maturation of antibodies displayed
on bacteriophage using a bacterial mutator strain." J Mol Biol
260(3): 359-68. [0254] 51. Wagner, S. D., A. V. Popov, et al.
(1994). "The diversity of antigen-specific monoclonal antibodies
from transgenic mice bearing human immunoglobulin gene miniloci."
Eur J Immunol 24(11): 2672-81. [0255] 52. Wagner, S. D., G. T.
Williams, et al. (1994). "Antibodies generated from human
immunoglobulin miniloci in transgenic mice." Nucleic Acids Res
22(8): 1389-93. [0256] 53. Wang, X. and B. D. Stollar (2000).
"Human immunoglobulin variable region gene analysis by single cell
RT-PCR." J Immunol Methods 244(1-2): 217-25. [0257] 54. Winter, G.,
A. D. Griffiths, et al. (1994). "Making antibodies by phage display
technology." Annu Rev Immunol 12: 433-55.
[0258] Having now fully described the invention, it will be
appreciated by those skilled in the art that the invention can be
performed within a range of equivalents and conditions without
departing from the spirit and scope of the invention and without
undue experimentation. In addition, while the invention has been
described in light of certain embodiments and examples, the
inventors believe that it is capable of further modifications. This
application is intended to cover any variations, uses, or
adaptations of the invention which follow the general principles
set forth above.
[0259] The specification includes recitation to the literature and
those literature references are herein specifically incorporated by
reference.
Sequence CWU 1
1
12 1 112 PRT Artificial Sequence Description of Artificial Sequence
Synthetic M130 light chain antibody 1 Asp Ile Lys Met Thr Gln Ser
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys
Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp
Gln Gly 85 90 95 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys 100 105 110 2 336 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA encoding M130
light chain antibody CDS (1)..(336) 2 gat att aag atg acc cag tct
cca ctc act ttg tcg gtt acc att gga 48 Asp Ile Lys Met Thr Gln Ser
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 caa cca gcc tcc atc
tct tgc aag tca agt cag agc ctc tta gat agt 96 Gln Pro Ala Ser Ile
Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 gat gga aag
aca tat ttg aat tgg ttg tta cag cgg cca ggc cag tct 144 Asp Gly Lys
Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 cca
aag cgc cta atc tat ctg gtg tct aaa ctg gac tct gga gtc cct 192 Pro
Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 gac agg ttc gct ggc agt gga tca ggg aca gat ttc aca ctg aaa atc
240 Asp Arg Phe Ala Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 agc aga gtg gag gct gag gat ttg gga gtt tat tat tgc tgg
caa ggt 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp
Gln Gly 85 90 95 aca cat ttt cct ctc acg ttc ggt gct ggg acc aag
ttg gaa ctg aaa 336 Thr His Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu Lys 100 105 110 3 119 PRT Artificial Sequence
Description of Artificial Sequence Synthetic M130 heavy chain
antibody 3 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Ile Leu Gln Pro
Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 Gly Met Ser Val Ser Trp Ile Arg Gln Pro
Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Phe Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr
Val Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile
Thr Ser Val Gly Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala
Arg Asn Tyr Asp Tyr Asp Trp Phe Val Tyr Trp Gly Gln Gly 100 105 110
Thr Leu Val Thr Val Ser Ala 115 4 357 DNA Artificial Sequence
Description of Artificial Sequence Synthetic DNA encoding M130
heavy chain antibody CDS (1)..(357) 4 cag gtt cag ctg cag cag tct
ggc cct ggg ata ttg cag ccc tcc cag 48 Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 acc ctc agt ctg act
tgt tct ttc tct ggg ttt tca ctg agc act tct 96 Thr Leu Ser Leu Thr
Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 ggt atg agt
gtg agc tgg att cgt cag cct tca gga aag ggt ctg gag 144 Gly Met Ser
Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 tgg
ctg gct cac att ttc tgg gat gat gac aag cgc tat aac cca tcc 192 Trp
Leu Ala His Ile Phe Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55
60 ctg aag agc cga ctc aca gtc tcc aag gat acc tcc agc aac cag gtc
240 Leu Lys Ser Arg Leu Thr Val Ser Lys Asp Thr Ser Ser Asn Gln Val
65 70 75 80 ttc ctc aag atc acc agt gtg ggc act gca gat act gcc aca
tac tac 288 Phe Leu Lys Ile Thr Ser Val Gly Thr Ala Asp Thr Ala Thr
Tyr Tyr 85 90 95 tgt gct cga aac tat gat tac gac tgg ttt gtt tac
tgg ggc caa ggg 336 Cys Ala Arg Asn Tyr Asp Tyr Asp Trp Phe Val Tyr
Trp Gly Gln Gly 100 105 110 act ctg gtc act gtc tct gca 357 Thr Leu
Val Thr Val Ser Ala 115 5 40 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 5 tgttttcgta cgtcttgtcc
caggtbcarc tkmarsartc 40 6 32 DNA Artificial Sequence Description
of Artificial Sequence Synthetic primer 6 taccgtaccg gtgayatyma
gatgacmcag wc 32 7 32 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 7 taccgtaccg gtsaaattgw
kctsacycag tc 32 8 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 8 gcacctccag atgttaactg ctc 23
9 22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic primer 9 ctggacaggg mtccakagtt cc 22 10 38 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 10
ataggattcg aaaagtgtac ttmcgtttca gytccarc 38 11 23 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 11
tgttttcgta cgtcttgtcc cag 23 12 35 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 12 ttttctgaat
tctgcagaga cagtgaccag agtcc 35
* * * * *