U.S. patent application number 17/491889 was filed with the patent office on 2022-03-24 for combinations of anti-staphylococcus aureus antibodies.
The applicant listed for this patent is MedImmune, LLC. Invention is credited to Taylor COHEN, Melissa DAMSCHRODER, Qun DU, Bret SELLMAN, Christine TKACZYK.
Application Number | 20220089699 17/491889 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220089699 |
Kind Code |
A1 |
TKACZYK; Christine ; et
al. |
March 24, 2022 |
COMBINATIONS OF ANTI-STAPHYLOCOCCUS AUREUS ANTIBODIES
Abstract
The present disclosure is directed to anti-Staphylococcus aureus
antibody combinations including combinations of antibodies that
bind to S. aureus alpha toxin (AT) protein, clumping factor A
protein (ClfA), and/or at least one leukotoxin protein. Methods of
treating and preventing infections comprising administering the
antibody combinations are also provided herein.
Inventors: |
TKACZYK; Christine;
(Gaithersburg, MD) ; SELLMAN; Bret; (Gaithersburg,
MD) ; DU; Qun; (Gaithersburg, MD) ;
DAMSCHRODER; Melissa; (Gaithersburg, MD) ; COHEN;
Taylor; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune, LLC |
Gaithersburg |
MD |
US |
|
|
Appl. No.: |
17/491889 |
Filed: |
October 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16596388 |
Oct 8, 2019 |
11168133 |
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17491889 |
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62743490 |
Oct 9, 2018 |
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62833297 |
Apr 12, 2019 |
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International
Class: |
C07K 16/12 20060101
C07K016/12 |
Claims
1. A method of preventing a Staphylococcus aureus (S. aureus)
infection in a subject comprising administering to the subject (a)
an antibody or antigen-binding fragment thereof that binds to S.
aureus alpha toxin (AT), (b) an antibody or antigen-binding
fragment thereof that binds to S. aureus clumping factor A (ClfA),
and (c) an antibody or antigen-binding fragment thereof that binds
to at least one S. aureus leukotoxin.
2. A method of treating or preventing a Staphylococcus aureus (S.
aureus) infection in a subject comprising administering to the
subject an antibody or antigen-binding fragment thereof that binds
to at least one S. aureus leukotoxin and (a) an antibody or
antigen-binding fragment thereof that binds to S. aureus alpha
toxin (AT) or (b) an antibody or antigen-binding fragment thereof
that binds to S. aureus clumping factor A (ClfA).
3. A composition comprising (a) an antibody or antigen-binding
fragment thereof that binds to S. aureus AT, (b) an antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA, and
(c) an antibody or antigen-binding fragment thereof that binds to
at least one S. aureus leukotoxin.
4. A composition comprising an antibody or antigen-binding fragment
thereof that binds to at least one S. aureus leukotoxin and (a) an
antibody or antigen-binding fragment thereof that binds to S.
aureus AT or (b) an antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA.
5-12. (canceled)
13. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus AT binds to the same S.
aureus AT epitope as an antibody comprising a VH comprising the
amino acid sequence of SEQ ID NO:19 and a VL comprising the amino
acid sequence of SEQ ID NO:33.
14. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus AT competitively inhibits
binding of an antibody comprising a VH comprising the amino acid
sequence of SEQ ID NO:19 and a VL comprising the amino acid
sequence of SEQ ID NO:33 to S. aureus AT.
15. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus AT comprises a variable
heavy chain (VH) complementarity determining region (CDR) 1
comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2
comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3
comprising the amino acid sequence of SEQ ID NO:3, a variable light
chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO:10,
a VL CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a
VL CDR3 comprising the amino acid sequence of SEQ ID NO:12.
16-19. (canceled)
20. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus AT comprises the VH CDR1,
VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of MEDI4893.
21-34. (canceled)
35. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus AT has an affinity of
80-100 pM for S. aureus AT.
36. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA binds to the same S.
aureus ClfA epitope as an antibody comprising a VH comprising the
amino acid sequence of SEQ ID NO:20 and a VL comprising the amino
acid sequence of SEQ ID NO:34.
37. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA competitively
inhibits binding of an antibody comprising a VH comprising the
amino acid sequence of SEQ ID NO:20 and a VL comprising the amino
acid sequence of SEQ ID NO:34 to S. aureus ClfA.
38. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA comprises a VH CDR1
comprising the amino acid sequence of SEQ ID NO:4, a VH CDR2
comprising the amino acid sequence of SEQ ID NO:5, a VH CDR3
comprising the amino acid sequence of SEQ ID NO:6, a VL CDR1
comprising the amino acid sequence of SEQ ID NO:13, a VL CDR2
comprising the amino acid sequence of SEQ ID NO:14, and a VL CDR3
comprising the amino acid sequence of SEQ ID NO:15.
39-40. (canceled)
41. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA comprises a heavy
chain constant domain comprising the amino acid sequence of CSYHLC
(SEQ ID NO:55).
42. The method of claim 41, wherein said heavy chain constant
domain comprises the amino acid sequence of MHEACSYHLCQKSLSLS (SEQ
ID NO:56).
43-45. (canceled)
46. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA comprises the VH
CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of
SAR114-N3Y, 11H10, SAR72, SAR80, SAR113, SAR132, SAR352, SAR372,
SAR510, SAR547, SAS1, SAS19, or SAS203.
47-65. (canceled)
66. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA has binding
affinities (K.sub.D) of less than 1 nM for all ClfA genotypes.
67-69. (canceled)
70. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
binds to the same S. aureus leukotoxin epitope as an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO:32
and a VL comprising the amino acid sequence of SEQ ID NO:46.
71. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
competitively inhibits binding of an antibody comprising a VH
comprising the amino acid sequence of SEQ ID NO:32 and a VL
comprising the amino acid sequence of SEQ ID NO:46 to the S. aureus
leukotoxin.
72. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
comprises a VHCDR1 comprising the amino acid sequence of SEQ ID
NO:7, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8,
a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9, a VL
CDR1 comprising the amino acid sequence of SEQ ID NO:16, a VL CDR2
comprising the amino acid sequence of SEQ ID NO:17, and a VL CDR3
comprising the amino acid sequence of SEQ ID NO:18.
73-76. (canceled)
77. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL
CDR3 of SAN481-SYT.
78-93. (canceled)
94. The method of claim 1, wherein the S. aureus infection is
sepsis, bacteremia, pneumonia, ICU pneumonia, skin or soft tissue
infection (SSTI), diabetic infection of the lower limbs, a diabetic
foot ulcer (DFU), a bone infection, joint infection, a device
infection, a wound infection, a surgical site infection,
osteomyelitis, and/or antibiotic-resistant S. aureus.
95-109. (canceled)
110. The method of claim 1, wherein the treating or preventing an
S. aureus infection comprises inhibiting S. aureus agglutination,
toxin neutralization, inducing opsonophagocytosis, inhibiting S.
aureus fibrinogen binding, inhibiting S. aureus agglutination,
inhibiting thromboembolic lesion formation, inhibiting S.
aureus-associated sepsis, or any combination of the foregoing.
111-118. (canceled)
119. The method of claim 1, wherein the antibody or antigen-binding
fragment thereof that binds to S. aureus AT, the antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA,
and/or the antibody or antigen-binding fragment thereof that binds
to at least one S. aureus leukotoxin are administered in the same
pharmaceutical composition.
120. A method of treating or preventing a S. aureus infection in a
subject with diabetes comprising administering to the subject an
antibody or antigen-binding fragment thereof that binds to S.
aureus AT.
121-160. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application No. 62/833,297, filed Apr. 12, 2019, and
U.S. Provisional Application No. 62/743,490, filed Oct. 9, 2018,
each of which is hereby incorporated by reference herein in its
entirety.
SEQUENCE LISTING
[0002] The content of the electronically submitted sequence listing
(Name: 2943_1020002_SeqListing_ST25.txt; Size: 81,662 bytes; and
Date of Creation: Oct. 8, 2019) is hereby incorporated by
reference.
BACKGROUND
[0003] Infections caused by antimicrobial resistant (AMR) bacterial
pathogens are an increasing threat to public health. The ongoing
AMR epidemic has been fueled, in part, by empiric broad spectrum
antibiotic therapy. This has led to the exploration of pathogen
specific methods, including monoclonal antibodies (mAbs), to
prevent or treat serious bacterial infections. Numerous monoclonal
antibodies are currently in development for the prevention or
treatment of antibiotic resistant bacterial infections (see, e.g.,
DiGiandomenico, A., and B. R. Sellman, Curr. Opin. Microbiol., 27:
78-85 (2015)). Such passive immunization strategies provide an
immediate and potent immunoglobulin response against the target
pathogen. Ideally, the monoclonal antibody or monoclonal antibody
cocktail provides multiple mechanisms of action to neutralize key
bacterial virulence mechanisms and augment the host innate immune
response, thus providing the greatest opportunity for clinical
success.
[0004] Staphylococcus aureus is a bacterial pathogen that causes a
wide array of diseases including skin and soft tissue infections,
endocarditis, osteomyelitis, pneumonia, and bacteremia (Lowy, F.
D., N. Engl. J. Med., 339(8): 520-32 (1998)). Preclinical studies
indicate monoclonal antibody-based approaches hold promise for
prophylaxis and adjunctive therapy against S. aureus infections
(see, e.g., Hazenbos et al., PLoS Pathog., 9(10):e1003653. doi:
10.1371/journal.ppat.10036532013 (2013); Rouha, H., MAbs, 7(1):
243-254 (2015); Foletti et al., J. Mol. Biol., 425(10): 1641-1654
(2013); Karauzum et al., J Biol Chem., 287(30): 25203-15 (2012);
and Hua et al., Antimicrob Agents Chemother., 58(2): 1108-17
(2014)). However, treatment with individual antibodies may not be
sufficient to address all Staphylococcus aureus infections. Thus,
there remains a need for compositions and methods for treating
Staphylococcus aureus infections, particularly infections that are
resistant to currently-available antibiotics and that provide broad
disease and strain coverage. The present disclosure provides such
compositions and methods.
BRIEF SUMMARY OF THE INVENTION
[0005] As demonstrated herein, combinations of antibodies that
target several different bacterial virulence factors via
complementary mechanism of action can provide broad strain coverage
and broad disease coverage. Exemplary animal models supporting the
breadth of strain and disease coverage encompassed by the
combinations of antibodies provided herein is provided in FIG.
1.
[0006] Provided herein are methods of treating or preventing a
Staphylococcus aureus (S. aureus) infection in a subject comprising
administering to the subject (a) an antibody or antigen-binding
fragment thereof that binds to S. aureus alpha toxin (AT), (b) an
antibody or antigen-binding fragment thereof that binds to S.
aureus clumping factor A (ClfA), and (c) an antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin.
[0007] Provided herein are also methods of treating or preventing a
S. aureus infection in a subject comprising administering to the
subject an antibody or antigen-binding fragment thereof that binds
to at least one S. aureus leukotoxin and (a) an antibody or
antigen-binding fragment thereof that binds to S. aureus alpha
toxin (AT) or (b) an antibody or antigen-binding fragment thereof
that binds to S. aureus clumping factor A (ClfA).
[0008] Provided herein are also compositions comprising (a) an
antibody or antigen-binding fragment thereof that binds to S.
aureus AT, (b) an antibody or antigen-binding fragment thereof that
binds to S. aureus ClfA, and (c) an antibody or antigen-binding
fragment thereof that binds to at least one S. aureus
leukotoxin.
[0009] Provided herein are also compositions comprising an antibody
or antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin and (a) an antibody or antigen-binding fragment
thereof that binds to S. aureus AT or (b) an antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA.
[0010] In certain instances, the composition is for use in treating
or preventing a S. aureus infection in a subject.
[0011] Provided herein are also antibodies and antigen-binding
fragments thereof that bind to S. aureus AT for use in treating or
preventing a S. aureus infection in a subject in combination with
an antibody or antigen-binding fragment thereof that binds to S.
aureus ClfA and an antibody or antigen-binding fragment thereof
that binds to at least one S. aureus leukotoxin.
[0012] Provided herein are also antibodies and antigen-binding
fragments thereof that bind to S. aureus ClfA for use in treating
or preventing a S. aureus infection in a subject in combination
with an antibody or antigen-binding fragment thereof that binds to
S. aureus AT and an antibody or antigen-binding fragment thereof
that binds to at least one S. aureus leukotoxin.
[0013] Provided herein are also antibodies and antigen-binding
fragments thereof that bind to at least one S. aureus leukotoxin
for use in treating or preventing a S. aureus infection in a
subject in combination with an antibody or antigen-binding fragment
thereof that binds to S. aureus AT and/or an antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA.
[0014] In certain instances, the composition is used in the
preparation of a medicament for treating or preventing a S. aureus
infection in a subject.
[0015] Provided herein are also uses of an antibody or
antigen-binding fragment thereof that binds to S. aureus AT in the
preparation of a medicament for treating or preventing a S. aureus
infection in a subject in combination with an antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA and
an antibody or antigen-binding fragment thereof that binds to at
least one S. aureus leukotoxin.
[0016] Provided herein are also uses of an antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA in
the preparation of a medicament for treating or preventing a S.
aureus infection in a subject in combination with an antibody or
antigen-binding fragment thereof that binds to S. aureus AT and an
antibody or antigen-binding fragment thereof that binds to at least
one S. aureus leukotoxin.
[0017] Provided herein are also uses of an antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin in the preparation of a medicament for treating
or preventing a S. aureus infection in a subject in combination
with an antibody or antigen-binding fragment thereof that binds to
S. aureus AT and/or an antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA.
[0018] In certain instances of the method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT binds to the same S. aureus AT epitope as an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO:19
and a VL comprising the amino acid sequence of SEQ ID NO:33. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus AT competitively inhibits binding of an
antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO:19 and a VL comprising the amino acid sequence of SEQ ID
NO:33 to S. aureus AT. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus AT
comprises a variable heavy chain (VH) complementarity determining
region (CDR) 1 comprising the amino acid sequence of SEQ ID NO:1, a
VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH
CDR3 comprising the amino acid sequence of SEQ ID NO:3, a variable
light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID
NO:10, a VL CDR2 comprising the amino acid sequence of SEQ ID
NO:11, and a VL CDR3 comprising the amino acid sequence of SEQ ID
NO:12. In certain instances, the antibody or antigen-binding
fragment thereof that binds to S. aureus AT comprises a VH
comprising the amino acid sequence of SEQ ID NO:19. In certain
instances, the antibody or antigen-binding fragment thereof that
binds to S. aureus AT comprises a VL comprising the amino acid
sequence of SEQ ID NO:33. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus AT
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:47. In certain instances, the antibody or antigen-binding
fragment thereof that binds to S. aureus AT comprises a light chain
comprising the amino acid sequence of SEQ ID NO:52. In certain
instances, the antibody or antigen-binding fragment thereof that
binds to S. aureus AT comprises the VH CDR1, VH CDR2, VH CDR3, VL
CDR1, VL CDR2, and VL CDR3 of MEDI4893. In certain instances, the
CDRs are the Kabat-defined CDRs, the Chothia-defined CDRs, or the
AbM-defined CDRs. In certain instances, the antibody or
antigen-binding fragment that binds to S. aureus AT further
comprises a heavy chain constant region. In certain instances, the
heavy chain constant region is selected from the group consisting
of human immunoglobulin IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4,
IgA.sub.1, and IgA.sub.2 heavy chain constant regions. In certain
instances, the heavy chain constant region is a human IgG.sub.1
constant region. In certain instances, the antibody or
antigen-binding fragment that binds to S. aureus AT further
comprises a light chain constant region. In certain instances, the
light chain constant region is selected from the group consisting
of human immunoglobulin IgG.kappa. and IgG.lamda. light chain
constant regions. In certain instances, the light chain constant
region is a human IgG.kappa. light chain constant region. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus AT is an IgG antibody or antigen-binding
fragment thereof. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus AT
comprises an Fc region that has been engineered to improve
half-life. In certain instances, the antibody or antigen-binding
fragment thereof that binds to S. aureus AT comprises an Fc region
with a YTE mutation. In certain instances, the antibody or
antigen-binding fragment that binds to S. aureus AT is a monoclonal
antibody or antigen-binding fragment. In certain instances, the
antibody or antigen-binding fragment that binds to S. aureus AT is
a full-length antibody. In certain instances, the antibody or
antigen-binding the antigen-binding fragment comprises a Fab, Fab',
F(ab').sub.2, single chain Fv (scFv), disulfide linked Fv,
intrabody, IgG.DELTA.CH2, minibody, F(ab').sub.3, tetrabody,
triabody, diabody, DVD-Ig, Fcab, mAb.sup.2, (scFv).sub.2, or
scFv-Fc. In certain instances, the antibody or antigen-binding
fragment thereof that binds to S. aureus AT has an affinity of
80-100 pM for S. aureus AT.
[0019] In certain instances of the method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus ClfA binds to the same S. aureus ClfA epitope as an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO:20
and a VL comprising the amino acid sequence of SEQ ID NO:34. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA competitively inhibits binding of an
antibody comprising a VH comprising the amino acid sequence of SEQ
ID NO:20 and a VL comprising the amino acid sequence of SEQ ID
NO:34 to S. aureus ClfA. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA
comprises a VH CDR1 comprising the amino acid sequence of SEQ ID
NO:4, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:5,
a VH CDR3 comprising the amino acid sequence of SEQ ID NO:6, a VL
CDR1 comprising the amino acid sequence of SEQ ID NO:13, a VL CDR2
comprising the amino acid sequence of SEQ ID NO:14, and a VL CDR3
comprising the amino acid sequence of SEQ ID NO:15. In certain
instances, the antibody or antigen-binding fragment thereof that
binds to S. aureus ClfA comprises a VH comprising the amino acid
sequence of SEQ ID NO:20. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA
comprises a VL comprising the amino acid sequence of SEQ ID NO:34.
In certain instances, the antibody or antigen-binding fragment
thereof that binds to S. aureus ClfA comprises a heavy chain
constant domain comprising the amino acid sequence of CSYHLC (SEQ
ID NO:55). In certain instances, the heavy chain constant domain
comprises the amino acid sequence of MHEACSYHLCQKSLSLS (SEQ ID
NO:56). In certain instances, the antibody or antigen-binding
fragment thereof that binds to S. aureus ClfA comprises a heavy
chain comprising the amino acid sequence of SEQ ID NO:49. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA comprises a light chain comprising the
amino acid sequence of SEQ ID NO:53. In certain instances, the
antibody or antigen-binding fragment thereof that binds to S.
aureus ClfA comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL
CDR2, and VL CDR3 of SAR114-N3Y. In certain instances, the antibody
or antigen-binding fragment thereof that binds to S. aureus ClfA
comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL
CDR3 of 11H10, SAR72, SAR80, SAR113, SAR132, SAR352, SAR372,
SAR510, SAR547, SAS1, SAS19, or SAS203. In certain instances, the
CDRs are the Kabat-defined CDRs, the Chothia-defined CDRs, or the
AbM-defined CDRs. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA
comprises a VH and a VL, wherein the VH comprises the amino acid
sequence set forth in any one of SEQ ID NOs:21-31 and 68. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA comprises a VH and a VL, wherein the
VL comprises the amino acid sequence set forth in any one of SEQ ID
NOs: 35-45 and 69. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA
comprises VH and VL sequences comprising the amino acid sequences
set forth in (a) SEQ ID NOs:21 and 35, respectively (b) SEQ ID
NOs:22 and 36, respectively, (c) SEQ ID NOs:23 and 37,
respectively, (d) SEQ ID NOs:24 and 38, respectively, (e) SEQ ID
NOs:25 and 39, respectively, (f) SEQ ID NOs:26 and 40,
respectively, (g) SEQ ID NOs:27 and 41, respectively, (h) SEQ ID
NOs:28 and 42, respectively (i) SEQ ID NOs:29 and 43, respectively,
(j) SEQ ID NOs:30 and 44, respectively, (k) SEQ ID NOs:31 and 45,
respectively, or (1) SEQ ID NOs: 68 and 69, respectively. In
certain instances, the antibody or antigen-binding fragment that
binds to S. aureus ClfA further comprises a heavy chain constant
region. In certain instances, the heavy chain constant region is
selected from the group consisting of human immunoglobulin
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2 heavy chain constant regions. In certain instances, the
heavy chain constant region is a human IgG.sub.1 constant region.
In certain instances, the antibody or antigen-binding fragment that
binds to S. aureus ClfA further comprises a light chain constant
region. In certain instances, the light chain constant region is
selected from the group consisting of human immunoglobulin
IgG.kappa. and IgG.lamda. light chain constant regions. In certain
instances, the light chain constant region is a human IgG.kappa.
light chain constant region. In certain instances, the antibody or
antigen-binding fragment that binds to S. aureus ClfA comprises a
mutation that extends half-life relative to the same antibody
without the mutation in human FcRn mice. In certain instances, the
antibody or antigen-binding fragment that binds to S. aureus ClfA
comprises a mutation that extends half-life relative to the same
antibody without the mutation, and wherein the mutation does not
inhibit OPK activity relative to the same antibody or
antigen-binding fragment the mutation. In certain instances, the
antibody or antigen-binding fragment that binds to S. aureus ClfA
is a monoclonal antibody or antigen-binding fragment. In certain
instances, the antibody or antigen-binding fragment that binds to
S. aureus ClfA is a full-length antibody. In certain instances, the
antibody or antigen-binding fragment that binds to S. aureus ClfA
is an antigen-binding fragment. In certain instances, the
antigen-binding fragment comprises a Fab, Fab', F(ab').sub.2,
single chain Fv (scFv), disulfide linked Fv, intrabody,
IgG.DELTA.CH2, minibody, F(ab').sub.3, tetrabody, triabody,
diabody, DVD-Ig, Fcab, mAb.sup.2, (scFv).sub.2, or scFv-Fc. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA has IC50s for ClfA001, ClfA002, and
ClfA004 in a fibrinogen binding inhibition assay that are within 2
.mu.g/ml of each other. In certain instances, the antibody or
antigen-binding fragment thereof that binds to S. aureus ClfA has
IC50s for ClfA001, ClfA002, and ClfA004 in a fibrinogen binding
inhibition assay that are all between 1 .mu.g/ml and 5 .mu.g/ml. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA has binding affinities (K.sub.D) for
ClfA001, ClfA002, and ClfA004 that are all between 200 and 350 pM.
In certain instances, the antibody or antigen-binding fragment
thereof that binds to S. aureus ClfA has binding affinities
(K.sub.D) of less than 1 nM for all ClfA genotypes. In certain
instances, the antibody or antigen-binding fragment that binds to
S. aureus ClfA has a monomer purity that decreases by no more than
5% after exposure of the antibody or antigen-binding fragment to
conventional white light at 2kLux/hr at 23.degree. C. for 14
days.
[0020] In certain instances of the method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to at least
one S. aureus leukotoxin binds to LukF, LukD, and/or HlgB, and/or
wherein the antibody or antigen-binding fragment thereof
neutralizes LukF, LukD, and/or HlgB. In certain instances, the
antibody or antigen-binding fragment thereof that binds to at least
one S. aureus leukotoxin binds to LukF, LukD, and HlgB, and/or
wherein the antibody or antigen-binding fragment thereof
neutralizes LukF, LukD, and HlgB. In certain instances, the
antibody or antigen-binding fragment thereof that binds to at least
one S. aureus leukotoxin binds to the same S. aureus leukotoxin
epitope as an antibody comprising a VH comprising the amino acid
sequence of SEQ ID NO:32 and a VL comprising the amino acid
sequence of SEQ ID NO:46. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin competitively inhibits binding of an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO:32
and a VL comprising the amino acid sequence of SEQ ID NO:46 to the
S. aureus leukotoxin. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin comprises a VHCDR1 comprising the amino acid
sequence of SEQ ID NO:7, a VH CDR2 comprising the amino acid
sequence of SEQ ID NO:8, a VH CDR3 comprising the amino acid
sequence of SEQ ID NO:9, a VL CDR1 comprising the amino acid
sequence of SEQ ID NO:16, a VL CDR2 comprising the amino acid
sequence of SEQ ID NO:17, and a VL CDR3 comprising the amino acid
sequence of SEQ ID NO:18. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin comprises a VH comprising the amino acid sequence
of SEQ ID NO:32. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin comprises a VL comprising the amino acid sequence
of SEQ ID NO:46. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:50. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin comprises a light chain comprising the amino acid
sequence of SEQ ID NO:54. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1,
VL CDR2, and VL CDR3 of SAN481-SYT. In certain instances, the CDRs
are the Kabat-defined CDRs, the Chothia-defined CDRs, or the
AbM-defined CDRs. In certain instances, the antibody or
antigen-binding fragment that binds to at least one S. aureus
leukotoxin further comprises a heavy chain constant region. In
certain instances, the heavy chain constant region is selected from
the group consisting of human immunoglobulin IgG.sub.1, IgG.sub.2,
IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2 heavy chain constant
regions. In certain instances, the heavy chain constant region is a
human IgG.sub.1 constant region. In certain instances, the antibody
or antigen-binding fragment that binds at least one S. aureus
leukotoxin further comprises a light chain constant region. In
certain instances, the light chain constant region is selected from
the group consisting of human immunoglobulin IgG.kappa. and
IgG.lamda. light chain constant regions. In certain instances, the
light chain constant region is a human IgG.kappa. light chain
constant region. In certain instances, the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin is an IgG antibody or antigen-binding fragment
thereof. In certain instances, the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
comprises an Fc region that has been engineered to improve
half-life. In certain instances, the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
comprises an Fc region with a YTE mutation. In certain instances,
the antibody or antigen-binding fragment that binds to at least one
S. aureus leukotoxin is a monoclonal antibody or antigen-binding
fragment. In certain instances, the antibody or antigen-binding
fragment that binds to at least one S. aureus leukotoxin is a
full-length antibody. In certain instances, the antibody or
antigen-binding fragment that binds to at least one S. aureus
leukotoxin is an antigen-binding fragment. In certain instances,
the antigen-binding fragment comprises a Fab, Fab', F(ab').sub.2,
single chain Fv (scFv), disulfide linked Fv, intrabody,
IgG.DELTA.CH2, minibody, F(ab').sub.3, tetrabody, triabody,
diabody, DVD-Ig, Fcab, mAb.sup.2, (scFv).sub.2, or scFv-Fc. In
certain instances, the antibody or antigen-binding fragment thereof
that binds to at least one S. aureus leukotoxin has an affinity of
less than 75 pM for S. aureus LukF, LukD, and HlgAB. In certain
instances, the antibody or antigen-binding fragment thereof that
binds to at least one S. aureus leukotoxin has similar binding
affinities for LukF, LukD, and HIgB.
[0021] In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the S.
aureus infection is sepsis. In certain instances of a method,
composition, antibody or antigen-binding fragment thereof, or use
provided herein, the S. aureus infection is bacteremia. In certain
instances of a method, composition, antibody or antigen-binding
fragment thereof, or use provided herein, the S. aureus infection
is pneumonia. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the S. aureus infection is pneumonia the S. aureus
infection is ICU pneumonia. In certain instances of a method,
composition, antibody or antigen-binding fragment thereof, or use
provided herein, the S. aureus infection is a skin or soft tissue
infection (SSTI). In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the S. aureus infection is a diabetic infection of the
lower limbs. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the S. aureus infection is a diabetic foot ulcer (DFU). In
certain instances, the DFU is uninfected. In certain instances, the
DFU is infected. In certain instances, the DFU is a grade 1, 2 or 3
DFU. In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the S.
aureus infection is a bone or joint infection. In certain instances
of a method, composition, antibody or antigen-binding fragment
thereof, or use provided herein, the S. aureus infection is a joint
infection, a device infection, a wound infection, a surgical site
infection, or osteomyelitis.
[0022] In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
subject is a surgical subject.
[0023] In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the S.
aureus infection comprises antibiotic-resistant S. aureus.
[0024] In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
subject has diabetes. In certain instances of a method,
composition, antibody or antigen-binding fragment thereof, or use
provided herein, the subject is human.
[0025] In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
treating or preventing an S. aureus infection comprises inhibiting
S. aureus agglutination, toxin neutralization, inducing
opsonophagocytosis, inhibiting S. aureus fibrinogen binding,
inhibiting S. aureus agglutination, inhibiting thromboembolic
lesion formation, inhibiting S. aureus-associated sepsis, or any
combination of the foregoing.
[0026] In certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT and the antibody or antigen-binding fragment thereof that
binds to S. aureus ClfA are administered in the same pharmaceutical
composition. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the antibody or antigen-binding fragment thereof that binds
to S. aureus AT and the antibody or antigen-binding fragment
thereof that binds to S. aureus ClfA are administered in the
separate pharmaceutical compositions. In certain instances of a
method, composition, antibody or antigen-binding fragment thereof,
or use provided herein, the antibody or antigen-binding fragment
thereof that binds to S. aureus AT and the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin are administered in the same pharmaceutical
composition. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the antibody or antigen-binding fragment thereof that binds
to S. aureus AT and the antibody or antigen-binding fragment
thereof that binds to at least one S. aureus leukotoxin are
administered in the separate pharmaceutical compositions. In
certain instances of a method, composition, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus ClfA and the antibody or antigen-binding fragment thereof
that binds to at least one S. aureus leukotoxin are administered in
the same pharmaceutical composition. In certain instances of a
method, composition, antibody or antigen-binding fragment thereof,
or use provided herein, the antibody or antigen-binding fragment
thereof that binds to S. aureus ClfA and the antibody or
antigen-binding fragment thereof that binds to at least one S.
aureus leukotoxin are administered in the separate pharmaceutical
compositions. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the separate pharmaceutical compositions are administered
simultaneously. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the separate pharmaceutical compositions are administered
sequentially. In certain instances of a method, composition,
antibody or antigen-binding fragment thereof, or use provided
herein, the antibody or antigen-binding fragment thereof that binds
to S. aureus AT, the antibody or antigen-binding fragment thereof
that binds to S. aureus ClfA, and the antibody or antigen-binding
fragment thereof that binds to at least one S. aureus leukotoxin
are administered in the same pharmaceutical composition.
[0027] Provided herein are also methods of treating or preventing a
S. aureus infection in a subject with diabetes comprising
administering to the subject an antibody or antigen-binding
fragment thereof that binds to S. aureus AT.
[0028] Provided herein are also antibodies or antigen-binding
fragments thereof that bind to S. aureus AT for use in treating or
preventing a S. aureus infection in a subject with diabetes.
[0029] Provided herein are also uses of an antibody or
antigen-binding fragment thereof that binds to S. aureus AT in the
preparation of a medicament for treating or preventing a S. aureus
infection in a subject with diabetes.
[0030] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT binds to the same S. aureus AT epitope as an antibody
comprising a VH comprising the amino acid sequence of SEQ ID NO:19
and a VL comprising the amino acid sequence of SEQ ID NO:33.
[0031] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT competitively inhibits binding of an antibody comprising
a VH comprising the amino acid sequence of SEQ ID NO:19 and a VL
comprising the amino acid sequence of SEQ ID NO:33 to S. aureus
AT.
[0032] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises a VH CDR1 comprising the amino acid sequence of
SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID
NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3,
a VL CDR1 comprising the amino acid sequence of SEQ ID NO:10, a VL
CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a VL
CDR3 comprising the amino acid sequence of SEQ ID NO:12.
[0033] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises a VH comprising the amino acid sequence of SEQ
ID NO:19.
[0034] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises a VL comprising the amino acid sequence of SEQ
ID NO:33.
[0035] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:47.
[0036] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises a light chain comprising the amino acid
sequence of SEQ ID NO:52.
[0037] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL
CDR2, and VL CDR3 of MEDI4893. In certain instances, the CDRs are
the Kabat-defined CDRs, the Chothia-defined CDRs, or the
AbM-defined CDRs.
[0038] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment that binds to S. aureus AT
further comprises a heavy chain constant region. In certain
instances, the heavy chain constant region is selected from the
group consisting of human immunoglobulin IgG.sub.1, IgG.sub.2,
IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2 heavy chain constant
regions. In certain instances, the heavy chain constant region is a
human IgG.sub.1 constant region.
[0039] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment that binds to S. aureus AT
further comprises a light chain constant region. In certain
instances, the light chain constant region is selected from the
group consisting of human immunoglobulin IgG.kappa. and IgG.lamda.
light chain constant regions. In certain instances, the light chain
constant region is a human IgG.kappa. light chain constant
region.
[0040] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT is an IgG antibody or antigen-binding fragment
thereof.
[0041] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises an Fc region that has been engineered to
improve half-life.
[0042] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT comprises an Fc region with a YTE mutation.
[0043] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment that binds to S. aureus AT is
a monoclonal antibody or antigen-binding fragment.
[0044] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment that binds to S. aureus AT is
a full-length antibody.
[0045] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment that binds to S. aureus AT is
an antigen-binding fragment. In certain instances, the
antigen-binding fragment comprises a Fab, Fab', F(ab').sub.2,
single chain Fv (scFv), disulfide linked Fv, intrabody,
IgG.DELTA.CH2, minibody, F(ab').sub.3, tetrabody, triabody,
diabody, DVD-Ig, Fcab, mAb.sup.2, (scFv).sub.2, or scFv-Fc.
[0046] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the
antibody or antigen-binding fragment thereof that binds to S.
aureus AT has an affinity of 80-100 pM for S. aureus AT.
[0047] In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the S.
aureus infection is sepsis. In certain instances of a method,
antibody or antigen-binding fragment thereof, or use provided
herein, the S. aureus infection is bacteremia. In certain instances
of a method, antibody or antigen-binding fragment thereof, or use
provided herein, the S. aureus infection is pneumonia. In certain
instances of a method, antibody or antigen-binding fragment
thereof, or use provided herein, the S. aureus infection is ICU
pneumonia. In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the S.
aureus infection is a SSTI. In certain instances of a method,
antibody or antigen-binding fragment thereof, or use provided
herein, the S. aureus infection is a diabetic infection of the
lower limbs. In certain instances of a method, antibody or
antigen-binding fragment thereof, or use provided herein, the S.
aureus infection is a DFU. In certain instances, the DFU is
uninfected. In certain instances, the DFU is infected. In certain
instances, the DFU is a grade 1, 2 or 3 DFU. In certain instances
of a method, antibody or antigen-binding fragment thereof, or use
provided herein, the S. aureus infection is a bone or joint
infection.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0048] FIG. 1 is a schematic showing that a range of animal models
supports the use of the combination of antibodies directed against
alpha toxin (AT), clumping factor A (ClfA), and leukotoxins to
achieve broad strain and disease coverage.
[0049] FIG. 2 is a graph showing the efficacy of the combination of
antibodies directed against AT, ClfA, and leukotoxins (MEDI6389) in
inhibiting red blood cell (RBC) hemolysis as compared to the
efficacy of an antibody directed against AT (MEDI4893*) alone and
the efficacy of a combination of antibodies directed against ClfA
(SAR114) and leukotoxins (SAN481-SYT*). (See Example 1.)
[0050] FIG. 3 is a graph showing the efficacy of the combination of
antibodies directed against AT, ClfA, and leukotoxins (MEDI6389) in
maintaining monocyte viability as compared to the efficacy an
antibody directed against leukotoxins (SAN481-SYT*) alone and the
efficacy of a combination of antibodies directed against AT
(MEDI4893*) and ClfA (SAR114). (See Example 1.)
[0051] FIG. 4 is a graph showing the efficacy of the combination of
antibodies directed against AT, ClfA, and leukotoxins (MEDI6389) in
inhibiting fibrinogen (Fg) binding as compared to the efficacy of
an antibody directed against ClfA (SAR114) alone and the efficacy
of a combination of antibodies directed against AT (MEDI4893*) and
leukotoxins (SAN481-SYT*). (See Example 1.)
[0052] FIG. 5 provides a graph and images showing that the
combination of SAN481-SYT* and MEDI4893* is superior to the
activity of either SAN481-SYT* or MEDI4893* alone in a
dermonecrosis model with a S. aureus wound isolate. (See Example
2.)
[0053] FIG. 6 provides graphs showing that neutralization of AT,
ClfA, and leukotoxins are necessary for protection in the rabbit
bacteremia model. (See Example 3.)
[0054] FIG. 7 provides graphs comparing the efficacy of the
combination of antibodies directed against AT, ClfA, and
leukotoxins (MEDI6389) against two different bacterial bloodstream
infections: HA-MRSA NRS382 (top panel) and CA-MRSA SF8300 (bottom
panel). (See Example 4).
[0055] FIG. 8 provides a graph and images showing that a mixed
infection of S. aureus (SA), Pseudomonas aeruginosa (PA), and
Streptococcus pyogenese (SP) resulted in delayed closure of skin
lesions in a diabetic mouse dermonecrosis model compared to an
infection by SA alone. The images show lesions at Day 43 post
intra-dermal challenge. (See Example 5.)
[0056] FIG. 9 provides graphs and images showing that the
combination of antibodies directed against AT, ClfA, and
leukotoxins (MEDI6389) improves the healing of wounds resulting
from mixed-bacteria infections. (See Example 5).
[0057] FIG. 10 provides a sequence alignment of HIgB (SEQ ID
NO:59), LukF (SEQ ID NO:60), and LukD (SEQ ID NO:61).
[0058] FIGS. 11A-G show that elevated glucose levels correlate with
more severe S. aureus infections. (A and B) After infection with S.
aureus, diabetic db/db (A) and STZ (B) mice had increased mortality
as compared to non-diabetic controls. (C) After infection with S.
aureus, diabetic db/db mice had similar levels of S. aureus in
their kidneys as non-diabetic controls. (D) After infection with S.
aureus, diabetic STZ mice had similar levels of S. aureus in their
kidneys as non-diabetic controls. (E, F, and G) Treatment with
Rosiglitazone for 1 week prior to infection with S. aureus reduced
circulating glucose (E) and increased survival (F), but did not
affect the bacterial burden in the kidney (G). (See Example 7.)
[0059] FIGS. 12A-D show that systemic infection of the diabetic
host lead to an AT-dependent increase in circulating NETs. (A)
After infection with S. aureus, ELISA detected increased serum NETs
in diabetic mice as compared to non-diabetic controls. (B)
Neutralization of S. aureus alpha toxin (AT) with the anti-alpha
toxin monoclonal antibody MEDI4893* significantly reduced the
number of NE-DNA complexes in the serum 48 hours post-infection in
diabetic mice. (C) After infection with S. aureus, Western blot
showed increased citrinulated Histone H3 (H3cit) in diabetic mice
as compared to non-diabetic controls. (D) Neutralization of S.
aureus AT increased survival of diabetic mice infected with S.
aureus. (See Example 8.)
[0060] FIGS. 13A-D show that diabetic db/db mice have increased low
density neutrophils (LDNs). (A) After infection with S. aureus, the
amount of LDNs in the blood of infected diabetic db/db mice was
significantly increased as compared to uninfected db/db mice or
non-diabetic controls. (B) Treatment with Rosiglitazone for 1 week
prior to infection with S. aureus reduced LDNs 48 hours
post-infection. (C and D) Neutralization of S. aureus AT prior to
infection reduced LDNs (C) but did not affect overall numbers of
neutrophils (D) in diabetic db/db mice. (See Example 9.)
[0061] FIG. 14 shows that, after infection with S. aureus, diabetic
STZ mice had increased low density neutrophils LDNs. (See Example
9.)
[0062] FIG. 15A-D shows that delivery of a TGF.beta. neutralizing
antibody prior to infection is protective in diabetic mice (A)
TGF.beta. significantly increased the number of LDNs in diabetic
db/db blood, but not in non-diabetic control blood. (B and C)
Delivery of a TGF.beta. neutralizing antibody provided prior to S.
aureus infection reduced LDNs in blood (B), but did not affect the
amount of bacteria in the kidney (C). (D) Delivery of a TGF.beta.
neutralizing antibody provided prior to infection increased
survival of diabetic db/db mice. (See Example 10.)
[0063] FIGS. 16A-E show that blocking the .alpha.V.beta.6/8 pathway
prior to infection is protective in diabetic mice. (A) .beta.8
positive inflammatory monocytes and dendritic cells (DCs) increased
in the livers of diabetic db/db mice, not C57BKS mice, following
infection. (B) Integrin expression increased on the surface of
monocytes, and the overall number of DCs (not the density of
.beta.8 on DCs) increased. (C) Neutralizing .alpha.V.beta.6/8 prior
to infection decreased LDNs in the blood stream as compared to
administration of an anti-.alpha.V.beta.6 antibody or a control
antibody (c-IgG). (D) Neutralizing .alpha.V.beta.6/8 prior to
infection did not affect the amount of bacteria in the kidney. (E)
Neutralizing .alpha.V.beta.6/8 prior to infection increased
survival as compared to administration of a control antibody
(c-IgG). (See Example 10.)
[0064] FIGS. 17A-C show that AT influences activation of TGF.beta.
independently of .alpha.V.beta.8 expression on innate immune cells.
(A) pSMAD levels were higher in the livers of infected diabetic
mice as compared with naive diabetic mice and infected non-diabetic
mice. (B) Neutralizing AT significantly reduced pSMAD levels in the
liver. (C) Neutralizing AT did not alter the numbers of
.alpha.V.beta.8 expressing innate immune cells. (See Example
11.)
DETAILED DESCRIPTION OF THE INVENTION
[0065] The present disclosure provides combinations of antibodies
and antigen-binding fragments thereof (e.g., monoclonal antibodies
and antigen-binding fragments thereof) that bind to Staphylococcus
aureus (S. aureus) alpha toxin (AT), clumping factor A (ClfA), and
at least one leukotoxin. The present disclosure also provides
methods of using such combinations, for example, in the treatment
or prevention of S. aureus infections.
I. Definitions
[0066] As used herein, the term "alpha toxin" or "AT" refers to
bacterial alpha toxin polypeptides including, but not limited to,
native alpha toxin polypeptides and isoforms of alpha toxin
polypeptides. "Alpha toxin" encompasses full-length, unprocessed
alpha toxin polypeptides as well as forms of alpha toxin
polypeptides that result from processing within the cell. As used
herein, the term "S. aureus alpha toxin" refers to a polypeptide
comprising the amino acid sequence of
TABLE-US-00001 (SEQ ID NO: 57)
adsdiniktgttdigsnttvktgdlvtydkengmhkkvfysfiddknhn
kkllvirtkgtiagqyrvyseeganksglawpsaflcvqlqlpdnevaq
isdyyprnsidtkeymstltygfngnvtgddtgkiggliganvsightl
kyvqpdfktilesptdkkvgwkvifnnmvnqnwgpydrdswnpvygnql
fmktrngsmkaadnfldpnkassllssgfspdfatvitmdrkaskqqtn
idviyervrddyqlhwtstnwkgtntkdkwtdrsserykidwekeemtn. The S. aureus
alpha toxin H35L mutant has the sequence (SEQ ID NO: 58)
adsdiniktgttdigsnttvktgdlvtydkengmlkkvfysfiddknhn
kkllvirtkgtiagqyrvyseeganksglawpsaflcvqlqlpdnevaq
isdyyprnsidtkeymstltygfngnvtgddtgkiggliganvsightl
kyvqpdfktilesptdkkvgwkvifnnmvnqnwgpydrdswnpvygnql
fmktrngsmkaadnfldpnkassllssgfspdfatvitmdrkaskqqtn
idviyervrddyqlhwtstnwkgtntkdkwtdrsserykidwekeemtn.
[0067] A "alpha toxin polynucleotide," "alpha toxin nucleotide," or
"alpha toxin nucleic acid" refer to a polynucleotide encoding alpha
toxin.
[0068] As used herein, the term "clumping factor A" or "ClfA"
refers to bacterial clumping factor A polypeptides including, but
not limited to, native clumping factor A polypeptides and isoforms
of clumping factor A polypeptides. "Clumping factor A" encompasses
full-length, unprocessed clumping factor A polypeptides as well as
forms of clumping factor A polypeptides that result from processing
within the cell. A "clumping factor A polynucleotide," "clumping
factor A nucleotide," or "clumping factor A nucleic acid" refer to
a polynucleotide encoding alpha toxin.
[0069] As used herein, the term "leukotoxin" refers to bacterial
leukotoxin polypeptides including, but not limited to, native
leukotoxin polypeptides and isoforms of leukotoxin polypeptides.
"Leukotoxin" encompasses a full-length, unprocessed leukotoxin
polypeptides as well as forms of leukotoxin polypeptides that
result from processing within the cell. Leukotoxins include LukSF,
leukotoxin ED (LukED), HlgAB, HlgCB), and leukotoxin AB (LukAB,
also known as LukGH). As used herein, the term "S. aureus HIgB"
refers to a polypeptide comprising the amino acid sequence of SEQ
ID NO:59. As used herein, the term "S. aureus LukF" refers to a
polypeptide comprising the amino acid sequence of SEQ ID NO:60. As
used herein, the term "S. aureus LukD" refers to a polypeptide
comprising the amino acid sequence of SEQ ID NO:61. As used herein,
the term "S. aureus HIgB" refers to a polypeptide comprising the
amino acid sequence of SEQ ID NO:59. (See FIG. 10.) A "leukotoxin
polynucleotide," "leukotoxin nucleotide," or "leukotoxin nucleic
acid" refer to a polynucleotide encoding a leukotoxin.
[0070] The term "antibody" means an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, chimeric
antibodies, humanized antibodies, human antibodies, fusion proteins
comprising an antibody, and any other modified immunoglobulin
molecule so long as the antibodies exhibit the desired biological
activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses
(isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2),
based on the identity of their heavy-chain constant domains
referred to as alpha, delta, epsilon, gamma, and mu, respectively.
The different classes of immunoglobulins have different and well
known subunit structures and three-dimensional configurations.
Antibodies can be naked or conjugated to other molecules such as
toxins, radioisotopes, etc.
[0071] The term "monoclonal antibodies," as used herein, refers to
antibodies that are produced by a single clone of B-cells and bind
to the same epitope. In contrast, the term "polyclonal antibodies"
refers to a population of antibodies that are produced by different
B-cells and bind to different epitopes of the same antigen.
[0072] The term "antibody fragment" refers to a portion of an
intact antibody. An "antigen-binding fragment," "antigen-binding
domain," or "antigen-binding region," refers to a portion of an
intact antibody that binds to an antigen. An antigen-binding
fragment can contain the antigenic determining regions of an intact
antibody (e.g., the complementarity determining regions (CDR)).
Examples of antigen-binding fragments of antibodies include, but
are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear
antibodies, and single chain antibodies. An antigen-binding
fragment of an antibody can be derived from any animal species,
such as rodents (e.g., mouse, rat, or hamster) and humans or can be
artificially produced.
[0073] A whole antibody typically consists of four polypeptides:
two identical copies of a heavy (H) chain polypeptide and two
identical copies of a light (L) chain polypeptide. Each of the
heavy chains contains one N-terminal variable (VH) region and three
C-terminal constant (CHI, CH2 and CH3) regions, and each light
chain contains one N-terminal variable (VL) region and one
C-terminal constant (CL) region. The variable regions of each pair
of light and heavy chains form the antigen binding site of an
antibody. The VH and VL regions have the same general structure,
with each region comprising four framework regions, whose sequences
are relatively conserved. The term "framework region," as used
herein, refers to the relatively conserved amino acid sequences
within the variable region which are located between the
hypervariable or complementary determining regions (CDRs). There
are four framework regions in each variable domain, which are
designated FR1, FR2, FR3, and FR4. The framework regions form the R
sheets that provide the structural framework of the variable region
(see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed.,
Garland Publishing, New York, N.Y. (2001)). The three CDRs, known
as CDR1, CDR2, and CDR3, form the "hypervariable region" of an
antibody, which is responsible for antigen binding.
[0074] The terms "VL" and "VL domain" are used interchangeably to
refer to the light chain variable region of an antibody.
[0075] The terms "VH" and "VH domain" are used interchangeably to
refer to the heavy chain variable region of an antibody.
[0076] The term "Kabat numbering" and like terms are recognized in
the art and refer to a system of numbering amino acid residues in
the heavy and light chain variable regions of an antibody or an
antigen-binding fragment thereof. In certain aspects, CDRs can be
determined according to the Kabat numbering system (see, e.g.,
Kabat E A & Wu T T (1971) Ann NY Acad Sci 190: 382-391 and
Kabat E A et al., (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242). Using the Kabat numbering
system, CDRs within an antibody heavy chain molecule are typically
present at amino acid positions 31 to 35, which optionally can
include one or two additional amino acids, following 35 (referred
to in the Kabat numbering scheme as 35A and 35B) (CDR1), amino acid
positions 50 to 65 (CDR2), and amino acid positions 95 to 102
(CDR3). Using the Kabat numbering system, CDRs within an antibody
light chain molecule are typically present at amino acid positions
24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino
acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs
of the antibodies described herein have been determined according
to the Kabat numbering scheme.
[0077] Chothia refers instead to the location of the structural
loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end
of the Chothia CDR-H1 loop when numbered using the Kabat numbering
convention varies between H32 and H34 depending on the length of
the loop (this is because the Kabat numbering scheme places the
insertions at H35A and H35B; if neither 35A nor 35B is present, the
loop ends at 32; if only 35A is present, the loop ends at 33; if
both 35A and 35B are present, the loop ends at 34). The AbM
hypervariable regions represent a compromise between the Kabat CDRs
and Chothia structural loops, and are used by Oxford Molecular's
AbM antibody modeling software.
TABLE-US-00002 Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2
L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B
H26-H35B H26-H32 . . . 34 (Kabat Numbering) H1 H31-H35 H26-H35
H26-H32 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102
H95-H102 H95-H102
[0078] As used herein, the term "constant region" or "constant
domain" are interchangeable and have its meaning common in the art.
The constant region is an antibody portion, e.g., a carboxyl
terminal portion of a light and/or heavy chain which is not
directly involved in binding of an antibody to antigen but which
can exhibit various effector functions, such as interaction with
the Fc receptor. The constant region of an immunoglobulin molecule
generally has a more conserved amino acid sequence relative to an
immunoglobulin variable domain.
[0079] As used herein, the term "heavy chain" when used in
reference to an antibody can refer to any distinct type, e.g.,
alpha (.alpha.), delta (.delta.), epsilon (.epsilon.), gamma
(.gamma.), and mu (.mu.), based on the amino acid sequence of the
constant domain, which give rise to IgA, IgD, IgE, IgG, and IgM
classes of antibodies, respectively, including subclasses of IgG,
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4. Heavy chain
amino acid sequences are well known in the art. In specific
embodiments, the heavy chain is a human heavy chain.
[0080] As used herein, the term "light chain" when used in
reference to an antibody can refer to any distinct type, e.g.,
kappa (.kappa.) or lambda (.lamda.) based on the amino acid
sequence of the constant domains. Light chain amino acid sequences
are well known in the art. In specific embodiments, the light chain
is a human light chain.
[0081] A "chimeric" antibody refers to an antibody or fragment
thereof comprising both human and non-human regions. A "humanized"
antibody is a antibody comprising a human antibody scaffold and at
least one CDR obtained or derived from a non-human antibody.
Non-human antibodies include antibodies isolated from any non-human
animal, such as, for example, a rodent (e.g., a mouse or rat). A
humanized antibody can comprise, one, two, or three CDRs obtained
or derived from a non-human antibody. A fully human antibody does
not contain any amino acid residues obtained or derived from a
non-human animal. It will be appreciated that fully human and
humanized antibodies carry a lower risk for inducing immune
responses in humans than mouse or chimeric antibodies (see, e.g.,
Harding et al., mAbs, 2(3): 256-26 (2010)).
[0082] As used herein, an "epitope" is a term in the art and refers
to a localized region of an antigen to which an antibody or
antigen-binding fragment thereof can specifically bind. An epitope
can be, for example, contiguous amino acids of a polypeptide
(linear or contiguous epitope) or an epitope can, for example, come
together from two or more non-contiguous regions of a polypeptide
or polypeptides (conformational, non-linear, discontinuous, or
non-contiguous epitope). In certain embodiments, the epitope to
which an antibody or antigen-binding fragment thereof binds can be
determined by, e.g., NMR spectroscopy, X-ray diffraction
crystallography studies, ELISA assays, hydrogen/deuterium exchange
coupled with mass spectrometry (e.g., liquid chromatography
electrospray mass spectrometry), array-based oligo-peptide scanning
assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis
mapping). For X-ray crystallography, crystallization can be
accomplished using any of the known methods in the art (e.g., Giege
R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):
339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen N E
(1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251:
6300-6303). Antibody/antigen-binding fragment thereof: antigen
crystals can be studied using well known X-ray diffraction
techniques and can be refined using computer software such as
X-PLOR (Yale University, 1992, distributed by Molecular
Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 &
115, eds Wyckoff H W et al.; U.S. 2004/0014194), and BUSTER
(Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):
37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W;
Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt
10): 1316-1323). Mutagenesis mapping studies can be accomplished
using any method known to one of skill in the art. See, e.g.,
Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham B
C & Wells J A (1989) Science 244: 1081-1085 for a description
of mutagenesis techniques, including alanine scanning mutagenesis
techniques.
[0083] An antibody that "binds to the same epitope" as a reference
antibody refers to an antibody that binds to the same amino acid
residues as the reference antibody. The ability of an antibody to
bind to the same epitope as a reference antibody can determined by
a hydrogen/deuterium exchange assay (see Coales et al. Rapid
Commun. Mass Spectrom. 2009; 23: 639-647) or x-ray
crystallography.
[0084] As used herein, the terms "immunospecifically binds,"
"immunospecifically recognizes," "specifically binds," and
"specifically recognizes" are analogous terms in the context of
antibodies or antigen-binding fragments thereof. These terms
indicate that the antibody or antigen-binding fragment thereof
binds to an epitope via its antigen-binding domain and that the
binding entails some complementarity between the antigen binding
domain and the epitope. Accordingly, for example, an antibody that
"specifically binds" to a first S. aureus leukotoxin may also bind
to other S. aureus leukotoxins, but the extent of binding to an
un-related, non-leukotoxin protein is less than about 10% of the
binding of the antibody to the first S. aureus leukotoxin as
measured, e.g., by a radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA), BiaCore or an octet binding assay.
[0085] An antibody is said to "competitively inhibit" binding of a
reference antibody to a given epitope if it preferentially binds to
that epitope or an overlapping epitope to the extent that it
blocks, to some degree, binding of the reference antibody to the
epitope. Competitive inhibition may be determined by any method
known in the art, for example, competition ELISA assays. An
antibody may be said to competitively inhibit binding of the
reference antibody to a given epitope by at least 90%, at least
80%, at least 70%, at least 60%, or at least 50%.
[0086] The term "nucleic acid sequence" is intended to encompass a
polymer of DNA or RNA, i.e., a polynucleotide, which can be
single-stranded or double-stranded and which can contain
non-natural or altered nucleotides. The terms "nucleic acid" and
"polynucleotide" as used herein refer to a polymeric form of
nucleotides of any length, either ribonucleotides (RNA) or
deoxyribonucleotides (DNA). These terms refer to the primary
structure of the molecule, and thus include double- and
single-stranded DNA, and double- and single-stranded RNA. The terms
include, as equivalents, analogs of either RNA or DNA made from
nucleotide analogs and modified polynucleotides such as, though not
limited to, methylated and/or capped polynucleotides. Nucleic acids
are typically linked via phosphate bonds to form nucleic acid
sequences or polynucleotides, though many other linkages are known
in the art (e.g., phosphorothioates, boranophosphates, and the
like).
[0087] An S. aureus infection can occur, for example, as a skin or
soft tissue infection (SSTI) or bacteremia. S. aureus bacteria can
travel through the bloodstream and infect a site in the body,
resulting in pneumonia, ICU pneumonia, a diabetic infection of the
lower limbs, diabetic foot ulcer (DFU), a bone or joint infection,
a device infection, a wound infection, a surgical site infection,
or osteomyelitis.
[0088] "Transfection," "transformation," or "transduction," as used
herein, refer to the introduction of one or more exogenous
polynucleotides into a host cell by using physical or chemical
methods. Many transfection techniques are known in the art and
include, for example, calcium phosphate DNA co-precipitation (see,
e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7,
Gene Transfer and Expression Protocols, Humana Press (1991));
DEAE-dextran; electroporation; cationic liposome-mediated
transfection; tungsten particle-facilitated microparticle
bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium
phosphate DNA co-precipitation (Brash et al, Mol. Cell Biol., 7:
2031-2034 (1987)). Phage or viral vectors can be introduced into
host cells, after growth of infectious particles in suitable
packaging cells, many of which are commercially available.
[0089] As used herein, the terms "treatment," "treating," and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. In one embodiment, the effect is therapeutic, i.e., the
effect partially or completely cures a disease and/or adverse
symptom attributable to the disease.
[0090] A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired therapeutic result (e.g., treatment of S. aureus
infection). The therapeutically effective amount may vary according
to factors such as the disease state, age, sex, and weight of the
individual, and the ability of the antibody or antigen-binding
fragment to elicit a desired response in the individual.
[0091] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
a desired prophylactic result (e.g., prevention of S. aureus
infection or disease onset).
[0092] The terms "administer", "administering", "administration",
and the like, as used herein, refer to methods that may be used to
enable delivery of a drug, e.g., a combination of anti-S. aureus
antibodies or antigen-binding fragments thereof to the desired site
of biological action (e.g., intravenous administration).
Administration techniques that can be employed with the agents and
methods described herein are found in e.g., Goodman and Gilman, The
Pharmacological Basis of Therapeutics, current edition, Pergamon;
and Remington's, Pharmaceutical Sciences, current edition, Mack
Publishing Co., Easton, Pa.
[0093] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) or
consecutive administration in any order.
[0094] As used in the present disclosure and claims, the singular
forms "a," "an," and "the" include plural forms unless the context
clearly dictates otherwise.
[0095] Unless specifically stated or obvious from context, as used
herein, the term "or" is understood to be inclusive. The term
"and/or" as used in a phrase such as "A and/or B" herein is
intended to include both "A and B," "A or B," "A," and "B."
Likewise, the term "and/or" as used in a phrase such as "A, B,
and/or C" is intended to encompass each of the following
embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and
C; A and B; B and C; A (alone); B (alone); and C (alone).
II. Anti-Staphylococcus aureus Antibodies and Combinations
Thereof
[0096] As provided herein, antibodies and antigen-binding fragments
thereof (e.g., monoclonal antibodies and fragments) that bind to S.
aureus proteins can be used in combination. In particular,
antibodies and antigen-binding fragments thereof that bind to S.
aureus alpha toxin (AT) protein, antibodies and antigen-binding
fragments thereof that bind to S. aureus clumping factor A (ClfA)
protein, and antibodies an antigen-binding fragments thereof that
bind to at least one S. aureus leukotoxin protein can
advantageously be used in combination.
[0097] Alpha toxin (AT) is a key virulence factor in several S.
aureus diseases, including pneumonia, skin and soft tissue
infections (SSTI), and bacteremia (Bubeck Wardenburg, J. and O.
Schneewind, J. Exp. Med., 205: 287-294 (2008); Inoshima et al., J.
Invest. Dermatol., 132: 1513-1516 (2012); and Foletti et al.,
supra). Passive immunization with anti-AT monoclonal antibodies
reduced disease severity in pneumonia and dermonecrosis models (Hua
et al., Antimicrob. Agents Chemother., 58: 1108-1117 (2014);
Tkaczyk et al., Clin. Vaccine Immunol., 19: 377-385 (2012); and
Ragle, B. E. and J. Wardenburg Bubeck, Infect. Immun., 77:
2712-2718 (2009)), and vaccination with an AT toxoid containing an
H35L mutation (ATH35L) protected against death in mouse lethal
bacteremia and pneumonia models (Bubeck Wardenburg, supra, Foletti
et al., supra, Hua et al., supra, Ragle, supra, Menzies, B. E. and
D. S Kernodle, Infect. Immun., 77: 2712-2718 (2009); and Adhikari
et al., PLoS One, 7: e38567 (2012)). AT contributes to multiple
aspects of S. aureus pathogenesis during bacteremia and sepsis,
including stimulating a hyperinflammatory response characteristic
of sepsis and activating ADAM10-mediated cleavage of endothelial
tight junctions, leading to a loss in vascular integrity (Powers et
al., J Infect. Dis., 206: 352-356 (2012); Wilke, G. A. and J.
Bubeck Wardenburg, Proc. Natl. Acad. Sci. USA, 107: 13473-13478
(2010); and Becker et al., J Innate Immun., 6: 619-631 (2014)). AT
also has been demonstrated to target platelets, which prevents
repair of the injured endothelial barrier and promotes organ
dysfunction through platelet-neutrophil aggregate formation (Powers
et al., Cell Host Microbe, 17: 775-787 (2015)). Alpha toxin
structure and function is described in detail in, for example,
Bhakdi, S. and J. Tranum-Jensen, Microbiol. Mol. Biol. Rev., 55(4):
733-751 (1991).
[0098] Monoclonal and polyclonal antibodies that bind AT are known
in the art (see, e.g., Hua et al., Antimicrob. Agents Chemother.,
58(2): 1108-1117 (2014); and Oganesyan et al., J. Biol. Chem., 289:
29874-29880 (2014)) and are commercially available from sources
such as, for example, Sigma Aldrich (St. Louis, Mo.) and AbCam
(Cambridge, Mass.). Exemplary antibodies that bind to AT are
disclosed, for example, in WO 2012/109285 and WO 2014/074540 (both
of which are herein incorporated by reference in their
entireties).
[0099] In one instance, an antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment) that specifically binds to
S. aureus alpha toxin (AT) comprises, consists essentially of, or
consists of (i) a heavy chain polypeptide comprising a CDR1 amino
acid sequence of SEQ ID NO:1, a CDR2 amino acid sequence of SEQ ID
NO:2, and a CDR3 amino acid sequence of SEQ ID NO:3, and (ii) a
light chain polypeptide comprising a CDR1 amino acid sequence of
SEQ ID NO:10, a CDR2 amino acid sequence of SEQ ID NO:11, and a
CDR3 amino acid sequence of SEQ ID NO:12. In another instance, the
heavy chain polypeptide of an antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment) that specifically binds to
S. aureus AT comprises, consists essentially of, or consists of a
variable region amino acid sequence of SEQ ID NO:19. In another
instance, the light chain polypeptide of an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to S. aureus AT comprises, consists
essentially of, or consists of a variable region amino acid
sequence of SEQ ID NO:33. In another instance, an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to S. aureus AT comprises, consists
essentially of, or consists of a variable heavy chain comprising,
consisting essentially of, or consisting of the amino acid sequence
of SEQ ID NO:19 and a light chain variable region comprising,
consisting essentially of, or consisting of the amino acid sequence
of SEQ ID NO:33. In another instance, an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to S. aureus AT comprises, consists
essentially of, or consists of a heavy chain comprising, consisting
essentially of, or consisting of the amino acid sequence of SEQ ID
NO:47 and/or a light chain variable region comprising, consisting
essentially of, or consisting of the amino acid sequence of SEQ ID
NO:52.
[0100] Among the many S. aureus surface adhesins, clumping factor A
(ClfA) has been demonstrated to play an important role in serious
bloodstream infections (Foster et al., Nat. Rev. Microbiol., 12:
49-62 (2014); and Murphy et al., Hum. Vaccin., 7(Suppl): 51-59
(2011)). ClfA binds fibrinogen and facilitates both bacterial
adherence to fibrinogen and bacterial clumping, both of which are
key attributes in the development of an S. aureus bloodstream
infection (Vaudaux et al., Infect. Immun., 63: 585-590 (1995);
McDevitt et al., Mol. Microbiol., 11: 237-248 (1994); and McDevitt
et al., Eur. J. Biochem., 247: 416-424 (1997)). ClfA bound to
fibrin or fibrinogen at a site of injury or coated on an indwelling
device can facilitate bacterial colonization (Foster et al., supra)
and bacterial clumping, which is thought to enhance bacterial
invasiveness (McDevitt et al., Eur. J. Biochem., 247: 416-424
(1997); McAdow et al., PLoS Pathog., 7:e1002307 (2011); Flick et
al., Blood, 121: 1783-1794 (2013); and Rothfork et al., J.
Immunol., 171: 5389-5395 (2003)). ClfA also has been reported to
impair complement deposition required for opsonophagocytic
bacterial killing (OPK) (Hair et al., Infect. Immun., 78: 1717-1727
(2010)). Consistent with these observations, isogenic .DELTA.clfA
mutants exhibited reduced virulence in infection models (McAdow et
al., supra; Josefsson et al., PLoS One, 3: e2206 (2008); and
Josefsson et al., J Infect. Dis., 184: 1572-1580 (2001)). In
addition, passive immunization with human anti-ClfA-enriched
intravenous (i.v.) immunoglobulin (Ig) (INH-A21 or Veronate) or a
monoclonal antibody (tefibazumab or Aurexis) improved disease
outcomes for patients with S. aureus bloodstream infections
(Vernachio et al., Antimcirob. Agents Chemother., 47: 3400-3406
(2003); and Vernachio et al., Antimicrob. Agents Chemother., 50:
511-518 (2006)). However, these antibody preparations failed to
improve outcomes in clinical studies of prophylaxis or adjunctive
therapy with vancomycin to prevent or treat S. aureus bacteremia in
very-low-birth-weight infants (DeJonge et al., J. Pediatr., 151:
260-265 (2007); Capparelli et al., Antimicrob. Agents Chemother.,
49: 4121-4127 (2005); and Bloom et al., Pediatr. Infect. Dis., 24:
858-866 (2005)). ClfA structure and function is described in detail
in, for example, McDevitt et al., Mol. Microbiol., 11: 237-248
(1994)).
[0101] Monoclonal and polyclonal antibodies which bind ClfA are
known in the art (see, e.g., U.S. Pat. No. 7,364,738; Hall et al.,
Infect. Immun., 71(12): 6864-6870 (2003); and Vernachio et al.,
Antimicrob. Agents Chemother., 47(11): 3400-3406 (2003)) and are
commercially available from sources such as, for example, Creative
Biolabs (Shirley, N.Y.). Exemplary antibodies that bind to ClfA are
disclosed, for example, in WO 2014/074540 and U.S. 62/702,762 (both
of which are herein incorporated by reference in their
entireties).
[0102] In one instance, an antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment) that specifically binds to
S. aureus clumping factor A (ClfA) comprises, consists essentially
of, or consists of (i) a heavy chain polypeptide comprising a CDR1
amino acid sequence of SEQ ID NO:4, a CDR2 amino acid sequence of
SEQ ID NO:5, and a CDR3 amino acid sequence of SEQ ID NO:6, and
(ii) a light chain polypeptide comprising a CDR1 amino acid
sequence of SEQ ID NO:13, a CDR2 amino acid sequence of SEQ ID
NO:14, and a CDR3 amino acid sequence of SEQ ID NO:15. In another
instance, the heavy chain polypeptide of an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to S. aureus ClfA comprises, consists
essentially of, or consists of a variable region amino acid
sequence of SEQ ID NO:20. In another instance, the light chain
polypeptide of an antibody or antigen-binding fragment (e.g.,
monoclonal antibody or fragment) that specifically binds to S.
aureus ClfA comprises, consists essentially of, or consists of a
variable region amino acid sequence of SEQ ID NO:34. In another
instance, an antibody or antigen-binding fragment (e.g., monoclonal
antibody or fragment) that specifically binds to S. aureus ClfA
comprises, consists essentially of, or consists of a variable heavy
chain comprising, consisting essentially of, or consisting of the
amino acid sequence of SEQ ID NO:20 and a light chain variable
region comprising, consisting essentially of, or consisting of the
amino acid sequence of SEQ ID NO:34. In certain instances, an
antibody or antigen-binding fragment (e.g., monoclonal antibody or
fragment) that specifically binds to S. aureus ClfA comprises a
heavy chain constant domain comprising the amino acid sequence of
CSYHLC (SEQ ID NO:55), MHEACSYHLCQKSLSLS (SEQ ID NO:56), or amino
acids 233-454 of SEQ ID NO:49. In another instance, an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to S. aureus ClfA comprises, consists
essentially of, or consists of a heavy chain comprising, consisting
essentially of, or consisting of the amino acid sequence of SEQ ID
NO:49 and/or a light chain variable region comprising, consisting
essentially of, or consisting of the amino acid sequence of SEQ ID
NO:53.
[0103] In another instance, an antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment) that specifically binds to
S. aureus ClfA (e.g., an antibody with the CDR, VH and/or VL, or
heavy and or light chains of SAR114-N3Y) has IC50's for ClfA001,
ClfA002, and ClfA004 in a fibrinogen binding inhibition assay that
are within 2 .mu.g/ml of each other. For example, the IC.sub.50's
of the antibody or antigen-binding fragment thereof for ClfA001,
ClfA002, and ClfA004 can all be between 1 .mu.g/ml and 5 .mu.g/ml.
The binding affinities (K.sub.D) of the antibody or antigen-binding
fragment thereof for ClfA001, ClfA002, and ClfA004 can all be all
between 200 and 350 pM.
[0104] In another instance, an antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment) that specifically binds to
S. aureus ClfA (e.g., an antibody with the CDR, VH and/or VL, or
heavy and or light chains of SAR114-N3Y) has a monomeric purity
that decreases by no more than 5% after exposure to conventional
white light at 2kLux/hr at 23.degree. C. for 14 days.
[0105] Leukotoxins are another type of S. aureus virulence factor.
Leukotoxins target a broad range of immune cells for destruction.
Leukotoxins include Panton-Valentine leukocidin (LukSF-PV also
known as LukSF), leukotoxin ED (LukED), gamma hemolysin (which
exists as two toxins: HlgAB and HlgCB), and leukotoxin AB (LukAB,
also known as LukGH). In certain instances, an antibody or
antigen-binding fragment thereof that binds to at least one
leukotoxin binds to LukF, LukD, and/or HIgAB. In certain instances,
an antibody or antigen-binding fragment thereof that binds to at
least one leukotoxin binds to LukF, LukD, and HIgB.
[0106] In one instance, an antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment) that specifically binds to
at least one S. aureus leukotoxin comprises, consists essentially
of, or consists of (i) a heavy chain polypeptide comprising a CDR1
amino acid sequence of SEQ ID NO:7, a CDR2 amino acid sequence of
SEQ ID NO:8, and a CDR3 amino acid sequence of SEQ ID NO:9, and
(ii) a light chain polypeptide comprising a CDR1 amino acid
sequence of SEQ ID NO:16, a CDR2 amino acid sequence of SEQ ID
NO:17, and a CDR3 amino acid sequence of SEQ ID NO:18. In another
instance, the heavy chain polypeptide of an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to at least one S. aureus leukotoxin
comprises, consists essentially of, or consists of a variable
region amino acid sequence of SEQ ID NO:32. In another instance,
the light chain polypeptide of an antibody or antigen-binding
fragment (e.g., monoclonal antibody or fragment) that specifically
binds to at least one S. aureus leukotoxin comprises, consists
essentially of, or consists of a variable region amino acid
sequence of SEQ ID NO:46. In another instance, an antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment)
that specifically binds to at least one S. aureus leukotoxin
comprises, consists essentially of, or consists of a variable heavy
chain comprising, consisting essentially of, or consisting of the
amino acid sequence of SEQ ID NO:32 and a light chain variable
region comprising, consisting essentially of, or consisting of the
amino acid sequence of SEQ ID NO:46. In another instance, an
antibody or antigen-binding fragment (e.g., monoclonal antibody or
fragment) that specifically binds to at least one S. aureus
leukotoxin comprises, consists essentially of, or consists of a
heavy chain comprising, consisting essentially of, or consisting of
the amino acid sequence of SEQ ID NO:50 and/or a light chain
variable region comprising, consisting essentially of, or
consisting of the amino acid sequence of SEQ ID NO:54.
[0107] Sequences of exemplary anti-AT, anti-ClFA, and
anti-leukotoxin antibodies are provided below. Additional anti-AT
antibodies are provided, for example, in U.S. Pat. No. 9,527,905,
which is herein incorporated by reference in its entirety.) In
certain instances, an antibody or antigen-binding fragment thereof
described herein binds to AT, ClfA, or at lease one leukotoxin and
comprises the six CDRs of an antibody listed in the two tables
below (i.e., the three VH CDRs of the antibody listed in the first
table and the three VL CDRs of the same antibody listed in the
second table).
[0108] The anti-AT antibody MEDI4893 is the half-life extended
(YTE) version of MEDI4893* or "LC10" described previously in
International Patent Application Publications WO 2012/109285 and WO
2014/074540 (both of which are herein incorporated by reference in
their entireties). The anti-ClfA antibody SAR114-N3Y is described
in U.S. Provisional Application No. 62/702,762. The anti-leukotoxin
antibody SAN481-SYT is the half-life extended (YTE) version of
SAN481-SYT*. SAN481-SYT* does not contain the YTE mutation.
TABLE-US-00003 VH CDR Amino Acid Sequences Antibody Antibody VH
CDR1 VH CDR2 VH CDR3 Name Target (SEQ ID NO:) (SEQ ID NO:) (SEQ ID
NO:) MEDI4893 AT SHDMH (SEQ GIGTAGDTYYPD DRYSPTGHYYGMDV and ID NO:
1) SVKG (SEQ ID (SEQ ID NO: 3) MEDI4893* NO: 2) SAR114 ClfA NSYWS
(SEQ YLYSSGRTNYTPS THLGGFHYGGGFWF and ID NO: 4) LKS (SEQ ID DP (SEQ
ID NO: 6) SAR114- NO: 5) N3Y 11H10 ClfA SFAMS (SEQ AISGSGGNTYYA
IAFDI ID NO: 62) DSVKG (SEQ ID (SEQ ID NO: 64) NO: 63) SAN481-SYT
and Leuko- TYAMH (SEQ VTSFDGSNEYYID DEYTGGWYSVGY SAN481- toxin ID
NO: 7) SVKG (SEQ ID (SEQ ID NO: 9) SYT* NO: 8)
TABLE-US-00004 VL CDR Amino Acid Sequences Antibody VL CDR1 VL CDR2
VL CDR3 Antibody Target (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)
MEDI4893 AT RASQSISSWLA KASSLES (SEQ ID KQYADYWT and (SEQ ID NO:
10) NO: 11) (SEQ ID NO: 12) MEDI4893* SAR114 ClfA RASQSITSYLN
ASSSLQS (SEQ ID QESYSTPPT (SEQ and (SEQ ID NO: 13) NO: 14) ID NO
15) SAR114- N3Y 11H10 ClfA RASQGIRNDL VASSLQS (SEQ ID LQHNSYPFT
(SEQ G (SEQ ID NO: 66) ID NO: 67) NO: 65) SAN481-SYT and Leuko-
SGSSYNIGSNY RSIQRPS (SEQ ID AAWDDSLRAWV SAN481- toxin VY (SEQ ID
NO: 17) (SEQ ID NO: 18) SYT * NO: 16)
[0109] In certain instances, an antibody or antigen-binding
fragment thereof described herein binds to AT, ClfA, or at least
one leukotoxin and comprises the VH of an antibody listed in the
following table, e.g., in combination with a VL.
TABLE-US-00005 Variable Heavy Chain (VH) Amino Acid Sequence
Antibody Antibody Target VH Amino Acid Sequence (SEQ ID NO)
MEDI4893 AT EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDIVIEIWVRQA and
TGKGLEWVSGIGTAGDTYYPDSVKGRFTISRENAKNSLYLQ MEDI4893*
MNSLRAGDTAVYYCARDRYSPTGHYYGMDVWGQGTTVT VSS (SEQ ID NO: 19) SAR114
ClfA QVQLQESGPGLVKPSETLSLTCTVSGGSIQNSYWSWIRQPPG and
KGLEWIGYLYSSGRTNYTPSLKSRVTISVDTSKNQFSLKLSS SAR114-
VTAADTAVYYCARTHLGGFHYGGGFWFDPWGQGTLVTVS N3Y S (SEQ ID NO: 20) 11H10
ClfA EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVR
QAPGKGLEWVSAISGSGGNTYYADSVKGRFTISRDNS
KNTLYLQMNSLRAEDTAVYYCAKIAFDIWGQGTMVT VSS (SEQ ID NO: 68) SAR72 ClfA
EVQLVESGGGLVKPGGSLRVSCAASGFSFRNALMSWVRQA
PGKGLEWVGRSKTDGGTTDYAAPVKGRFTISRDDSKNTLY
LQMNSLKTEDTAVYYCTTGPGGGPPGDYYYDGMDVWGQG TTVTVSS (SEQ ID NO: 21)
SAR80 ClfA EVQLVESGGDLVKPGGSLRLSCAASGFTFSDAWMTWVRQA
PGKGLEWVGRIRSKTAGGTTDYAAPVKGRFTISRDDSKNTL
YLQMTSLKIEDTALYYCMTDGLGLLNFGDSDPHEYWGQGT RVTVSS (SEQ ID NO: 22)
SAR113 ClfA EVQLVQSGAEVKKPGESLKISCKAXGYXFTSYWIGWVRQV
PGKGLEWMGIIYPGDSDTRHSPSFQGQVTISVDKSISTAYLQ
WSSLKASDSAMYYCARHQSGSHGFDAFEIWGQGTMVTVSS (SEQ ID NO: 23) SAR132
ClfA EVQLVQSGAEVKKPGESLKISCKGSGYNFTNYWIAWVRQM
PGKGLEWMGIIYSGDSDTRYSPSFLGQVSISVDKSFTTAYLQ
WRSLKASDTAMYYCARRPGGQKPYDYWGQGTLVTVSS (SEQ ID NO: 24) SAR352 ClfA
EVQLVESGGGLVKPGGSLRLSCAASGFTFNNAWMSWVRQA
PGKGLEWVGRIKSETAGGTTDYAAPVKGRFSISRDDSRNTL
YLEMNSLKTEDTAVYYCTTDSYTPLEEPCPNGVCYTYYYY GMDVWGQGTTVTVSS (SEQ ID
NO: 25) SAR372 ClfA EVQLVESGGGLVQPGGSLRLSCAASGFIFNRYSMNWVRQA
PGKGLEWVSYISSSSSPIYYADSVKGRFTISRDNAKNSLYLQ
MNSLRDEDTAVYYCASRVTLGLEFDFWGQGTLVTVSS (SEQ ID NO: 26) SAR510 ClfA
QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVGWIRQP
PGKALEWLALIEWDDDKYYNTSLKTRLSISKDTSKNQVVLT
MTNMDPVDTGTYYCARHSSSSRGFDYWGQGALVTVSS (SEQ ID NO: 27) SAR547 ClfA
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWIAWVRQMP
GKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSTATAYLQ
WSSLNASDSAMYYCARQGGSHGYDAFHMWGQGTMVTVS S (SEQ ID NO: 28) SAS1 ClfA
EVQLLESGGGLVQPGGSLRLSCTASGFTFSTYALNWVRQAP
GKGLEWVAGINGTGYNTYYADSVRGRFTISRDNSKNTVTLE
MNSLRVEDTATYYCHKVPWWGQGTLVSVSS (SEQ ID NO: 29) SAS19 ClfA
QVQLQESGPRLVKPSETLSLTCFVSGGSINNSYWTWIRQPPG
QGLEWIGFVFSSGRTNYSPSLKSRVTISVDTSKNLFSLRLTSV
TAADTAVYFCARQVHYDFWSGYSLTKTNWFDPWGQGTLV TVSS (SEQ ID NO: 30) SAS203
ClfA QVQLQESGPGLVKPSETLSLTCVVSGGSINNSYWTWIRQPPG
QGLEWIGFVYSSGRTYYSPSLKSRVTISVDTSKNFFSLRLNS
VTAADTAVYFCARQVHYDLWSGYSLTKTNWFDPWGQGTL VTVSS (SEQ ID NO: 31)
SAN481- Leuko- QLQLVESGGGAVQPGRSLKLSCAASGFTFSTYAMHWVRQA SYT and
toxin PGRGLEWVAVTSFDGSNEYYIDSVKGRFTISRDNTKNTLYL SAN481-
QMTGLRVEDTALYFCARDEYTGGWYSVGYWGQGTLVTVS SYT* S (SEQ ID NO: 32)
[0110] In certain instances, an antibody or antigen-binding
fragment thereof described herein binds to AT, ClfA, or at least
one leukotoxin and comprises the VL of an antibody listed in the
following table, e.g., in combination with a VH, optionally the VH
of the same antibody listed in the preceding table.
TABLE-US-00006 Variable Light Chain (VL) Amino Acid Sequence
Antibody Antibody Target VL Amino Acid Sequence (SEQ ID NO)
MEDI4893 AT DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPG and
KAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFAT MEDI4893*
YYCKQYADYWTFGQGTKVEIK (SEQ ID NO: 33) SAR114 ClfA
DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQKPGK and
APKLLIYASSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY SAR114-
YCQESYSTPPTFGQGTKVEIK (SEQ ID NO: 34) N3Y 11H10 ClfA
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQ
KPGKAPKRLIYVASSLQSGVPSRFSGSGSGTEFTLTISSL
QPEDFATYYCLQHNSYPFTFGPGTKVDIK (SEQ ID NO: 69) SAR72 ClfA
SYELTQPPSVSVSPGQTARITCSGDAVPKKYAYWYQQKSGQ
APVLVIYEDKKRPSGIPERFSGSSSGTMATLTISGAQVEDEA DYYCYSTDSSEGVFGGGTKLTVL
(SEQ ID NO: 35) SAR80 ClfA
SYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQ
APVLVIHEDTKRPSGIPERFSGSSSGTMATLTISGAQVEDEAD YHCYSTDSSGVVFGGGTKLTVL
(SEQ ID NO: 36) SAR113 ClfA
DIVLTQSPDSLAVSLGERATINCKSSQGVLSRSNNKNYLAW
YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSL
QAEDVAVYYCQQYYNNLRTFGQGTKVEIR (SEQ ID NO: 37) SAR132 ClfA
DIQMTQSPSTLSASVGDRVTITCRASQRISNWLAWYQKKPG
KAPKLLIYKASTLESEVPSRFSGSGSGTEFTLTISSLQPDDLAT YYCHQYISYYTFGQGTKLEIK
(SEQ ID NO: 38) SAR352 ClfA
QSVLTQPPSVSAAPGEKVTISCSGSSSNIGANSVSWYQQFPG
TAPKLLIYDNDKRPSGVPDRFSGSKSGTSATLGITGLQTGDE
ADYYCGTWVGILSAGWVFGGGTKLTVL (SEQ ID NO: 39) SAR372 ClfA
EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQ
APRLLIYDASNRATGIPDRFSGSGSGTDFTLTISSLKPEDFAV YYCQLRSNWAYTFGQGTKLEIK
(SEQ ID NO: 40) SAR510 ClfA
SYGLTQPPSVSVSPGQTARITCSGDALAKQYVYWYQQKPG
QAPVLVIDKDRERPSGIPERFSGSSSGTTVTLTISGVQAEDEA
DYYCQSADSSRTYVFGTGTKVTVL (SEQ ID NO: 41) SAR547 ClfA
DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWF
QQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRV
EAEDVGVYYCMQGTHLTWTFGQGTKVEIK (SEQ ID NO: 42) SAS1 ClfA
DIVLTQSPESLAVSLGERATISCKSSQSLFFKSNNKNYLAWY
QQKPGQPPKVIIYWASTRESGVPARFSGSGSGTDFTLTISSLQ
AEDVAVYFCHQYYSTQYSFGQGTKLEIK (SEQ ID NO: 43) SAS19 ClfA
DIQMTQSPSSLSASVGDTVTITCRTSQSISNFLNWYQQKPGK
APKLLIYAASSLQSGVPSRVNGSTSGTEFTLTLSSLQPEDFAT YYCQQSYSTPWTFGQGTKVEIK
(SEQ ID NO: 44) SAS203 ClfA
DIQMTQSPSSLSASVGDTVTITCRTSQSISNFLNWYQQKPGK
APKLLIYAASSLQSGVPSRFNGSTSGTDFTLTLSSLQPEDFAT YYCQQSYSTPWTFGQGTKVEIK
(SEQ ID NO: 45) SAN481- Leuko-
QSVLTQPPSASGTPGQRVTISCSGSSYNIGSNYVYWYQQFPG SYT and toxin
TAPKLLISRSIQRPSGVPDRFSGSKSVTSASLAISGLRSEDEAD SAN481-
YYCAAWDDSLRAWVFGGGTKLTVL (SEQ ID NO: 46) SYT*
[0111] In certain instances, an antibody or antigen-binding
fragment thereof described herein binds to AT, ClfA, or at least
one leukotoxin and comprises the heavy chain of an antibody listed
in the following table, e.g., in combination with a light
chain.
TABLE-US-00007 Full-length heavy chain amino acid sequences
Antibody Full-Length Heavy Chain Amino Acid Sequence (SEQ ID
Antibody Target NO) MEDI4893 AT
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMIEIWVRQA
TGKGLEWVSGIGTAGDTYYPDSVKGRFTISRENAKNSLYL
QMNSLRAGDTAVYYCARDRYSPTGHYYGMDVWGQGTTV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 47)
MEDI4893* AT EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHDMIEIWVRQA
TGKGLEWVSGIGTAGDTYYPDSVKGRFTISRENAKNSLYLQ
MNSLRAGDTAVYYCARDRYSPTGHYYGMDVWGQGTTVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 48) SAR114- ClfA
QVQLQESGPGLVKPSETLSLTCTVSGGSIQNSYWSWIRQPPG N3Y
KGLEWIGYLYSSGRTNYTPSLKSRVTISVDTSKNQFSLKLSS
VTAADTAVYYCARTHLGGFHYGGGFWFDPWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEACSYHLCQKSLSLSP GK (SEQ ID NO: 49) SAR114
ClfA QVQLQESGPGLVKPSETLSLTCTVSGGSIQNSYWSWIRQPPG
KGLEWIGYLYSSGRTNYTPSLKSRVTISVDTSKNQFSLKLSS
VTAADTAVYYCARTHLGGFHYGGGFWFDPWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 70) SAN481-
Leuko- QLQLVESGGGAVQPGRSLKLSCAASGFTFSTYAMIHWVRQA SYT toxin
PGRGLEWVAVTSFDGSNEYYIDSVKGRFTISRDNTKNTLYL
QMTGLRVEDTALYFCARDEYTGGWYSVGYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 50) SAN481-
Leuko- QLQLVESGGGAVQPGRSLKLSCAASGFTFSTYAMIHWVRQA SYT* toxin
PGRGLEWVAVTSFDGSNEYYIDSVKGRFTISRDNTKNTLYL
QMTGLRVEDTALYFCARDEYTGGWYSVGYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK (SEQ ID NO: 51)
[0112] In certain instances, an antibody or antigen-binding
fragment thereof described herein binds to AT, ClfA, or at least
one leukotoxin and comprises the light chain of an antibody listed
in the following table, e.g., in combination with a heavy chain,
optionally the heavy chain of the same antibody listed in the
preceding table.
TABLE-US-00008 Full-length light chain amino acid sequences
Antibody Full-Length Light Chain Amino Acid Sequence Antibody
Target (SEQ ID NO) MEDI4893 AT
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQK and
PGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQP MEDI4893*
DDFATYYCKQYADYWTFGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGE (SEQ ID
NO: 52) SAR114- ClfA DIQMTQSPSSLSASVGDRVTITCRASQSITSYLNWYQQK N3Y
PGKAPKLLIYASSSLQSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQESYSTPPTFGQGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID
NO: 53) SAN481- Leuko- QSVLTQPPSASGTPGQRVTISCSGSSYNIGSNYVYWYQQ SYT
and toxin FPGTAPKLLISRSIQRPSGVPDRFSGSKSVTSASLAISGLR SAN481-
SEDEADYYCAAWDDSLRAWVFGGGTKLTVLGQPKAA SYT*
PSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD
SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTVAPTECS (SEQ
ID NO: 54)
[0113] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
Chothia numbering scheme, which refers to the location of
immunoglobulin structural loops (see, e.g., Chothia C & Lesk A
M, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J
Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227:
799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and
U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering
convention, the Chothia CDR-H1 loop is present at heavy chain amino
acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at
heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is
present at heavy chain amino acids 95 to 102, while the Chothia
CDR-L1 loop is present at light chain amino acids 24 to 34, the
Chothia CDR-L2 loop is present at light chain amino acids 50 to 56,
and the Chothia CDR-L3 loop is present at light chain amino acids
89 to 97. The end of the Chothia CDR-H1 loop when numbered using
the Kabat numbering convention varies between H32 and H34 depending
on the length of the loop (this is because the Kabat numbering
scheme places the insertions at H35A and H35B; if neither 35A nor
35B is present, the loop ends at 32; if only 35A is present, the
loop ends at 33; if both 35A and 35B are present, the loop ends at
34).
[0114] In certain aspects, provided herein are combinations of
antibodies and antigen-binding fragments thereof that comprise the
Chothia VH and VL CDRs of the MEDI4893, SAR114-N3Y, and/or
SAN481-SYT antibodies. In certain embodiments, antibodies or
antigen-binding fragments thereof comprise one or more CDRs, in
which the Chothia and Kabat CDRs have the same amino acid sequence.
In certain embodiments, provided herein are antibodies and
antigen-binding fragments thereof comprise combinations of Kabat
CDRs and Chothia CDRs.
[0115] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
IMGT numbering system as described in Lefranc M-P, (1999) The
Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic
Acids Res 27: 209-212. According to the IMGT numbering scheme,
VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57,
VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to
32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions
89 to 97. In a particular embodiment, provided herein are
combinations of antibodies and antigen-binding fragments thereof
that comprise the IMGT VH and VL CDRs of MEDI4893, SAR114-N3Y,
and/or SAN481-SYT antibodies, for example, as described in Lefranc
M-P (1999) supra and Lefranc M-P et al., (1999) supra).
[0116] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to
MacCallum R M et al., (1996) J Mol Biol 262: 732-745. See also,
e.g., Martin A. "Protein Sequence and Structure Analysis of
Antibody Variable Domains," in Antibody Engineering, Kontermann and
Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin
(2001). In a particular embodiment, provided herein are
combinations of antibodies or antigen-binding fragments thereof
comprise the VH and VL CDRs of the MEDI4893, SAR114-N3Y, and/or
SAN481-SYT antibodies determined by the method in MacCallum R M et
al.
[0117] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
AbM numbering scheme, which refers AbM hypervariable regions which
represent a compromise between the Kabat CDRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software (Oxford Molecular Group, Inc.). In a particular
embodiment, provided herein are combinations of antibodies or
antigen-binding fragments that and comprise VH and VL CDRs of the
MEDI4893, SAR114-N3Y, and/or SAN481-SYT antibodies as determined by
the AbM numbering scheme.
[0118] In another aspect, the antibody or antigen-binding fragment
thereof (e.g., monoclonal antibody or fragment) described herein
can comprise a constant region (Fc) of any suitable class (e.g.,
IgG, IgA, IgD, IgM, and IgE) that has been modified in order to
improve the half-life of the antibody or antigen-binding fragment
(e.g., monoclonal antibody or fragment). For example, the antibody
or antigen-binding fragment thereof (e.g., monoclonal antibody or
fragment) described herein can comprise an Fc that comprises a
mutation that extends half-life relative to the same antibody
without the mutation.
[0119] Fc region engineering is widely used in the art to extend
the half-life of therapeutic antibodies and protect from
degradation in vivo. In some embodiments, the Fc region of an IgG
antibody or antigen-binding fragment can be modified in order to
increase the affinity of the IgG molecule for the Fc
Receptor-neonate (FcRn), which mediates IgG catabolism and protects
IgG molecules from degradation. Suitable Fc region amino acid
substitutions or modifications are known in the art and include,
for example, the triple substitution M252Y/S254T/T256E (referred to
as "YTE") (see, e.g., U.S. Pat. No. 7,658,921; U.S. Patent
Application Publication 2014/0302058; and Yu et al., Antimicrob.
Agents Chemother., 61(1): e01020-16 (2017)). In certain aspects, an
antibody or antigen-binding binding fragment (e.g., monoclonal
antibody or fragment) that binds to S. aureus AT comprises an Fc
region comprising the YTE mutation. In certain aspects, an antibody
or antigen-binding binding fragment (e.g., monoclonal antibody or
fragment) that binds to at least one S. aureus leukotoxin comprises
an Fc region comprising the YTE mutation. In certain aspects, an
antibody or antigen-binding binding fragment (e.g., monoclonal
antibody or fragment) that binds to S. aureus AT comprises an Fc
region comprising the YTE mutation and an antibody or
antigen-binding binding fragment (e.g., monoclonal antibody or
fragment) that binds to at least one S. aureus leukotoxin comprises
an Fc region comprising the YTE mutation.
[0120] In another aspect, the Fc region can comprise the sequence
CSYHLC (referred to as "N3Y"; SEQ ID NO:55). In certain aspects, an
antibody or antigen-binding binding fragment (e.g., monoclonal
antibody or fragment) that binds to S. aureus ClfA comprises an Fc
region comprising the N3Y Fc variant.
[0121] In another aspect, the antibody or antigen-binding fragment
thereof (e.g., monoclonal antibody or fragment) described herein
can comprise a constant region (Fc) of any suitable class (IgG,
IgA, IgD, IgM, and IgE) that has been modified in order to improve
effector functions (e.g., opsonophagocytic bacterial killing
(OPK)), optionally wherein the half-life of the antibody or
antigen-binding fragment (e.g., monoclonal antibody or fragment) is
also improved. For example, the antibody or antigen-binding
fragment thereof (e.g., monoclonal antibody or fragment) described
herein may comprise an Fc that comprises a mutation that extends
half-life relative to the same antibody without the mutation, and
wherein the mutation does not inhibit OPK activity relative to the
same antibody or antigen-binding fragment the mutation. The N3Y Fc
variant, in particular, exhibits enhanced pharmacokinetic (PK)
properties (e.g., serum persistence) and effector functions (e.g.,
opsonophagocytic bacterial killing (OPK)) in certain antibodies as
compared to the YTE variants.
[0122] An antibody or antigen-binding fragment (e.g. monoclonal
antibody or fragment) described herein can be, or can be obtained
from, a human antibody, a humanized antibody, a non-human antibody,
or a chimeric antibody. In one aspect, an antibody described
herein, or antigen-binding fragment thereof, is a fully human
antibody.
[0123] A human antibody, a non-human antibody, a chimeric antibody,
or a humanized antibody can be obtained by any means, including via
in vitro sources (e.g., a hybridoma or a cell line producing an
antibody recombinantly) and in vivo sources (e.g., rodents, human
tonsils). Methods for generating antibodies are known in the art
and are described in, for example, Kohler and Milstein, Eur. J.
Immunol., 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A
Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.),
Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).
In certain embodiments, a human antibody or a chimeric antibody can
be generated using a transgenic animal (e.g., a mouse) wherein one
or more endogenous immunoglobulin genes are replaced with one or
more human immunoglobulin genes. Examples of transgenic mice
wherein endogenous antibody genes are effectively replaced with
human antibody genes include, but are not limited to, the Medarex
HUMAB-MOUSE.TM., the Kirin TC MOUSE.TM., and the Kyowa Kirin
KM-MOUSE.TM. (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25
(2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A
humanized antibody can be generated using any suitable method known
in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal
Antibodies: From Bench to Clinic, John Wiley & Sons, Inc.,
Hoboken, N.J. (2009)), including, e.g., grafting of non-human CDRs
onto a human antibody scaffold (see, e.g., Kashmiri et al.,
Methods, 36(1): 25-34 (2005); and Hou et al., J. Biochem., 144(1):
115-120 (2008)). In one embodiment, a humanized antibody can be
produced using the methods described in, e.g., U.S. Patent
Application Publication 2011/0287485 A1.
III. Nucleic Acids, Vectors, and Host Cells
[0124] Also provided herein are one or more isolated nucleic acid
sequences that encode the antibody or antigen-binding fragment
thereof that binds to AT, the antibody or antigen-binding fragment
thereof that binds to ClfA, or the antibody or antigen-binding
fragment thereof that binds to at least one leukotoxin (optionally
wherein one or more of the antibodies or antigen-binding fragments
thereof is a monoclonal antibody or fragment).
[0125] The disclosure further provides one or more vectors
comprising one or more nucleic acid sequences encoding antibody or
antigen-binding fragment thereof that binds to AT, the antibody or
antigen-binding fragment thereof that binds to ClfA, and/or the
antibody or antigen-binding fragment thereof that binds to at least
one leukotoxin (optionally wherein one or more of the antibodies or
antigen-binding fragments thereof is a monoclonal antibody or
fragment). The vector can be, for example, a plasmid, episome,
cosmid, viral vector (e.g., retroviral or adenoviral), or phage.
Suitable vectors and methods of vector preparation are well known
in the art (see, e.g., Sambrook et al., Molecular Cloning, a
Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols
in Molecular Biology, Greene Publishing Associates and John Wiley
& Sons, New York, N.Y. (1994)).
[0126] In addition to the nucleic acid sequence encoding the
antibody or antigen-binding fragment thereof that binds to AT, the
antibody or antigen-binding fragment thereof that binds to ClfA,
and/or the antibody or antigen-binding fragment thereof that binds
to at least one leukotoxin (optionally wherein one or more of the
antibodies or antigen-binding fragments thereof is a monoclonal
antibody or fragment), the vector desirably comprises expression
control sequences, such as promoters, enhancers, polyadenylation
signals, transcription terminators, internal ribosome entry sites
(IRES), and the like, that provide for the expression of the coding
sequence in a host cell. Exemplary expression control sequences are
known in the art and described in, for example, Goeddel, Gene
Expression Technology: Methods in Enzymology, Vol. 185, Academic
Press, San Diego, Calif. (1990).
[0127] The vector(s) comprising the nucleic acid(s) encoding the
antibody or antigen-binding fragment thereof that binds to AT, the
antibody or antigen-binding fragment thereof that binds to ClfA, or
the antibody or antigen-binding fragment thereof that binds to at
least one leukotoxin (optionally wherein one or more of the
antibodies or antigen-binding fragments thereof is a monoclonal
antibody or fragment) can be introduced into a host cell that is
capable of expressing the polypeptides encoded thereby, including
any suitable prokaryotic or eukaryotic cell. As such, the present
disclosure provides an isolated cell comprising the vector. Host
cells that may be used include those that can be easily and
reliably grown, have reasonably fast growth rates, have well
characterized expression systems, and can be transformed or
transfected easily and efficiently. Examples of suitable
prokaryotic cells include, but are not limited to, cells from the
genera Bacillus (such as Bacillus subtilis and Bacillus brevis),
Escherichia (such as E. coli), Pseudomonas, Streptomyces,
Salmonella, and Erwinia. Particularly useful prokaryotic cells
include the various strains of Escherichia coli (e.g., K12, HB101
(ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).
Suitable eukaryotic cells are known in the art and include, for
example, yeast cells, insect cells, and mammalian cells. In one
embodiment, the vector is expressed in mammalian cells. A number of
suitable mammalian host cells are known in the art, and many are
available from the American Type Culture Collection (ATCC,
Manassas, Va.). Examples of suitable mammalian cells include, but
are not limited to, Chinese hamster ovary cells (CHO) (ATCC No.
CCL61), CHO DHFR- cells (Urlaub et al, Proc. Natl. Acad. Sci. USA,
97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T
cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other
suitable mammalian cell lines are the monkey COS-1 (ATCC No.
CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the
CV-1 cell line (ATCC No. CCL70). The mammalian cell desirably is a
human cell. For example, the mammalian cell can be a human lymphoid
or lymphoid derived cell line, such as a cell line of pre-B
lymphocyte origin, a PER.C6.RTM. cell line (Crucell Holland B.V.,
The Netherlands), or human embryonic kidney (HEK) 293 or 293T cells
(ATCC No. CRL1573).
[0128] A nucleic acid sequence encoding amino acids of any of the
antibodies or antigen-binding fragments (optionally monoclonal
antibodies or fragments) described herein can be introduced into a
cell by transfection, transformation, or transduction.
IV. Pharmaceutical Compositions and Methods of Using Combinations
of Anti-Staphylococcus aureus Antibodies
[0129] The present disclosure provides a composition comprising an
effective amount of any one or combination of the antibodies or
antigen-binding fragments thereof described herein and a
pharmaceutically acceptable carrier. In one embodiment, for
example, the composition may comprise a first antibody or
antigen-binding fragment thereof (optionally monoclonal) that
specifically binds to S. aureus alpha toxin protein, as described
above, a second antibody or antigen-binding fragment thereof
(optionally monoclonal) that specifically binds to S. aureus ClfA
protein, and a third antibody or antigen-binding fragment thereof
(optionally monoclonal) that specifically binds to at least one S.
aureus leukotoxin, as described above, and a pharmaceutically
acceptable carrier. Alternatively, the composition can comprise a
pharmaceutically acceptable carrier and any one or any two of (i)
an antibody or antigen-binding fragment thereof that specifically
binds to S. aureus AT, (ii) an antibody or antigen-binding fragment
thereof that specifically binds to S. aureus ClfA, (iii) an
antibody or antigen-binding fragment thereof that specifically
binds to at least one S. aureus leukotoxin.
[0130] In another aspect, the composition may comprise the nucleic
acid sequences encoding the AT-binding antibody or antigen-binding
fragment, the ClfA-binding antibody or antigen-binding fragment,
and/or the leukotoxin-binding antibody or antigen-binding fragment,
or one or more vectors comprising such nucleic acid sequences. In
one aspect, the composition is a pharmaceutically acceptable (e.g.,
physiologically acceptable) composition, which comprises a carrier,
such as a pharmaceutically acceptable (e.g., physiologically
acceptable) carrier and the AT-binding antibody or antigen-binding
fragment, the ClfA-binding antibody or antigen-binding fragment,
and/or the anti-leukotoxin antibody or antigen-binding fragment
nucleic acid sequence(s), or vector(s).
[0131] Any suitable carrier can be used within the context of the
disclosure, and such carriers are well known in the art. The choice
of carrier will be determined, in part, by the particular site to
which the composition may be administered and the particular method
used to administer the composition. The composition optionally can
be sterile. The composition can be frozen or lyophilized for
storage and reconstituted in a suitable sterile carrier prior to
use. The compositions can be generated in accordance with
conventional techniques described in, e.g., Remington: The Science
and Practice of Pharmacy, 21st Edition, Lippincott Williams &
Wilkins, Philadelphia, Pa. (2001).
[0132] The composition desirably comprises the AT-binding antibody
or antigen-binding fragment, the ClfA-binding antibody or
antigen-binding fragment, and the leukotoxin-binding antibody or
antigen-binding fragment in an amount that is effective to treat or
prevent a S. aureus infection. In another aspect, the composition
comprises the AT-binding antibody or antigen-binding fragment in an
amount that is effective to treat or prevent a S. aureus infection
in combination with the ClfA-binding antibody or antigen-binding
fragment, and the leukotoxin-binding antibody or antigen-binding
fragment. In another aspect, the composition comprises the
ClfA-binding antibody or antigen-binding fragment in an amount that
is effective to treat or prevent a S. aureus infection in
combination with the AT-binding antibody or antigen-binding
fragment, and the leukotoxin-binding antibody or antigen-binding
fragment. In another aspect, the composition comprises the
leukotoxin-binding antibody or antigen-binding fragment in an
amount that is effective to treat or prevent a S. aureus infection
in combination with the AT-binding antibody or antigen-binding
fragment, and the ClfA-binding antibody or antigen-binding
fragment. In another aspect, the composition comprises the
AT-binding antibody or antigen-binding fragment and the
ClfA-binding antibody or antigen-binding fragment in an amount that
is effective to treat or prevent a S. aureus infection in
combination with the leukotoxin-binding antibody or antigen-binding
fragment. In another aspect, the composition comprises the
AT-binding antibody or antigen-binding fragment and the
leukotoxin-binding antibody or antigen-binding fragment in an
amount that is effective to treat or prevent a S. aureus infection
in combination with the ClfA-binding antibody or antigen-binding
fragment. In another aspect, the composition comprises the
ClfA-binding antibody or antigen-binding fragment and the
leukotoxin-binding antibody or antigen-binding fragment in an
amount that is effective to treat or prevent a S. aureus infection
in combination with the AT-binding antibody or antigen-binding
fragment.
[0133] To this end, the disclosed method comprises administering a
therapeutically effective amount or prophylactically effective
amount of an AT-binding antibody or antigen-binding fragment
thereof, a ClfA-binding antibody or antigen-binding fragment
thereof, and a leukotoxin-binding antibody or antigen-binding
fragment thereof or a composition comprising any one or any
combination of the aforementioned antibodies or fragments
(including monoclonal antibodies or fragments).
[0134] The disclosure provides a method of treating or preventing a
Staphylococcus aureus (S. aureus) infection in a subject (e.g., a
human), which comprises administering the AT-binding antibody or
antigen-binding fragment, the ClfA-binding antibody or
antigen-binding fragment, and/or the leukotoxin-binding antibody or
antigen-binding fragment described herein to a subject in need
thereof, whereupon the S. aureus infection is treated or prevented
in the subject. The disclosure also provides use of the AT-binding
antibody or antigen-binding fragment, the ClfA-binding antibody or
antigen-binding fragment, and/or the leukotoxin-binding antibody or
antigen-binding fragment, described herein, or the composition
comprising any one or combination of the antibodies or fragments
thereof described herein, in the manufacture of a medicament for
treating or preventing a S. aureus infection.
[0135] As discussed herein, Staphylococcus aureus is a major human
pathogen that causes a wide range of clinical infections. S. aureus
is a leading cause of bacteremia and infective endocarditis as well
as osteoarticular, skin and soft tissue, pleuropulmonary, and
device-related infections. Approximately 30% of the human
population is colonized with S. aureus (Wertheim et al., Lancet
Infect. Dis., 5: 751-762 (2005)). The symptoms of S. aureus skin
infections include, for example, boils, cellulits, and impetigo. S.
aureus also may cause food poisoning, blood poisoning (also known
as bacteremia), toxic shock syndrome, and septic arthritis. The
epidemiology, pathophysiology, and clinical manifestations of S.
aureus infections are described in detail in, e.g., Tong et al.,
Clin. Microbiol. Rev., 28(3): 603-661 (2015), and the genomes of
several different S. aureus strains have been sequenced (see, e.g.,
GenBank/EMBL Accession Nos. BX571856, BX571857, BX571858, FN433596,
FN433597, FN433598, HE681097, FR821777, FR821778, FR821779, and
FR821780). As discussed herein, the subject (e.g., human subject)
can have diabetes.
[0136] In certain instances, a therapeutically effective amount of
the AT-binding antibody or antigen-binding fragment, the
ClfA-binding antibody or antigen-binding fragment, and/or the
leukotoxin-binding antibody or antigen-binding fragment, is an
amount which inhibits S. aureus-associated sepsis, inhibits S.
aureus agglutination, inhibits thromboembolic lesion formation,
neutralizes alpha toxin, neutralizes LukSF, HlgAB, HlgCB and LukED,
induces opsonophagocytosis, inhibits S. aureus fibrinogen binding,
inhibits S. aureus agglutination, or any combination of the
foregoing, in a human.
[0137] Alternatively, the pharmacologic and/or physiologic effect
may be prophylactic, i.e., the effect completely or partially
prevents a disease or symptom thereof. In this respect, the
disclosed method comprises administering a "prophylactically
effective amount" of the AT-binding antibody or antigen-binding
fragment, the ClfA-binding antibody or antigen-binding fragment,
and/or the leukotoxin-binding antibody or antigen-binding fragment,
(including monoclonal antibodies or fragments).
[0138] Therapeutic or prophylactic efficacy can be monitored by
periodic assessment of treated patients. For repeated
administrations over several days or longer, depending on the
condition, the treatment can be repeated until a desired
suppression of disease symptoms occurs. However, other dosage
regimens may be useful and are within the scope of the present
disclosure. The desired dosage can be delivered by a single bolus
administration of the composition, by multiple bolus
administrations of the composition, or by continuous infusion
administration of the composition.
[0139] The method of treating or preventing a S. aureus infection
can comprise administering the AT-binding antibody or
antigen-binding fragment, the ClfA-binding antibody or
antigen-binding fragment, and/or the leukotoxin-binding antibody or
antigen-binding fragment in the same composition or in separate
compositions. When separate compositions are administered to the
subject, each of the compositions can be administered
simultaneously or sequentially in any order.
[0140] The composition(s) comprising an effective amount of any one
or combination of the antibodies described herein, or
antigen-binding fragments thereof, the nucleic acid sequence(s)
encoding any of the foregoing, or the vector comprising the nucleic
acid sequence can be administered to a subject, such as a human,
using standard administration techniques, including intravenous,
intraperitoneal, subcutaneous, and intramuscular administration
routes. The composition may be suitable for parenteral
administration. The term "parenteral," as used herein, includes
intravenous, intramuscular, subcutaneous, and intraperitoneal
administration. In some embodiments, the composition is
administered to a subject using peripheral systemic delivery by
intravenous, intraperitoneal, or subcutaneous injection.
[0141] The AT-binding antibody or antigen-binding fragment, the
ClfA-binding antibody or antigen-binding fragment, and/or the
leukotoxin-binding antibody or antigen-binding fragment or
composition(s) comprising same, can be administered alone or in
combination with other drugs (e.g., as an adjuvant) conventionally
used for treating S. aureus infections. The composition(s)
comprising the AT-binding antibody or antigen-binding fragment, the
ClfA-binding antibody or antigen-binding fragment, and/or the
leukotoxin-binding antibody or antigen-binding fragment can be used
in combination with, for example, one or more antibiotics, such as
a penicillinase-resistant .beta.-lactam antibiotic (e.g., oxacillin
or flucloxacillin). Gentamicin can be used to treat serious
infections, such as endocarditis. Most strains of S. aureus,
however, are now resistant to penicillin, and two in 100 people
carry methicillin-resistant strains of S. aureus (MRSA). MRSA
infections typically are treated with vancomycin, and minor skin
infections can be treated with triple antibiotic ointment.
[0142] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0143] This example demonstrates that antibodies that bind to alpha
toxin (AT), clumping factor A (ClfA), and leukotoxins do not
interfere with each other's in vitro activities when used in
combination.
[0144] Several experiments were conducted to determine if using
antibodies that bind to AT, ClfA, and leukotoxins in combination
would interfere with the activity of any of these individual
assays. In these experiments, the MEDI4893*, SAR114, and
SAN481-SYT* antibodies were used in combination and are
collectively referred to as "MEDI6389."
[0145] A red blood cell (RBC) hemolysis inhibition assay was
performed to determine if the anti-ClfA SAR114 or the
anti-leukotoxin SAN481-SYT* antibodies interfered with the activity
of MEDI4893*. Washed rabbit red blood cells (50 .mu.l) were
incubated with native alpha toxin (0.1 .mu.g/ml in 25 .mu.l) and
serial dilution of 25 .mu.l of MEDI4893*, SAN481_SYT*+SAR114 or mAb
trio combination (MEDI6389) as indicated on FIG. 2. Irrelevant mAb
c-IgG was used as negative control. After 2 hrs incubation at
37.degree. C., hemoglobin release was measured in 50m1 supernatants
at OD450 nm. % hemolysis inhibition was measured as:
100*[(OD.sub.AT+mAb)/(OD.sub.AT alone)]. The results, shown in FIG.
2 and in Table 1, below, demonstrate that the use of the three
antibodies in combination (MED6389) was about equally effective in
inhibiting RBC hemolysis as MEDI4893* alone.
[0146] A monocyte viability assay was performed to determine if the
anti-AT MEDI4893* or the anti-ClfA SAR114 antibodies interfered
with the activity of SAN481-SYT*. Human monocytic cell line HL-60
(5e4/well/25 .mu.l) were incubated for 2 hrs at 37.degree. C. with
a mix of LukS+LukF (100 ng/ml each) and serial dilution as
indicated on FIG. 3 of SAN481_SYT*, MEDI4893*+SAR114, or mAb trio
combination (MEDI6389). Irrelevant mAb c-IgG was used as negative
control. Cell viability was quantified by measuring luminescent
signal in a Cell Glo assay (Promega) following company
instructions. % viability was calculated as followed:
100*[(OD.sub.cells+LukSF+mAb)/(OD.sub.cells alone)]. The results,
shown in FIG. 3 and in Table 1, below, demonstrate that the use of
the three antibodies in combination (MED6389) was about equally
effective in maintaining monocyte viability as SAN481-SYT*
alone.
[0147] A fibrinogen-binding inhibition assay was performed to
determine if the anti-AT MEDI4893* or the anti-leukotoxin
SAN481-SYT* antibodies interfered with the activity of SAR114.
Fibrinogen coated 96 well plate (4 .mu.g/ml) was blocked with PBS
2% BSA, and after washes incubated for 1 hr at room temperature
with a biotinylated ClfA001 (2 .mu.g/ml) and serial dilution of
SAR114, MEDI4893*+SAN481_SYT*, or mAb trio combination (MEDI6389)
as indicated on FIG. 4. After 3 washes, plates were incubated with
streptavidin-phycoerythrin at 1:10 000 for 1 hr, and OD.sub.450 nm
read following addition of 100 .mu.l TMB, and then 100 .mu.l of
H.sub.2SO.sub.4 0.2M. Irrelevant mAb c-IgG was used as negative
control. The percentage (%) of fibrinogen binding inhibition was
calculated as: 100*[(OD.sub.ClfA+mAb)/(OD.sub.ClfA alone)]. The
results, shown in FIG. 4 and in Table 1, below, demonstrate that
the use of the three antibodies in combination (MED6389) was about
equally effective in inhibiting fibrinogen binding as SAR114
alone.
TABLE-US-00009 TABLE 1 IC50 (.mu.g/ml) MEDI4893* SAN481-SYT* SAR114
MEDI6389 RBC assay 0.1731 0.1635 Monocyte viability 0.225 0.2246 Fg
binding 3.02 2.63
[0148] The use of the combination of the three antibodies
(MEDI6389) did not inhibit the activity of MEDI4893 in the RBC
assay, the activity of SAN481* in the monocyte viability assay, or
the Fg binding of SAR114.
Example 2
[0149] This example demonstrates that the combination of antibodies
that bind to alpha toxin (AT) and leukotoxins is superior to either
antibody alone in a wound healing model.
[0150] In these experiments, 6-7 week old female Balb/c mice (n=5)
were immunized intra-peritoneally with (i) 0.5 mg/kg a control
antibody (c-IgG), (ii) 0.1 mg/kg of the anti-AT antibody MEDI4893*,
(iii) 0.5 mg/kg of the anti-leukotoxin antibody SAN481-SYT*, or
(iv) both MEDI4893* (0.1 mg/kg) and SAN481-SYT* (0.5 mg/kg). The
mice were then intradermally infected 24 hrs later with a wound
isolate 1447526 (5e7 cfu in 50 .mu.l PBS).
[0151] Lesions were monitored over 17 days, and the results are
shown in FIG. 5. Lesion sizes were significantly smaller in mice
treated with the combination of anti-AT and anti-leukotoxin
antibodies than in mice treated with either antibody alone
(p<0.05 and indicated with a (*). Pictures on FIG. 5 shows
lesions at day 7 post-infection.
Example 3
[0152] This example demonstrates that neutralization of alpha toxin
(AT), clumping factor A (ClfA), and leukotoxins are all necessary
for in vivo protection in the rabbit bacteremia model.
[0153] In these experiments, 3-month old female rabbits (n=7)
received intravenous administration of (i) a control IgG antibody,
(ii) the anti-leukotoxin antibody SAN481-SYT*, (iii) SAN481-SYT*
and the anti-ClfA antibody SAR114, (iv) SAR114 and the anti-AT
antibody MEDI4893*, (v) SAN481-SYT* and MEDI4893*, or (vi)
SAN481-SYT*, SAR114 and MEDI4893*, i.e., MEDI6389. All antibodies
were administered at 5 mg/kg, other than the control antibody,
which was administered at 15 mg/kg. The rabbits were then infected
12 hours later with intravenous CA-MRSA SF8300.
[0154] Survival was monitored over four days after challenge, and
the combination of SAN481-SYT*, SAR114 and MEDI4893* (MEDI6389) or
MEDI4893*+SAN481_SYT* significantly improved survival over c-IgG as
showed by a Log Rank Mantel-Cox statistical test (p=0.0001). The
results are shown in FIG. 6. Notably, neither targeting AT and ClfA
nor targeting leukotoxins is sufficient for protection in this
rabbit lethal bacteremia model.
Example 4
[0155] This example demonstrates that neutralization of alpha toxin
(AT), clumping factor A (ClfA), and leukotoxins are all necessary
for in vivo protection in the rabbit bloodstream infection
model.
[0156] In these experiments, 3-month old female rabbits (n=12)
received intravenous administration of 15 mg/kg of (i) a control
IgG antibody, (ii) the anti-leukotoxin antibody SAN481-SYT*, (iii)
the anti-ClfA antibody SAR114 and the anti-AT antibody MEDI4893*,
(iv) SAN481-SYT* and MEDI4893*, or (vi) SAN481-SYT*, SAR114 and
MEDI4893*, i.e., MEDI6389. The rabbits were then infected 12 hours
later with intravenous HA-MRSA NRS382 or CA-MRSA SF8300.
[0157] Survival was monitored over four days after challenge, and
the results are shown in FIG. 7. The combination of SAN481-SYT*,
SAR114 and MED14893*(MEDI6389) or MEDI4893*+SAN481_SYT*
significantly improved survival over c-IgG as showed by a Log Rank
Mantel-Cox statistical test (p=0.0015 for NRS382 and p=0.0001 for
SF8300) was most effective in increasing survival as a result with
either HA-MRSA NRS382 or CA-MRSA SF8300 bacteria.
Example 5
[0158] This example demonstrates that a combination of antibodies
that bind to alpha toxin (AT), clumping factor A (ClfA), and
leukotoxins (MEDI6389) improves wound healing resulting from
mixed-bacterial infections in a diabetic mouse dermonecrosis
model.
[0159] Mixed-bacterial infections were compared to infections
caused by a single bacteria in seven week male (n=10) type 2
diabetic mice (BKS.Cg-m+/+Lepr.sup.db) mice. The mice were infected
intra-dermally with a mixture of S. aureus (SA; 5e6 cfu),
Pseudomonas aeruginosa (A; 5 cfu) and Streptococcus pyogenese (SP;
1e1 cfu) under 50 .mu.l in PBS, or with SA (5e6 cfu). The lesion
sizes were monitored over 43 days. The results, shown in FIG. 8,
demonstrate that the mixed infections result in delay in the time
of wound closure in this diabetic mouse dermonecrosis model as
compared to infections that result from SA alone.
[0160] The effect of the MEDI6389 combination (comprising anti-AT
mAb MEDI4893*, anti-ClfA mAb SAR114, and anti-leukotoxin mAb
SAN481_SYT*) on the time of wound closure and bacteria load was
examined. Mice were passively immunized intra-peritoneally with
MEDI6389 (each mAb at 15 mg/kg) or control IgG c-IgG (15 mg/kg) and
infected intra-dermally 24 hrs later with SA/SP/PA. Lesions were
followed over 43 days, and bacteria counts were enumerated at days
7, 14, and 21 in skin lesions. The results, shown in FIG. 9,
demonstrate that MED6389 increases wound healing and decreases
bacteria counts in mixed-bacterial skin lesions in this diabetic
mouse dermonecrosis model.
Example 6
[0161] This example provides the materials and methods used in
Examples 7-11.
[0162] In Vivo Model of Systemic Infection
[0163] Frozen stock cultures of S. aureus USA300 strain SF8300 were
thawed and diluted to the appropriate inoculum in sterile PBS, pH
7.2 (Invitrogen) (Hua et al., Antimicrob Agents Chemother.
58:1108-17 (2014)). Specific-pathogen-free 7- to 8-week-old female
BKS.Cg-Dok7<m>+/+Lepr,db>/J (db/db), C57BKS, C57BL/6J-STZ,
and C57BL/6J mice (The Jackson Laboratory) were briefly
anesthetized and maintained in 3% isoflurane (Butler Schein.TM.
Animal Health) with oxygen at 3 L/min and infected intravenously.
All bacterial suspensions were administered in 100 .mu.L of PBS. In
select experiments, neutralizing antibodies MEDI4893*,
anti-.alpha.V.beta.6/8, anti-.alpha.V.beta.6, c-IgG (MedImmune
antibodies), anti-TGF.beta. (clone 1D11.16.8, BioXcell), or control
mouse IgG1 were administered (15 mg/kg) in 0.5 mL intraperitoneally
(IP) 24 hours prior to infection. Rosiglitazone (Sigma-Aldrich) was
administered (10 mg/kg) orally for 7 days. Mice were infected 24
hours following the final dose of rosiglitazone. Animals were
euthanized with CO.sub.2 at the indicated time points, and blood,
liver, or kidneys were collected for analysis. The bacterial load
in kidneys was determined by plating serial dilutions on TSA.
[0164] NET ELISA
[0165] To measure NETs, a hybrid of 2 different ELISA kits were
used. Plates were initially coated with anti-elastase capture
antibody (R&D Systems). Fresh serum samples were added to the
coated wells, then incubated, and washed. Next, anti-DNA-POD
antibody (Roche) was used to detect DNA in the captured proteins in
the wells. Plates were developed with ABTS solution and ABTS stop
solution. Absorbances were measured at 405 nm on a plate reader
using SoftMax Pro software.
[0166] HDN and LDN Purification
[0167] High and low density neutrophils (HDN and LDN) were isolated
from whole blood. Following sacrifice, blood was collected and
layered over with histopaque 1077 (Sigma-Aldrich). Cells were
separated by centrifugation (500 g, 30 minutes). The lower fraction
was treated with ACK lysis buffer (Thermo Fisher Scientific) to
remove red blood cells from the high density neutrophils. The upper
(PBMC) fraction was washed 2.times. with PBS, and low density
neutrophils were isolated with the EasySep Mouse Neutrophil
Enrichment Kit (Stemcell Technologies). Purified cell populations
were lysed for protein or RNA analysis.
[0168] Flow Cytometry
[0169] Either whole blood or purified low density cells, were
washed twice in ice-cold FACs buffer (PBS with 5% fetal bovine
serum, and 0.1% sodium azide). Fc receptors were blocked with
anti-mouse CD16/CD32 (eBioscience), and cells were stained with
antibodies against mouse CD45 (PE conjugated, clone FA-11), CD11c
(APC-Cy5.5 or FITC conjugated, clone N418), CD11b (BV605
conjugated, clone M1/70), Ly6-G (BV421 or PE-Cy7 conjugated, clone
1A8), and Ly6-C. Cells were imaged using the LSR II Flow Cytometer
(BD Biosciences) and analyzed with FlowJo. A known concentration of
counting beads (Bangs Laboratories) was added to each sample to
calculate the number of cells.
[0170] Western Blotting
[0171] Cells were lysed with Ripa buffer (ThermoFisher Scientific)
containing complete protease inhibitor (Sigma) and frozen. In
select experiments, IP3R was immunoprecipitated using anti-IP3R
(Abcam cat #ab5804) and the Dynabeads protein G immunoprecipitation
kit (ThermoFisher Scientific). Equal amounts of protein were
separated on 4-12% bis-Tris NuPage gels and transferred to PVDF
membranes (ThermoFisher Scientific). Immunodetection was performed
using anti-H3Cit (Abcam cat #ab5103), anti-lactoferrin (Abcam cat
#ab77705), anti-MMP9 (Abcam cat #ab38898), anti-IP3R (Abcam cat
#ab5804), anti-P-Ser/Thr (Abcam cat #ab17464), and anti-actin
(Sigma cat #A3854). Proteins were visualized with the Odyssey
imaging system (Li-COR).
Example 7
[0172] This example demonstrates that elevated glucose levels
correlate with more severe S. aureus infections.
[0173] Two models of murine diabetes, STZ induced and db/db, were
used to study the effect of diabetes on the systemic response to
systemic infection with S. aureus. In each model, the diabetic mice
had a non-fasting glucose level greater than 450 dg/mL, while
non-diabetic control levels were less than 200 dg/mL. Mice were
infected with 5e7 CFU S. aureus (USA300, SF8300). CFU were
collected from the kidney 48 hours post infection, and mortality
was monitored for 14 days. Increased mortality was observed in both
STZ (P=0.0011) and db/db (P=0.0241) models as compared with
non-diabetic control (FIGS. 11A and 11B). Of note, this did not
correlate with a difference in bacterial CFU recovered from the
kidneys 48 hours post-infection (FIGS. 11C and 11D). To confirm
that increased mortality was a consequence of elevated glucose in
the diabetic host, mice were treated with Rosiglitazone for 1 week
prior to infection to reduce circulating glucose levels (FIG. 11E).
Rosiglitazone significantly reduced mortality (P=0.0041) following
infection with S. aureus, however the bacterial burden in the
kidney was unaffected (FIGS. 11F and 11G).
[0174] It is notable that no clearance defect was observed in the
diabetic mice as compared with non-diabetic controls. This
highlights the contribution of excessive inflammation or
exaggerated host response to the increase in mortality.
Example 8
[0175] This example demonstrates that enhanced NEToisis occurs in
diabetic mice.
[0176] Neutrophils in a diabetic host, or in the presence of
elevated glucose levels, are increasingly prone to NETosis. In the
diabetic population, NET release has been shown to impair wound
healing in mice, and the presence of NETs in the serum correlates
with non-healing wounds in patients (Fadini, G. P. et al., Diabetes
65: 1061-1071 (2016) and Wong, S. L. et al., Nat Med 21: 815-819
(2015)). Neutrophils also release NETs in response to bacterial
infection, therefore it was hypothesized that S. aureus infection
would result in increased systemic NET release in diabetic mice.
Complexes of neutrophil elastase and double stranded DNA are used
as a measurement of NET formation and quantified by ELISA (Fadini,
G. P. et al., Diabetes 65: 1061-1071 (2016)). Significant increases
(P=0.0003) in serum NETs were observed in diabetic mice
intravenously infected with S. aureus for 24 hours, while
significant increases were not observed in non-diabetic control
mice (FIG. 12A). Levels of circulating NETs were not different in
uninfected diabetic and non-diabetic mice.
[0177] Alpha toxin (AT), once released by S. aureus, binds to the
receptor ADAM10 on the surface of platelets. (Neutrophils do not
express ADAM10.) In response to AT, platelets aggregate and bind to
circulating neutrophils, resulting in activation of caspase-1
mediated signaling and NET production (Powers, M. E. et al., Cell
Host Microbe 17: 775-787 (2015) and Surewaard, B. G. J. et al. Cell
Host Microbe 24: 271-284 (2018)). Consistent with these findings,
neutralization of AT with monoclonal antibody MEDI4893*
significantly reduced the number of NE-DNA complexes in the serum
48 hours post-infection in diabetic animals (FIG. 12B). Increased
AT-dependent NET production was confirmed 48 hours post-infection
by increased citrinulated Histone H3 (H3cit) in the liver as
detected by western blot (FIG. 12C) Visualization of liver sections
immunohistochemically stained with anti-Ly6G to mark neutrophils
and anti-H3cit also showed increased AT-dependent NET (i.e., less
anti-H3 cit staining in the livers of mice that received MEDI4893*)
(Cohen T S, et al. Staphylococcus aureus drives expansion of low
density neutrophils in diabetic mice. JCI 2019 IN PRESS).
Neutralization of AT significantly increased survival (P=0.0255) of
diabetic mice infected with S. aureus (FIG. 12D). These data
indicate that systemic infection of the diabetic host lead to an
AT-dependent increase in circulating NETs that can be inhibited by
MEDI4893*.
Example 9
[0178] This example demonstrates that low density neutrophils
correlate with increased NETosis.
[0179] Similar to macrophages, neutrophils can be separated into
different classes based on functional characteristics. Severe burns
have been shown to alter the phenotype of circulating neutrophils
and to alter TLR expression, cytokine production, and their ability
to drive macrophage polarization (Tsuda, Y. et al. Immunity 21:
215-226 (2004)). Neutrophils are unique in that they can also be
separated by cell density. High density neutrophils are anti-tumor,
phagocytic cells, while low density neutrophils are considered
pro-tumor phagocytic defective cells (Sagiv, J. Y. et al. Cell Rep
10: 562-573 (2015)). While Tsuda et. al. did not measure the
density of neutrophils isolated from mice susceptible to S. aureus
infection, the shape of the nuclei in these neutrophils was similar
to the shape of nuclei in low density cells (Sagiv, J. Y. et al.
Cell Rep 10: 562-573 (2015) and Fridlender, Z. G. et al. Cancer
Cell 16: 183-194 (2009)). The shapes of the nuclei in neutrophils
taken from non-diabetic mice and diabetic mice also had striking
differences. The nucleus in cells isolated from non-diabetic mice
were multilobular or round, while large numbers of cells with
ringed nuclei were observed in the blood of diabetic mice (Cohen T
S, et al. Staphylococcus aureus drives expansion of low density
neutrophils in diabetic mice. JCI 2019 IN PRESS). These structures
were similar to those reported by Tsuda et. al to be found in the
cells isolated from S. aureus susceptible mice, indicating that
diabetic mice could have an increased number of low density, or
immune impaired neutrophils.
[0180] Hyper NET production is a characteristic of low density
neutrophils (LDN), and it was hypothesized that higher numbers of
LDNs in infected diabetic mice were responsible for the increases
in NETs (Villanueva, E. et al. J Immunol 187: 538-552 (2011)).
Blood was collected from C57BKS and db/db mice 48 hours post-IV
infection and was analyzed for presence of LDNs. The amount of LDNs
in the blood of infected db/db mice was significantly increased
compared to uninfected db/db mice (P<0.0001) as well as infected
C57BKS control mice (P=0.0003) (FIG. 13A). Increases in LDNs were
not observed in C57BKS mice (FIG. 13A). Similar increases were
observed in STZ induced diabetic mice and not in C57BL/6 controls
(FIG. 14). Lowering glucose levels with Rosiglitazone prior to
infection significantly (P=0.0116) reduced LDNs 48 hours
post-infection (FIG. 13B).
[0181] To ensure that the observations were not based on
degranulated neutrophils, LDNs and high density neutrophils (HDNs)
were isolated from the blood of infected db/db mice, and the
amounts of lactoferrin (secondary granules) and MMP9 (tertiary
granules) were measured by western blot. Equivalent amounts of both
were observed, indicating that LDNs have similar granular content
as compared to HDNs (Cohen T S, et al. Staphylococcus aureus drives
expansion of low density neutrophils in diabetic mice. JCI 2019 IN
PRESS). Neutralizing AT prevented systemic NET release, therefore
the influence of AT on the number of LDNs was assessed. LDNs in the
blood of db/db mice treated 24 hours prior to infection with c-IgG
or MEDI4893* and infected with S. aureus for 48 hours were
measured. A significant reduction in LDNs in mice prophylactically
treated with MEDI4893* (FIG. 13C) was observed, while overall
numbers of neutrophils were not affected (FIG. 13D), indicating
that AT contributes to the increase in LDNs.
[0182] These data indicate that LDNs contribute to the pathology
associated with diabetic S. aureus infection and that these LDNs
are associated with excessive NET release in both the liver, a key
target organ of systemic infections, and systemically in the blood.
Moreover, MEDI4893* reduces LDNs in diabetic mice.
Example 10
[0183] This example demonstrates that TGF.beta. drives expansion of
LDNs.
[0184] TGF.beta. has been implicated as a central regulator of
neutrophil phenotype, and in tumor models it can drive a phenotypic
switch from high to low density neutrophil (Sagiv, J. Y. et al.
Cell Rep 10: 562-573 (2015) and Fridlender, Z. G. et al. Cancer
Cell 16: 183-194 (2009). Sagiv et. al. demonstrated that the
addition of TGF.beta. to blood taken from tumor bearing mice, not
naive mice, will increase numbers of LDNs in vitro (id.). This
study was repeated with blood from non-diabetic and diabetic mice.
The addition of TGF.beta. to diabetic blood significantly increased
(P=0.0021) the number of LDNs (FIG. 15A). The same was not observed
in non-diabetic blood. Based on this in vitro evidence
demonstrating that TGF.beta. can increase numbers of LDNs, its
necessity for their induction by blocking in vivo was tested.
Diabetic mice were prophylactically treated with neutralizing
TGF.beta. antibody 24 hours prior to infection with S. aureus. The
numbers of LDNs in the bloodstream was significantly reduced
(P=0.0003) by inhibition of TGF.beta., while numbers of bacteria in
the kidneys were similar between groups (FIGS. 15B and 15C).
Survival was significantly improved (P=0.0072) by neutralizing
TGF.beta. (FIG. 15D). Visualization of NETs in the liver
demonstrated a loss of NETs when TGF.beta. was neutralized (Cohen T
S, et al. Staphylococcus aureus drives expansion of low density
neutrophils in diabetic mice. JCI 2019 IN PRESS). These data
suggest that reducing LDNs by blocking TGF.beta. could promote
survival.
[0185] TGF.beta. is secreted as a pro-form protein (pro-TGF.beta.)
and requires cleavage to be activated. Binding of pro-TGF.beta. by
.alpha.V.beta.8 integrin has been linked to its activation and
prevention of colitis, and its expression on dendritic cell and
monocyte subsets is increased in response to inflammation (Travis,
M. A. et al. Nature 449: 361-365 (2007) and Kelly, A. et al. J Exp
Med, doi:10.1084/jem.20171491 (2018)). To determine if S. aureus
infection influences expression of .alpha.V.beta.8 integrin, innate
immune cells were isolated from the liver and spleen of C57BKS and
db/db mice 24 hours post-infection, and the expression of
.alpha.V.beta.8 was analyzed by flow cytometry. Numbers of 38
positive inflammatory monocytes and dendritic cells increased in
the livers of db/db mice, not C57BKS mice, following infection
(FIG. 16A). Interestingly, while integrin expression increased on
the surface of monocytes, it was the overall number of DCs that
increased, not the density of 38 (FIG. 16B). To demonstrate the
functional relevance of .alpha.V.beta.8 in this model, mice were
prophylactically treated with antibodies neutralizing
.alpha.V.beta.6/8, .alpha.V.beta.6 or c-IgG and infected with S.
aureus. Forty-eight hours post infection LDNs were significantly
decreased (P=0.0090) in the bloodstream in the mice treated with
.alpha.V.beta.6/8 neutralizing antibody compared with c-IgG (FIG.
16C). Neutralization of .alpha.V.beta.6 alone did not reduce the
numbers of these cells. Integrin inhibition did not affect the
numbers of bacteria in the kidneys 48 hours post-infection (FIG.
16D). Survival was significantly improved in mice treated with
anti-.alpha.V 6/8 antibody as compared with c-IgG treated mice
(FIG. 16E). Therefore, consistent with directly neutralizing
TGF.beta., blocking the integrin responsible for activating this
pathway was protective in diabetic mice.
[0186] These data show that neutralization of either .alpha.V 6/8
or TGF.beta. prevents LDN increases and reduces mortality. These
data also show that dendritic cells play a central role in the
pathogenesis of diabetic infection due to their ability to activate
TGF.beta. and promote expansion of LDNs.
Example 11
[0187] This example demonstrates that AT drives TGF.beta.
activation.
[0188] It was hypothesized that AT was influencing LDN numbers by
affecting the TGF.beta. pathway. Following its activation, TGF
binds to its receptor complex, activates SMAD transcription
factors, and drives expression of downstream genes. Therefore,
activation of SMAD signaling is commonly used as a surrogate
measurement of TGF.beta. activation. pSMAD levels were analyzed in
the livers of diabetic and non-diabetic mice that were infected (24
hours) with S. aureus. Significantly increased pSMAD was observed
in the livers of infected diabetic mice as compared to naive
diabetic mice (P<0.0001) and infected non-diabetic mice
(P=0.0338) (FIG. 17A). In diabetic mice, MEDI4893* significantly
reduced (P<0.0001) pSMAD levels in the liver, indicating that AT
was contributing to activation of TGF.beta. signaling (FIG. 17B).
Neutralizing AT did not alter the numbers of .alpha.V.beta.8
expressing innate immune cells (FIG. 17C). These data indicate that
AT influences activation of TGF.beta. through a mechanism that is
independent of .alpha.V.beta.8 expression on innate immune cells.
Accordingly, neutralization of AT, which is a key S. aureus
virulence factor, limits activation of TGF.beta. signaling, and
subsequently reduces LDN numbers and NET release.
[0189] These data indicate that, in addition to binding to ADAM10
on platelets, AT can act through a second pathway that alters the
neutrophil phenotype and subsequent response to S. aureus
infection. In the diabetic host, AT-dependent activation of TGF
signaling drives expansion of LDNs. Thus, AT is both promoting the
expansion of the LDN population which spontaneously release NETs
and activating platelets, which can bind and further activate
neutrophils.
[0190] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0191] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents in the context of describing the
invention (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0192] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
7015PRTArtificial SequenceVH CDR1 1Ser His Asp Met His1
5216PRTArtificial SequenceVH CDR2 2Gly Ile Gly Thr Ala Gly Asp Thr
Tyr Tyr Pro Asp Ser Val Lys Gly1 5 10 15314PRTArtificial SequenceVH
CDR3 3Asp Arg Tyr Ser Pro Thr Gly His Tyr Tyr Gly Met Asp Val1 5
1045PRTArtificial SequenceVH CDR1 4Asn Ser Tyr Trp Ser1
5516PRTArtificial SequenceVH CDR2 5Tyr Leu Tyr Ser Ser Gly Arg Thr
Asn Tyr Thr Pro Ser Leu Lys Ser1 5 10 15616PRTArtificial SequenceVH
CDR3 6Thr His Leu Gly Gly Phe His Tyr Gly Gly Gly Phe Trp Phe Asp
Pro1 5 10 1575PRTArtificial SequenceVH CDR1 7Thr Tyr Ala Met His1
5817PRTArtificial SequenceVH CDR2 8Val Thr Ser Phe Asp Gly Ser Asn
Glu Tyr Tyr Ile Asp Ser Val Lys1 5 10 15Gly912PRTArtificial
SequenceVH CDR3 9Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr1 5
101011PRTArtificial SequenceVL CDR1 10Arg Ala Ser Gln Ser Ile Ser
Ser Trp Leu Ala1 5 10117PRTArtificial SequenceVL CDR2 11Lys Ala Ser
Ser Leu Glu Ser1 5128PRTArtificial SequenceVL CDR3 12Lys Gln Tyr
Ala Asp Tyr Trp Thr1 51311PRTArtificial SequenceVL CDR1 13Arg Ala
Ser Gln Ser Ile Thr Ser Tyr Leu Asn1 5 10147PRTArtificial
SequenceVL CDR2 14Ala Ser Ser Ser Leu Gln Ser1 5159PRTArtificial
SequenceVL CDR3 15Gln Glu Ser Tyr Ser Thr Pro Pro Thr1
51613PRTArtificial SequenceVL CDR1 16Ser Gly Ser Ser Tyr Asn Ile
Gly Ser Asn Tyr Val Tyr1 5 10177PRTArtificial SequenceVL CDR2 17Arg
Ser Ile Gln Arg Pro Ser1 51811PRTArtificial SequenceVL CDR3 18Ala
Ala Trp Asp Asp Ser Leu Arg Ala Trp Val1 5 1019122PRTArtificial
SequenceVH Amino Acid Sequence 19Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30Asp Met His Trp Val Arg
Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Gly Ile Gly Thr
Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60Gly Arg Phe Thr
Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu65 70 75 80Gln Met
Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Asp Arg Tyr Ser Pro Thr Gly His Tyr Tyr Gly Met Asp Val Trp 100 105
110Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
12020124PRTArtificial SequenceVH Amino Acid Sequence 20Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Gln Asn Ser 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Leu Tyr Ser Ser Gly Arg Thr Asn Tyr Thr Pro Ser Leu Lys
50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Thr His Leu Gly Gly Phe His Tyr Gly Gly Gly
Phe Trp Phe Asp 100 105 110Pro Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 12021126PRTArtificial SequenceVH Amino Acid Sequence
21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Ser Phe Arg Asn
Ala 20 25 30Leu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Arg Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala
Ala Pro Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Thr Gly Pro Gly Gly Gly Pro Pro
Gly Asp Tyr Tyr Tyr Asp Gly 100 105 110Met Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser 115 120 12522127PRTArtificial
SequenceVH Amino Acid Sequence 22Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Ala 20 25 30Trp Met Thr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Arg Ser
Lys Thr Ala Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr
Leu Gln Met Thr Ser Leu Lys Ile Glu Asp Thr Ala Leu Tyr 85 90 95Tyr
Cys Met Thr Asp Gly Leu Gly Leu Leu Asn Phe Gly Asp Ser Asp 100 105
110Pro His His Tyr Trp Gly Gln Gly Thr Arg Val Thr Val Ser Ser 115
120 12523122PRTArtificial SequenceVH Amino Acid
Sequencemisc_feature(25)..(25)Xaa can be any naturally occurring
amino acidmisc_feature(28)..(28)Xaa can be any naturally occurring
amino acid 23Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Ala Xaa Gly Tyr Xaa
Phe Thr Ser Tyr 20 25 30Trp Ile Gly Trp Val Arg Gln Val Pro Gly Lys
Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr
Arg His Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Val Asp
Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys
Ala Ser Asp Ser Ala Met Tyr Tyr Cys 85 90 95Ala Arg His Gln Ser Gly
Ser His Gly Phe Asp Ala Phe Glu Ile Trp 100 105 110Gly Gln Gly Thr
Met Val Thr Val Ser Ser 115 12024119PRTArtificial SequenceVH Amino
Acid Sequence 24Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn
Phe Thr Asn Tyr 20 25 30Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys
Gly Leu Glu Trp Met 35 40 45Gly Ile Ile Tyr Ser Gly Asp Ser Asp Thr
Arg Tyr Ser Pro Ser Phe 50 55 60Leu Gly Gln Val Ser Ile Ser Val Asp
Lys Ser Phe Thr Thr Ala Tyr65 70 75 80Leu Gln Trp Arg Ser Leu Lys
Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Gly Gly
Gln Lys Pro Tyr Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr
Val Ser Ser 11525136PRTArtificial SequenceVH Amino Acid Sequence
25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn
Ala 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Arg Ile Lys Ser Glu Thr Ala Gly Gly Thr Thr Asp
Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asp
Ser Arg Asn Thr65 70 75 80Leu Tyr Leu Glu Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr Asp Ser Tyr Thr Pro
Leu Glu Glu Pro Cys Pro Asn 100 105 110Gly Val Cys Tyr Thr Tyr Tyr
Tyr Tyr Gly Met Asp Val Trp Gly Gln 115 120 125Gly Thr Thr Val Thr
Val Ser Ser 130 13526119PRTArtificial SequenceVH Amino Acid
Sequence 26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe
Asn Arg Tyr 20 25 30Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Ser Ser Pro Ile Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Val Thr Leu Gly
Leu Glu Phe Asp Phe Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 11527120PRTArtificial SequenceVH Amino Acid Sequence 27Gln
Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10
15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30Gly Met Cys Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45Trp Leu Ala Leu Ile Glu Trp Asp Asp Asp Lys Tyr Tyr Asn
Thr Ser 50 55 60Leu Lys Thr Arg Leu Ser Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val65 70 75 80Val Leu Thr Met Thr Asn Met Asp Pro Val Asp
Thr Gly Thr Tyr Tyr 85 90 95Cys Ala Arg His Ser Ser Ser Ser Arg Gly
Phe Asp Tyr Trp Gly Gln 100 105 110Gly Ala Leu Val Thr Val Ser Ser
115 12028121PRTArtificial SequenceVH Amino Acid Sequence 28Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser
Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25
30Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser
Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Thr Ala Thr
Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Asn Ala Ser Asp Ser Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Gln Gly Gly Ser His Gly Tyr Asp Ala
Phe His Met Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val Ser Ser
115 12029112PRTArtificial SequenceVH Amino Acid Sequence 29Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Gly Ile Asn Gly Thr Gly Tyr Asn Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Val Thr65 70 75 80Leu Glu Met Asn Ser Leu Arg Val Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95His Lys Val Pro Trp Trp Gly Gln Gly Thr Leu
Val Ser Val Ser Ser 100 105 11030128PRTArtificial SequenceVH Amino
Acid Sequence 30Gln Val Gln Leu Gln Glu Ser Gly Pro Arg Leu Val Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Phe Val Ser Gly Gly Ser
Ile Asn Asn Ser 20 25 30Tyr Trp Thr Trp Ile Arg Gln Pro Pro Gly Gln
Gly Leu Glu Trp Ile 35 40 45Gly Phe Val Phe Ser Ser Gly Arg Thr Asn
Tyr Ser Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Leu Phe Ser Leu65 70 75 80Arg Leu Thr Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Phe Cys Ala 85 90 95Arg Gln Val His Tyr Asp
Phe Trp Ser Gly Tyr Ser Leu Thr Lys Thr 100 105 110Asn Trp Phe Asp
Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12531128PRTArtificial SequenceVH Amino Acid Sequence 31Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Val Val Ser Gly Gly Ser Ile Asn Asn Ser 20 25 30Tyr
Trp Thr Trp Ile Arg Gln Pro Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Phe Val Tyr Ser Ser Gly Arg Thr Tyr Tyr Ser Pro Ser Leu Lys
50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Phe Phe Ser
Leu65 70 75 80Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Phe Cys Ala 85 90 95Arg Gln Val His Tyr Asp Leu Trp Ser Gly Tyr Ser
Leu Thr Lys Thr 100 105 110Asn Trp Phe Asp Pro Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 120 12532121PRTArtificial SequenceVH
Amino Acid Sequence 32Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala
Val Gln Pro Gly Arg1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro
Gly Arg Gly Leu Glu Trp Val 35 40 45Ala Val Thr Ser Phe Asp Gly Ser
Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Thr Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Thr Gly
Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95Ala Arg Asp Glu
Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115 12033106PRTArtificial SequenceVL
Amino Acid Sequence 33Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr
Tyr Tyr Cys Lys Gln Tyr Ala Asp Tyr Trp Thr 85 90 95Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 10534107PRTArtificial SequenceVL Amino
Acid Sequence 34Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Thr Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ser Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Glu Ser Tyr Ser Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10535106PRTArtificial SequenceVL Amino Acid
Sequence 35Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro
Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Val Pro Lys
Lys Tyr Ala 20 25 30Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val
Leu Val Ile Tyr 35 40 45Glu Asp Lys Lys Arg Pro Ser Gly Ile Pro Glu
Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Thr Met Ala Thr Leu Thr Ile
Ser Gly Ala Gln Val Glu65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Tyr
Ser Thr Asp Ser Ser Glu Gly Val 85 90 95Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu 100 10536106PRTArtificial SequenceVL Amino Acid
Sequence 36Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro
Gly Gln1 5 10 15Thr Ala Arg
Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Lys Tyr Ala 20 25 30Tyr Trp
Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val Leu Val Ile His 35 40 45Glu
Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55
60Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val Glu65
70 75 80Asp Glu Ala Asp Tyr His Cys Tyr Ser Thr Asp Ser Ser Gly Val
Val 85 90 95Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
10537113PRTArtificial SequenceVL Amino Acid Sequence 37Asp Ile Val
Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys Lys Ser Ser Gln Gly Val Leu Ser Arg 20 25 30Ser
Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr
Cys Gln Gln 85 90 95Tyr Tyr Asn Asn Leu Arg Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile 100 105 110Arg38106PRTArtificial SequenceVL Amino
Acid Sequence 38Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg
Ile Ser Asn Trp 20 25 30Leu Ala Trp Tyr Gln Lys Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Thr Leu Glu Ser Glu Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Leu Ala Thr Tyr Tyr
Cys His Gln Tyr Ile Ser Tyr Tyr Thr 85 90 95Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 10539111PRTArtificial SequenceVL Amino Acid
Sequence 39Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro
Gly Glu1 5 10 15Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile
Gly Ala Asn 20 25 30Ser Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45Ile Tyr Asp Asn Asp Lys Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu
Gly Ile Thr Gly Leu Gln65 70 75 80Thr Gly Asp Glu Ala Asp Tyr Tyr
Cys Gly Thr Trp Val Gly Ile Leu 85 90 95Ser Ala Gly Trp Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 11040107PRTArtificial
SequenceVL Amino Acid Sequence 40Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Lys Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Leu Arg Ser Asn Trp Ala Tyr 85 90 95Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 10541107PRTArtificial
SequenceVL Amino Acid Sequence 41Ser Tyr Gly Leu Thr Gln Pro Pro
Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Ser
Gly Asp Ala Leu Ala Lys Gln Tyr Val 20 25 30Tyr Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Val Leu Val Ile Asp 35 40 45Lys Asp Arg Glu Arg
Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Thr
Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu65 70 75 80Asp Glu
Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Ser Ser Arg Thr Tyr 85 90 95Val
Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 10542112PRTArtificial
SequenceVL Amino Acid Sequence 42Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asp Gly Asn Thr Tyr Leu
Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45Pro Arg Arg Leu Ile
Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95Thr
His Leu Thr Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
11043113PRTArtificial SequenceVL Amino Acid Sequence 43Asp Ile Val
Leu Thr Gln Ser Pro Glu Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Phe Phe Lys 20 25 30Ser
Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45Pro Pro Lys Val Ile Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe
Cys His Gln 85 90 95Tyr Tyr Ser Thr Gln Tyr Ser Phe Gly Gln Gly Thr
Lys Leu Glu Ile 100 105 110Lys44107PRTArtificial SequenceVL Amino
Acid Sequence 44Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Thr Val Thr Ile Thr Cys Arg Thr Ser Gln Ser
Ile Ser Asn Phe 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Val Asn Gly 50 55 60Ser Thr Ser Gly Thr Glu Phe Thr Leu
Thr Leu Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Tyr Ser Thr Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10545107PRTArtificial SequenceVL Amino Acid
Sequence 45Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Thr Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile
Ser Asn Phe 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Asn Gly 50 55 60Ser Thr Ser Gly Thr Asp Phe Thr Leu Thr
Leu Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Ser Thr Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 10546110PRTArtificial SequenceVL Amino Acid
Sequence 46Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro
Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Tyr Asn Ile
Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45Ile Ser Arg Ser Ile Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Val Thr Ser Ala Ser Leu
Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr
Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Arg Ala Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 11047452PRTArtificial
SequenceFull-Length Heavy Chain Amino Acid Sequence 47Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30Asp
Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Asp Arg Tyr Ser Pro Thr Gly His Tyr Tyr Gly
Met Asp Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 245 250 255Tyr Ile Thr Arg Glu Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310
315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly Lys 45048452PRTArtificial
SequenceFull-Length Heavy Chain Amino Acid Sequence 48Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30Asp
Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Asp Arg Tyr Ser Pro Thr Gly His Tyr Tyr Gly
Met Asp Val Trp 100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310
315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly Lys 45049454PRTArtificial
SequenceFull-Length Heavy Chain Amino Acid Sequence 49Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Gln Asn Ser 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Leu Tyr Ser Ser Gly Arg Thr Asn Tyr Thr Pro Ser Leu Lys
50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Thr His Leu Gly Gly Phe His Tyr Gly Gly Gly
Phe Trp Phe Asp 100 105 110Pro Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Pro 210 215 220Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu225 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290
295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp305 310 315 320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 325 330 335Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410
415Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430Ser Val Met His Glu Ala Cys Ser Tyr His Leu Cys Gln Lys
Ser Leu 435 440 445Ser Leu Ser Pro Gly Lys 45050451PRTArtificial
SequenceFull-Length Heavy Chain Amino Acid Sequence 50Gln Leu Gln
Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg1 5 10 15Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala
Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40
45Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu
Tyr Phe Cys 85 90 95Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val
Gly Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Tyr 245 250 255Ile Thr Arg Glu Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310
315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445Pro Gly Lys 45051451PRTArtificial SequenceFull-Length
Heavy Chain Amino Acid Sequence 51Gln Leu Gln Leu Val Glu Ser Gly
Gly Gly Ala Val Gln Pro Gly Arg1 5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met His Trp Val Arg
Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45Ala Val Thr Ser Phe
Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95Ala
Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly Lys
45052212PRTArtificial SequenceFull-Length Light Chain Amino Acid
Sequence 52Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr Cys
Lys Gln Tyr Ala Asp Tyr Trp Thr 85 90 95Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150
155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu
21053214PRTArtificial SequenceFull-Length Light Chain Amino Acid
Sequence 53Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Thr Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ser Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Glu Ser Tyr Ser Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21054216PRTArtificial SequenceFull-Length Light Chain Amino Acid
Sequence 54Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro
Gly Gln1 5 10 15Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Tyr Asn Ile
Gly Ser Asn 20 25 30Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala
Pro Lys Leu Leu 35 40 45Ile Ser Arg Ser Ile Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60Gly Ser Lys Ser Val Thr Ser Ala Ser Leu
Ala Ile Ser Gly Leu Arg65 70 75 80Ser Glu Asp Glu Ala Asp Tyr Tyr
Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95Arg Ala Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys145 150
155 160Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
Tyr 165 170 175Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser His 180 185 190Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu Lys 195 200 205Thr Val Ala Pro Thr Glu Cys Ser 210
215556PRTArtificial SequenceFc region 55Cys Ser Tyr His Leu Cys1
55617PRTArtificial Sequenceheavy chain constant domain 56Met His
Glu Ala Cys Ser Tyr His Leu Cys Gln Lys Ser Leu Ser Leu1 5 10
15Ser57293PRTArtificial SequenceS. aureus alpha toxin 57Ala Asp Ser
Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser1 5 10 15Asn Thr
Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30Gly
Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40
45Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln
50 55 60Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala
Trp65 70 75 80Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn
Glu Val Ala 85 90 95Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp
Thr Lys Glu Tyr 100 105 110Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly
Asn Val Thr Gly Asp Asp 115 120 125Thr Gly Lys Ile Gly Gly Leu Ile
Gly Ala Asn Val Ser Ile Gly His 130 135 140Thr Leu Lys Tyr Val Gln
Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro145 150 155 160Thr Asp Lys
Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175Gln
Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly 180 185
190Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp
195 200 205Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser
Gly Phe 210 215 220Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg
Lys Ala Ser Lys225 230 235 240Gln Gln Thr Asn Ile Asp Val Ile Tyr
Glu Arg Val Arg Asp Asp Tyr 245 250 255Gln Leu His Trp Thr Ser Thr
Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270Lys Trp Thr Asp Arg
Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285Glu Glu Met
Thr Asn 29058293PRTArtificial SequenceS. aureus alpha toxin H35L
mutant 58Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile
Gly Ser1 5 10 15Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp
Lys Glu Asn 20 25 30Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp
Asp Lys Asn His 35 40 45Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly
Thr Ile Ala Gly Gln 50 55 60Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn
Lys Ser Gly Leu Ala Trp65 70 75 80Pro Ser Ala Phe Lys Val Gln Leu
Gln Leu Pro Asp Asn Glu Val Ala 85 90 95Gln Ile Ser Asp Tyr Tyr Pro
Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110Met Ser Thr Leu Thr
Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125Thr Gly Lys
Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135 140Thr
Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro145 150
155 160Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val
Asn 165 170 175Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro
Val Tyr Gly 180 185 190Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser
Met Lys Ala Ala Asp 195 200 205Asn Phe Leu Asp Pro Asn Lys Ala Ser
Ser Leu Leu Ser Ser Gly Phe 210 215 220Ser Pro Asp Phe Ala Thr Val
Ile Thr Met Asp Arg Lys Ala Ser Lys225 230 235 240Gln Gln Thr Asn
Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255Gln Leu
His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265
270Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys
275 280 285Glu Glu Met Thr Asn 29059300PRTArtificial SequenceS.
aureus HIgB 59Gly Glu Gly Lys Ile Thr Pro Val Ser Val Lys Lys Val
Asp Asp Lys1 5 10 15Val Thr Leu Tyr Lys Thr Thr Ala Thr Ala Asp Ser
Asp Lys Phe Lys 20 25 30Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys
Asp Lys Ser Tyr Asp 35 40 45Lys Asp Thr Leu Val Leu Lys Ala Thr Gly
Asn Ile Asn Ser Gly Phe 50 55 60Val Lys Pro Asn Pro Asn Asp Tyr Asp
Phe Ser Lys Leu Tyr Trp Gly65 70 75 80Ala Lys Tyr Asn Val Ser Ile
Ser Ser Gln Ser Asn Asp Ser Val Asn 85 90
95Val Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln
100 105 110Asn Thr Leu Gly Tyr Thr Phe Gly Gly Asp Ile Ser Ile Ser
Asn Gly 115 120 125Leu Ser Gly Gly Leu Asn Gly Asn Thr Ala Phe Ser
Glu Thr Ile Asn 130 135 140Tyr Lys Gln Glu Ser Tyr Arg Thr Thr Leu
Ser Arg Asn Thr Asn Tyr145 150 155 160Lys Asn Val Gly Trp Gly Val
Glu Ala His Lys Ile Met Asn Asn Gly 165 170 175Trp Gly Pro Tyr Gly
Arg Asp Ser Phe His Pro Thr Tyr Gly Asn Glu 180 185 190Leu Phe Leu
Ala Gly Arg Gln Ser Ser Ala Tyr Ala Gly Gln Asn Phe 195 200 205Ile
Ala Gln His Gln Met Pro Leu Leu Ser Arg Ser Asn Phe Asn Pro 210 215
220Glu Phe Leu Ser Val Leu Ser His Arg Gln Asp Gly Ala Lys Lys
Ser225 230 235 240Lys Ile Thr Val Thr Tyr Gln Arg Glu Met Asp Leu
Tyr Gln Ile Arg 245 250 255Trp Asn Gly Phe Tyr Trp Ala Gly Ala Asn
Tyr Lys Asn Phe Lys Thr 260 265 270Arg Thr Phe Lys Ser Thr Tyr Glu
Ile Asp Trp Glu Asn His Lys Val 275 280 285Lys Leu Leu Asp Thr Lys
Glu Thr Glu Asn Asn Lys 290 295 30060302PRTArtificial SequenceS.
aureus LukF 60Gly Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val
Asp Asp Lys1 5 10 15Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Ser
Asp Lys Leu Lys 20 25 30Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys
Asp Lys Ser Tyr Asp 35 40 45Lys Asp Thr Leu Ile Leu Lys Ala Ala Gly
Asn Ile Tyr Ser Gly Tyr 50 55 60Thr Lys Pro Asn Pro Lys Asp Thr Ile
Ser Ser Gln Phe Tyr Trp Gly65 70 75 80Ser Lys Tyr Asn Ile Ser Ile
Asn Ser Asp Ser Asn Asp Ser Val Asn 85 90 95Val Val Asp Tyr Ala Pro
Lys Asn Gln Asn Glu Glu Phe Gln Val Gln 100 105 110Gln Thr Val Gly
Tyr Ser Tyr Gly Gly Asp Ile Asn Ile Ser Asn Gly 115 120 125Leu Ser
Gly Gly Gly Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn 130 135
140Tyr Lys Gln Glu Ser Tyr Arg Thr Ser Leu Asp Lys Arg Thr Asn
Phe145 150 155 160Lys Lys Ile Gly Trp Asp Val Glu Ala His Lys Ile
Met Asn Asn Gly 165 170 175Trp Gly Pro Tyr Gly Arg Asp Ser Tyr His
Ser Thr Tyr Gly Asn Glu 180 185 190Met Phe Leu Gly Ser Arg Gln Ser
Asn Leu Asn Ala Gly Gln Asn Phe 195 200 205Leu Glu Tyr His Lys Met
Pro Val Leu Ser Arg Gly Asn Phe Asn Pro 210 215 220Glu Phe Ile Gly
Val Leu Ser Arg Lys Gln Asn Ala Ala Lys Lys Ser225 230 235 240Lys
Ile Thr Val Thr Tyr Gln Arg Glu Met Asp Arg Tyr Thr Asn Phe 245 250
255Trp Asn Gln Leu His Trp Ile Gly Asn Asn Tyr Lys Asp Glu Asn Arg
260 265 270Ala Thr His Thr Ser Ile Tyr Glu Val Asp Trp Glu Asn His
Thr Val 275 280 285Lys Leu Ile Asp Thr Gln Ser Lys Glu Lys Asn Pro
Met Ser 290 295 30061302PRTArtificial SequenceS. aureus LukD 61Gly
Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp Lys1 5 10
15Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Asn Asp Lys Leu Asn
20 25 30Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr
Asp 35 40 45Lys Asp Thr Leu Val Leu Lys Ala Ala Gly Asn Ile Asn Ser
Gly Tyr 50 55 60Lys Lys Pro Asn Pro Lys Asp Tyr Asn Tyr Ser Gln Phe
Tyr Trp Gly65 70 75 80Gly Lys Tyr Asn Val Ser Val Ser Ser Glu Ser
Asn Asp Ala Val Asn 85 90 95Val Val Asp Tyr Ala Pro Lys Asn Gln Asn
Glu Glu Phe Gln Val Gln 100 105 110Gln Thr Leu Gly Tyr Ser Tyr Gly
Gly Asp Ile Asn Ile Ser Asn Gly 115 120 125Leu Ser Gly Gly Leu Asn
Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn 130 135 140Tyr Lys Gln Glu
Ser Tyr Arg Thr Thr Ile Asp Arg Lys Thr Asn His145 150 155 160Lys
Ser Ile Gly Trp Gly Val Glu Ala His Lys Ile Met Asn Asn Gly 165 170
175Trp Gly Pro Tyr Gly Arg Asp Ser Tyr Asp Pro Thr Tyr Gly Asn Glu
180 185 190Leu Phe Leu Gly Gly Arg Gln Ser Ser Ser Asn Ala Gly Gln
Asn Phe 195 200 205Leu Pro Thr His Gln Met Pro Leu Leu Ala Arg Gly
Asn Phe Asn Pro 210 215 220Glu Phe Ile Ser Val Leu Ser His Lys Gln
Asn Asp Thr Lys Lys Ser225 230 235 240Lys Ile Lys Val Thr Tyr Gln
Arg Glu Met Asp Arg Tyr Thr Asn Gln 245 250 255Trp Asn Arg Leu His
Trp Val Gly Asn Asn Tyr Lys Asn Gln Asn Thr 260 265 270Val Thr Phe
Thr Ser Thr Tyr Glu Val Asp Trp Gln Asn His Thr Val 275 280 285Lys
Leu Ile Gly Thr Asp Ser Lys Glu Thr Asn Pro Gly Val 290 295
300625PRTArtificial SequenceVH CDR1 62Ser Phe Ala Met Ser1
56317PRTArtificial SequenceVH CDR2 63Ala Ile Ser Gly Ser Gly Gly
Asn Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly645PRTArtificial
SequenceVH CDR3 64Ile Ala Phe Asp Ile1 56511PRTArtificial
SequenceVL CDR1 65Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly1 5
10667PRTArtificial SequenceVL CDR2 66Val Ala Ser Ser Leu Gln Ser1
5679PRTArtificial SequenceVL CDR3 67Leu Gln His Asn Ser Tyr Pro Phe
Thr1 568114PRTArtificial SequenceVH Amino Acid Sequence 68Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Ile Ala Phe Asp Ile Trp Gly Gln Gly
Thr Met Val Thr Val 100 105 110Ser Ser69107PRTArtificial SequenceVL
Amino Acid Sequence 69Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Val Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Phe 85 90 95Thr Phe Gly Pro
Gly Thr Lys Val Asp Ile Lys 100 10570454PRTArtificial
SequenceFull-length light chain amino acid sequecne 70Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Gln Asn Ser 20 25 30Tyr
Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Tyr Leu Tyr Ser Ser Gly Arg Thr Asn Tyr Thr Pro Ser Leu Lys
50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser
Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Arg Thr His Leu Gly Gly Phe His Tyr Gly Gly Gly
Phe Trp Phe Asp 100 105 110Pro Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro145 150 155 160Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170 175Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 180 185
190Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Pro 210 215 220Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu225 230 235 240Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp 260 265 270Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310
315 320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro 325 330 335Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425
430Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445Ser Leu Ser Pro Gly Lys 450
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